ПОЛИНУКЛЕОТИДНАЯ МОЛЕКУЛА, КОДИРУЮЩАЯ БЕЛОК-РЕГУЛЯТОР ВЫРАБОТКИ АВЕРМЕКТИНА В Streptomyces avermitilis (ВАРИАНТЫ), СОДЕРЖАЩИЕ ЕЕ ВЕКТОР КЛОНИРОВАНИЯ И ШТАММ; СПОСОБЫ СКРИНИНГА МУТАЦИИ В РЕГУЛЯТОРНОМ ГЕНЕ, ПОЛУЧЕНИЯ ГЕНЕТИЧЕСКИ МОДИФИЦИРОВАННЫХ КЛЕТОК И ПОВЫШЕНИЯ СОДЕРЖАНИЯ АВЕРМЕКТИНОВ В КУЛЬТУРЕ

Изобретение относится к области биотехнологии и может быть использовано в медико-биологической промышленности при получении авермектинов. Идентифицированы и охарактеризованы два новых гена (обозначены aveR1 и aveR2) Streptomyces avermitilis, кодирующих полипептиды, участвующие в регуляции экспрессии фермента авермектинполикетидсинтазы (РКS) и соответственно биосинтеза авермектинов. С помощью мутаций, приводящих к инактивации одного или обоих названных генов, получены культуры клеток Streptomyces avermitilis, характеризующиеся повышенным по сравнению со штаммами дикого типа уровнем выработки авермектинов. Использование изобретения позволяет повысить продуктивность штаммов стрептомицетов, применяемых в производстве антибиотиков. 8 с. и 13 з.п. ф-лы, 7 ил., 1 табл.

ПЕПТИДЫ С БОЛЬШИМ ЧИСЛОМ МОСТИКОВЫХ СВЯЗЕЙ, ВЫДЕЛЯЕМЫЕ ИЗ ACTINOMADURA NAMIBIENSIS

Группа изобретений относится к биохимии. Предложены соединения лабиринтопептинов А1, А2, или А3 формулы (I), где {A}, {B}, {C}, R-R, m и n имеют значения, указанные в формуле изобретения. Соединения получают при ферментации штаммаDSM 6313 в приемлемых условиях в культуральной среде до образования одного или нескольких соединений формулы (I). Предложены ДНК, кодирующая препролабиринтопептин А2, и ДНК, кодирующая препролабиринтопептин А1, а также препролабиринтопептины А1 и А2, пролабиринтопептины А1 и А2. Лабиринтопептины формулы (I) применяют для лечения инфекций, вызываемых грамположительными бактериями, вирусных инфекций и/или невропатической боли, вызываемой воспалением. 11 н. и 13 з. п. ф-лы, 4 табл., 20 пр.

АНТИБАКТЕРИАЛЬНЫЕ И ПРОТИВОВИРУСНЫЕ ПЕПТИДЫ ИЗ ACTINOMADURA NAMIBIENSIS

... 1. Соединение формулы (I) !! где R1 представляет собой H, C(O)-(C1-C6)алкил или C(O)-O-(C1-C6)алкил; ! R2 представляет собой OH, NH2, NH-(C1-C6)алкил, NH-(C1-C4)алкилен-фенил или NH-(C1-C4)алкилен-пиридил; ! R3 и R4 независимо друг от друга представляют собой H или OH, или R3 и R4 вместе представляют собой =O; и ! m и n независимо друг от друга равны 0, 1 или 2; ! в любой стереохимической форме, или в смеси любых стереохимических форм при любом отношении, или его физиологически переносимая соль. ! 2. Соединение формулы (I) по п.1, представленное формулой (II) ! ! 3. Соединение формулы (I) по любому из пп.1 или 2, в котором R1 представляет собой H. ! 4. Соединение формулы (I) по п.1, в котором R2 представляет собой OH. ! 5. Соединение формулы (I) по п.1, в котором R3 и R4 представляют собой H или OH, где, если R3 представляет собой OH, тогда R4 представляет собой H, или если R3 представляет собой H, тогда R4 представляет собой OH, или R3 и R4 вместе представляют собой =O. ! 6. Соединение ...

Rekombinantes Core-Streptavidin

The invention concerns a method of producing recombinant core-streptavidin, the method calling for host cells to be transformed with DNA coding for core-streptavidin, for the transformed host cells to be cultured under suitable conditions, for the transformed cells to be made to express DNA coding for core-streptavidin and for the recombinant core-streptavidin to be isolated from the host cells or the culture medium. Used as the DNA coding for core-streptavidin is DNA which has (a) the nucleotide sequence shown in SEQ ID No. 1 or (b) a nucleotide sequence which corresponds to sequence (a) for degeneracy of the genetic code.

METHODEN UND MATERIALIEN FUR GENEXPRESSION

Herstellung von Melanin.

MODIFIZIERTES AVIDIN UND STREPTAVIDIN UND METHODEN ZU IHRER VERWENDUNG

Rekombinante inaktive Core-Streptavidin Mutanten

Streptomyces aveermitilis gene directing the ratio of b2:b1

Microorganisms with increased production of clavulanic acid.

Novel bacterial genes, microorganisms and processes for improving the manufacture of 5R clavans, e.g. clavulanic acid.

Streptomyces avermitilis gene directing the ratio of B2:B1 avermectins.

The present invention relates to polynucleotide molecules comprising nucleotide sequences encoding an avec gen product which polynucleotide moleules can be used to alter the ratio or amount of class 2:1 avermectins producted in fermentation cultures of s.AVermitilis. The present invention further relates to vectors, host cells, and mutant strains of s.AVermitilis in which the avec gene has beeen inactivated, or mutated so as to change the ratio or amount of class 2:1 avermections produced.

Microorganisme with increased production of clavulanic acid

Microorganisms with increased production of clavulanic acid

Streptomyces avermitilis gene directing the ratio of B2:B1 avermectins.

Microorganisme with increased production of clavulanic acid

SHIGA TOXIN SUBUNIT B AS VECTOR FOR THE TUMOR DIAGNOSIS AND TO ARZNEIMTTELVERABREICHUNG AT GB3-EXPRIMIERENDEN TUMORS

MODIFIED AVIDIN AND STREPTAVIDIN AND METHODS TO YOUR USE

BINDING ONE PEPTIDOMIMETIKA AND THEIR USES

ADP RIBOSYLIERENDE BACTERIAL TOXINE

BICYCLI OLIGOPEPTIDE AND THEIR USE AS GLUCAGON A RECEPTOR ANTAGONIST

INTRODUCTION OF THE TRANSKRIPTION ADJUSTMENT NUCLEOTIDE SEQUENCE.

SIGNALPEPTID FOR THE EXKRETION OF PEPTIDEN IN STREPTOMYCETEN.

COLOR MARKER FOR KLONIERUNGEN INTO STREPTOMYCES LIVIDANS.

SAF POLYPEPTID, GENE, PROMOTER AND USE FOR EXPRESSION INTO STREPTOMYCES

PRODUCTION OF MELANIN.

ISOLATION AND CLEANING OF LANTIBIOTIKA

NEW POLYPEPTIDE WITH ALPHA AMYLASE RESTRAINING EFFECT, PROCEDURE FOR THEIR PRODUCTION, THEIR USE AND PHARMACEUTICAL PREPARATIONS.

REKOMBINANTES MERSACIDIN AND PROCEDURES FOR ITS PRODUCTION

PROCEDURE FOR THE CONNECTION OF A CONNECTION TO A SURFACE

PROCEDURE FOR THE REDUCTION OF THE FOAMING POWER DURING THE CULTIVATION OF A MICROORGANISM

MELANINPRODUKTION BY TRANSFORMED MICROORGANISMS

VACCINES ON THE BASIS OF STREPTOKINASE

PROCESS FOR PRODUCING FOREIGN PROTEINS IN STREPTOMYCES

Replikin peptides and uses thereof

Methods of preparing purified lipopeptides

Polypeptide having antihuman immunodeficiency virus activity, gene encoding the polypeptide and process for producing the polypeptide

Novel compounds

COMPOSITIONS, METHODS AND SYSTEMS FOR DISCOVERY OF LIPOPEPTIDES

Recombinant Mersacidin and a method for production

Methods for introducing genes into mammalian subjects

POLYPEPTIDES WITH AN ALPHA-AMLASE-INHIBITING ACTION

POLYPEPTIDES WITH AN ALPHA-AMLASE-INHIBITING ACTION

Isolated nucleic acids from micromonospora rosaria plasmid pMR2 and vectors made therefrom

STREPTOMYCES AVERMITILIS GENE DIRECTING THE RATIO OF B2:B1 AVERMECTINS

The present invention relates to polynucleotide molecules comprising nucleotide sequences encoding an aveC gene product, which polynucleotide molecules can be used to alter the ratio or amount of class 2:1 avermectins produced in fermentation cultures of S. avermitilis. The present invention further relates to vectors, host cells, and mutant strains of S. avermitilis in which the aveC gene has been inactivated, or mutated so as to change the ratio or amount of class 2:1 avermectins produced.

DNA CODING FOR POLYPEPTIDE PARTICIPATING IN BIOSYNTHESIS OF PLADIENOLIDE

GENES AND PROTEINS INVOLVED IN THE BIOSYNTHESIS OF LIPOPEPTIDES

Genes and proteins involved in the biosynthesis of lipopeptides by microorganisms, in particular the nucleic acids forming the biosynthetic locus for the A54145 lipopeptide from Streptomyces fradiae and the A54145-like lipopeptide from Streptomyces refuineus. These nucleic acids can be used to make expression constructs and transformed host cells for the production of lipopeptides. The genes and proteins allow direct manipulation of lipopeptides and related chemical structures via chemical engineering of the proteins involved in the biosynthesis of A54145.

HIGH PURITY LIPOPEPTIDES, LIPOPEPTIDE MICELLES AND PROCESSES FOR PREPARING SAME

The invention discloses highly purified daptomycin and to pharmaceutical compositions comprising this compound. The invention discloses a method of purifying daptomycin comprising the sequential steps of anion exchange chromatography, hydrophobic interaction chromatography and anion exchange chromatography. The invention also discloses a method of purifying daptomycin by modified buffer enhanced anion exchange chromatography. The invention also discloses an improved method for producing daptomycin by fermentation of Streptomyces roseosporus. The invention also discloses high pressure liquid chromatography methods for analysis of daptomycin purity. The invention also discloses lipopeptide micelles and methods of making the micelles. The invention also discloses methods of using lipopeptide micelles for purifying lipopeptide antibiotics, such as daptomycin. The invention also discloses using lipopeptide micelles therapeutically.

NEW EXPRESSION SYSTEM FROM RHODOCOCCUS

Title: The present invention provides an isolated polynucleotide comprising the kstD promoter from Rhodococcus erythropolis. The polynucleotide can very advantageously be used as a controllable transcription activator. Said controlling function can be provided by providing said isolated polynucleotide with a nucleotide sequence encoding a transcription regulator of said promoter. In the present invention, such a transcription regulator may be externally induced, such as by introduction of steroidal compounds. In an alternative embodiment of the present invention the isolated polynucleotide may comprise the kstR gene or a homologue or a functional part thereof as the transcription regulator of the kstD promoter.

REDUCING BRANCHING AND ENHANCING FRAGMENTATION IN CULTURING FILAMENTOUS MICROORGANISMS

The invention relates to the field of microorganisms and in particular to the culturing of microorganisms. The invention provides means and methods for enhancing the culturing properties of filamentous microorganisms, in particular filamentous fungi. The means and methods according to the invention comprise reducing the branching and/or enhancing the fragmentation of said filamentous microorganisms, whereby their liquid culturing properties are improved. In one embodiment this is achieved by providing said microorganisms with activity capable of enhancing fragmentation and/or reducing branching such as the activity which in e.g. Streptomyces griseus is encoded by ssgA.

PROCEDE D'EXTRACTION D'UN INHIBITEUR DE L'(ALPHA)-AMYLOSE A PARTIR DE STREPTOMYCETES

GENES AND PROTEINS FOR THE BIOSYNTHESIS OF POLYKETIDES

Genes and proteins involved in the biosynthesis of polyketides by microorganisms, including the genes and proteins forming the biosynthetic loci for the polyketide dorrigocin from Streptomyces platensis subsp. rosaceus and the polyketide lactimidomycin from Streptomyces amphibiosporus. The genes and proteins allow direct manipulation of dorrigocin, lactimidomycin and related chemical structures via chemical engineering of the enzymes involved in the biosynthesis of dorrigocin and lactimidomycin.

METHODS AND MATERIALS RELATING TO GENE EXPRESSION

An expression cassette for expressing a nucleic acid of interest derived from the regulatory region of the methylenomycin gene cluster of the SCP1 plasmid of Streptomyces coelicolor A3(2), and related materials and methods.

COMBINATION OF REVERSIBLE AND IRREVERSIBLE CELL LABELING FOR ANALIZING RECEPTOR-LIGAND KOFF RATE

The invention relates to a method of determining the dissociation rate constant (koff) of a receptor molecule R on a target cell using a combination of reversible and irreversible cell labeling. The invention further relates to a cell comprising such a receptor molecule R, wherein the cell has bound to it such a combination of cell labeling. The invention further relates to a kit and an apparatus useful in performing the methods of the invention. The invention further relates to a method of isolation a high-avidity T cell.

CLONING OF THE STREPTOMYCES AVERMITILIS GENES FOR GLYCOSYLATION OF AVERMECTIN AGLYCONES

A cluster of genes involved in the synthesis and/or addition of oleandrose to avermectin aglycones has been cloned. A 11-kb PstI clone complemented 28 avermectin glycosylation mutants in seven complementation classes. Sequencing of an 10-kb region identified 9 ORFs and an additional partial ORF. Eight of the ORFs were correlated to the seven glycosylation complementation classes. Sequence comparison to Genbank databases identified 6 genes: dTDP- glucose synthase; dTDP-glucose 4,6 dehydrase; dTDP-4-keto-hexose reductase; dTDP-hexose 3,5 epimerase; dTDP-hexose 3' O-methylase; and an avermectin aglycone-dTDP-oleandrose glycosyltransferase. The ninth ORF was essential for biosynthesis of the avermectin aglycones. The partial ORF encoded part of an avermectin polyketide synthase module 7.

POLYPEPTIDES INVOLVED IN SPIRAMYCIN BIOSYNTHESIS, NUCLEOTIDE SEQUENCES ENCODING SAID POLYPEPTIDES AND USES THEREOF

... ²²²La présente invention concerne l'isolement et l'identification de nouveaux ²gènes de la voie de biosynthèse des spiramycines et de nouveaux polypeptides ²impliqués dans cette biosynthèse. L'invention est également relative à un ²procédé de production de ces polypeptides. Elle est également relative à ²l'utilisation de ces gènes dans le but d'augmenter les taux de production et ²la pureté de la spiramycine produite. L'invention concerne notamment un ²microorganisme produisant de la spiramycine I mais ne produisant pas de ²spiramycine II et III et l'utilisation d'un tel micoorganisme. L'invention a ²également trait à l'utilisation des gènes de la voie de biosynthèse des ²spiramycines pour la construction de mutants pouvant conduire à la synthèse de ²nouveaux antibiotiques ou à des formes dérivées de spiramycines. L'invention ²concerne encore les molécules produites grâce à l'expression de ces gènes et ²des compositions pharmacologiquement actives d'une molécule produite grâce à ²l'expression ...

CIRCULARLY PERMUTED BIOTIN BINDING PROTEINS

Circularly permuted proteins are described wherein the natural termini of the polypeptide are joined and the resulting circular protein is opened at another point to create new C- and N-termini. The resulting protein exhibits some altered characteristic such as reduced substrate binding, for example. Fusion proteins can be made from the circularly permuted protein by attaching the second polypeptide to these newly created termini. These fusion proteins will have altered properties from a fusion protein made by attaching the second polypeptide to the natural termini. For example, the second peptide or protein can be attached at a position where it is more accessible to its substrate or intended target. In the preferred embodiment, the base polypeptide is streptavidin. Circular permutation of streptavidin results in a circularly permuted biotin binding protein. In one embodiment, a flexible polypeptide loop important for the binding of biotin was opened by creation of the circularly permuted ...

DNA CODING FOR POLYPEPTIDE PARTICIPATING IN BIOSYNTHESIS OF PLADIENOLIDE

The present invention provides polypeptides that participate in the biosynthesis of the pladienolide macrolide compounds, DNA that encodes these polypeptides and variants of this DNA, transformants that maintain all or a portion of this DNA or variant thereof, and a method of producing the pladienolide macrolide compounds using these transformants. More particularly, it provides an isolated pure DNA that contains at least one region encoding a polypeptide that participates in pladienolide biosynthesis; polypeptide encoded by this DNA; a self-replicating or integrated-replicating recombinant plasmid carrying this DNA; a transformant maintaining this DNA; and a method of producing a pladienolide, characterized by culturing this transformant on culture medium and collecting pladienolide from this culture medium.

IDENTIFICATION OF TRANSGLUTAMINASE SUBSTRATES AND USES THEREFOR

According to one aspect, the present disclosure provides a method of identifying a substrate of a transglutaminase using a peptide array comprising a plurality of peptides. The method includes the steps of contacting the peptides in the peptide array with the transglutaminase, allowing the transglutaminase to bind to the peptides, and identifying the substrate of the transglutaminase.

METHODS FOR NON-COVALENT FC-DOMAIN-CONTAINING PROTEIN DISPLAY ON THE SURFACE OF CELLS AND METHODS OF SCREENING THEREOF

The present invention provides methods for the non-covalent surface display of proteins of interest (POI), in particular for Fc-domain containing proteins such as antibodies. The inventive method may be used to screen and select proteins of interest of a desired phenotype. The present invention further discloses polynucleotides and proteins and methods of producing the same, which may be used in carrying out the inventive method.

STREPTAVIDIN MUTEINS AND METHODS OF USING THEM

The invention concerns novel streptavidin muteins. In one embodiment such a mutein (a) contains at least one mutation in the region of the amino acid positions 115 to 121 with reference to the amino acid sequence of wild type streptavidin as set forth at SEQ ID NO: 15 and (b) has a higher binding affinity than each of (i) a streptavidin mutein that comprises the amino acid sequence Val44 -Thr45- Ala46-Arg47 (SEQ ID NO: 98), or (ii) a streptavidin mutein that comprises the amino acid sequence He44-Gly45- Ala46-Arg47 (SEQ ID NO: 99) at amino acid positions 44 to 47, or (iii) wild type-streptavidin (SEQ ID NO: 15) for peptide ligands comprising the amino acid sequence Trp-Ser-His-Pro-Gln- Phe-Glu-Lys (SEQ ID NO: 100).

ANTIVIRAL ANTIBIOTIC BU-4724V AND PREPARATION THEREOF

There is provided a new antiviral antibiotic designated herein as BU-4724V which is produced by fermentation of a BU-4724V-producing strain of a new microorganism, Streptomyces sp. ATCC 55290. Antibiotic BU-4724V is recovered and purified from the fermentation broth by use of extraction and chromatography techniques. BU-4724V has been found to have some antibacterial activity and inhibits the growth of HIV and HSV viruses.

DNA-SEQUENCE CONFERRING RESISTANCE TO THE ANTIBIOTIC THERMORUBIN

The present invention relates to a new DNA fragment or a sub-fragment thereof which is capable of conferring resistance to Thermorubin, upon introduction into a suitable microbial host by a proper vector.

GENES ENCODING BRANCHED-CHAIN ALPHA-KETOACID DEHYDROGENASE COMPLEX FROM STREPTOMYCES AVERMITILIS

The present invention relates to novel DNA sequences that encode for the branched-chain alpha-ketoacid dehydrogenase complex of an organism belonging to the genus Streptomyces and to novel polypeptides produced by the expression of such sequences. It also relates to novel methods of enhancing the production of natural avermectin, of producing a Streptomyces avermitilis bkd mutant and of producing novel avermectins through fermentation.

Verfahren zur Herstellung eines neuen Antibiotikums

[...] DE [...] D'UN [...] DE [...][...] - [...].

OF NEOCARZINOSTATINE DERIVATIVES AND PROCESS FOR THEIR PREPARATION.

GENE CLUSTER FOR BIOSYNTHESIS OF GRISELIMYCIN AND METHYLGRISELIMYCIN

КЛАСТЕР ГЕНОВ БИОСИНТЕЗА ГРИЗЕЛИМИЦИНА И МЕТИЛГРИЗЕЛИМИЦИНА

Настоящее изобретение относится к кластеру генов и генам, входящим в состав кластера генов, которые вовлечены в биосинтез гризелимицина и метилгризелимицина, и к применению кластера генов, входящих в него генов, и белков, кодируемых ими, для получения агентов-антибиотиков.

ПОЛИПЕПТИД ДЛЯ ОЖИВЛЕНИЯ ПОКОЯЩИХСЯ, ПОГИБАЮЩИХ ИЛИ ЛАТЕНТНЫХ БАКТЕРИАЛЬНЫХ КЛЕТОК, КОДИРУЮЩАЯ ЕГО НУКЛЕИНОВАЯ КИСЛОТА И СПОСОБЫ ИСПОЛЬЗОВАНИЯ ПОЛИПЕПТИДА И НУКЛЕИНОВОЙ КИСЛОТЫ

Настоящее изобретение относится к выделенному полипептиду, стимулирующему оживление покоящихся, погибающих или латентных бактериальных клеток, к кодирующей его нуклеиновой кислоте, к вектору, содержащему эту нуклеиновую кислоту, к антителу или производному антитела, специфичному в отношении выделенного полипептида, к фармацевтической композиции, содержащей указанный полипептид, нуклеиновую кислоту и вектор, и к их применению в терапии, например в иммунотерапии, диагностике или профилактике инфекции, а также к рецептору выделенного полипептида. Также настоящее изобретение включает диагностический набор для выявления оживших или неоживших покоящихся, погибающих или латентных бактериальных клеток, культуральную среду и среду для транспортировки бактериальных клеток, которые в своем составе содержат полипептид.

BACTERIAL PHEROMONES AND THEIR FIELD

Gene cluster for biosynthesis of griselimycin and methylgriselimycin

The present invention refers to the gene cluster and genes comprised by the gene cluster which are involved in the biosynthesis of griselimycin and methylgriselimycin and to the use of the gene cluster, genes comprised thereby and proteins encoded thereby for the production of antibiotic agents.

NEW INHIBITOR OF GLYCO-HYDROLASES AND SA PREPARATION BY CULTURE Of STREPTOMYCES

INHIBITEUR D'A-AMYLASE, SON PROCEDE DE PREPARATION A PARTIR D'UN STREPTOMYCETE ET SON APPLICATION EN THERAPEUTIQUE

L'invention a pour objet un inhibiteur peptidique de glycosidehydrolase, caractérisé en ce qu'il a un poids moléculaire de 5 000 à 10 000, un maximum d'absorption en lumière UV à 276 nm, un point isoélectrique de 4,4 et la composition suivante en acides aminés : Acide asparaginique 5-6 Isoleucine 1-2 Thréonine 5-6 Leucine 3-4 Serine 3-5 Tyrosine 4-5 Acide glutaminique 5-6 Phénylalanine 0-2 Proline 2-3 Histidine 1-2 Glycine 5-6 Lysine 0-1 Alanine 5-6 Arginine 2-3 Cystéine 3-4 Tryptophane 1-2 Valine 5-6 Cet inhibiteur est utilisable notamment comme régulateur du taux de sucre dans le sang.

Recombinant core-streptavidin

NOVEL METHOD OF PROTEIN PURIFICATION

HERBICIDE-METABOLIZING CYTOCHROME P450 MONOOXYGENASE

A method for metabolizing PPO-inhibiting herbicides, comprising contacting the PPO-inhibiting herbicide with a CytP450 monooxygenase, is provided. Transgenic plants comprising the CytP450 monooxygenase polypeptide of the invention, as well as methods for producing the same are provided. A method for controlling weeds, employing the CytP450 monooxygenase polypeptide of the invention, a method for producing a polypeptide with enhanced ability to metabolize PPO-inhibiting herbicide, and a method for screening for a microorganism capable of metabolizing PPO-inhibiting herbicide are also provided.

ACTINOBACILLUS PLEUROPNEUMONIAE RECOMBINANT TOXIN PROTEIN AND APPLICATION THEREOF

The present invention provides an actinobacillus pleuropneumoniae recombinant toxin protein, comprising at least one antigenic determinant of an actinobacillus pleuropneumoniae toxin protein; and when there is a plurality of antigenic determinants, connexons can exist among the antigenic determinants. A recombinant protein may comprise at least one amino acid sequence of a complement fragmentation C3d, and connexons can exist between antigenic determinants and the amino acid sequences of the C3d. The present invention also provides a nucleotide sequence for encoding a protein, and an immune composition containing the protein.

5R CLAVAM PRODUCING MICROORGANISMS WITH DELETED LAT AND CVM GENES

The invention relates to organisms producing increased levels of clavulanic acid and other 5R clavams, including those with strong β-lactamase inhibitory activity, in combination with low or undetectable levels of 5S clavams.

i(STREPTOMYCES AVERMITILIS) GENE DIRECTING THE RATIO OF B2:B1 AVERMECTINS

The present invention relates to polynucleotide molecules comprising nucleotide sequences encoding an aveC gene product, which polynucleotide molecules can be used to alter the ratio or amount of class 2:1 avermectins produced in fermentation cultures of i(S. avermitilis). The present invention further relates to vectors, host cells, and mutant strains of i(S. avermitilis) in which the aveC gene has been inactivated, or mutated so as to change the ratio or amount of class 2:1 avermectins produced.

BIOTIN VARIANT, STREPTAVIDIN MUTANT, AND USES THEREOF

The problem addressed by the present invention is: to provide a streptavidin mutant having a reduced affinity to natural biotin; and furthermore to provide a biotin variant that has a high affinity to the streptavidin mutant having reduced affinity to natural biotin. The present invention provides a compound comprising a biotin variant dimer, a streptavidin mutant, and uses thereof.

GENE CLUSTER FOR RABELOMYCIN BIOSYNTHESIS AND ITS USE TO GENERATE COMPOUNDS FOR DRUG SCREENING

This invention relates to the gene cluster for angucycline biosynthesis, derived from Streptomyces, and use of the genes therein to obtain antibiotics for drug screening.

Method of producing a fibrin monomer using a biotinylated enzyme and immobilized avidin

Compositions and methods for avidin immobilized on an inert support material, e.g. agarose, are disclosed. The compositions have high activity levels of avidin and may further include a bulking agent, e.g., maltose, and a protectant to maintain the stability and integrity of the avidin agarose during lyophilization and terminal sterilization processes. These compositions have applicability in any instance where avidin agarose and/or the avidin/biotin technology are useful. In particular, the present compositions are useful in an enzyme capture system to prepare fibrin monomer useful for fibrin sealants. The fibrin is prepared by subjecting a fibrinogen-containing composition to a biotinylated enzyme to convert the fibrinogen in the composition to a corresponding fibrin monomer. A fibrin monomer/biotinylated enzyme mixture is formed. A material comprising an avidin/inert support composition having 1000 biotin binding units of activity per gram of the composition, a bulking agent and a protectant ...

Recombinant core streptavidin

The present invention concerns a process for the isolation of recombinant core streptavidin in which host cells are transformed with a DNA coding for core streptavidin, the transformed host cells are cultured under suitable conditions, the DNA coding for core streptavidin is expressed and the recombinant core streptavidin is isolated from the host cells or the culture medium, wherein a DNA coding for core streptavidin is used which has (a) the nucleotide sequence shown in SEQ ID NO. 1 or (b) a nucleotide sequence corresponding to the nucleotide sequence (a) within the scope of the degeneracy of the genetic code.

Replikin peptides and antibodies therefore

The present invention provides a new class of peptides related to rapid replication and their use in diagnosing, preventing and treating disease.

Compositions for lipid matrix-assisted chemical ligation

The present invention relates to methods and compositions for lipid matrix-assisted chemical ligation and synthesis of membrane polypeptides that are incorporated in a lipid matrix. The invention is exemplified in production of a prefolded membrane polypeptide embedded within a lipid matrix via stepwise chemoselective chemical ligation of unprotected peptide segments, where at least one peptide segment is embedded in a lipid matrix. Any chemoselective reaction chemistry amenable for ligation of unprotected peptide segments can be employed. Suitable lipid matrices include liposomes, micelles, cell membrane patches and optically isotropic cubic lipidic phase matrices. Prefolded synthetic and semi-synthetic membrane polypeptides synthesized according to the methods and compositions of the invention also permit site-specific incorporation of one or more detectable moieties, such as a chromophore, which can be conveniently introduced during synthesis. The methods and compositions of the invention ...

Metal radionuclide labeled proteins for diagnosis and therapy

Protein, ligand and anti-ligand conjugated chelated metal radionuclides are provided for use in vivo. Intermediates are provided for preparing the compositions efficiently.

Biosensor system for the rapid detection of analytes

A system, device, and method for rapid detection of analytes that includes a living, engineered biosensor cell that is typically a component of the mammalian immune system; a reporter protein that is engineered into and expressed by the living, engineered biosensor cell, wherein the reporter protein emits a detectable signal in response to certain predetermined changes in the cytosol of the living, engineered cell; a signal transduction pathway expressed by the living, engineered biosensor cell, wherein the signal transduction pathway controls a biological process within the cytosol of the living, engineered biosensor cell, and wherein the biochemical process, when it occurs, causes the reporter protein to emit a detectable signal; at least one type of detector molecule that is adapted to bind to a specific analyte; at least one analyte that binds to the detector molecule that is specific to that analyte; a plurality of non-antibody signal transducing elements that are either expressed ...

Shiga toxin B-subunit as a vector for tumor diagnosis and drug delivery to Gb3 expressing tumors

The invention relates to new compounds for cancer therapy or diagnosis and to the use of a non-toxic B subunit of Shiga toxin mutant as a vector for diagnostic products or drugs in over-expressing Gb3 receptor cells, such compounds having the following formula: STxB-Z(n)-Cys-Y(m)-T wherein-STxB is the Shiga Toxin B subunit or a functional equivalent thereof, -Z(n) wherein n is 0 or 1, Z is an amino-acid residue devoid of sulfydryl groups, or is a polypeptide, -T is a molecule linked by a covalent bound to the S part of Cys, selected from: agents for in vivo diagnosis, cytotoxic agents, prodrugs, or enzymes for the conversion of a prodrug to a drug, - Y(m) wherein m is 0 or 1, Y is a linker between T and Cys, which is either cleavable or not cleavable for the release of T after the internalization of the hybrid compound into cells.

Monovalent streptavidin compositions

The invention relates, in part, to monovalent streptavidin compositions. The invention also relates to methods of preparing and using monovalent streptavidin compositions. In some aspects of the invention, the compositions are monovalent streptavidin with a single femtomolar biotin-binding site.

Isolated DNA comprising one or more genes specific for 5S clavam biosynthesis, vectors comprising such DNA and Streptomyces hosts capable of improved clavulanic acid production

Novel bacterial genes, microorganisms and processes for improving the manufacture of 5R clavams, eg. clavulanic acid.

Actinomycetes polynucleotides

Novel polynucleotides derived from microorganisms belonging to actinomycetes and fragments thereof, polypeptides encoded by the polynucleotides and fragments thereof, polynucleotide arrays comprising the polynucleotides and fragments thereof, recording media in which the nucleotide sequences of the polynucleotide and fragments thereof have been recorded which are readable in a computer, and use of them.

METHOD OF BINDING A COMPOUND TO A SURFACE

Polypeptide inhibitor of the activity of alpha-amylase, process for its preparation, its use, and pharmaceutical preparations

Cyclic polypeptides of the formula I I in which Z, S, Y and AS have the meanings given, a process for their preparation and their use are described. The compounds have an alpha -amylase-inhibiting action.

MELANIN PRODUCTION BY TRANSFORMED MICROORGANISMS

The present invention is directed to a process for producing melanins, their precursors and their analogs, hereinafter referred to generically as melanins. According to the invention, melanins are produced in amounts greater than about 0.2 grams dry weight per liter of growth medium. The enhanced production of melanin can be achieved by manipulating the constituents of the growth medium, and/or attenuating fermentation conditions, and/or by genetically engineering microorganism to produce melanins, and/or mutating the microorganisms.

Process for production of stranger protein in streptomycetes

INTERFERENCE SUPPRESSION AGENT FOR USE IN IMMUNO ASSAYING

Recombinant mersacidin and a method for production

Reactive derivatives of sulforhodamine 101 with enhanced hydrolytic stability

The invention describes reactive dyes having an alkyl spacer attached via a sulfonamide bond to a sulforhodamine 101 fluorophore, and a variety of conjugates prepared therefrom which are useful in the detection of a complementary member of a binding pair. The increased length of the covalent linkage due to the alkyl spacer results in dye-conjugates having a number of surprisingly advantageous properties relative to previous sulforhodamine 101-labelled conjugates, including enhanced solubility and increased fluorescence. The reactive dyes of the present invention are more stable than the known compound sulforhodamine 101 sulfonyl chloride. Novel reactive dyes are described for selective modification of groups other than amines, including thiols and photoreactive derivatives. The reactive dyes are of the formula or of the formula wherein R 2 is H, C 1 -C 6 alkyl or C 1 -C 6 acyl, is selected from certain reactive groups and n is an integer which, depending on R, may be from 2 to 8 or from ...

AMYLASE INHIBITOR FROM A STREPTOMYCETE AND PROCESS FOR ITS PREPARATION

Process for the production of arachidonic acid and/or eicosapentaenoic acid

The present invention relates to a new process for the production of arachidonic acid and/or eicosapentaenoic acid in plants through the co-expression of a Δ-12-/Δ-15-desaturase, Δ-9-elongase, Δ-8-desaturase and a Δ-5-desaturase and a process for the production of lipids or oils having an increased content of unsaturated fatty acids, in particular ω-3 and ω-6 fatty acids having at least two double bonds and a 18 or 20 carbon atom chain length. Preferably the arachidonic acid and eicosapentaenoic acid are produced in at least a 1:2 ratio. The invention furthermore relates to the production of a transgenic plants, preferably a transgenic crop plant, having an increased content of arachidonic acid and/or eicosapentaenoic acid, oils or lipids containing C 18 - or C 20 -fatty acids with a double bond in position Δ5, 8, 9, 11, 12, 14, 15 or 17 of the fatty acid produced, respectively due to the expression of the Δ-12-/Δ-15-desaturase, of the Δ-9-elongase, of the Δ-8-desaturase and of the Δ-5-desaturase in the plant. The expression of the inventive Δ-12-/Δ-15-desaturase leads preferably to linoleic acid and linolenic acid as products having a double bond in the position

Method for obtaining a singular cell model capable of reproducing in vitro the metabolic idiosyncrasy of humans

The method is based on the use of expression vectors coding for the sense and anti-sense mRNA of the Phase I and Phase II drug biotransformation enzymes showing a greatest variability in humans for transforming cells expressing reductase activity. Such vectors can modulate (increase or decrease) the individualized expression of an enzyme without affecting the other enzymes. This singular cell model can reproduce in vitro the metabolic idiosyncrasy of humans. It is applicable in the study of development of new drugs, specifically in the study of metabolism, potential idiosyncratic hepatotoxicity, medicament interactions, etc., of new drugs.

Extracellular yaluronidase from streptomyces koganeiensis

The invention relates to Streptomyces koganeiensis ATCC 31394 hyaluronidase having molecular weight of 21.6 kDalton, which has hyaluronidase activity and stability markedly higher than those of the hyaluronidase obtained from such microorganism to date. The invention further relates to a process for the isolation and purification of said hyaluronidase and its use for the preparation of pharmaceutical compositions or as an analytical reagent.

Subtilases

The present invention relates to novel JP170 like subtilases from wild-type bacteria, hybrids thereof and to methods of construction and production of these proteases. Further, the present invention relates to use of the claimed subtilases in detergents, such as a laundry or an automatic dishwashing detergent.

Nucleic acid molecule

The invention relates to an isolated nucleic acid molecule encoding a polypeptide capable of producing a triterpenoid hydrocarbon. The invention also relates to the encoded polypeptide, a vector comprising the nucleic acid molecule, a recombinant non-human organism comprising the nucleic acid molecule, and to methods of producing a triterpenoid hydrocarbon or an intermediate of biofuel using the nucleic acid molecule, polypeptide or recombinant organism.

Sequentially arranged streptavidin-binding modules as affinity tags

The present invention relates to sequentially arranged streptavidin-binding binding modules which may in particular be used as affinity tags. The affinity tags comprise at least two individual modules capable of mediating avidic binding to streptavidin.

Filamentous fungi and methods for producing trichodiene from lignocellulosic feedstocks

The present invention relates to the production of a C-15 fuel from lignocellulosic or other feedstock. Specifically at least double mutant of filamentous fungi having the isoprenoid pathway results in production of trichodiene in commercial quantities. One embodiment of the invention relates to producing the fuel at the site of the lignocellulosic feedstock to reduce costs of shipping the feedstock.

Method for a rational cell culturing process

Biopharmaceutical process development with recombinant protein producing mammalian cells has realized a tremendous increase in both productivity and product yields in the past years. These achievements can be mainly attributed to the advancements in cell line development, media, and process optimization. Only recently, genome-scale technologies enable a system-level analysis to elucidate the complex biomolecular basis of protein production in mammalian cells promising an increased process understanding and the deduction of knowledge-based approaches for further process optimization. The present invention describes a method for a rational cell culturing process using such a knowledge-based approach.

Transgenic animal overexpressing luciferase and preparation method thereof

The present disclosure provides a vector comprising a promoter and a luciferase gene having a nucleic acid sequence as disclosed in SEQ ID NO: 1; a fertilized egg transformed with the present vector; and a transgenic non-human animal overexpressing a luciferase gene from the vector and a method for preparing it. The vector and the animal of the present disclosure have a high expression rate for the luciferase gene, which confers high sensitivity for detection and thus useful for imaging analysis in a variety of research areas.

Process for preparation of tacrolimus

Genetically modified strains of Streptomyces tsukubaensis ( S. tsukubaensis ) can be used for an improved fermentation process for the preparation of tacrolimus or a salt or derivative thereof by cultivation of these genetically modified strains. Novel genes allowing biosynthesis of allylmalonyl-CoA can be used for polyketide production with allylmalonyl extender unit.

METHOD FOR PRODUCING 1,5-PENTANEDIAMINE

A method of producing 1,5-pentanediamine includes culturing coryneform bacterium having a gene encoding lysine decarboxylase in its chromosome, which coryneform bacterium maintains lysine decarboxylase activity of not less than 50 mU/mg protein during culturing and the gene encoding lysine decarboxylase is linked downstream of a promoter that functions during the logarithmic growth phase. 1. A method of producing 1 ,5-pentanediamine comprising culturing a coryneform bacterium having a gene encoding lysine decarboxylase in its chromosome , which coryneform bacterium maintains lysine decarboxylase activity of not less than 50 mU/mg protein during culturing.2. A method for producing 1 ,5-pentanediamine comprising culturing a coryneform bacterium having a gene encoding lysine decarboxylase in its chromosome , said gene encoding lysine decarboxylase being linked downstream of a promoter that functions during the logarithmic growth phase.3. The method according to claim 2 , wherein said promoter is divIVA gene promoter.4. The method according to claim 2 , wherein said promoter is a promoter selected from (A) to (D) below:(A) a promoter having a base sequence shown in SEQ ID NO:2;(B) a promoter having the same base sequence as the base sequence shown in SEQ ID NO:2 except that one or several bases are substituted, deleted, inserted and/or added;(C) a promoter that entirely or partially hybridizes under stringent conditions with the promoter having the base sequence shown in SEQ ID NO:2 or a complementary strand thereof; and(D) a promoter having a base sequence having a sequence identity of not less than 80% to the base sequence shown in SEQ ID NO:2.5E. coli.. The method according to claim 1 , wherein said gene encoding lysine decarboxylase is a gene derived from6. The method according to claim 1 , wherein said gene encoding lysine decarboxylase is a gene selected from (A) to (D) below and encodes a protein having lysine decarboxylase activity:(A) a gene having a base ...

Isolated luciferases and the use thereof

The invention relates to the nucleotide and amino acid sequences, and to the activity and use, of the luciferases LuAL, Lu164, Lu16, Lu39, Lu45, Lu52 and Lu22.

VECTORS AND SEQUENCES FOR THE TREATMENT OF DISEASES

The present invention provides new sequences, gene constructions, vectors and pharmaceutical compositions for the treatment of diseases and specially, for the treatment of mucopolysaccharidoses. 126-. (canceled)27. An isolated nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 1 that codifies for the protein SEQ ID NO: 2.28. An isolated nucleotide sequence wherein said sequence is SEQ ID NO: 1.29. A gene construction comprising the nucleotide sequence according to .30. The gene construction according to claim 29 , wherein said gene construction is an expression vector.31. An expression vector as defined in claim 30 , wherein said vector is an adeno-associated vector.32. The expression vector according to claim 31 , wherein the serotype of the adeno-associated vector is 1 claim 31 , 2 claim 31 , 5 claim 31 , 7 claim 31 , 8 or 9.33. The expression vector according to claim 32 , wherein the serotype is 9.34. The expression vector according to claim 31 , comprising a CAG promoter operably linked to SEQ ID NO: 1.35. The expression vector according to claim 34 , wherein the expression vector is a plasmid vector AAV9-CAG-co-hu-SFMD and is serotype 9.36. The plasmid vector pAAV-CAG-co-hu-SFMD according to with accession number DSM 24817.37. The expression vector as defined in comprising a hAAT promoter operably linked to SEQ ID NO: 1.38. The expression vector according to claim 37 , wherein the expression vector is a plasmid vector pAAV-hAAT-co-hu-SFMD and is serotype 8 or 9.39. The plasmid vector pAAV9-hAAT-co-hu-SFMD according to wherein the vector is serotype 9.40. A pharmaceutical composition comprising the nucleotide sequence according to .41. A pharmaceutical composition comprising the gene construction according to .42. A pharmaceutical composition comprising the expression vector according to .4342. A method for increasing the sulfamidase activity in the body comprising administering to a subject in need thereof the pharmaceutical composition ...

Heterologous Expression of Urease in Anaerobic, Thermophilic Hosts

The invention is directed to the heterologous expression of urease in anaerobic thermophilic hosts, such as , and other related genera. For example, the anaerobic thermophilic host can be . The host cells express the catalytic subunits of the urease enzyme together with the accessory proteins ureDEFG that facilitate protein folding and nickel activation. The invention further relates to the use of urea as a nitrogen source in the growth of microorganisms involved in consolidated bioprocessing systems. 1. A recombinant anaerobic , thermophilic host cell comprising one or more heterologous polynucleotides encoding (a) at least two catalytic subunits of a urease enzyme and (b) four urease accessory proteins.2ThermoanaerobacterThermoananerbacterium.. The recombinant anaerobic claim 1 , thermophilic host cell of claim 1 , wherein said host is of the genus or3T. saccharolyticum.. The recombinant anaerobic claim 2 , thermophilic host cell of claim 2 , wherein said host is4. The recombinant anaerobic claim 1 , thermophilic host cell of claim 1 , wherein said host heterologously expresses three catalytic subunits of a urease enzyme.5. The recombinant anaerobic claim 1 , thermophilic host cell of claim 1 , wherein said catalytic subunits are selected from group consisting of urease α claim 1 , β and γ.6. The recombinant anaerobic claim 1 , thermophilic host cell of claim 1 , wherein said accessory proteins are urease D claim 1 , E claim 1 , F claim 1 , and G.7. The recombinant anaerobic claim 1 , thermophilic host cell of claim 1 , wherein said urease catalytic subunits and accessory proteins are derived from an anaerobic claim 1 , thermophilic organism that natively expresses the urease enzyme.8Clostridium thermocellum.. The recombinant anaerobic claim 1 , thermophilic host cell of claim 1 , wherein said urease catalytic subunits and accessory proteins are derived from9. The recombinant anaerobic claim 1 , thermophilic host cell of claim 1 , wherein nickel in the host cell ...

Methods for Incorporating Unnatural Amino Acids in Eukaryotic Cells

The invention relates to a nucleic acid comprising a nucleotide sequence encoding a tRNA orthogonal to a eukaryotic cell, said nucleotide sequence operably linked to a promoter capable of directing transcription by eukaryotic RNA polymerase III. The invention also relates to methods for incorporating unnatural amino acids in eukaryotic cells using same. 1. A nucleic acid comprising a nucleotide sequence encoding a tRNA orthogonal to a eukaryotic cell , said nucleotide sequence operably linked to a promoter capable of directing transcription by eukaryotic RNA polymerase III.2. A nucleic acid according to wherein said orthogonal tRNA is tRNA.3. A nucleic acid according to wherein said promoter is claim 1 , or is derived from claim 1 , the eukaryotic U6 promoter.4. A nucleic acid according to wherein said eukaryotic cell is a yeast cell and wherein the tRNAcomprises sequence at positions 3 and 70 which do not form a 3-70 base pair.5. A nucleic acid according to wherein the tRNAcomprises adenosine at position 3.6Saccharomyces cerevisiae.. A nucleic acid according to wherein the yeast is7. A nucleic acid according to wherein the promoter comprises A and B box consensus sequences.8. A nucleic acid according to wherein the promoter comprises the yeast sequence encoding tRNA.9. A nucleic acid according to wherein the tRNAis tRNA.10M. mazei. A nucleic acid according to wherein the sequence encoding tRNAcomprises the tRNAsequence.11M. barkeri. A nucleic acid according to wherein the sequence encoding tRNAcomprises the tRNAsequence having a G3A substitution.12. An expression system comprising a nucleic acid according to ; said system further comprising a nucleotide sequence encoding a PylRS capable of aminoacylating the tRNA.13M. barkeri. An expression system according to wherein the PylRS comprises PylRS or AcKRS or TfaKRS or PcKRS.14. A eukaryotic cell comprising a nucleic acid according to .15S. cerevisiae.. The eukaryotic cell according to claim 14 , wherein the eukaryotic ...

Polypeptides and biosynthetic pathways for the production of monatin and its precursors

Methods and compositions that can be used to make monatin from glucose, tryptophan, indole-3-lactic acid, indole-3-pyruvate, and 2-hydroxy 2-(indol-3-ylmethyl)-4-keto glutaric acid, are provided. Methods are also disclosed for producing the indole-3-pyruvate and 2-hydroxy2-(indol-3-ylmethyl)-4-keto glutaric acid intermediates. Compositions provided include nucleic acid molecules, polypeptides, chemical structures, and cells. Methods include in vitro and in vivo processes, and the in vitro methods include chemical reactions.

Method of Genome Surgery with Paired, Permeant Endonuclease Excision

The use of P2E2 constructs in genome surgery includes a cell penetration component, a DNA binding component and a restriction endonuclease. The method for performing genome surgery includes: 1. A method for performing genome surgery comprising:a) providing one or more recombinant P2E2 constructs comprising a cell penetration component, a DNA binding component and a endonuclease;b) penetrating a cell with the recombinant P2E2 protein construct;c) forming a protein product in the cell by the processes of transcription and translation or by direct introduction of the P2E2 protein construct to the cell;d) attaching the protein product of the P2E2 construct to one or more targeted genomic sequences within the cell; ande) the endonuclease of the P2E2 construct cutting both strands of the genome at target locations.2E. coli. The method of wherein the cell is penetrated by the recombinant P2E2 constructs comprising a purified P2E2 protein through a process selected from the group consisting of i) introduction to cells with a viral vector encoding the P2E2 construct claim 1 , ii) transfection of cells with the P2E2 construct using a transfection strategies and iii) application of a recombinant protein purified from claim 1 , yeast claim 1 , insect claim 1 , or mammalian cells transfected claim 1 , transformed claim 1 , or infected with a vector encoding the P2E2 construct.3. The method of wherein the cell is penetrated by one or more P2E2 proteins through a cell penetration process in which the recombinant protein is delivered by direct application or is bound to a carrier molecule and delivered.4. The method of wherein cutting of both strands is at site(s) within the genome that are within genome segments that include targeted regions that contain some base pair mismatches.5. A method for performing genome surgery comprising:a) providing a P2E2 protein comprising, a cell penetration component, a DNA binding component and a endonuclease;b) penetrating a cell with the ...

SCREENING AND ENRICHMENT SYSTEM FOR PROTEIN EXPRESSION IN EUKARYOTIC CELLS USING A TRICISTRONIC EXPRESSION CASSETTE

The present invention provides a polynucleotide and a method for screening and enriching a recombinant protein expressed in a eukaryotic cell, the polynucleotide comprising a tricistronic expression cassette comprising a) a promoter, b) a gene of interest (GOI), c) a reporter gene, d) a selection marker gene, e) an internal ribosome entry site (IRES) element, and f) a 2A element. Said method comprises a) transfecting or transducing suitable eukaryotic host cells with the polynucleotides of the present invention, b) culturing the eukaryotic host cells under conditions so as to express the protein of interest, the reporter protein and the selection marker protein in a cell culture selection medium suitable for selecting positively-transfected/transduced cells by means of said selection marker, and c) screening, sorting and/or enriching the cells expressing high levels of protein of interest by means of the reporter protein. 2. A polynucleotide according to claim 1 , wherein the order in 5′ to 3′ direction within said tricistronic expression cassette is: promoter—GOI—IRES element—reporter gene—2A element—selection marker gene.3. A polynucleotide according to claim 1 , wherein said selection marker gene encodes for a metabolic enzyme or an antibiotic drug resistance protein.4. A polynucleotide according to claim 1 , wherein the selection marker gene is P5CS gene or zeocin resistance gene.5. A polynucleotide according to claim 1 , wherein the reporter gene encodes for a fluorescent protein.6. A method for screening and enriching stable eukaryotic cells expressing high levels of a protein(s) of interest claim 1 , the method comprising{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'a) transfecting or transducing suitable eukaryotic host cells with the polynucleotide(s) according to'}b) culturing the eukaryotic host cells under conditions so as to express the protein(s) of interest, the reporter protein(s) and the selection marker protein(s) in a cell culture selection ...

METHODS AND COMPOSITIONS FOR TREATMENT OF A GENETIC CONDITION

Methods and compositions for a genetic disease are provided. 1. A non-naturally occurring fusion protein comprising a TALE DNA binding domain that binds to an endogenous BCL11A , KLF1 or globin gene and a cleavage domain , wherein the fusion protein modifies the endogenous BCL11A , KLF1 or globin gene.2. The fusion protein of claim 1 , wherein the TALE DNA binding protein binds to a sequence as shown in a single row of Table 1C or Table 3.3. The fusion protein of wherein the cleavage domain or cleavage half-domain is a wild-type domain.4. The fusion protein of claim 1 , wherein the cleavage domain or cleavage half-domain is an engineered domain.5. A polynucleotide encoding one or more proteins of .6. An isolated cell comprising one or more fusion proteins according to .7. An isolated cell comprising one or more polynucleotides according to .8. The cell of claim 7 , wherein the cell is a red blood cell (RBC) precursor cell.9. The cell of wherein the cell is a red blood cell (RBC).10. The cell of claim 8 , wherein the RBC precursor cell is a CD34+ hematopoietic stem cell.11. A kit comprising a protein according to .12. A kit comprising a polynucleotide according to .13. A method of altering globin gene expression in a cell claim 5 , the method comprising:{'claim-ref': {'@idref': 'CLM-00005', 'claim 5'}, 'introducing, into the cell, one or more polynucleotides according to , under conditions such that the one or more proteins are expressed and expression of the globin gene is altered.'}14. The method of claim 13 , wherein the proteins increase expression of a globin gene.15. The method of claim 14 , wherein the globin gene is a gamma globin or beta globin gene.16. The method of claim 13 , further comprising integrating a donor sequence into the genome of the cell.17. The method of claim 16 , wherein the donor sequence is introduced to the cell using a viral vector claim 16 , as an oligonucleotide or on a plasmid.18. The method of claim 13 , wherein the cell is a red ...

ENGINEERING THE RRM2 SUBUNIT OF RIBONUCLEOTIDE REDUCTASE TO RESIST DEGRADATION

Provided herein are isolated nucleic acids that encode a stable form of Rrm2 for the use of increasing the intracellular Rrm2 protein levels and cytosolic 2-deoxy-ATP (dATP) levels. Further provided herein are methods for treating a cardiac disease or disorder, e.g., myocardial infarction or myocardial ischemia, by administering the isolated nucleic acids, a polypeptide encoded by the isolated nucleic acids, or composition comprising the isolated nucleic acids to a subject in need thereof. 1. An isolated nucleic acid molecule encoding an Rrm2 polypeptide that , together with Rrm1 polypeptide comprises ribonucleotide reductase activity , the encoded Rrm2 polypeptide comprising a mutation that increases the intracellular level of the polypeptide as compared to wild-type Rrm2 polypeptide.2. The isolated nucleic acid of claim 1 , wherein the mutation increases the intracellular stability of the Rrm2 polypeptide relative to wild-type.3. The isolated nucleic acid molecule of claim 1 , wherein the mutation is in a ubiquitin-binding degron.4. The isolated nucleic acid molecule of claim 3 , wherein the mutation in the ubiquitin-binding degron prevents or inhibits ubiquitin-mediated degradation of its gene product.5. The isolated nucleic acid molecule of claim 1 , wherein the Rrm2 gene sequence encodes an Rrm2 polypeptide having 2 to 10 amino acid changes as compared to wild-type Rrm2 polypeptide sequence.6. The isolated nucleic acid molecule of claim 3 , wherein the ubiquitin-binding degron is encoded at nucleotides 88-96 claim 3 , 97-99 claim 3 , and/or 145-153 of SEQ ID NO: 3 or SEQ ID NO: 5.7. The isolated nucleic acid molecule of claim 1 , wherein the mutation is a mutation of a nucleotide selected from the group consisting of: 88-96 claim 1 , 97-99 claim 1 , and/or 145-153 positions of SEQ ID NO: 3 or SEQ ID NO: 5.8. The isolated nucleic acid molecule of claim 1 , wherein the sequence is selected from SEQ ID NO: 16-20.9. The isolated nucleic acid molecule of claim 1 , ...

High-affinity human ace2 construct for use in diagnosing and treating coronaviruses

Provided herein, in some aspects, are polypeptide monomers comprising an angiotensin-converting enzyme 2 (ACE2) ectodomain and an oligomerization domain. Also provided herein are oligomeric complexes comprising ACE2 monomers. Methods of using such to monomers and oligomeric complexes for the diagnosis, prevention, and treatment of viral infections such as the coronavirus are also provided.

COMPOSITIONS AND METHODS FOR PRODUCTION OF MYRCENE

Provided herein are compositions and methods for producing myrcene by culturing genetically modified microbial host cells that express a myrcene synthase and optionally a geranyl pyroplosphate synthase. Also provided herein are isolated nucleic acid molecules that encode myrcene synthase variants derived from the species myrcene synthase, which comprise one or more amino acid substitutions that improve in vivo performance of myrcene synthase in genetically modified microbial host cells. Also provided herein are isolated myrcene synthase variants that exhibit an improved activity for converting geranyl diphosphate into myrcene. 137.-. (canceled)38. A genetically modified microbial host cell comprising:{'i': 'Ocimum', 'claim-text': (i) an amino acid sequence of SEQ ID NO: 2; or', '(ii) an amino acid sequence that has at least 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%), at least about 97%, at least about 8%, at least about 99% sequence identity to SEQ ID NO: 2; and, '(a) a heterologous nucleic acid molecule encoding an species myrcene synthase that comprises(b) a heterologous nucleic acid molecule encoding a geranyl pyrophosphate synthase.39. The genetically modified microbial host cell of claim 38 , wherein the heterologous nucleic acid encodes a geranyl pyrophosphate synthase derived from a bacterium.40Streptomyces aculeolatus. The genetically modified host cell of claim 39 , wherein the geranyl pyrophosphate synthase is derived from a geranyl pyrophosphate synthase.41Streptomyces aculeolatus. The genetically modified host cell of claim 40 , wherein the geranyl pyrophosphate synthase comprises an amino acid sequence having SEQ ID NO: 7 or a variant thereof that has at least about 70% claim 40 , at least about 75% claim 40 , at least about 80% claim 40 , at least about 85% claim 40 , at least about 90% claim 40 , at least about 95% claim 40 , at least about 96% claim 40 , at least about ...

NOVEL P450-BM3 VARIANTS WITH IMPROVED ACTIVITY

The present invention provides improved P450-BM3 variants with improved activity. In some embodiments, the P450-BM3 variants exhibit improved activity over a wide range of substrates. 1. A recombinant cytochrome P450-BM3 variant having at least 90% sequence identity to a polypeptide sequence comprising the sequence set forth in SEQ ID NOS: 6 , 8 , 12 , 14 , or 16.2. The recombinant cytochrome P450-BM3 variant of claim 1 , wherein said variant oxidizes at least three organic substrates.3. The recombinant cytochrome P450-BM3 variant of claim 2 , wherein said organic substrate is selected from loratadine claim 2 , imatinib claim 2 , geftinib claim 2 , and diclofenac.4. A recombinant polynucleotide sequence encoding a recombinant cytochrome p450-BM3 variant having at least 90% sequence identity to a polynucleotide sequence comprising the sequence set forth in SEQ ID NO: 5 claim 2 , 7 claim 2 , 11 claim 2 , 13 claim 2 , or 15.5. A recombinant polynucleotide sequence encoding a recombinant cytochrome P450-BM3 variant of .6. The recombinant polynucleotide sequence of claim 4 , wherein said sequence comprises SEQ ID NO: 5 claim 4 , 7 claim 4 , 11 claim 4 , 13 claim 4 , or 15.7. An expression vector comprising the polynucleotide sequence of .8. The vector of claim 7 , wherein said polynucleotide sequence is operably linked with regulatory sequences suitable for expression of said polynucleotide sequence in a suitable host cell.9. The vector of claim 8 , wherein said host cell is a prokaryotic or eukaryotic cell.10. The vector of claim 9 , wherein said host cell is a prokaryotic cell.11E. coli.. The vector of claim 10 , wherein said host cell is12. A host cell comprising the vector of .13. A method for producing at least one recombinant cytochrome P450-BM3 variant comprising culturing a host cell under conditions such that at least one recombinant cytochrome P450-BM3 variant of is produced by said host cell.14. The method of claim 13 , further comprising the step of ...

POLYPEPTIDES WITH IMPROVED ARABINOSE TRANSPORT SPECIFICITY

The invention relates to a variant of a parent polypeptide which is preferably a hexose transporter, wherein the variant comprises an amino acid sequence which, when aligned with the amino acid sequence of SEQ ID NO: 1, comprises a substitution of amino acids N376 and T89, the positions of said amino acids being defined with reference to the amino acid sequence of SEQ ID NO: 1. Said polypeptide is suitable in the production of ethanol from arabinose-rich biomass such as corn stover and corn starch. 1. A variant of a parent polypeptide which is optionally a hexose transporter , wherein the variant comprises an amino acid sequence which , when aligned with the amino acid sequence of SEQ ID NO: 1 , comprises a substitution of amino acids N376 and T89 , the positions of said amino acids being defined with reference to the amino acid sequence of SEQ ID NO: 1.2. A variant according to wherein the parent polypeptide comprises the amino acid sequence of SEQ ID NO: 1.3. A variant according to claim 1 , wherein the substitution of amino acid N376 comprises any one of N376T claim 1 , N376C claim 1 , N376V claim 1 , N376M claim 1 , N376L claim 1 , N376I claim 1 , or N376F.4. A variant according to wherein substitution of amino acid T89 comprises any one of T89I claim 1 , T89V claim 1 , T89L claim 1 , T89S claim 1 , T89M claim 1 , or T89F.5. A variant according to claim 1 , wherein substitution of amino acid N376 comprises any one of N376T claim 1 , N376S claim 1 , or N376I claim 1 , and wherein substitution of amino acid T89 comprises T89I.6. A variant according to which has at least 80% sequence identity with the amino acid sequence of the parent polypeptide.7. A variant according to which is a man-made polypeptide.8. A nucleic acid sequence encoding the variant according to claim 1 , optionally wherein said nucleic acid sequence is man-made.9. A nucleic acid construct comprising the nucleic acid sequence of operably linked to one or more control sequences capable of directing ...

NUCLEIC ACID MOLECULE

The disclosure relates to a nucleic acid molecule isolated from a cultivar that produces the opiate alkaloid noscapine which comprises 10 genes involved in the biosynthesis of opiate alkaloids. 1. An isolated nucleic acid molecule that comprises a gene cluster that encodes two or more polypeptides involved in the biosynthesis of opiate alkaloids or intermediates , wherein one of said two polypeptides is encoded by a nucleotide sequence selected from the group consisting of:i) a nucleotide sequence as set forth in SEQ ID NO: 8;ii) a nucleotide sequence, wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);iii) a nucleic acid molecule, the complementary strand of which hybridizes under stringent hybridization conditions to the nucleotide sequence in SEQ ID NO: 8 and which encodes a polypeptide that has carboxylesterase activity; andiv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 18 or a nucleotide sequence that encodes a polypeptide that has 46% amino acid sequence identity across the full length amino acid sequence set forth in SEQ ID NO: 18 wherein said polypeptide has carboxylesterase activity.2. An isolated nucleic acid molecule that comprises a gene cluster that encodes two or more polypeptides involved in the biosynthesis of opiate alkaloids or intermediates , wherein one of said two polypeptides is encoded by a nucleotide sequence selected from the group consisting of;i) a nucleotide sequence as set forth in SEQ ID NO: 9;ii) a nucleotide sequence, wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);iii) a nucleic acid molecule, the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 9 and which encodes a polypeptide that has short-chain dehydrogenase/reductase activity; andiv) a nucleotide sequence that encodes a polypeptide ...

METHODS AND COMPOSITIONS FOR TREATING AND PREVENTING VIRAL INFECTION

The disclosure provides recombinant polypeptides for treating or preventing viral infection comprising an immunoglobulin Fc fragment and at least one viral receptor or fragment thereof. Also provided are RNA molecules, therapeutic compositions, and expression systems comprising such recombinant polypeptides, along with methods of preventing or treating a viral infection in a subject in need thereof, comprising administering such recombinant polypeptides to a subject or patient. 196-. (canceled)97. A recombinant polypeptide comprising:a) an immunoglobulin (Ig) Fc fragment;b) a first viral receptor, wherein the receptor is ACE2 or fragment thereof; andc) a second viral receptor.98. The recombinant polypeptide of claim 97 , wherein the second viral receptor is selected from the group consisting of HSPG claim 97 , CD147 claim 97 , sialic acid claim 97 , and SRB1 claim 97 , and is used for the treatment of SARS-CoV or SARS-CoV-2.99. The recombinant polypeptide of claim 98 , wherein the polynucleotide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth as SEQ ID NOs:31 claim 98 , 37 claim 98 , or 45.100. The recombinant polypeptide of claim 99 , wherein the polynucleotide comprises an amino acid sequence set forth as SEQ ID NOs:31 claim 99 , 37 claim 99 , or 45.101. A polynucleotide encoding the recombinant polypeptide of claim 98 , wherein the polynucleotide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth as:SEQ ID NOs:25-30; orSEQ ID NOs:25, 28, 30, 34-36, and 38; orSEQ ID NOs:25, 28, 30, and 39-44.102. A polynucleotide encoding the recombinant polypeptide of claim 98 , wherein the polynucleotide comprises an amino acid sequence set forth as SEQ ID NOs:25-30; or SEQ ID NOs:25 claim 98 , 28 claim 98 , 30 claim 98 , 34-36 claim 98 , and 38; or SEQ ID NOs:25 claim 98 , 28 claim 98 , 30 claim 98 , and 39-44.103. The recombinant polypeptide of claim 97 , further comprising a sulfated ...

COMPOSITIONS, METHODS AND THERAPIES FOR ADMINISTERING ANTIGEN PEPTIDE

The invention relates to compositions, methods and therapies for the treatment of inflammation caused by infection with . The compositions include a combination of peptide and anti-TNF. The peptide consists of a specific amino acid sequence or a peptide consisting of an amino acid sequence derived the specific amino acid sequence by deletion, substitution, insertion or addition of one or more amino acids. The administration of the peptide and anti-TNF in therapeutically effective amounts to a patient is effective to suppress, by immune response, inflammation caused by infection with 1Propionibacterium acnes. A method of suppressing inflammation caused by infection with in a patient , comprising:introducing into the patient a peptide consisting of an amino acid sequence indicated by SEQ NO. 3, andintroducing into the patient a TNF inhibitor.2. The method of claim 1 , wherein the peptide and the TNF inhibitor are introduced simultaneously.3. The method of claim 2 , wherein the peptide and the TNF inhibitor are in the form of a composition.4. The method of claim 1 , wherein the peptide and the TNF inhibitor are introduced sequentiallly.5Propionibacterium acnes. A method of suppressing inflammation caused by infection with in a patient claim 1 , comprising:introducing into the patient a peptide consisting of an amino acid sequence indicated by SEQ NO. 1, andintroducing into the patient a TNF inhibitor.6. The method of claim 5 , wherein the peptide and the TNF inhibitor are introduced simultaneously.7. The method of claim 6 , wherein the peptide and the TNF inhibitor are in the form of a composition.8. The method of claim 5 , wherein the peptide and the TNF inhibitor are introduced sequentially.9Propionibacterium acnes. A composition to reduce inflammation in a patient with a infection claim 5 , comprising:a peptide consisting of an amino acid sequence indicated by SEQ NO. 1;a peptide consisting of an amino acid sequence indicated by SEQ NO. 3; anda TNF inhibitor.10. The ...

MICROORGANISMS AND METHODS FOR THE BIOSYNTHESIS OF BUTADIENE

The invention provides non-naturally occurring microbial organisms having a butadiene pathway. The invention additionally provides methods of using such organisms to produce butadiene. 1. A non-naturally occurring microbial organism , comprising a microbial organism having a butadiene pathway comprising at least one exogenous nucleic acid encoding a butadiene pathway enzyme expressed in a sufficient amount to produce butadiene , said butadiene pathway comprising a butadiene synthase , an acetyl-CoA:acetyl-CoA acyltransferase , an acetoacetyl-CoA reductase , a 3-hydroxybutyryl-CoA dehydratase , a crotonyl-CoA reductase (aldehyde forming) , a crotonaldehyde reductase (alcohol forming) , a crotyl alcohol kinase , a 2-butenyl-4-phosphate kinase , a crotonyl-CoA hydrolase , synthetase , or transferase , a crotonate reductase , a crotonyl-CoA reductase (alcohol forming) , a glutaconyl-CoA decarboxylase , a glutaryl-CoA dehydrogenase , an 3-aminobutyryl-CoA deaminase , a 4-hydroxybutyryl-CoA dehydratase or a crotyl alcohol diphosphokinase.2. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises two exogenous nucleic acids each encoding a butadiene pathway enzyme.3. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises three exogenous nucleic acids each encoding a butadiene pathway enzyme.4. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises four exogenous nucleic acids each encoding a butadiene pathway enzyme.5. The non-naturally occurring microbial organism of claim 1 , wherein said butadiene pathway comprises an acetyl-CoA:acetyl-CoA acyltransferase claim 1 , an acetoacetyl-CoA reductase claim 1 , a 3-hydroxybutyryl-CoA dehydratase claim 1 , a crotonyl-CoA reductase (aldehyde forming) claim 1 , a crotonaldehyde reductase (alcohol forming) claim 1 , a crotyl alcohol kinase claim 1 , a 2-butenyl-4-phosphate kinase and a ...

FLUORESCENT POLYMERASE ENZYME SUBSTRATES HAVING PROTEIN SHIELDS

Compositions, methods, and systems are provided for fluorescent polymerase enzyme substrates comprising protein shields for improving enzyme photostability in single molecule real time sequencing. Fluorescent polymerase enzyme substrates of the invention have a protein shield between the fluorescent dye moieties and nucleotide moieties of the polymerase enzyme substrate. The polymerase enzyme substrates have a nucleotide component and a dye component, each attached to a protein. The attachments can be covalent. The protein can, for example, prevent the direct interaction of the fluorescent dye moiety with the enzyme when carrying out nucleotide synthesis, preventing photodamage to the enzyme. The polymerase enzyme substrates of the invention can have multiple dyes and multiple nucleotide moieties. 1. A polymerase enzyme substrate comprising:a protein comprising at least 60 amino acids;a nucleotide component comprising at least one nucleoside polyphosphate attached through its phosphate portion to a first position on the protein;a dye component comprising at least one fluorescent dye moiety attached to a second position on the protein.2. The polymerase enzyme substrate of wherein the substrate is connected through covalent attachments.3. The polymerase enzyme substrate of wherein the protein comprises 60 to 1 claim 1 ,000 amino acids.4. The polymerase enzyme substrate of wherein the protein comprises 80 to 600 amino acids.5. The polymerase enzyme substrate of wherein the nucleotide component and dye component are covalently attached to the protein.6. The polymerase enzyme substrate of wherein the nucleotide component comprises two or more nucleoside phosphates.7. The polymerase enzyme substrate of wherein the substrate has 2 claim 1 , 3 claim 1 , 4 claim 1 , 5. 6 claim 1 , 7 claim 1 , or 8 nucleotide phosphates.8. The polymerase enzyme substrate of wherein the dye component comprises two or more fluorescent dye moieties.9. The polymerase enzyme substrate of wherein ...

URIDINE DIPHOSPHATE-DEPENDENT GLYCOSYLTRANSFERASE ENZYME

In various aspects, the present invention provides uridine diphosphate-dependent glycosyltransferase (UGT) enzymes capable of catalyzing the transfer of a monosaccharide moiety from a NDP-sugar to the 3′ carbon of a sugar moiety of a substrate, such as a terpenoid glycan, thereby functioning as a “1-3 UGT.” In other aspects, the invention provides polynucleotides encoding the 1-3 UGT, and host cells comprising the same. In still other aspects, the invention provides methods for preparing glycosylated substrates, including steviol glycosides, using the enzyme and host cells of this disclosure. 1. A uridine diphosphate-dependent glycosyltransferase (UGT) enzyme comprising an amino acid sequence that is at least about 75% identical to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.2. The enzyme of claim 1 , wherein the amino acid sequence is at least about 80% identical to SEQ ID NO: 5 or SEQ ID NO: 6.3. The enzyme of claim 1 , wherein the amino acid sequence is at least about 85% identical to SEQ ID NO: 5 or SEQ ID NO: 6.4. The enzyme of claim 1 , wherein the amino acid sequence is at least about 90% identical to SEQ ID NO: 5 or SEQ ID NO: 6.5. The enzyme of claim 1 , wherein the amino acid sequence is at least about 95% identical to SEQ ID NO: 5 or SEQ ID NO: 6.6. The enzyme of claim 1 , wherein the amino acid sequence is at least about 98% identical to SEQ ID NO: 5 or SEQ ID NO: 6.7. The enzyme of claim 1 , wherein the amino acid sequence is the amino acid of SEQ ID NO: 5 or SEQ ID NO: 6.8. The enzyme of claim 1 , wherein the amino acid sequence has from 1 to 20 amino acid modifications independently selected from substitutions claim 1 , deletions claim 1 , and insertions claim 1 , with respect to the amino acid sequence SEQ ID NO: 5 or SEQ ID NO: 6.9. The enzyme of claim 8 , wherein the amino acid sequence has from 1 to 10 amino acid modifications independently selected from substitutions claim 8 , deletions claim 8 , and insertions claim 8 , with respect ...

Plastic degrading proteases

The present invention relates to novel proteases, more particularly to protease variants having improved activity compared to the protease of SEQ ID N° 1 and the uses thereof for degrading polyester containing material, such as plastic products. The proteases of the invention are particularly suited to degrade polylactic acid, and material containing polylactic acid.

HETEROLOGOUS PRODUCTION OF PSILOCYBIN

A recombinant host cell is disclosed for producing psilocybin and related compounds, such as metabolic intermediates of the psilocybin biosynthesis. Also provided is a method of producing psilocybin and its synthesis intermediates and related compounds, such as metabolic intermediates of the psilocybin biosynthesis, in the host cell, as well as a production system for producing them. 118.-. (canceled)19. A recombinant host cell comprising:heterologous polynucleotides encoding PsiD, PsiH, PsiK, and PsiM;wherein the heterologous polynucleotides are operably linked to at least one promoter which is capable of directing expression of said heterologous polynucleotides in the host cell;wherein the recombinant host cell further comprises at least one further genetic element encoding Trp2 and Trp3 arranged to increase biosynthetic production of L-tryptophan in the host cell, wherein the further genetic element is operably linked to at least one promoter which is capable of directing expression of said further genetic element in the host cell; and wherein the recombinant host cell is capable of producing psilocybin.20. The host cell of claim 19 , wherein the at least one promoter provides production of the at least one heterologous polynucleotide.21. The host cell of claim 19 , wherein the at least one heterologous polynucleotide is operably linked to a single promoter claim 19 , which controls the expression of each of PsiD claim 19 , PsiH claim 19 , PsiK claim 19 , and PsiM.22. The recombinant host cell of claim 19 , wherein the promoter is controlled by a synthetic transcription factor.23. The host cell of claim 19 , wherein the further genetic element encoding Trp2 is genetically modified to inhibit its allosteric regulation.24. The recombinant host cell of claim 23 , comprising at least two further genetic elements that are controlled by a single synthetic transcription factor.25. The recombinant host cell of claim 24 , wherein the synthetic transcription factor is the ...

Polypeptides Having Arabinofuranosidase Activity And Polynucleotides Encoding Same

The present invention relates to isolated polypeptides having arabinofuranosidase activity, catalytic domains, carbohydrate binding modules and polynucleotides encoding the polypeptides, catalytic domains or carbohydrate binding modules. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or carbohydrate binding modules. 1. An isolated polypeptide having arabinofuranosidase activity , selected from the group consisting of:(a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 2 or 92% sequence identity to the mature polypeptide of SEQ ID NO: 4;(b) a polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions with SEQ ID NO: 1 or the mature polypeptide coding sequence of SEQ ID NO: 3, or the full-length complement thereof;(c) a polypeptide encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 1 or at least 92% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 3;(d) a variant of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 4 comprising a substitution, deletion, and/or insertion at one or more positions;(e) a fragment of the polypeptide of (a), (b), (c), or (d) that has arabinofuranosidase activity;(f) a polypeptide comprising a catalytic domain having at least 90% sequence identity to amino acids 4 to 303 of SEQ ID NO: 2 or amino acids 173 to 475 of SEQ ID NO: 4;(g) a polypeptide comprising a catalytic domain encoded by a polynucleotide that hybridizes under very high stringency conditions with nucleotides 10 to 909 of SEQ ID NO: 1 or nucleotides 517 to 1425 of SEQ ID NO: 3, or the full-length complement thereof;(h) a polypeptide comprising a catalytic domain encoded by a polynucleotide having at least 90% sequence identity to nucleotides 10 to 909 of SEQ ID NO: 1 or at least 92% sequence identity to nucleotides 517 to 1425 of ...

Pullulanase and Use Thereof

The present application relates to the field of enzyme engineering, especially relates to a pullulanase as well as preparation and use thereof. The pullulanase and coding gene thereof were obtained by random mutation by using the Error-prone PCR technique on the gene of wild-type pullulanase to obtain a mutant PLUM. The enzyme activity of the mutant PLUM was improved by 57.03% compared with the wild-type pullulanase PLUM. 1. A pullulanase mutant , wherein , the amino acid sequence of said mutant is shown in SEQ ID NO. 4.2. The pullulanase mutant according to claim 1 , wherein its expression vector is pGAPZαC.3Pichia Pastoris. The pullulanase mutant according to claim 1 , wherein its host cell is GS 115.4. A coding gene of the pullulanase mutant according to .5. The coding gene according to claim 4 , wherein the nucleotide sequence of said gene is shown in SEQ ID NO. 3.6. A use of the pullulanase mutant according to .7. A use of the coding gene of the pullulanase mutant according to . The contents of the electronic sequence listing (16029647sequenceListing20180930.txt; Size: 24,000 bytes; and Date of Creation: Sep. 30, 2018) is herein incorporated by reference in its entirety.This application claims the priority of Chinese Patent No. 201810527491.3 filed on May 29, 2018 and entitled “Pullulanase and Use Thereof”.The present disclosure relates to the enzyme engineering technical field, especially relates to a pullulanase as well as preparation and use thereof.A pullulanase is a starch debranching enzyme, which can hydrolyze alpha-1,6 glucosidic bond of polysaccharides so that the amylose can be converted into amylopectin to the maximum extent. The pullulanase has very important application in the starch processing industry, and it can improve on a large scale the utilization rate and the production efficiency of the starch, and is relatively well applied to production of glucose syrup, maltose syrup and beer.In the production of glucose, pullulanase and glucoamylase ...

ENZYME COMPLEX COMPRISING BETA-AGARASE, KAPPA-CARRAGEENASE AND ANHYDRO-GALACTOSIDASE, AND USE THEREOF

The present invention relates to an enzyme complex which the following (i) to (iv) are combined: (i) chimeric beta-agarase formed by a fusion of beta-agarase and the dockerin module of endo-β-1,4-glucanase-B; (ii) chimeric kappa-carrageenase formed by a fusion of kappa-carrageenase and the dockerin module of endo-β-1,4-glucanase-B; (iii) chimeric anhydro-galactosidase formed by a fusion of anhydro-galactosidase and the dockerin module of endo-β-1,4-glucanase-B; and (iv) mini cellulose-binding protein A, and to a method of degrading red algal biomass using the same. According to the present invention, an enzymatic degradation process is applied for the production of agar degradation products, deviating from conventional methods that relied on physical and chemical pretreatment processes. Thus, the present invention will greatly contribute to efficiently converting marine algae into valuable products by use of a convenient, cost-effective, highly efficient and environmentally friendly degradation system while controlling the products. 1. An enzyme complex which the following (i) to (iv) are combined:(i) chimeric beta-agarase formed by a fusion of beta-agarase and the dockerin module of endo-β-1,4-glucanase-B;(ii) chimeric kappa-carrageenase formed by a fusion of kappa-carrageenase and the dockerin module of endo-β-1,4-glucanase-B;(iii) chimeric anhydro-galactosidase formed by a fusion of anhydro-galactosidase and the dockerin module of endo-β-1,4-glucanase-B; and(iv) mini cellulose-binding protein A.2. The enzyme complex of claim 1 , wherein the beta-agarase has the amino acid sequence of SEQ ID NO: 1.3. The enzyme complex of claim 1 , wherein the kappa-carrageenase has the amino acid sequence of SEQ ID NO: 2.4. The enzyme complex of claim 1 , wherein the anhydro-galactosidase has the amino acid sequence of SEQ ID NO: 3.5. The enzyme complex of claim 1 , wherein the endo-β-1 claim 1 ,4-glucanase-B has the amino acid sequence of SEQ ID NO: 4.6. The enzyme complex of ...

ENGINEERED PHOSPHOPENTOMUTASE VARIANT ENZYMES

The present invention provides engineered phosphopentomutase (PPM) enzymes, polypeptides having PPM activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing PPM enzymes are also provided. The present invention further provides compositions comprising the PPM enzymes and methods of using the engineered PPM enzymes. The present invention finds particular use in the production of pharmaceutical compounds. 1. An engineered phosphopentomutase comprising a polypeptide sequence having at least 85% , 86% , 87% , 88% , 89% , 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% , or more sequence identity to SEQ ID NO: 2 , 4 , 118 , 266 , 420 , 562 , 656 , 790 , and/or 846 , or a functional fragment thereof , wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2 , 4 , 118 , 266 , 420 , 562 , 656 , 790 , and/or 846.2. The engineered phosphopentomutase of claim 1 , wherein said polypeptide sequence has at least 85% claim 1 , 86% claim 1 , 87% claim 1 , 88% claim 1 , 89% claim 1 , 90% claim 1 , 91% claim 1 , 92% claim 1 , 93% claim 1 , 94% claim 1 , 95% claim 1 , 96% claim 1 , 97% claim 1 , 98% claim 1 , 99% claim 1 , or more sequence identity to SEQ ID NO:2 claim 1 , and wherein the polypeptide sequence of said engineered phosphopentomutase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 173 claim 1 , 65 claim 1 , 77/118/147/154/231/357 claim 1 , 98 claim 1 , 99 claim 1 , 114 claim 1 , 117 claim 1 , 118/147/154/225/231 claim 1 , 118/147/154/225/233/257/357 claim 1 , 118/147/154/225/257/357 claim 1 , 118/147/154/231/309 claim 1 , 118/147/225/231/257/309/357 claim 1 , 118/147/225/231/357 claim 1 , 118/147/225/234/257/ ...

LONG CHAIN DIBASIC ACID WITH LOW CONTENT OF LONG CHAIN DIBASIC ACID IMPURITY OF SHORTER CARBON-CHAIN AND PREPARATION METHOD THEREOF

The present invention relates to a long-chain dibasic acid with low content of long-chain dibasic acid impurity of shorter carbon chain, to the preparation of a long-chain dibasic acid producing strain by directed evolution of POX gene and homologous recombination, and to the production of a long-chain dibasic acid with low content of long-chain dibasic acid impurity of shorter carbon chain by using the strain. The present invention also relates to a strain containing a mutated promoter, wherein, when a long-chain dibasic acid is produced by fermentation of this strain, the content of the acid impurity of shorter carbon chain in the fermentation product is significantly reduced. 1. A product , which is one of the following products I) to V): relative to GenBank Accession Number M12161, taking the first base upstream of the start codon ATG as −1, has base mutation −182_−191 AAAAAAAAAA>AAAAAAAAA; or', 'relative to SEQ ID NO: 25, has base mutation 266_275 AAAAAAAAAA>AAAAAAAAA;, 'I) an isolated mutated promoter whichII) an isolated mutated POX gene, homologous gene or variant thereof, which, relative to GenBank Accession Number M12161, taking the first base upstream of the start codon ATG as −1, has base mutation −182_−191AAAAAAAAAA>AAAAAAAAA in its promoter region, wherein the variant has at least 70% sequence identity with the mutated POX gene or a homologous gene thereof;III) a microorganism containing the mutated POX gene, homologous gene or variant thereof of II), which, relative to a microorganism containing a non-mutated POX gene, homologous gene or variant thereof, produces a long chain dibasic acid with significantly decreased content of long-chain dibasic acid impurity of shorter carbon-chain, wherein the number of carbon atoms in the long-chain dibasic acid impurity of shorter carbon-chain is less than the number of carbon atoms in the long-chain dibasic acid;IV) a long-chain dibasic acid with low content of long-chain dibasic acid impurity of shorter carbon- ...

ENGINEERED GALACTOSE OXIDASE VARIANT ENZYMES

The present invention provides engineered galactose oxidase (GOase) enzymes, polypeptides having GOase activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing GOase enzymes are also provided. The present invention further provides compositions comprising the GOase enzymes and methods of using the engineered GOase enzymes. The present invention finds particular use in the production of pharmaceutical and other compounds. 1. An engineered galactose oxidase comprising a polypeptide sequence having at least 85% , 86% , 87% , 88% , 89% , 90% , 91% , 92% , 93% , 94% , 95% , 96% , 97% , 98% , 99% , or more sequence identity to SEQ ID NO: 2 , 4 , 166 , 272 , 928 , 932 , 1264 , 1416 , 1598 , 1866 , 1912 , 2080 , 2300 , and/or 2424 , or a functional fragment thereof , wherein said engineered galactose oxidase comprises at least one substitution or substitution set in said polypeptide sequence , and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2 , 4 , 166 , 272 , 928 , 932 , 1264 , 1416 , 1598 , 1866 , 1912 , 2080 , 2300 , and/or 2424.2. The engineered galactose oxidase of claim 1 , wherein said polypeptide sequence has at least 85% claim 1 , 86% claim 1 , 87% claim 1 , 88% claim 1 , 89% claim 1 , 90% claim 1 , 91% claim 1 , 92% claim 1 , 93% claim 1 , 94% claim 1 , 95% claim 1 , 96% claim 1 , 97% claim 1 , 98% claim 1 , 99% claim 1 , or more sequence identity to SEQ ID NO: 2 claim 1 , and wherein said engineered galactose oxidase comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 331/406/407/465 and 331/406/465 claim 1 , wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.3. The engineered galactose oxidase of claim 1 , wherein said polypeptide sequence has at least 85% claim 1 , 86% claim ...

FLUORESCENT PREMIX PARTICLES, FLUORESCENT STAIN CONTAINING SAME, AND FLUORESCENT STAINING METHOD IN WHICH THESE ARE USED

The present invention relates to fluorescent premix particles, a fluorescent stain containing the same, and a fluorescent staining method in which these are used, and the fluorescent premix particles are particles including: phosphor integrated particles that are modified with a first reactive substance; and at least one target protein-binding substance that is modified with a second reactive substance and is selected from the group consisting of an antibody and an aptamer, wherein the phosphor integrated particles and the at least one target protein-binding substance are linked by interaction between the first reactive substance and the second reactive substance.

SYSTEMS AND METHODS FOR DISCOVERING AND OPTIMIZING LASSO PEPTIDES

Provided herein are lasso peptides libraries, and particularly molecular display libraries of lasso peptides. Also provided herein are related methods and systems for producing the libraries and for screening the libraries to identify candidate lasso peptides having desirable properties. 1. A lasso peptide display library comprising a plurality of members , wherein each member comprises a lasso peptide or a functional fragment of lasso peptide; and wherein each member is associated with a unique identification mechanism for distinguishing the plurality of members from one another , wherein the unique identification mechanism is a unique nucleic acid molecule or a unique location.2. The lasso peptide display library of claim 1 , wherein the library further comprises a solid support.3. The lasso peptide display library of claim 2 , wherein each member is associated with the unique identification mechanism through the solid support.4. The lasso peptide display library of claim 2 , wherein the solid support comprises a plurality of unique locations claim 2 , and each member is associated with one of the plurality of unique locations.5. The lasso peptide display library of any one of - claim 2 , wherein at least one of the lasso peptide and/or functional fragment of lasso peptide forms part of a fusion protein.6. The lasso peptide display library of any one of - claim 2 , wherein at least one of the lasso peptide and/or functional fragment of lasso peptide forms part of a protein complex.7. The lasso peptide display library of any one of - claim 2 , wherein at least one of the lasso peptide and/or functional fragment of lasso peptide forms part of a conjugate.8. The lasso peptide display library of any one of - claim 2 , wherein the unique identification mechanism is a unique nucleic acid molecule.9. The lasso peptide display library of claim 8 , wherein the lasso peptide or functional fragment of lasso peptide is fused to a first binding partner; and wherein the unique ...

GENETICALLY ENGINEERED CYANOBACTERIA FOR GROWTH IN UNSTERILIZED CONDITIONS USING ANTIBIOTIC-FREE SELECTION

The present invention relates to methods of metabolic engineering cells to increase their ability to compete with contaminating microorganisms without the need for antibiotics. More particularly, the invention provides methods to engineer cyanobacteria to utilize melamine as nitrogen source, phosphite as phosphorous source, optionally also utilizing NADP+ over NAD+, and also provides genetically engineered cells made using such methods. 2. The isolated genetically engineered cyanobacterium of claim 1 , wherein the triA gene encodes an amino acid sequence selected from the group comprising SEQ ID NO: 56 claim 1 , SEQ ID NO: 58 claim 1 , SEQ ID NO: 60 claim 1 , SEQ ID NO: 62 claim 1 , SEQ ID NO: 64 claim 1 , SEQ ID NO: 66 and SEQ ID NO: 68; and/orwherein the triA gene comprises a polynucleotide sequence which has at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence selected from the group comprising SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69 and SEQ ID NO: 70.3. (canceled)4. The isolated genetically engineered cyanobacterium of claim 1 , wherein the heterologous gene trzE comprises a polynucleotide sequence which has at least 80% claim 1 , at least 85% claim 1 , at least 90% claim 1 , at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence set forth in SEQ ID NO: 71 or SEQ ID NO: 72;trzC comprises a polynucleotide sequence which has at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence set forth in SEQ ID NO: 73 or SEQ ID NO: 74;DUR1,2 comprises a polynucleotide sequence which has at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence set forth in SEQ ID NO: 75 or SEQ ID NO: 76;atzD comprises a polynucleotide sequence which has at least 80%, at least 85 ...

Modulating Laccase Enzyme to Regulate Cell Wall Biosynthesis and Recalcitrance in Plants

This disclosure provides genetically modified plants having desirable levels of sugar release and syringyl/guaiacyl (S/G) ratio; methods of genetically modifying plants to modulate sugar release and S/G ratio; and uses of such plants. The inventors have determined that genetic modification of a laccase gene (LAC2) from , encoded by locus Potri.008G064000 resulted in transgenic trees with changes in syringyl/guaiacyl ratios as well as altered sugar release phenotypes. Plants with altered sugar release, and S/G ratio, based on modulation of the expression or activity of the LAC2 gene, have divergent uses including pulp and paper production, and biofuel and bioproducts production. 1. A genetically modified plant characterized by a reduced expression of the LAC2 gene as compared to a control plant.2Populus, Manihot, Gossypium, Eucalyptus, Medicago, Arabidopsis, Solanum, OryzaZea.. The genetically modified plant of claim 1 , wherein said genetically modified plant belongs to a genus selected from the group consisting of and3Populus balsamifera, Populus deltoides, Populus trichocarpa, Populus tremuloides, Populus tremula, Populus albaPopulus maximowiczii.. The genetically modified plant of claim 2 , wherein the plant is selected from the group consisting of and4. The genetically modified plant of claim 1 , wherein the reduced expression of the LAC2 gene is achieved by a method selected from the group consisting of introducing a nucleic acid inhibitor claim 1 , the CRISPR/Cas system claim 1 , the Cre/Lox system claim 1 , the TALEN system claim 1 , and homologous recombination.5. The genetically modified plant of claim 4 , wherein said nucleic acid inhibitor is selected from the group consisting of an antisense RNA claim 4 , a small interfering RNA claim 4 , an RNAi microRNA claim 4 , an artificial microRNA claim 4 , and a ribozyme.6. A method for biofuel production claim 1 , comprising using the plant of in a biofuel fermentation process.7. An expression vector claim 1 , ...

POLYPEPTIDE HAVING ENDONUCLEASE ACTIVITY AND METHOD FOR PRODUCING THE SAME

A polypeptide includes an amino acid sequence selected from the group consisting of: an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1; and an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution, deletion, and/or addition of one or more amino acid residues. A yeast host expressing the polypeptide in a secretory production system does not add an N-linked sugar chain to the polypeptide, and the polypeptide has endonuclease activity. 1. A polypeptide comprising an amino acid sequence selected from the group consisting of:an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1; andan amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution, deletion, and/or addition of one or more amino acid residues,wherein a yeast host expressing the polypeptide in a secretory production system does not add an N-linked sugar chain to the polypeptide, andwherein the polypeptide has endonuclease activity.2. The polypeptide according to claim 1 , wherein amino acid residues of the polypeptide corresponding to N20 and N33 of SEQ ID NO: 1 are not modified with the N-linked sugar chain.3. The polypeptide according to claim 2 , comprising at least one of:an amino acid residue other than asparagine at a position corresponding to N20 of SEQ ID NO: 1; oran amino acid residue other than serine or threonine at a position corresponding to S22 of SEQ ID NO: 1,wherein the polypeptide further comprises at least one of:proline at a position corresponding to S34 of SEQ ID NO: 1; oran amino acid residue other than serine or threonine at a position corresponding to T35 of SEQ ID NO: 1.4. The polypeptide according to claim 3 ,wherein the amino acid residue at the position corresponding to N20 of SEQ ID NO: 1 is glycine, proline, aspartic acid, glutamic acid, threonine, alanine, valine, histidine, arginine, methionine, isoleucine, leucine ...

MODIFIED BIOTIN, STREPTAVIDIN MUTANT, AND USAGE OF THEM

An object of this invention is to provide a streptavidin mutant reduced in affinity to the naturally-occurring biotin, and to provide a modified biotin which shows a high affinity to such streptavidin mutant reduced in affinity to the naturally-occurring biotin. This invention can provide a compound composed of a dimer of modified biotin, a streptavidin mutant, angsd usage of them. 1. A streptavidin mutant comprising an amino acid sequence in which Asn at amino acid residue 37 in the amino acid sequence represented by SEQ ID NO: 3 is substituted with other amino acid residue.2. A streptavidin mutant comprising an amino acid sequence represented by SEQ ID NO: 4.3. A DNA encoding the streptavidin mutant described in .4. A streptavidin mutant-molecular probe conjugate claim 1 , obtained by combining the streptavidin mutant described in with a molecular probe.5. The streptavidin mutant-molecular probe conjugate of claim 4 , wherein the molecular probe is an anti-human CD20 antibody.6. The streptavidin mutant-molecular probe conjugate of claim 4 , wherein the molecular probe is rituximab.7. The streptavidin mutant-molecular probe conjugate of claim 4 , wherein the molecular probe is an anti-epiregulin single chain antibody.8. A therapeutic agent claim 4 , or in vivo or in vitro diagnostic agent claim 4 , comprising the streptavidin mutant-molecular probe conjugate described in . The present application is a Divisional of U.S. application Ser. No. 15/119,441, filed Aug. 17, 2016, which is a National stage of International Patent Application No. PCT/JP2015/054431, filed Feb. 18, 2015, which claims priority to Japanese Application No. 2014-212861, filed Oct. 17, 2014 and Japanese Application No. 2014-028525, filed Feb. 18, 2014, the contents of which are expressly incorporated by reference herein in their entireties.This invention relates to compound composed of a dimer of modified biotin, a streptavidin mutant, and usage of them.Avidin and biotin, or, streptavidin and ...

XYLOSE METABOLIZING YEAST

Described herein are microorganisms, in particular yeast, which have been transformed with one or more expression construct(s) for i) the overexpression of the native genes encoding xylulose kinase (XKS1), transaldolase (TAL1), transketolase 1 (TKL1) and transketolase 2 (TKL2) and ii) the expression of a functional heterologous gene encoding a xylose isomerase (XI), where the xylose isomerase (XI) gene is derived from a microorganism selected from the group consisting of and . Also described herein are expression constructs, methods for fermenting pentose sugars using the microorganisms and methods for producing such microorganisms. 1. A microorganism which has been transformed with one or more expression construct(s) fori) the overexpression of native genes encoding xylulose kinase (XKS1), transaldolase (TAL1), transketolase 1 (TKL1) and transketolase 2 (TKL2) and{'i': Thermotoga neapolitana, Anditalea andensis', 'Clostridium clariflavum., 'ii) the expression of a functional heterologous gene encoding a xylose isomerase (XI), wherein the xylose isomerase (XI) gene is derived from a microorganism selected from the group consisting of and'}2. The microorganism according to claim 1 , wherein the xylose isomerase (XI) is encoded by a nucleic acid sequence having at least 66% sequence identity to SEQ ID No 21 claim 1 , SEQ ID No 5 or SEQ ID No 25.3. The microorganism according to claim 1 , wherein the xylose isomerase (XI) is represented by an amino acid sequence having at least 80% sequence identity to SEQ ID No 22 claim 1 , SEQ ID No 6 or SEQ ID No 26.4. The microorganism according to claim 1 , wherein the xylulose kinase (XKS1) is encoded by a nucleic acid sequence having at least 80% sequence identity to SEQ ID No 74 claim 1 , the transaldolase (TAL1) is encoded by a nucleic acid sequence having at least 80% sequence identity to SEQ ID No 77 claim 1 , the transketolase 1 (TKL1) is encoded by a nucleic acid sequence having at least 80% sequence identity to SEQ ID No ...

DIAT GENE DERIVED FROM ORYZA SATIVA CONTROLLING DROUGHT STRESS TOLERANCE OF A PLANT AND USES THEREOF

A method for controlling drought stress tolerance of a plant includes transforming a cell of the plant with a recombinant vector which includes a gene encoding -derived DIAT (Drought-Induced AminoTransferase) protein to control expression of the gene encoding the DIAT protein. As the drought stress tolerance of a plant can be enhanced by the DIAT gene of the present invention, it is expected that a plant having drought stress tolerance is developed and used for enhancement of the productivity of crops. 1. A method for controlling drought stress tolerance of a plant , the method comprising:{'i': 'Oryza sativa', 'transforming a cell of the plant with a recombinant vector which includes a gene encoding -derived DIAT (Drought-Induced AminoTransferase) protein to control expression of the gene encoding the DIAT protein.'}2Oryza sativa. The method of claim 1 , wherein the -derived DIAT protein consists of the amino acid sequence of SEQ ID NO: 2.3Oryza sativa. The method of claim 1 , wherein the drought stress tolerance of a plant is increased by overexpressing a gene encoding -derived DIAT protein.4. A method for producing a transformed plant with controlled drought stress tolerance claim 1 , the method comprising:{'i': 'Oryza sativa', 'transforming a plant cell with a recombinant vector containing a gene encoding -derived DIAT (Drought-Induced AminoTransferase) protein; and'}regenerating a plant from the transformed plant cells.5. A transformed plant with controlled drought stress tolerance produced by the method of .6. A transformed seed of the transformed plant of .7Oryza sativa. The method of claim 4 , wherein the drought stress tolerance of a plant is enhanced by overexpressing a gene encoding -derived DIAT protein.8. A transformed plant with controlled drought stress tolerance produced by the method of .9. A transformed seed of the transformed plant of .10Oryza sativa. A composition for controlling drought stress tolerance of a plant which contains claim 8 , as an ...

Genome Editing without Nucleases

Methods and compositions are provided for editing the genome of a cell without the use of an exogenously supplied nuclease. Aspects of the methods include contacting a cell with a targeting vector comprising nucleic acid sequence to be integrated into the target locus, where the cell is not also contacted with a nuclease. In addition, reagents, devices and kits thereof that find use in practicing the subject methods are provided. 1. A method for the targeted integration of a transgene into the genome of a cell in the absence of an exogenously provided nuclease , the method comprising:contacting a cell with a recombinant viral vector, the recombinant viral vector comprising:i. a polynucleotide comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes the transgene; and the second nucleic acid sequence is positioned 5′ or 3′ to the first nucleic acid sequence and promotes the production of two independent gene products upon integration into the target integration site in the genome of the cell;ii. a third nucleic acid sequence positioned 5′ to the polynucleotide and comprising sequence that is substantially homologous to genomic sequence 5′ of a target integration site in the genome of the cell; andiii. a fourth nucleic acid sequence positioned 3′ of the polynucleotide and comprising sequence that is substantially homologous to genomic sequence 3′ of a target integration site in the genome of the cell;wherein the cell is not contacted with a nuclease or nucleic acid encoding a nuclease.2. The method according to claim 1 , wherein the cell is a non-dividing cell.3. The method according to claim 1 , wherein the contacting occurs in vivo.4. The method according to claim 3 , wherein the method finds use in treating a medical condition associated with a gene deficiency.5. The method according to claim 4 , wherein the medical condition is selected from the group consisting of hemophilia claim 4 , hemophilia ...

Method for Awakening Silent Gene Clusters in Bacteria and Discovery of Cryptic Metabolites

The majority of clinically used antibiotics and anticancer agents are derived from bacterial small molecules. These molecules are produced by dedicated biosynthetic gene clusters, sets of genes that are responsible for the step-wise generation of the target small molecule. Recent investigations have indicated, to the surprise of many experts, that the majority of these biosynthetic genes are inactive or ‘silent’ for unknown reasons. Thus under typical bacterial culturing conditions, these genes are not expressed and consequently the bioactive small molecule products are not synthesized. Disclosed is a method for high throughput screening of elicitors of cryptic metabolites, a method for producing cryptic metabolites, and a new family of cryptic metabolites, the acybolins, as well as their complete structural elucidation.

NOVEL P450-BM3 VARIANTS WITH IMPROVED ACTIVITY

The present invention provides improved P450-BM3 variants with improved activity. In some embodiments, the P450-BM3 variants exhibit improved activity over a wide range of substrates. 1. A recombinant cytochrome P450-BM3 variant having at least 90% sequence identity to a polypeptide sequence comprising the sequence set forth in SEQ ID NOS: 2 , 6 , 8 , 12 , 14 , or 16.2. The recombinant cytochrome P450-BM3 variant of claim 1 , wherein said variant oxidizes at least three organic substrates.3. The recombinant cytochrome P450-BM3 variant of claim 2 , wherein said organic substrate is selected from loratadine claim 2 , imatinib claim 2 , geftinib claim 2 , and diclofenac.4. A recombinant polynucleotide sequence encoding a recombinant cytochrome p450-BM3 variant having at least 90% sequence identity to a polynucleotide sequence comprising the sequence set forth in SEQ ID NO: 1 claim 2 , 5 claim 2 , 7 claim 2 , 11 claim 2 , 13 claim 2 , or 15.5. A recombinant polynucleotide sequence encoding a recombinant cytochrome P450-BM3 variant of .6. The recombinant polynucleotide sequence of claim 4 , wherein said sequence comprises SEQ ID NO: 1 claim 4 , 5 claim 4 , 7 claim 4 , 11 claim 4 , 13 claim 4 , or 15.7. An expression vector comprising the polynucleotide sequence of .8. The vector of claim 7 , wherein said polynucleotide sequence is operably linked with regulatory sequences suitable for expression of said polynucleotide sequence in a suitable host cell.9. The vector of claim 7 , wherein said host cell is a prokaryotic or eukaryotic cell.10. The vector of claim 9 , wherein said host cell is a prokaryotic cell.11E. coli.. The vector of claim 10 , wherein said host cell is12. A host cell comprising the vector of .13. A method for producing at least one recombinant cytochrome P450-BM3 variant comprising culturing a host cell under conditions such that at least one recombinant cytochrome P450-BM3 variant of is produced by said host cell.14. The method of claim 13 , further ...

Mini-III RNases, Methods for Changing the Specificity of RNA Sequence Cleavage by Mini-III RNases, and Uses Thereof

The object of the invention is a Mini-III RNase with amino acid sequence comprising an acceptor part, and a transplantable a4 helix, and a transplantable a5b-a6 loop, which form structures of a4 helix and a5b-a6 loop, respectively, in the Mini-III RNase structure, wherein the fragments which form structures of a4 helix and a5b-a6 loop, respectively, correspond structurally to respective structures of a4 helix and a5b-a6 loop formed by amino acid sequence fragments 46-52 and 85-98, respectively, of Mini-III RNase from shown in SEQ ID NO: 1, wherein the said Mini-III RNase exhibits sequence specificity in dsRNA cleavage being dependent only on a ribonucleotide sequence of the substrate, and independent from an occurrence of secondary structures in the substrate's structure, and independent from a presence of other assisting proteins, and wherein the Mini-III RNase is not the Mini-III protein from of SEQ ID NO: 1, nor SEQ ID NO: 1 with D94R mutation. The invention also relates to a method of obtaining a chimeric Mini-III RNase, a Mini-III RNase encoding construct, a cell with a Mini-III RNase encoding gene, use of Mini-III RNase for dsRNA cleavage, as well as a method of dsRNA cleavage depending only on a ribonucleotide sequence. 1. A Mini-III RNase with amino acid sequence comprising an acceptor part , and a transplantable α4 helix , and a transplantable α5b-α6 loop , which form structures of α4 helix and α5b-α6 loop , respectively , in the Mini-III RNase structure ,{'i': 'Bacillus stubtilis', 'wherein the fragments which form structures of α4 helix and α5b-α6 loop, respectively, correspond structurally to respective structures of α4 helix and α5b-α6 loop formed by amino acid sequence fragments 46-52 and 85-98, respectively, of Mini-III RNase from shown in SEQ ID NO: 1,'}{'i': 'Bacillus stubtilis', "wherein said Mini-III RNase exhibits sequence specificity in dsRNA cleavage that is dependent only on a ribonucleotide sequence of a substrate, and independent from an ...

OLIGOMERIC PARTICLE REAGENTS AND METHODS OF USE THEREOF

Provided herein are oligomeric reagents, including oligomeric reagents of streptavidin or a streptavidin mutein, compositions thereof, and methods for manufacturing oligomeric reagents, including methods for reliably manufacturing oligomeric particle reagents of a desired size. In some cases, the reagents are oligomeric particle reagents containing a plurality of binding sites for agents, and thus the one or more agents are multimerized by reversibly binding to the oligomeric particle reagent, e.g., thereby creating a multimerized oligomeric particle reagent, having stimulatory agents multimerized thereon. Also provided are methods for using the oligomeric reagents for incubation or culturing, such as to induce stimulation of expansion, activation, and/or survival, of a composition of cells such as a population of lymphocytes. In some aspects, the disclosure provides methods and reagents for the stimulation, survival, persistence, activation, or other effect of cell populations that involve binding of agents to a molecule on the cell surface. 1. An oligomeric particle reagent comprising a plurality of streptavidin or streptavidin mutein molecules , wherein the size of the oligomeric particle reagent comprises i) a radius of greater than 50 nm , ii) a molecular weight of at least 5×10g/mol; and/or (iii) at least 100 streptavidin or streptavidin mutein tetramers per oligomeric particle reagent.2. The oligomeric particle reagent of claim 1 , wherein each of the plurality of streptavidin or streptavidin mutein molecules reversibly bind to or are capable of reversibly binding to biotin claim 1 , a biotin analog or a streptavidin-binding peptide.3. The oligomeric particle reagent of claim 1 , wherein the oligomeric particle reagent comprises a plurality of streptavidin mutein molecules and each of the streptavidin mutein molecules comprises the amino acid sequence Val-Thr-Ala-Argor Ile-Gly-Ala-Argat sequence positions corresponding to positions 44 to 47 with reference to ...

DOXA PROTEIN MUTANT, AND CODING GENE AND APPLICATIONS THEREOF

The present invention relates to a DoxA protein mutant having an amino acid sequence set forth in SEQ ID No. 16, and coding gene thereof. The protein mutant or the coding gene thereof can be used for producing epirubicin. The present invention further relates to a capable of efficiently expressing epirubicin, which is constructed by replacing the dnmV gene of a starting in situ with the avrE gene and mutating the doxA gene of the starting into a gene encoding the protein set forth in SEQ ID No. 16. The fermentation broth of this has an epirubicin potency of up to 102.0 μg/ml. 1. A protein comprising the amino acid sequence set forth in SEQ ID No. 16.2. A biological material related to the protein according to claim 1 , which is the following B1) or B2):{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'B1) nucleic acid molecule encoding the protein according to ;'}B2) expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of B1).3. The biological material according to claim 2 , wherein the nucleic acid molecule of B1) is a gene represented by the following 1) or 2) or 3):1) a DNA molecule set forth in SEQ ID No. 15;{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, '2) a DNA molecule that hybridizes to the DNA molecule as defined in 1) under stringent conditions and encodes the protein according to ;'}{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, '3) a DNA molecule having 90% or more identity to the DNA molecule as defined in 1) or 2) and encoding the protein according to .'}4StreptomycesStreptomycesStreptomyces. A method for constructing epirubicin-expressing claim 2 , comprising the steps of: replacing the dnmV gene of a starting in situ with avrE gene claim 2 , and mutating the doxA gene of the starting into a gene sequence encoding the protein set forth in SEQ ID No. 16.5Streptomyces. The method according to claim 4 , wherein the preservation number of the starting is CGMCC No. 48276. ...

MODIFIED HOST CELLS AND USES THEREOF

Described herein are modified host cells useful in the production of bioconjugates that can be used to vaccinate subjects against infection with . The genomes of the modified host cells described herein comprise genes that encode proteins involved in glyosylation of proteins as well as genes specific to the production of -specific antigens. 1102-. (canceled)103. A host cell comprising:{'i': 'Pseudomonas,', '(i) a nucleic acid that encodes a glycosyltransferase derived from an rfb cluster of'} (a) a polypeptide comprising a formyltransferase enzyme, wherein said nucleic acid encodes a protein having about or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or homology to SEQ ID NO:2, or wherein said nucleic acid encodes SEQ ID NO:2;', '(b) a wzy polymerase, wherein said nucleic acid encodes a protein having about or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or homology to SEQ ID NO:3, or wherein said nucleic acid encodes SEQ ID NO:3; and', {'i': 'Pseudomonas', '(c) a glycosyltransferase derived from an rfb cluster of , wherein said nucleic acid has been stably inserted into the genome of the host cell;'}], '(ii) a nucleic acid that encodes a polypeptide selected from the group consisting of(iii) a nucleic acid that encodes an oligosaccharyl transferase, and(iv) a nucleic acid that encodes a carrier protein comprising an N-glycosylation consensus, wherein if the polypeptide is (a) or (b), then the N-glycosylation consensus sequence is D/E-X-N-X-S/T and X is any amino acid except proline.104Pseudomonas aeruginosa. The host cell of claim 103 , wherein the glycotransferase is derived from an rfb cluster of serotype 6.105E. coli.. The host cell of claim 103 , wherein said host cell is106Pseudomonas. A method of producing a bioconjugate that comprises a antigen linked to a carrier protein claim 103 , said method comprising culturing the host cell of under conditions suitable for the production of proteins.107Pseudomonas. A bioconjugate produced ...

STREPTAVIDIN MUTEINS AND METHODS OF USING THEM

The invention concerns novel streptavidin muteins and methods of determining, immobilizing, isolating or purifying proteins under denaturing conditions. In one embodiment such a mutein has an Cys residue at sequence position 127 of the wild-type sequence of streptavidin and comprises at least one mutation in the region of the amino acid positions 117 to 121 with reference to the amino acid sequence of wild type streptavidin. 1. A mutein , selected from muteins of streptavidin , wherein the mutein(a) has a Cys residue at sequence position 127 with reference to the amino acid sequence of wild-type streptavidin as set forth in SEQ ID NO: 1, and{'sup': ['44', '45', '46', '47', '44', '45', '46', '47'], '#text': '(b) wherein the sequence Val-Thr-Ala-Arg(SEQ ID NO: 10) or the sequence IleGlyAlaArg(SEQ ID NO: 11) is present in the region of amino acid positions 44 to 47 with reference to the amino acid sequence of wild-type streptavidin.'}3. A mutein according to claim 1 , wherein the mutein is a mutein of a minimal streptavidin which begins N-terminally in the region of the amino acids 10 to 16 of wild type streptavidin and terminates C-terminally in the region of the amino acids 133-142 of wild type streptavidin.4. A mutein according to claim 1 , wherein the mutein comprises the sequence of the mutein m302C (SEQ ID NO: 14).5. The mutein according to claim 4 , wherein the mutein consists of the sequence of mutein m1-9C (SEQ ID NO: 16) or m302C (SEQ ID NO: 14).6. A nucleic acid molecule claim 1 , comprising a sequence coding for a streptavidin mutein as defined in .7. A method of isolating claim 1 , purifying or determining under denaturing conditions a protein that is fused with a) a peptide sequence of the formula Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa (SEQ ID NO: 8) in which Xaa represents an arbitrary amino acid and Yaa and Zaa either both denote Gly or Yaa denotes Glu and Zaa denotes Arg or Lys claim 1 , or b) with a peptide sequence that comprises a sequential arrangement of ...

LIVER-SPECIFIC NUCLEIC ACID REGULATORY ELEMENTS AND METHODS AND USE THEREOF

Described are nucleic acid regulatory elements that are able to enhance liver-specific expression of genes, methods employing these regulatory elements and uses of these elements. Expression cassettes and vectors containing these nucleic acid regulatory elements are also disclosed. These are particularly useful for applications using gene therapy. 120.-. (canceled)21. A nucleic acid expression cassette comprising: wherein the nucleic acid regulatory element comprises SEQ ID NO:14 or a polynucleotide having at least 95% identity to SEQ ID NO:14, and', 'wherein the nucleic acid regulatory element is operably linked to a promoter and a heterologous transgene., 'a nucleic acid regulatory element of 200 nucleotides or less that enhances liver-specific gene expression,'}22. The nucleic acid expression cassette of claim 21 , further comprising:at least one additional nucleic acid regulatory element that enhances liver-specific gene expression comprising a polynucleotide having at least 95% identity to a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, wherein said second nucleic acid regulatory element is 600 nucleotides or less.23. The nucleic acid expression cassette of claim 22 , wherein the total length of the combined nucleic acid regulatory elements does not exceed 700 nucleotides.24. The nucleic acid expression cassette of claim 22 , wherein the cassette comprises at least two identical nucleic acid regulatory elements.25. The nucleic acid expression cassette of claim 22 , wherein the cassette comprises repeats of polynucleotides having at least 95% identity to SEQ ID NO:14.26. The nucleic acid expression cassette of claim 25 , wherein the cassette comprises three repeats of polynucleotides having at least 95% identity to SEQ ID NO:14.27. The nucleic acid expression cassette of ...

METHOD OF PRODUCING RNA FROM CIRCULAR DNA AND CORRESPONDING TEMPLATE DNA

The present invention is concerned with a method of producing a target RNA using a circular DNA, wherein said method does not comprise a step of linearizing said circular DNA. The present invention further relates to a circular DNA comprising an RNA polymerase promoter sequence, followed by a sequence encoding a target RNA, followed by a sequence encoding a self-cleaving ribozyme, followed by an RNA polymerase terminator sequence element, wherein the latter element may comprise several RNA polymerase terminator sequences. Multimeric DNA obtained by rolling circle amplification of said circular DNA is also within the scope of the present invention. 2. The method according to claim 1 , wherein said circular DNA provided in step a) comprises an RNA polymerase terminator sequence element iv which comprises at least one Class II termination sequence and fails to comprise a Class I termination sequence.3. The method according to or claim 1 , wherein said circular DNA provided in step a) comprises an RNA polymerase terminator sequence element iv which comprises at least one VSV terminator sequence and/or at least one 1R11 variant rrnB T1 downstream terminator sequence.4. The method according to or claim 1 , wherein said circular DNA provided in step a) comprises an RNA polymerase terminator sequence element iv which comprises at least one VSV terminator sequence.5. The method according to any one of to claim 1 , wherein the in vitro transcription in step b) is carried out in the presence of naturally occurring nucleotides and at least one modified nucleotide claim 1 , wherein said at least one modified nucleotide at least partially replaces at least one naturally occurring nucleotide.6. The method according to any one of to claim 1 , wherein the in vitro transcription in step b) is carried out in the presence of a cap analog.7. The method according to any one of to claim 1 , wherein said target RNA is purified in step c) by at least one first and at least one second ...

COMPOSITIONS AND METHODS OF NUCLEIC ACID-TARGETING NUCLEIC ACIDS

This disclosure provides for compositions and methods for the use of nucleic acid-targeting nucleic acids and complexes thereof. 126-. (canceled)27. An engineered nucleic acid targeting nucleic acid , comprising:an engineered CRISPR RNA (crRNA), an engineered trans-activating CRISPR RNA (tracrRNA), or a combination thereof, wherein (i) the engineered nucleic acid targeting nucleic acid can associate with a Cas9 protein to form a nucleic acid targeting nucleic acid-Cas9 complex capable of binding a target DNA, and (ii) the engineered crRNA, the engineered tracrRNA, or the combination thereof is altered relative to a corresponding wild-type crRNA, tracrRNA, or both; andthe engineered nucleic acid targeting nucleic acid comprises at least one of the following: a duplex variant, a spacer extension, a mid-tracr modification, or a tracr extension.28. The engineered nucleic acid targeting nucleic acid of claim 27 , wherein the duplex variant comprises at least one of the following modifications: a modification of a first stem claim 27 , a modification of a bulge claim 27 , a modification of a first duplex claim 27 , or an affinity tag.29. The engineered nucleic acid targeting nucleic acid of claim 28 , wherein the modification of the first stem comprises a Csy4 binding sequence to which a Csy4 protein binds claim 28 , a Cas5 binding sequence to which a Cas5 protein binds claim 28 , or a Cas6 binding sequence to which a Cas6 protein binds.30. The engineered nucleic acid targeting nucleic acid of claim 27 , wherein the spacer extension comprises at least one of the following: a spacer extension less than 10 nucleotides in length claim 27 , a 5′ cap claim 27 , a riboswitch sequence claim 27 , a sequence that forms a hairpin claim 27 , a sequence that targets the nucleic acid targeting nucleic acid to a subcellular location claim 27 , a sequence that provides for tracking claim 27 , a sequence that provides a protein binding site for a protein claim 27 , a primer binding site ...

A CANCER VACCINE FOR DOGS

The present invention provides an immunogenic composition comprising a nucleic acid that comprises a sequence encoding a dog telomerase deprived of telomerase catalytic activity, or a fragment thereof. 119-. (canceled)20. An immunogenic composition comprising a nucleic acid that comprises a sequence encoding (i) a dog telomerase reverse transcriptase (TERT) deprived of telomerase catalytic activity , or (ii) a fragment thereof.21. The composition of claim 20 , wherein the nucleic acid further encodes a non-dog TERT antigenic fragment.22. The composition of claim 20 , wherein the nucleic acid is a DNA claim 20 , preferably a DNA plasmid.23. The composition of claim 20 , wherein the sequence that encodes dog TERT contains mutations that provide inactivation of the catalytic activity.24. The composition of claim 23 , wherein the mutations are deletion of at least one amino acid.25. The composition of claim 24 , wherein the sequence is deleted of amino acids VDD.26. The composition of claim 25 , wherein the sequence encoding dog TERT is further deprived of a nucleolar localization signal.27. The composition of claim 26 , wherein the sequence encoding dog TERT is deleted of N-terminal 47 amino acids with respect to full-length dog TERT sequence.28. The composition of claim 27 , wherein the nucleic acid encodes a protein comprising claim 27 , or consisting of claim 27 , SEQ ID NO: 6.29. The composition of claim 21 , wherein the nucleic acid further comprises a sequence that encodes a non-dog TERT antigenic fragment claim 21 , which non-dog TERT antigenic fragment originates from a cat TERT sequence.30. A nucleic acid that comprises a sequence encoding (i) a dog TERT deprived of telomerase catalytic activity claim 21 , or (ii) an antigenic fragment thereof.31. The nucleic acid of claim 30 , which further comprises a non-dog TERT antigenic fragment.32. The nucleic acid of claim 30 , which encodes a protein sequence selected from the group consisting of SEQ ID NO: 2 claim 30 ...

METHOD FOR PRODUCING COENZYME AND TRANSFORMANT SET FOR COENZYME PRODUCTION

The present invention provides a method for synthesizing NAD or NADH in an enzyme reaction system requiring NAD or NADH so as to supplement NAD lost due to thermal decomposition in the enzyme reaction system, and a transformant set for coenzyme production used in the method. That is, the present invention relates to a method for producing a coenzyme including performing synthesis of NAD or NADH in an enzyme reaction system requiring NAD or NADH by adding one or more thermostable enzymes required for a reaction for synthesizing NAD from nicotinamide, to the enzyme reaction system, and a transformant set for coenzyme production including one or more transformants into which one or more genes encoding thermostable enzymes required for a reaction for synthesizing NAD or NADH from nicotinamide are introduced by using a non-thermostable microorganism as a host. 1. A method for producing a coenzyme , the method comprising:{'sup': +', '+', '+, 'performing synthesis of NAD in an enzyme reaction system requiring NAD by adding one or more thermostable enzymes required for a reaction for synthesizing NAD from ADP-ribose and nicotinamide, to the enzyme reaction system.'}2. The method for producing a coenzyme according to claim 1 , wherein the thermostable enzymes required for a reaction for synthesizing NAD are nicotinamidase claim 1 , nicotinate phosphoribosyltransferase claim 1 , nicotinate-nucleotide adenylyltransferase claim 1 , NAD synthase claim 1 , ADP-ribose pyrophosphatase claim 1 , and ribose-phosphate pyrophosphokinase.3. A method for producing a coenzyme claim 1 , the method comprising:performing synthesis of NADH in an enzyme reaction system requiring NADH by adding one or more thermostable enzymes required for a reaction for synthesizing NADH from ADP-ribose and nicotinamide, to the enzyme reaction system.4. The method for producing a coenzyme according to claim 3 , wherein the thermostable enzymes required for a reaction for synthesizing NADH are nicotinamidase ...

INSECTICIDAL PROTEINS

Compositions and methods for controlling insect pests are disclosed. In particular, novel insecticidal proteins having toxicity to at least coleopteran insect pests are provided. Nucleic acid molecules encoding the novel insecticidal proteins are also provided. Methods of making the insecticidal proteins and methods of using the insecticidal proteins and nucleic acids encoding the insecticidal proteins of the invention, for example in transgenic plants to confer protection from insect damage, are also disclosed. 1. A nucleic acid molecule comprising a nucleotide sequence that encodes a protein that is toxic to an insect pest , wherein the nucleotide sequence (a) encodes a protein comprising an amino acid sequence that has at least 80% to at least 99% sequence identity with any of SEQ ID NOs:1-17 , or a toxin fragment thereof; (b) has at least 80% to at least 99% sequence identity with any of SEQ ID NOs:18-40 , or a toxin-encoding fragment thereof; or (c) is a synthetic sequence of (a) or (b) that has codons optimized for expression in a transgenic organism.2. The nucleic acid molecule of claim 1 , wherein the insecticidal protein comprises an amino acid sequence of any of SEQ ID NOs:1-17 claim 1 , or a toxic fragment thereof.34.-. (canceled)5. A chimeric gene comprising a heterologous promoter operably linked to the nucleic acid molecule of .611.-. (canceled)12. A protein that is toxic to an insect pest claim 1 , wherein the protein comprises (a) an amino acid sequence that has at least 80% to at least 99% sequence identity with an amino acid sequence of any of SEQ ID NOs:1-17 claim 1 , or a toxin fragment thereof; (b) an amino acid sequence that comprises any of SEQ ID NOs:1-17 claim 1 , or a toxin fragment thereof; (c) an amino acid sequence that is encoded by a nucleotide sequence that has at least 80% to at least 99% sequence identity with a nucleotide sequence of any of SEQ ID NOs:18-40 claim 1 , or a toxin-encoding fragment thereof; or (d) an amino acid ...

LIVER-SPECIFIC NUCLEIC ACID REGULATORY ELEMENTS AND METHODS AND USE THEREOF

Described are nucleic acid regulatory elements that are able to enhance liver-specific expression of genes, methods employing these regulatory elements and uses of these elements. Expression cassettes and vectors containing these nucleic acid regulatory elements are also disclosed. These are particularly useful for applications using gene therapy. 119.-. (canceled)20. A nucleic acid expression cassette comprising:a nucleic acid regulatory element of 100 nucleotides or less that enhances liver-specific gene expression,wherein the nucleic acid regulatory element comprises SEQ ID NO:7, or a polynucleotide having at least 95% identity to SEQ ID NO:7, andwherein the nucleic acid regulatory element is operably linked to a promoter and a heterologous transgene.21. The nucleic acid expression cassette of claim 20 , wherein the nucleic acid regulatory element does not form part of a larger regulatory region.22. A nucleic acid expression cassette comprising: a first polynucleotide of 100 nucleotides or less comprising SEQ ID NO:7 or a polynucleotide having at least 95% identity to SEQ ID NO:7; and', 'a second nucleic acid regulatory element of 90 nucleotides or less comprising SEQ ID NO:3 or a polynucleotide having at least 95% identity to SEQ ID NO:3., 'at least two nucleic acid regulatory elements that enhance liver-specific gene expression, each of the at least two nucleic acid regulatory elements comprising23. The nucleic acid expression cassette of claim 22 , wherein the at least two nucleic acid regulatory elements do not form part of a larger regulatory region.24. The nucleic acid expression cassette of claim 22 , wherein the length of the total of the at least two regulatory elements does not exceed 700 nucleotides.25. The nucleic acid expression cassette of claim 22 , wherein the at least two nucleic acid regulatory elements are identical.26. The nucleic acid expression cassette of claim 22 , wherein the at least two nucleic acid regulatory elements comprise repeats ...

Protein Layers And Their Use In Electron Microscopy

Protein layers ( 1 ) repeating regularly in two dimensions comprise protein protomers ( 2 ) which each comprise at least two monomers ( 5 ), ( 6 ) genetically fused together. The monomers ( 5 ), ( 6 ) are monomers of respective oligomer assemblies ( 3 ), ( 4 ) into which the monomers are assembled to assembly of the protein layer. The first oligomer assembly ( 3 ) belongs to a dihedral point group of order O, where O equals ( 3 ), ( 4 ) or ( 6 ) and has a set of O rotational symmetry axes of order ( 2 ). The second oligomer assembly ( 4 ) has a rotational symmetry axis of order ( 2 ). Due to the symmetry of the oligomer assemblies ( 3 ), ( 4 ), the rotational symmetry axes of each second oligomer assembly ( 4 ) is aligned with one of said set of O rotational symmetry axes of a first oligomer assembly ( 3 ) with ( 2 ) protomers being arranged symmetrically therearound. Thus, an 2-fold fusion between the oligomer assemblies ( 3 ), ( 4 ) is produced and the arrangements of the rotational symmetry axes of the oligomer assemblies ( 3 ), ( 4 ) cause the protein layer to repeat regularly. The protein layer has many uses, for example to support molecular entities for biosensing, x-ray crystallography or electron microscopy.

METHOD FOR PREPARING CRYSTALS OF IsPETase PROTEIN AND IsPETase VARIANTS

Disclosed are a method for preparing crystals of IsPETase protein, a method for screening an IsPETase protein activity regulator and IsPETase variants using a conformation of the protein crystal, a method for screening, IsPETase variants with increased PETase activity, and a method for decomposing PET using the variants. According to exemplary embodiments of the present invention, it is possible to determine a method for effectively preparing a crystal of the IsPETase protein and to obtain the resulting crystal thereof. Further, according to exemplary embodiments of the present invention, it is possible to identify a tertiary structure of the IsPETase from the crystal thereof and to prepare the variant with an increased PETase activity based on this structure. The IsPETase variant may be used effectively in the PET decomposition field. 1. An IsPETase variant having one or more amino acid substitutions in the amino acid sequence represented by SEQ ID NO: 1 , wherein the IsPETase variant includes an amino acid substitution at position 280-th from N-terminal of SEQ ID NO: 1 with alanine.2. The IsPETase variant of claim 1 , wherein the IsPETase variant further includes an amino acid substitution at position 121-th from N-terminal of SEQ ID NO: 1 with aspartic acid or an amino acid substitution at position 186-th from N-terminal of SEQ ID NO: 1 claim 1 , wherein the substitution of the 186th amino acid is replaced by any one of histidine claim 1 , phenylalanine claim 1 , isoleucine claim 1 , leucine claim 1 , and valine.3. The IsPETase variant of claim 1 , wherein the IsPETase variant further includes an amino acid substitution at position 121-th from N-terminal of SEQ ID NO: 1 with aspartic acid and an amino acid substitution at position 186-th from N-terminal of SEQ ID NO: 1 claim 1 , wherein the substitution of the 186th amino acid is replaced by any one of histidine claim 1 , phenylalanine claim 1 , isoleucine claim 1 , leucine claim 1 , and valine.4. The IsPETase ...

CULTURE MODIFIED TO CONVERT METHANE OR METHANOL TO 3-HYDROXYPROPRIONATE

Provided are engineered organisms which can convert methane or methanol to 3-hydroxypropionate. 1. A synthetic culture comprising one or more microorganisms comprising one or more modifications that improve the production of a product from a substrate , wherein the substrate comprises methane and/or methanol.2. The synthetic culture according to claim 1 , wherein the substrate comprises methane.3. The synthetic culture according to claim 2 , wherein the product comprises 3-hydroxyproprionate.4. The synthetic culture according to claim 1 , wherein the product comprises 3-hydroxyproprionate.5. The synthetic culture according to claim 1 , wherein the product comprises a substance derived from acetyl-CoA and/or malonyl-CoA.6Escherichia coli.. The synthetic culture according to claim 1 , wherein at least one of the one or more microorganisms comprises7. The synthetic culture according to claim 1 , wherein the one or more microorganisms comprises a first at least one microorganism and a second at least one microorganism claim 1 , wherein the first at least one microorganism produces methanol from methane and the second at least one microorganism produces 3-hydroxypropionate from methanol.8. The synthetic culture according to claim 1 , wherein the one or more modifications comprise exogenous polynucleotides or deletion of one or more genes.9. The synthetic culture according to claim 8 , wherein the exogenous polynucleotides encode polypeptides selected from one or more polypeptides comprising methane monooxygenase (EC 1.14.13.25) claim 8 , malonyl-CoA reductase (EC 1.2.1.75) claim 8 , acetyl-CoA carboxylase (EC 6.4.1.2) claim 8 , methanol dehydrogenase (EC 1.1.1.244 or EC 1.1.2.7) claim 8 , 3-hexulose-6-phosphate synthase (EC 4.1.2.43) claim 8 , and/or 6-phospho-3-hexuloisomerase (EC 5.3.1.27).10Bacillus methanolicus, Bacillus stearothermophilusCorynebacterium glutamicum.. The synthetic culture according to claim 9 , wherein the methanol dehydrogenase comprises a methanol ...

MUTANT MICROORGANISM PRODUCING L-ASPARTIC ACID DERIVATIVES, AND METHOD FOR PRODUCING L-ASPARTIC ACID DERIVATIVES USING SAME

The present invention relates to a mutant organism having the ability to produce aspartic acid derivatives, wherein a gene encoding the glyoxylate shunt regulator and a gene encoding fumarase are deleted and a gene encoding aspartase is overexpressed compared to that in a wild-type strain, and to a method for producing L-aspartic acid derivatives using the same. According to the present invention, various aspartic acid derivatives, including L-alanine, 3-aminopropionic acid, threonine, 1,3-diaminopropane, lysine, methionine, 3-hydroxypropionic acid, cadaverine, 5-aminovaleric acid, etc., can be produced by biological methods. 1. A mutant microorganism having the ability to produce L-aspartic acid derivatives from sugar , wherein a gene encoding the glyoxylate shunt regulator and a gene encoding fumarase are deleted , and a gene encoding aspartase is overexpressed compared to that in a wild-type strain.2. The mutant microorganism of claim 1 , wherein the overexpression of the gene encoding aspartase is performed by introducing a vector comprising the gene encoding aspartase or replacing a wild-type promoter with a strong promoter in the chromosome of the host strain.3. The mutant microorganism of claim 1 , wherein the microorganism is selected from the group consisting of bacteria claim 1 , yeasts claim 1 , and fungi.4. The mutant microorganism of claim 1 , wherein the gene encoding the glyoxylate shunt regulator is an iclR gene.5. The mutant microorganism of claim 1 , wherein the gene encoding fumarase is selected from the group consisting of fumA claim 1 , fumB and fumC.6. The mutant microorganism of claim 1 , wherein the gene encoding aspartase is an aspA gene.7. The mutant microorganism of claim 1 , wherein the strong promoter is selected from the group consisting of a trc promoter claim 1 , a tac promoter claim 1 , a T7 promoter claim 1 , an lac promoter claim 1 , and a trp promoter.8. The mutant microorganism of claim 1 , wherein the L-aspartic acid derivatives ...

FUSION PROTEIN OF ANTIBODY THAT RECOGNIZES CANCER CELLS AND MUTANT STREPTAVIDIN

It is an object of the present invention to provide a fusion protein of an antibody that recognizes cancer cells and a mutant streptavidin, which is for use in the treatment or diagnosis of cancer. According to the present invention, provided is a fusion protein having the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 7, a linker sequence, and the amino acid sequence as set forth in SEQ ID NO: 1 (provided that the amino acid sequence portion consisting of 6 histidine residues at the C-terminus thereof may be partially or entirely deleted), from the N-terminal side to the C-terminal side, in this order. 1. A fusion protein having the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 7 , a linker sequence , and the amino acid sequence as set forth in SEQ ID NO: 1 (provided that the amino acid sequence portion consisting of 6 histidine residues at the C-terminus thereof may be partially or entirely deleted) , from the N-terminal side to the C-terminal side , in this order.2. The fusion protein according to claim 1 , wherein the number of amino acids of the linker sequence is 5 to 15.3. The fusion protein according to claim 1 , wherein the linker sequence consists of 4 to 14 glycine residues and 1 cysteine residue.4. The fusion protein according to claim 1 , wherein the linker sequence is Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly or Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly.5. A fusion protein having the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 8 (provided that the amino acid sequence portion consisting of 6 histidine residues at the C-terminus thereof may be partially or entirely deleted).6. The fusion protein according to claim 1 , further having a secretory signal sequence.7. A fusion protein having the amino acid sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 9 (provided that the amino acid sequence portion consisting of 6 histidine residues at the C-terminus thereof may be partially or entirely deleted).8. A ...

RECOMBINANT MICROORGANISMS HAVING A METHANOL ELONGATION CYCLE (MEC)

Provided are microorganisms that catalyze the synthesis of chemicals and biochemicals from a methanol, methane and/or formaldehyde. Also provided are methods of generating such organisms and methods of synthesizing chemicals and biochemicals using such organisms. 1. A recombinant microorganism comprising a metabolic pathway for the synthesis of acetyl phosphate from formaldehyde using a pathway comprising (i) an enzyme having phosphoketolase activity and (ii) (a) a hexulose-6-phosphate synthase , or (b) dihdroxyacetone synthase , wherein the microorganism has an acetyl-phosphate yield better than a wild-type or parental organism.2. The recombinant microorganism of claim 1 , wherein the microorganism is a prokaryote.3E. coli. The recombinant microorganism of claim 2 , wherein the microorganism is derived from an microorganism.4E. coli. The recombinant microorganism of claim 3 , wherein the is engineered to express a phosphoketolase.5. The recombinant microorganism of claim 4 , wherein the phosphoketolase is Fpk claim 4 , Xpk or a bifunctional F/Xpk enzyme or homolog thereof.6. The recombinant microorganism of claim 1 , wherein the microorganism is engineered to heterologously expresses one or more of the following enzymes:(a) a phosphoketolase;(b) a transaldolase;(c) a transketolase;(d) a ribose-5-phosphate isomerase;(e) a ribulose-5-phosphate epimerase;(f) a hexulose-6-phosphate synthase;(g) a hexulose-6-phosphate isomerase;(h) a dihydroxyacetone synthase; and(i) a fructose-6-phosphate aldolase.7Bifidobaceterium adolescentis.. The recombinant microorganism of claim 1 , wherein the microorganism is engineered to express a phosphoketolase derived from8. The recombinant microorganism of claim 7 , wherein the phosphoketolase is a bifunctional F/Xpk.9. The recombinant microorganism of claim 7 , wherein the phosphoketolase comprises a sequence that is at least 49% identical to SEQ ID NO:2 and has phosphoketolase activity.10. The recombinant microorganism of claim 1 , ...

NRPS-PKS GENE CLUSTER AND ITS MANIPULATION AND UTILITY

A nucleic acid molecule comprises a nucleotide sequence: as shown in SEQ ID No. 1, which is the complement of SEQ ID No. 1, which is degenerate with SEQ ID No. 1, or which has at least 85% sequence identity with SEQ ID No. 1, or which is a part of such a sequence. The nucleic acid molecule encodes or is a complementary to a nucleic acid molecule encoding one or more polypeptides, or comprises or is complementary to a nucleic acid molecule comprising one or more genetic elements, having functional activity in the synthesis of a polyketide-based or macrolactam molecule. The nucleic acid molecule may be used to prepare a modified BE-14106 biosynthetic gene cluster for the preparation of a modified BE-14106 molecule. 1. A nucleic acid molecule comprising:(a) a nucleotide sequence as shown in SEQ ID No. 1; or (b) a nucleotide sequence which is the complement of SEQ ID No. 1; or(c) a nucleotide sequence which is degenerate with SEQ ID No. 1; or(d) a nucleotide sequence having at least 85% sequence identity (preferably at least 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) with SEQ ID No. 1; or(e) a part of any one of (a) to (d), wherein said nucleic acid molecule encodes or is a complementary to a nucleic acid molecule encoding one or more polypeptides, or comprises or is complementary to a nucleic acid molecule comprising one or more genetic elements, having functional activity in the synthesis of a polyketide-based or macrolactam molecule.2. The nucleic acid molecule of claim 1 , wherein said molecule encodes an NRPS-PKS biosynthetic system for synthesis of a polyketide-based or macrolactam molecule.3. The nucleic acid molecule of claim 1 , wherein said molecule comprises a nucleotide sequence as shown in any one of SEQ ID Nos 2 claim 1 , 4 claim 1 , 6 claim 1 , 8 claim 1 , 10 claim 1 , 12 claim 1 , 14 claim 1 , 16 claim 1 , 18 claim 1 , 20 claim 1 , 22 claim 1 , 24 claim 1 , 26 claim 1 , 28 claim 1 , 30 claim 1 , 32 claim 1 , 34 claim 1 , ...

COMPOSITIONS AND METHODS COMPRISING SEQUENCES HAVING HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) ACTIVITY

Compositions and methods comprising polynucleotides and polypeptides having 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity and having insensitivity to an HPPD inhibitor are provided. Further provided are nucleic acid constructs, plants, plant cells, explants, seeds and grain having the HPPD sequences. Various methods of employing the HPPD sequences are provided. Such methods include, for example, methods for producing an HPPD inhibitor tolerant plant, plant cell, explant or seed and methods of controlling weeds in a field containing a crop employing the plants and/or seeds disclosed herein. Methods are also provided to identify additional HPPD variants. Further provided are various methods and compositions that allow the various HPPD polypeptides and variant and fragments thereof to be expressed in a chloroplast or transported to a chloroplast. 2. The polypeptide of claim 1 , wherein the polypeptide further comprises substitution of one or more conservative amino acids claim 1 , insertion of one or more amino acids claim 1 , deletion of one or more amino acids claim 1 , and combinations thereof.3. The polypeptide of claim 1 , wherein the polypeptide has an ON rate ratio of at least about 0.5; wherein the ON rate ratio is the ratio of the reaction rate with herbicidal inhibitor to the reaction rate without herbicidal inhibitor; and wherein the reaction rates are determined in an in vitro assay.4. The polypeptide of claim 3 , wherein the herbicidal inhibitor is mesotrione or tembrione.5. The polypeptide of claim 4 , wherein in vitro assay is carried out in the presence of 60 or 120 nM of the 4-hydroxyphenylpyruvate dioxygenase protein; and 100 μM 4-hydroxyphenylpyruvate.6. The polypeptide of claim 3 , wherein the polypeptide has an OFF rate ratio of at least about 0.3; wherein the OFF rate ratio is the ratio of the steady state rate in the presence of inhibitor to the initial reaction rate in the absence of inhibitor; and wherein the reaction rates are determined ...

PYRUVATE CARBOXYLASE AND PYRUVATE CARBOXYLASE-ENCODING DNA, PLASMID CONTAINING SAID DNA AND MICROORGANISM FOR THE PRODUCTION THEREOF, AND METHODS FOR THE PRODUCTION OF PRODUCTS THE BIOSYNTHESIS OF WHICH INCLUDES OXALOACETATE AS PRECURSOR, AND CHROMOSOME

A DNA sequence that includes at least 70% identity with respect to SEQ ID NO: 1 and further includes a triplet at position 1027-1029 that codes for alanine. 1. A DNA sequence , comprising:at least 70% identity with respect to SEQ ID NO: 1,wherein a triplet at position 1027-1029 codes for alanine.2. The DNA sequence according to claim 1 , wherein the DNA has identity of at least 80% with respect to SEQ ID NO: 1.3. The DNA sequence according to claim 1 , wherein the DNA sequence is a DNA according to SEQ ID NO: 1.4. The DNA sequence according to claim 1 , further comprising claim 1 , at position 3034-3036 claim 1 , a triplet which encodes for serine.5. The DNA sequence according to claim 4 , wherein the DNA has identity of at least 80% with respect to SEQ ID NO: 2.6. The DNA sequence according to claim 4 , wherein the DNA is according to SEQ ID NO: 2.7. A pyruate carboxylase having at least 90% identity with respect to a pyruvate carboxylase according to SEQ ID NO: 3 claim 4 , wherein alanine is present at position 343.8. The pyruvate carboxylase according to claim 7 , wherein the pyruvate carboxylase has identity of at least 95% with respect to the pyruvate carboxylase according to SEQ ID NO: 3.9. The pyruvate carboxylase according to claim 7 , wherein the pyruvate carboxylase is a pyruvate carboxylase according to SEQ ID NO: 3.10. The pyruvate carboxylase according to claim 6 , further comprising serine at position 1012.11. The pyruvate carboxylase according to claim 10 , wherein the pyruvate carboxylase has identity of at least 95% with respect to the pyruvate carboxylase according to SEQ ID NO: 4.12. The pyruvate carboxylase according to claim 10 , wherein the pyruvate carboxylase is a pyruvate carboxylase according to SEQ ID NO: 4.13. A vector claim 1 , wherein the vector comprises a DNA sequence according to .14. The vector according to claim 13 , wherein the vector is a plasmid.15. A microorganism claim 1 , wherein the microorganism comprises a DNA sequence ...

METHODS FOR PRODUCING CARBO SUGARS AND APPLICATIONS THEREOF

Described herein are biological devices and methods for using the same to produce carbo sugars. The biological devices include microbial cells transformed with a DNA construct containing genes for producing a cellulose synthase and galactomannan galactosyltransferase. In some instances, the biological devices also include a gene for lipase. Methods for altering the viscosity of petroleum oil using the carbo sugars are also described herein. Finally, methods for degreasing or decontaminating water mixed with petroleum oil or other fatty substances or a surface coated with petroleum oil or other fatty substances using the carbo sugars are described herein. 1. A DNA construct comprising the following genetic components:a. a gene that expresses cellulose synthase; andb. a gene that expresses galactomannan galactosyltransferase.2. The construct of claim 1 , wherein the gene that expresses cellulose synthase is SEQ ID NO. 1 or 70% homology thereof.3. The construct of claim 1 , wherein the gene that expresses galactomannan galactosyltransferase is SEQ ID NOS. 2-4 or at least 70% homology thereof.4. The construct of claim 1 , wherein the construct further comprises a promoter positioned before the gene that expresses cellulose synthase.5. (canceled)6. (canceled)7. The construct of claim 1 , wherein the construct further comprises a gene that expresses lipase.8. The construct of claim 7 , wherein the gene that expresses lipase is SEQ ID NO. 9 or at least 70% homology thereof.9. (canceled)10. (canceled)11. The construct of claim 1 , wherein the construct comprises from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses cellulose synthase and (b) a gene that expresses galactomannan galactosyltransferase.12. The construct of claim 1 , wherein the construct comprises from 5′ to 3′ the following genetic components in the following order: (a) a gene that expresses lipase claim 1 , (b) a gene that expresses cellulose synthase claim 1 , and ( ...

PHOSPHORYLATION-BASED MIRNA SENSOR

Provided herein are genetic circuits and cell state classifiers for detecting the microRNA profile of a cell. The cell state classifiers of the present disclosure utilize phosphorylation state of a transcription factor to control classifier output. Kinases and phosphatase pairs that function in phosphorylating or dephosphorylating the transcription factor are integrated into the circuit, their expression tuned by the presence of microRNAs of interest (e.g., in a cell). The genetic circuits and cell state classifiers may be used in various applications (e.g., therapeutic or diagnostic applications). 1. A cell state classifier , comprising:(i) a first sensor circuit comprising a constitutive promoter operably linked to a nucleotide sequence encoding an activator, and a constitutive promoter operably linked to a nucleotide sequence encoding a kinase that phosphorylates the activator and produces a phosphorylated activator, and one or more target sites for a first microRNA;(ii) a second sensor circuit comprising a constitutive promoter operably linked to a nucleotide sequence encoding a phosphatase that de-phosphorylates the phosphorylated activator, and one or more target sites for a second microRNA; and(iii) a signal circuit comprising an activatable promoter that is activated by the phosphorylated activator, operably linked to a nucleotide sequence encoding an output molecule, and one or more target sites for the first microRNA.23.-. (canceled)4. The cell state classifier of claim 1 , wherein the kinase claim 1 , the phosphatase claim 1 , and/or the activator are members of a bacterial two-component signaling system.5. The cell state classifier of claim 4 , wherein bacterial two-component system comprises a histidine kinase comprises an amino acid sequence motif of HEXXN claim 4 , HEXXT claim 4 , or HDXXXP claim 4 , wherein X is any amino acid claim 4 , and a response regulator.620.-. (canceled)21. The cell state classifier of claim 5 , wherein the activator ...

HPPD VARIANTS AND METHODS OF USE

In the present invention, HPPD polypeptides and plants containing them showing a full tolerance against one or more HPPD inhibitor herbicides belonging to various chemical classes are described. A set of mutant HPPD polypeptides have been designed which have either no or only a significantly reduced affinity to HPPD inhibitor herbicides and, at the same time, the rate of dissociation of the HPPD inhibitors of the mutant HPPD polypeptide is increased to such an extent that the HPPD inhibitors no longer act as slow-binding or slow, tight-binding inhibitors but, instead of this, have become fully reversible inhibitors. In particular, isolated polynucleotides encoding mutant HPPD polypeptides conferring tolerance to HPPD inhibitor herbicides belonging to various chemical classes are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. 1. A recombinant nucleic acid molecule encoding an enzymatically active 4-hydroxyphenylpyruvate dioxygenase (HPPD) polypeptide whose amino acid sequence (1) comprises the substitutions (a) an alanine at the amino acid position corresponding to amino acid position 268 of SEQ ID NO:1 , (b) a proline at the amino acid position corresponding to amino acid position 335 of SEQ ID NO:1 , (c) a histidine or an aspartic acid at the position corresponding to amino acid position 336 of SEQ ID NO:1 , and (d) a serine at the position corresponding to amino acid position 337 of SEQ ID NO:1; or i. a lysine or leucine at the amino acid position corresponding to amino acid position 213 of SEQ ID NO:1; and/or', 'ii. an alanine at the amino acid position corresponding to amino acid position 215 of SEQ ID NO:1; and/or', 'iii. an arginine, asparagine, leucine, glutamic acid, proline or serine at the amino acid position corresponding to amino acid position 270 of SEQ ID NO:1; and/or', 'iv. an arginine, lysine, glutamine, methionine or histidine at the amino acid position corresponding to amino acid position 315 of ...

EXPRESSION VECTOR SYSTEM COMPRISING TWO SELECTION MARKERS

The invention pertains to an expression vector or a combination of at least two expression vectors comprising at least 2. The method according to claim 1 , wherein an expression vector or a combination of expression vectors comprising a polynucleotide according to a) claim 1 , b) claim 1 , and c) is introduced into the host cell.3. A method for selecting at least one host cell capable of expressing a product of interest claim 1 , comprising (i) an introduced polynucleotide encoding a product of interest;', '(ii) an introduced polynucleotide encoding a first selectable marker (sm I); and', '(iii) an introduced polynucleotide encoding a second selectable marker (sm II), which differs from the first selectable marker (sm I);', 'wherein the activity of the selectable marker (sm I) or (sm II) at least partially depends on the activity of the other selectable marker and wherein the selectable markers (sm I) and (sm II) are involved in folate metabolism; and, '(a) providing a plurality of host cells, comprising'}(b) culturing said plurality of host cells under conditions selective for the selectable markers (sm I) and (sm II), thereby obtaining a host cell expressing the product of interest.4. The method according to claim 3 , comprising additionally a step of(c) selecting at least one host cell which expresses the product of interest.5. The method according to claim 3 , wherein said culturing is in a selective culture medium comprising folate in a limiting concentration and an antifolate.6. The method according to claim 5 , wherein the selective culture medium comprises folate in a concentration of 500 nM or less claim 5 , or 100 nM or less claim 5 , or wherein the selective culture medium comprises an antifolate in a concentration of 500 nM or less claim 5 , or in a concentration of 200 nM or less.7. The process according to claim 3 , comprising at least one of the following steps:(a) isolating the product of interest from said cell culture medium and/or from said host ...

METHODS OF REDUCING ODOR

The present invention relates to reduced odor. More specifically the invention relates to a method for hydrolyzing a lipase substrate comprising: adding to said substrate a lipase variant of SEQ ID NO: 3, which variant comprises a substitution at one or more positions corresponding to positions 4; 44; 45; 49; 58; 84; 88; 93; 95; 97; 104; 108; 131; 140; 147; 152; 158; 165; 176; 177; 187; 195; 199; 208; 21 1; 221; 222; 224; 232; 244; 247; 248; 249; 251; 255; and 259 of SEQ ID NO: 3, has lipase activity, and has at least 75% but less than 100% sequence identity to SEQ ID NO: 3 or a fragment thereof with lipase activity, in which method odor is reduced when compared to the method wherein SEQ ID NO: 3 is added to the lipase substrate. The present invention also relates lipase variants, polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing and using the variants. 1. A method for hydrolyzing a lipase substrate comprising: adding to said substrate a lipase variant of SEQ ID NO: 3 , which variant comprises one or more of the substitutions: D4H , D4K , D4R; V44Y; A45V , A45I , A45L; Q49A , Q49G , Q49H , Q49K , Q49R , Q49S; I58K; I84P; I88A , I88P; F93L; P95V; N97D; A104A , A104G , A104S; K108A , K108G , K108S; R131A , R131G , R131S; T140A; A147G; S152A , S152G; H158Q; E165A , E165G , E165S; T176N; P177C , P177L , P177I , P177/V; K187N; H195D , H195S; I199G , I199V; F208G , F208K; L211C; K221A , K221G , K221P , K221S; D222P; S224G; I232H , I232K , I232N , I232R; F244T; V247A , V247G , V247S , V247D , V247L , V247P , V247I , V247Y; I248K , I248S; D249I , D249L , D249V; L251V , L251I , L251L; D255A , D255G , D255/S , G259I , G259L , and G259V of SEQ ID NO: 3 , has lipase activity , and has at least 75% but less than 100% sequence identity to SEQ ID NO: 3 or a fragment thereof with lipase activity , in which method odor is reduced when compared to the method wherein SEQ ID NO: 3 is added to ...

METHOD FOR DIRECTLY PRODUCING CARDIAC PRECURSOR CELL OR MYOCARDIAL CELL FROM FIBROBLAST

Provided is a method of inducing cardiac progenitor cells or cardiomyocytes from fibroblasts. The present invention provides a method for producing cardiac progenitor cells, comprising introducing one cardiac reprogramming factor into fibroblasts, or a method for producing cardiomyocytes, comprising introducing three cardiac reprogramming factors into fibroblasts. 1. A method for producing cardiac progenitor cells , comprising a step of introducing a Tbx6 gene into fibroblasts.2. A method for producing cardiomyocytes , comprising a step of introducing a Tbx6 gene , an SRF gene and a Myocardin gene into fibroblasts.3. A cardiac progenitor cell derived from a fibroblast , comprising an exogenous Tbx6 gene.4. A cardiomyocyte derived from a fibroblast , comprising an exogenous Tbx6 gene , an exogenous SRF gene and an exogenous Myocardin gene.5. An inducer for inducing cardiac progenitor cells from fibroblasts , wherein the inducer comprises a Tbx6 gene.6. An inducer for inducing cardiomyocytes from fibroblasts , wherein the inducer comprises a Tbx6 gene , an SRF gene and a Myocd gene.7. An inducer for inducing smooth muscle cells from fibroblasts , wherein the inducer comprises a Tbx6 gene , an SRF gene and a Myocd gene.8. An inducer for inducing vascular endothelial cells from fibroblasts , wherein the inducer comprises a Tbx6 gene , an SRF gene and a Myocd gene. The present invention relates to a method for producing cardiac progenitor cells and cardiomyocytes from fibroblasts; and fibroblast-derived cardiac progenitor cells and fibroblast-derived cardiomyocytes, which are produced by the aforementioned method.Heart disease has steadily increased with aging, and the incidence of heart failure in men aged 80 or over is high (14.7%). The heart is composed of cells such as cardiomyocytes and fibroblasts. Since cardiomyocytes having a beating function have almost no or completely no regeneration ability, the method for treating heart disease has been restricted so far.To ...

BENZYLISOQUINOLINE ALKALOIDS (BIA) PRODUCING MICROBES, AND METHODS OF MAKING AND USING THE SAME

Aspects of the invention include host cells that are engineered to produce benzylisoquinoline alkaloids (BIAs). The host cells include heterologous coding sequences for a variety of enzymes involved in synthetic pathways from starting compounds to BIAs of the host cell. Also provided are methods of producing the BIAs of interest by culturing the host cells under culture conditions that promote expression of enzymes encoded by the heterologous coding sequences of the host cells. Aspects of the invention further include compositions, e.g., host cells, starting compounds and kits, etc., that find use in methods of the invention. 1. A benzylisoquinoline alkaloid active pharmaceutical ingredient comprising a benzylisoquinoline alkaloid product selected from the group consisting of:a) codeine, wherein the benzylisoquinoline alkaloid active pharmaceutical ingredient does not contain a compound selected from the group consisting of morphine, oripavine, and morphinone;b) hydrocodone, wherein the benzylisoquinoline alkaloid active pharmaceutical ingredient does not contain a compound selected from the group consisting of codeine, morphine, oripavine, and morphinone;c) hydromorphone, wherein the benzylisoquinoline alkaloid active pharmaceutical ingredient does not contain a compound selected from the group consisting of codeine and morphine;d) oxycodone, wherein the benzylisoquinoline alkaloid active pharmaceutical ingredient does not contain a compound selected from the group consisting of codeine, morphine, oripavine, and morphinone;e) oxymorphone, wherein the benzylisoquinoline alkaloid active pharmaceutical ingredient does not contain a compound selected from the group consisting of codeine and morphine; andf) morphine, wherein the benzylisoquinoline alkaloid active pharmaceutical ingredient contains neomorphine.2. The benzylisoquinoline alkaloid active pharmaceutical ingredient of claim 1 , wherein the benzylisoquinoline alkaloid product comprises codeine claim 1 , and ...

TRANSAMINASE BIOCATALYSTS

The present disclosure relates to polypeptides having transaminase activity, polynucleotides encoding the polypeptides, and methods of using the polypeptides. 1. A recombinant transaminase polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2.2. The recombinant transaminase polypeptide of claim 1 , wherein the amino acid sequence of the recombinant transaminase polypeptide comprises a residue difference as compared to SEQ ID NO: 2 at one or more residue positions selected from: X8 claim 1 , X94 claim 1 , X136 claim 1 , and X209.3. The recombinant transaminase polypeptide of claim 2 , wherein the amino acid residue differences of the recombinant transaminase polypeptide are selected from the following: X8 is proline; X94 is leucine claim 2 , isoleucine claim 2 , or valine; X136 is leucine; and X209 is leucine.4. The recombinant transaminase polypeptide of claim 1 , wherein said recombinant transaminase polypeptide is capable of converting 4-oxo-4-[3-(trifluoromethyl)-5 claim 1 ,6-dihydro[1 claim 1 ,2 claim 1 ,4]triazolo[4 claim 1 ,3-a]pyrazin-7(8H)-yl]-1-(2 claim 1 ,4 claim 1 ,5-trifluorophenyl)butan-2-one (“ketoamide substrate”) to (2R)-4-oxo-4-[3-(trifluoromethyl)-5 claim 1 ,6-dihydro[1 claim 1 ,2 claim 1 ,4]triazolo[4 claim 1 ,3-a]pyrazin-7(8H)-yl]-1-(2 claim 1 ,4 claim 1 ,5-trifluorophenyl)butan-2-amine (“product”) under a defined reaction condition in the presence of an amino group donor isopropylamine to levels of the product detectable by HPLC-UV at 210 nm claim 1 , where the reaction condition comprises about 2 g/L ketoamide substrate claim 1 , about 0.5 M isopropylamine claim 1 , about 22° C. claim 1 , about pH 7.5 claim 1 , about 5% DMSO claim 1 , about 100 μM pyridoxal phosphate claim 1 , and about 20 mg/mL of transaminase polypeptide.5. The recombinant transaminase polypeptide of claim 1 , wherein said recombinant transaminase polypeptide is capable of converting the ketoamide substrate to the product with an activity that ...

URIDINE DIPHOSPHATE-DEPENDENT GLYCOSYLTRANSFERASE ENZYME

In various aspects, the present invention provides uridine diphosphate-dependent glycosyltransferase (UGT) enzymes capable of catalyzing the transfer of a monosaccharide moiety from a NDP-sugar to the 3′ carbon of a sugar moiety of a substrate, such as a terpenoid glycan, thereby functioning as a “1-3 UGT.” In other aspects, the invention provides polynucleotides encoding the 1-3 UGT, and host cells comprising the same. In still other aspects, the invention provides methods for preparing glycosylated substrates, including steviol glycosides, using the enzyme and host cells of this disclosure. 1. A uridine diphosphate-dependent glycosyltransferase (UGT) enzyme comprising an amino acid sequence that is at least about 75% identical to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.210-. (canceled)11. The enzyme of claim 1 , wherein the enzyme comprises an amino acid substitution at positions corresponding to one or more of positions 29 claim 1 , 200 claim 1 , 357 claim 1 , and 414 of SEQ ID NO: 1.12. The enzyme of claim 1 , comprising one or more of:a glycine (G) at the position corresponding to position 54 of SEQ ID NO: 5;a leucine (L) at the position corresponding to position 111 of SEQ ID NO: 5; anda methionine (M) at the position corresponding to position 183 of SEQ ID NO: 5.13. The enzyme of claim 12 , wherein the enzyme has Gly at the position corresponding to position 54 of SEQ ID NO: 5 claim 12 , Leu at the position corresponding to position 111 of the SEQ ID NO: 5 claim 12 , and a Met at the position corresponding to position 183 of SEQ ID NO: 5.14. The enzyme of claim 1 , wherein the enzyme comprises an insertion of from about 6 to about 12 amino acids after the position corresponding to position 155 of SEQ ID NO: 5.15. The enzyme of claim 14 , wherein the insertion is a flexible and hydrophilic sequence that is predominately Glycine and Serine residues claim 14 , and is optionally GSGGSG (SEQ ID NO: 7) or GSGGSGGSG (SEQ ID NO: 8).16. The enzyme of ...

TESTOSTERONAN, A NOVEL HEPAROSAN ANALOG, TESTOSTERONAN SYNTHASE, AND METHODS OF PRODUCTION AND USE THEREOF

Testosteronan, a heparosan analog having the structure [-4-D-GlcUA-α1,4-D-GlcNAc-α1-], is produced by testosteronan synthase, a single protein that is a dual-action catalyst that utilizes UDP-GlcUA and UDP-GlcNAc to synthesize a polysaccharide having the structure [-4-D-GlcUA-α1,4-D-GlcNAc-α1-]. 14-. (canceled)5. A recombinant vector comprising an isolated nucleotide sequence encoding an enzymatically active testosteronan synthase wherein the testosteronan synthase is a single protein that is a dual-action catalyst that utilizes UDP-GlcUA and UDP-GlcNAc to synthesize a polysaccharide having the repeat structure [-4-D-GlcUA-α1 ,4-D-GlcNAc-α1-] , the isolated nucleotide sequence comprising at least one of:(a) the nucleotide sequence of SEQ ID NO:2;(b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1;(c) a nucleotide sequence that is at least 90% identical to SEQ ID NO:2;(d) a nucleotide sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:1; ["(1) hybridization at a temperature of 68° C. in 5×SSC/5×Denhardt's solution/1.0% SDS, followed with washing in 3×SSC at 42° C.; and", "(2) hybridization at a temperature of 30° C. in 5×SSC, 5×Denhardt's reagent, 30% formamide for about 20 hours followed by washing twice in 2×SSC, 0.1% SDS at about 30° C. for about 15 minutes followed by 0.5×SSC, 0.1% SDS at about 30° C. for about 30 minutes;"], '(e) a nucleotide sequence capable of hybridizing to a complement of SEQ ID NO:2 under hybridization conditions of at least one of(f) a nucleotide sequence capable of hybridizing to a complement of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1 under hybridization conditions of at least one of (1) and (2) of (e) above; and(g) a nucleotide sequence encoding an amino acid sequence having up to 50 amino acid insertions, deletions or substitutions when compared to SEQ ID NO:1; and(h) a nucleotide sequence encoding an amino acid sequence having up to 25 amino acid ...

LIGHT-SWITCHABLE POLYPEPTIDE AND USES THEREOF

The present invention relates to a light-switchable polypeptide. In particular, the present invention relates to a polypeptide comprising a light-responsive element, wherein the configuration (i.e. the configurational state) of the light-responsive element can be switched between a trans and cis isomer by irradiating the polypeptide with (a) particular wavelength(s) of light, and wherein the switch of said configuration alters the conformation and binding activity of said polypeptide to a ligand (e.g. molecule of interest). Also, the present invention comprises using said light-switchable polypeptide for isolating and/or purifying a molecule of interest. The present invention further provides an affinity matrix, an affinity chromatography column, and an affinity chromatography apparatus comprising the light-switchable polypeptide of the invention. 1. A polypeptide comprising a light-responsive element ,wherein the light-responsive element can be switched between two isomers by irradiating the polypeptide with a particular wavelength of light, thereby altering the binding activity of the polypeptide to a ligand.23-. (canceled)4. A method for isolating and/or purifying a molecule of interest , comprising:{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'claim-text': wherein the polypeptide is part of a solid phase,', 'and wherein the light-responsive element is in a first configuration so that the polypeptide has high affinity to the molecule of interest; and, 'contacting a liquid phase comprising the molecule of interest with the polypeptide of ,'}(ii) irradiating the polypeptide with a wavelength that changes the light-responsive element to a second configuration so that the polypeptide has a decreased affinity to the molecule of interest as compared to the affinity of step (i); and(iii) eluting the molecule of interest from the solid phase.5. The polypeptide of claim 1 , wherein the polypeptide is streptavidin or a variant or mutein thereof comprising a light- ...

METHODS OF REDUCING ODOR

The present invention relates to reduced odor. More specifically the invention relates to a method for hydrolyzing a lipase substrate comprising: adding to said substrate a lipase variant of SEQ ID NO: 3, which variant comprises a substitution at one or more positions corresponding to positions 4; 44; 45; 49; 58; 84; 88; 93; 95; 97; 104; 108; 131; 140; 147; 152; 158; 165; 176; 177; 187; 195; 199; 208; 211; 221; 222; 224; 232; 244; 247; 248; 249; 251; 255; and 259 of SEQ ID NO: 3, has lipase activity, and has at least 75% but less than 100% sequence identity to SEQ ID NO: 3 or a fragment thereof with lipase activity, in which method odor is reduced when compared to the method wherein SEQ ID NO: 3 is added to the lipase substrate. The present invention also relates lipase variants, polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing and using the variants. 1. A method for hydrolyzing a lipase substrate comprising: adding to said substrate a lipase variant of SEQ ID NO: 3 , which variant comprises one or more of the substitutions: D4H , D4K , D4R; V44Y; A45V , A451 , A45L; Q49A , Q49G , Q49H , Q49K , Q49R , Q49S; I58K; I84P; I88A , I88P; F93L; P95V; N97D; A104A , A104G , A104S; K108A , K108G , K108S; R131A , R131G , R131S; T140A; A147G; S152A , S152G; H158Q; E165A , E165G , E165S; T176N; P177C , P177L , P1771 , P177/V; K187N; H195D , H195S; I199G , I199V; F208G , F208K; L211C; K221A , K221G , K221P , K221S; D222P; S224G; I232H , I232K , I232N , I232R; F244T; V247A , V247G , V247S , V247D , V247L , V247P , V2471 , V247Y; I248K , I248S; D2491 , D249L , D249V; L251V , L2511 , L251L; D255A , D255G , D255/S , G2591 , G259L , and G259V of SEQ ID NO: 3 , has lipase activity , and has at least 75% but less than 100% sequence identity to SEQ ID NO: 3 or a fragment thereof with lipase activity , in which method odor is reduced when compared to the method wherein SEQ ID NO: 3 is added to ...

BIOLOGICAL METHODS FOR PREPARING TERPENES

The technology relates in part to biological methods for producing terpenes and to engineered cells and microorganisms capable of such production. 1162-. (canceled)163. A genetically modified microorganism , comprising:one or more heterologous nucleic acids encoding one or more terpene biosynthesis polypeptides, wherein expression of at least one of the heterologous nucleic acids is regulated by a nucleic acid that provides for fatty acid or alkane induction of expression of the terpene biosynthesis polypeptide:wherein the one or more heterologous nucleic acids that encode one or more terpene biosynthesis polypeptides is chosen from terpene synthase, phytoene synthase, geranylgeranyl diphosphate synthase, phytoene desaturase, lycopene cyclase, bifunctional lycopene cyclase/phytoene synthase, β-carotene ketolase, β-carotene hydroxylase, astaxanthin synthase, zeaxanthin glucosyltransferase and valencene synthase, orwherein the one or more heterologous nucleic acids is a cytochrome P450 reductase and is regulated by a nucleic acid that provides for alkane induction of expression of the cytochrome P450 reductase.164CandidaYarrowiaRhodotorulaRhodosporidiumCryptococcusTrichosporonLipomycesBlastobotrys. The microorganism of claim 163 , wherein the microorganism is a yeast selected from spp claim 163 , spp claim 163 , spp claim 163 , spp claim 163 , spp claim 163 , spp claim 163 , spp claim 163 , and spp.165. The microorganism of claim 164 , wherein the fatty acid is a saturated fatty acid or an unsaturated fatty acid selected from one or more of oleic acid claim 164 , palmitoleic acid claim 164 , erucic acid claim 164 , linoleic acid claim 164 , palmitic acid claim 164 , caproic acid claim 164 , enanthic acid claim 164 , caprylic acid pelargonic acid claim 164 , capric acid claim 164 , undecylic acid claim 164 , lauric acid claim 164 , myristic acid claim 164 , pentadecanoic acid claim 164 , margaric acid claim 164 , stearic acid arachidic acid claim 164 , behenic acid ...

GENETIC SCREENING METHOD OF NEGATIVE REGULATORY FACTORS OF STREPTOMYCES BIOSYNTHESIS GENE CLUSTER

The present invention provides a screening method of negative regulatory factors of a biosynthesis gene cluster, the method including: constructing a reporter system in a cell, which is mediated by a promoter of a self-owned target gene of the cell, and then randomly mutating with the reporter system by using a random mutation system constructed based on a transposon Himar1; intensively screening strains that have been subjected to random mutation to obtain a strain with high expression of the target gene; performing phage packaging on a genome of the strain with high expression of the target gene and screening out a cosmid with a random insert; and determining the position of the random insert in the genome of the strain with high expression of the target gene by sequencing DNAs of the cosmid. 1Streptomyces. A screening method of negative regulatory factors of a biosynthesis gene cluster , comprising:{'i': Streptomyces', 'Streptomyces, 'constructing a reporter system mediated by a promoter of a self-owned target gene in a cell, and then randomly mutating with the reporter system by using a random mutation system constructed based on a transposon Himar1;'}{'i': Streptomyces', 'Streptomyces, 'intensively screening strains that have been subjected to random mutation to obtain a strain with high expression of the target gene;'}{'i': 'Streptomyces', 'packaging a genome of the strain with high expression of the target gene by a phage packaging method and screening out a cosmid with a random insert; and'}{'i': 'Streptomyces', 'finally determining an accurate position of the random insert in the genome of the strain with high expression of the target gene by sequencing DNAs of the cosmid.'}2Streptomyces. The screening method of negative regulatory factors of a biosynthesis gene cluster according to claim 1 , wherein the method comprises the following specific steps:{'i': 'Streptomyces', '(1) selecting a target gene, which needs to be screened for a regulatory factor, in a ...

MODIFIED DNA POLYMERASES

Modified X family DNA polymerases engineered to be capable of incorporating 3′-O-blocked nucleotide 5′-triphosphates during template-independent polynucleotide synthesis, and methods for synthesizing polynucleotides using said modified X family DNA polymerases. 1. A modified X family DNA polymerase comprising SEQ ID NO:1 inserted into a loop 1 region , wherein the modified X family DNA polymerase is other than a terminal deoxynucleotidyl transferase or human DNA polymerase mu.2. The modified X family DNA polymerase of claim 1 , wherein the modified X family DNA polymerase is capable of accommodating a nucleotide 5′-triphosphate comprising a removable 3′-O-blocking group.3. The modified X family DNA polymerase of claim 2 , wherein the removable 3′-O-blocking group is chosen from (CO)R claim 2 , (CO)OR claim 2 , (CO)CHOR claim 2 , (CO)NHR claim 2 , (CO)CHNHR claim 2 , (CO)SR claim 2 , CHOR claim 2 , CHN claim 2 , CHCH═CH claim 2 , CHCN claim 2 , or NH claim 2 , wherein R is alkyl or alkenyl.4. The modified X family DNA polymerase of claim 1 , wherein the modified X family DNA polymerase is capable of adding a 3′-O-blocked nucleotide to a free hydroxyl group in the absence of a nucleic acid template.5. The modified X family DNA polymerase of claim 1 , wherein the modified X family DNA polymerase is chosen from:(i) a polypeptide of less than about 400 amino acids that has at least about 90% sequence identity to SEQ ID NO:16, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39; or(iii) a polypeptide having at least about 90% sequence identity to SEQ ID NO:18, 19, 21, or 23.6. The modified X family DNA polymerase of claim 5 , wherein (i) has at least about 95% sequence identity to SEQ ID NO:16 claim 5 , 27 claim 5 , 28 claim 5 , 29 claim 5 , 30 claim 5 , 31 claim 5 , 32 claim 5 , 33 claim 5 , 34 claim 5 , 35 claim 5 , 36 claim 5 , 37 claim 5 , 38 claim 5 , or 39.7. The modified X family DNA polymerase of claim 6 , wherein (i) consists of SEQ ID NO:16 claim 6 , 27 claim ...

ENGINEERED BENZYLISOQUINOLINE ALKALOID EPIMERASES AND METHODS OF PRODUCING BENZYLISOQUINOLINE ALKALOIDS

The present disclosure provides systems and methods for increasing production of an alkaloid product through the epimerization of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benyzlisoquinoline alkaloid via an engineered epimerase in an engineered host cell. A (S)-1-benzylisoquinoline alkaloid is contacted with said engineered epimerase. Contacting said (S)-1-benzylisoquinoline alkaloid with said engineered epimerase converts said (S)-1-benzylisoquinoline alkaloid to said (R)-1-benzylisoquinoline alkaloid. 1. A method of increasing production of an alkaloid product through the epimerization of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benyzlisoquinoline alkaloid via an engineered split epimerase in an engineered host cell in comparison to the epimerization of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benyzlisoquinoline alkaloid via a fused epimerase in similar conditions , said method comprising:contacting said (S)-1-benzylisoquinoline alkaloid with said engineered split epimerase in said engineered host cell, thereby increasing production of an alkaloid product,wherein contacting said (S)-1-benzylisoquinoline alkaloid with said engineered split epimerase converts said (S)-1-benzylisoquinoline alkaloid to said (R)-1-benzylisoquinoline alkaloid within said engineered host cell, wherein contacting said (S)-1-benzylisoquinoline alkaloid with said engineered split epimerase increases production of the (R)-1-benyzlisoquinoline alkaloid as compared to the epimerization of a (S)-1-benzylisoquinoline alkaloid to a (R)-1-benyzlisoquinoline alkaloid via a fused epimerase in similar conditions.2. The method of claim 1 , wherein said fused epimerase is a wildtype epimerase.3. The method of claim 1 , wherein said fused epimerase is a parent epimerase of said engineered split epimerase.4. The method of claim 1 , wherein said fused epimerase is an engineered fused epimerase comprising an oxidase domain with a same sequence as said oxidase component of said engineered ...

METHOD FOR PRODUCTION OF MUTANT-TYPE HUMAN ERYTHROPOIETIN

Disclosed is a highly efficient method for production of highly pure mutant-type human erythropoietin. The method is for production of mutant-type human erythropoietin, in which a transformed mammalian cell is allowed to produce the mutant-type human erythropoietin, and the supernatant of the culture is subjected to hydrophobic column chromatography, multimodal anion exchange column chromatography, anion exchange column chromatography, phosphate group affinity column chromatography, and gel filtration column chromatography, in this order. 1. A method for production of a mutant-type human erythropoietin comprising the amino acid sequence set forth as SEQ ID NO:2 , the method comprising;(a) a step of providing a mutant-type human erythropoietin-producing mammalian cell which produces the mutant-type human erythropoietin,(b) a step of culturing the mutant-type human erythropoietin-producing mammalian cell in a serum-free medium to let the cell secrete the mutant-type human erythropoietin into the culture,(c) a step of preparing a supernatant by removing the cell from the culture,(d) a step of subjecting the supernatant to hydrophobic column chromatography to collect a fraction containing the mutant-type human erythropoietin,(e) a step of subjecting the fraction collected in the directly preceding step to multimodal anion exchange column chromatography to collect a fraction containing the mutant-type human erythropoietin,(f) a step of subjecting the fraction collected in the directly preceding step to anion exchange column chromatography to collect a fraction containing the mutant-type human erythropoietin,(g) a step of subjecting the fraction collected in the directly preceding step to column chromatography that employs a solid-phase material having affinity to phosphate group to collect a fraction containing the mutant-type human erythropoietin, and(h) a step of subjecting the fraction collected in the directly preceding step to gel filtration column chromatography to ...

Production of non-caloric sweeteners using engineered whole-cell catalysts

Disclosed are whole-cell catalysts, methods of making the whole-cell catalysts, and methods of using the whole-cell catalysts to produce steviol glycosides.

ALTERATION OF CELL MEMBRANE FOR NEW FUNCTIONS

Methods and compositions are provided for the persistent modification of cell membranes with exogenous proteins so as to alter the function of the cell to achieve effects similar to those of gene therapy, without the introduction of exogenous DNA. DNA sequences, the proteins and polypeptides embodying these sequences are disclosed for modulating the immune system. The modulations include down-regulation, up-regulation and apoptosis. 120-. (canceled)21. A method of attaching an immunomodulatory agent to a cell surface , comprising: contacting a biotinylated cell with a chimeric protein comprising (i) the immunomodulatory agent and (ii) avidin , streptavidin , or core streptavidin , wherein the immunomodulatory agent is selected from the group consisting of CD40L and IL-2.22. The method of claim 21 , wherein the method is effected by administering the chimeric protein to a subject containing the biotinylated cell.23. The method of claim 21 , further comprising the step of biotinylating a cell to form the biotinylated cell.24. The method of claim 21 , wherein the cell is selected from the group consisting of splenocytes claim 21 , tumor cells claim 21 , bone marrow cells claim 21 , endothelial cells claim 21 , islet cells and T cells.25. A method of modulating the immune system of a subject in need thereof claim 21 , comprising administering to the subject a chimeric protein comprising (i) an immunomodulatory agent and (ii) avidin claim 21 , streptavidin claim 21 , or core streptavidin claim 21 , wherein the immunomodulatory agent is selected from the group consisting of CD40L and IL-2.26. The method of claim 25 , wherein the immunomodulatory agent is CD40L.27. The method of claim 26 , wherein the subject is suffering from neoplasia or infectious disease.28. The method of claim 26 , wherein the chimeric protein is encoded by a nucleic acid comprising SEQ ID NO:6.29. The method of claim 25 , wherein the immunomodulatory agent is IL-2.30. The method of claim 29 , wherein ...

SPIDER SILK FUSION PROTEIN STRUCTURES WITHOUT REPETITIVE FRAGMENT FOR BINDING TO AN ORGANIC TARGET

A recombinant fusion protein comprising the moieties Band CT is provided. B is a non-spidroin moiety which provides the capacity of selective interaction with an organic target. CT is a moiety of from 70 to 120 amino acid residues and is derived from the C-terminal fragment of a spider silk protein. The fusion protein is not comprising any moiety derived from the repetitive fragment of a spider silk protein. 147-. (canceled)49. The recombinant fusion protein according to claim 48 , wherein the CT moiety has at least 50% identity to SEQ ID NO: 9 or at least 80% identity to SEQ ID NO: 7.50. The recombinant fusion protein according to claim 48 , wherein the B moiety has less than 30% identity to any of SEQ ID NOS: 6-10.51. The recombinant fusion protein according to claim 48 , wherein the organic target is selected from the group consisting of immunoglobulins and molecules comprising immunoglobulin or derivatives thereof.52. The recombinant fusion protein according to claim 51 , wherein the immunoglobulins are selected from the immunoglobulin subclasses IgG1 claim 51 , IgG2 claim 51 , IgG4 claim 51 , IgA and IGM from human.53. The recombinant fusion protein according to claim 51 , wherein the B moiety is selected from the group consisting of the Z domain derived from staphylococcal protein A and the E claim 51 , D claim 51 , A claim 51 , B and C domains thereof claim 51 , streptococcal protein G and the C1 claim 51 , C2 and C3 domains thereof; and protein fragments having at least 70% identity to any of these amino acid sequences.54. The recombinant fusion protein according to claim 55 , wherein the B moiety is selected from the group consisting of the Z domain derived from staphylococcal protein A claim 55 , the B domain of staphylococcal protein A claim 55 , and the C2 domain of streptococcal protein G; and protein fragments having at least 70% identity to any of these amino acid sequences.55. The recombinant fusion protein according to claim 54 , wherein the B ...

IN SITU EXPRESSION OF LIPASE FOR ENZYMATIC PRODUCTION OF ALCOHOL ESTERS DURING FERMENTATION

Disclosed herein are methods of producing alcohol esters during a fermentation by providing alcohol-producing microorganisms which further comprise an engineered polynucleotide encoding a polypeptide having lipase activity. 123-. (canceled)24. A recombinant host cell comprising:(a) an engineered alcohol production pathway; and(b) an engineered polynucleotide encoding a polypeptide having lipase activity.25. The recombinant host cell of wherein the polypeptide having lipase activity comprises a sequence having at least about 70% identity to SEQ ID NO:2 claim 24 , 4 claim 24 , 6 claim 24 , 256 claim 24 , 47 claim 24 , 49 claim 24 , 51 claim 24 , 53 claim 24 , 55 claim 24 , 241 claim 24 , 242 claim 24 , 243 claim 24 , 244 claim 24 , 245 claim 24 , 246 claim 24 , 247 claim 24 , 248 claim 24 , 272 claim 24 , or 274 or a fragment thereof having lipase activity.26. The recombinant host cell of wherein the polypeptide having lipase activity further comprises a sequence having at least about 70% identity to anyone of SEQ ID NOs: 249 claim 24 , 250 claim 24 , 251 claim 24 , 252 claim 24 , 253 or a fragment thereof having lipase activity.27. The recombinant host cell of wherein the polypeptide having lipase activity does not contain a glycosylation motif.28. The recombinant host cell of wherein the polypeptide having lipase activity is not glycosylated.29. The recombinant host cell of wherein the engineered polynucleotide encoding a polypeptide having lipase activity comprises a sequence having at least about 70% identity to SEQ ID NO: 1 claim 24 , 3 claim 24 , 5 claim 24 , 7 claim 24 , 8 claim 24 , 9 claim 24 , 46 claim 24 , 48 claim 24 , 50 claim 24 , 52 claim 24 , 54 claim 24 , 255 claim 24 , 271 or 273.30. A recombinant host cell comprising:(a) an alcohol production pathway; and(b) an engineered polynucleotide encoding a polypeptide having lipase activity wherein the polypeptide having lipase activity comprises a sequence having at least about 70% identity to SEQ ID NO: 2, ...

MODIFYING MESSENGER RNA STABILITY IN PLANT TRANSFORMATIONS

Materials and methods for genome engineering through transient expression of a targeted nuclease are described herein. For example, the methods described herein can include introducing into a cell a messenger RNA (mRNA) that encodes a nuclease targeted to a selected sequence within the cell, where the stability of the mRNA is modified by the addition of untranslated regions (UTRs). 1. A nucleic acid comprising:(a) a structural coding sequence encoding a rare-cutting endonuclease or a rare-cutting endonuclease subunit; and(b) a 5′ untranslated region (UTR), a 3′ UTR, or both a 5′ UTR and a 3′ UTR, wherein the 5′ UTR, 3′ UTR, or 5′ UTR and 3′ UTR are operably linked to the structural coding sequence.2. The nucleic acid of claim 1 , wherein the 5′ UTR comprises the nucleic acid sequence set forth in SEQ ID NO:10 claim 1 , or a nucleic acid sequence having at least 95 percent identity to SEQ ID NO:10.3. The nucleic acid of claim 1 , wherein the 3′ UTR comprises the nucleic acid sequence set forth in SEQ ID NO:11 claim 1 , or a nucleic acid sequence having at least 95 percent identity to SEQ ID NO:11.4. The nucleic acid of claim 1 , wherein the 5′ UTR comprises the nucleic acid sequence set forth in SEQ ID NO:10 claim 1 , or a nucleic acid sequence having at least 95 percent identity to SEQ ID NO:10 claim 1 , and wherein the 3′ UTR comprises the nucleic acid sequence set forth in SEQ ID NO:11 claim 1 , or a nucleic acid sequence having at least 95 percent identity to SEQ ID NO:11.5. The nucleic acid of claim 1 , wherein the 5′ UTR comprises the nucleic acid sequence set forth in SEQ ID NO:12 claim 1 , or a nucleic acid sequence having at least 95 percent identity to SEQ ID NO:12.6. The nucleic acid of claim 1 , wherein the 3′ UTR comprises the nucleic acid sequence set forth in SEQ ID NO:13 claim 1 , or a nucleic acid sequence having at least 95 percent identity to SEQ ID NO:13.711-. (canceled)12. An expression vector comprising the nucleic acid of .13. A method for ...

MOENOMYCIN BIOSYNTHESIS-RELATED COMPOSITIONS AND METHODS OF USE THEREOF

The methods and compositions described herein relate to the identification, isolation, and characterization of genes which encode proteins useful for the biosynthesis of transglycosylase inhibitors such as moes. The methods and compositions also relate to the production of such proteins, and their use in the synthesis of moes, the expression of moes, and the production of modified moes. 111-. (canceled)12. A method of synthesizing a moenomycin , a moenomycin derivative , or a moenomycin intermediate wholly or partially in vitro comprising: reacting a one or more moenomycin precursor , derivative and/or moenomycin intermediate with a one or more polypeptide selected from the group consisting of: moeA4 , moeB4 , moeC4 , moeB5 , moe A5 , moeD5 , moeJ5 , moeE5 , moeF5 , moeH5 , moeK5 , moeM5 , moeN5 , moe05 , moeX5 , moeP5 , moeR5 , moeS5 , moeGT1 , moeGT2 , moeGT3 , moeGT4 , and moeGT5 , under conditions wherein the moenomycin , the moenomycin derivative , or the intermediate is wholly or partially synthesized.13. A method of modifying a moenomycin wholly or partially in vitro comprising: reacting a moenomycin , a moenomycin derivative or a moe intermediate with a one or more polypeptide selected from the group consisting of: moeA4 , moeB4 , moeC4 , moeB5 , moe A5 , moeD5 , moeJ5 , moeE5 , moeF5 , moeH5 , moeK5 , moeM5 , moeN5 , moeO5 , moeX5 , moeP5 , moeR5 , moeS5 , moeGT1 , moeGT2 , moeGT3 , moeGT4 , and moeGT5 , under conditions wherein the moenomycin , the moenomycin derivative , or the moenomycin intermediate is modified.1415-. (canceled)17. The moenomycin derivative of claim 16 , wherein R and Rindependently are —NH.19. (canceled)2122-. (canceled)24. The moenomycin derivative of claim 23 , wherein R3 is hydrogen or hydroxyl.25. (canceled)2728-. (canceled)3031-. (canceled)3334-. (canceled)3637-. (canceled)3943-. (canceled)44. A pharmaceutical composition comprising the moenomycin derivative of and a pharmaceutically acceptable carrier.4648-. (canceled)49. A ...

RECOMBINANT PRODUCTION OF STEVIOL GLYCOSIDES

Recombinant polypeptides having UDP-glycosyltransferase activities, including a 1,2-19-O-glucose glycosylation activity and a 1,2-13-O-glucose glycosylation activity for synthesizing of steviol glucosides, are provided. A method of producing a steviol glycoside composition using such recombinant polypeptide is also provided. Also disclosed are steviol glycosides referred to as rebaudioside Z1 and rebaudioside Z2. 111.-. (canceled)12. A method of producing a steviol glycoside composition , the method comprising incubating a substrate with a recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO:6.13. The method of claim 12 , further comprising incubating a recombinant sucrose synthase with the substrate and the recombinant polypeptide.14. The method of claim 13 , wherein the recombinant sucrose synthase comprises an amino acid sequence having at least 80% identity to SEQ ID NO:9.15. The method of claim 13 , further comprising incubating a recombinant UDP-glycosyltransferase with the sucrose synthase claim 13 , the substrate claim 13 , and the recombinant polypeptide.16. The method of claim 15 , wherein the recombinant UDP-glycosyltransferase comprises an amino acid sequence having at least 80% identity to SEQ ID NO:11.17. The method of claim 12 , wherein the substrate is selected from the group consisting of stevioside claim 12 , rebaudioside A claim 12 , rebaudioside E claim 12 , and combinations thereof.1820.-. (canceled)21. A method of producing rebaudioside Z claim 12 , the method comprising incubating a substrate with a recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO:622. The method of claim 21 , wherein the substrate is selected from the group consisting of rubusoside claim 21 , stevioside claim 21 , and combinations thereof.23. The method of claim 21 , further comprising incubating a recombinant sucrose synthase with the substrate and the recombinant polypeptide.24 ...

NOVEL CYCLOPROPANE COMPOUNDS AND GENETICALLY MODIFIED HOST CELLS AND METHODS USEFUL FOR PRODUCING THEREOF

The present invention provides for a cyclopropane compound having the following chemical formula: 2. The cyclopropane compound of claim 1 , wherein the cyclopropane compound has a longest carbon chain with 8 to 24 carbon atoms.3. The cyclopropane compound of claim 2 , wherein the cyclopropane compound has a longest carbon chain with 12 to 24 carbon atoms.4. The cyclopropane compound of claim 3 , wherein the cyclopropane compound has a longest carbon chain with 14 to 22 carbon atoms.5. The cyclopropane compound of claim 4 , wherein the cyclopropane compound has a longest carbon chain with 16 to 20 carbon atoms.6. The cyclopropane compound of claim 1 , wherein n is 2 claim 1 , 4 claim 1 , 5 claim 1 , 6 claim 1 , 7 claim 1 , 8 claim 1 , 9 claim 1 , 10 claim 1 , or 11 claim 1 , or any range of two integers thereof.12. The cyclopropane compound of claim 1 , wherein the cyclopropane compound comprises one of the cyclopropane compounds shown in or Table 3.13. A system capable of producing a cyclopropane compound of comprising: (a) one or more biosynthetic gene clusters (BGCs) each BGC capable of synthesizing a polyketide comprising one or more cyclopropane groups claim 1 , (b) one or more genes encoding a thioesterase and/or a reductase claim 1 , and optionally (c) one or more genes encoding an O-methyltransferase claim 1 , an ethyl transferases claim 1 , a decarboxylase claim 1 , and/or a decarbonylase; wherein one or more claim 1 , or all claim 1 , of the BGC claim 1 , thioesterase claim 1 , reductase claim 1 , O-methyltransferase claim 1 , ethyl transferases claim 1 , decarboxylase claim 1 , and decarbonylase are heterologous to the genetically modified host cell and/or each other.14. A genetically modified host cell capable of producing a cyclopropane compound of comprising: (a) one or more biosynthetic gene clusters (BGCs) each BGC capable of synthesizing a polyketide comprising one or more cyclopropane groups claim 1 , (b) one or more genes encoding a thioesterase ...

CRYPTIC METABOLITES AND METHOD FOR ACTIVATING SILENT BIOSYNTHETIC GENE CLUSTERS IN ACTINOMYCETE BACTERIA

Disclosed is a high-throughput transcriptional assay format in Actinomycete bacteria, and spp. in particular, that leverages eGFP, inserted both at a neutral site and inside the biosynthetic cluster of interest, as a read-out for secondary metabolite synthesis. Using this approach, a silent gene cluster in J1074 was induced. The cytotoxins etoposide and ivermectin were revealed as potent inducers, allowing the isolation and structural characterization of nearly 20 novel small molecule products of the chosen cluster. One of these molecules is a novel antifungal, while several others inhibit a cysteine protease implicated in cancer. Studies addressing the mechanism of induction by the two elicitors led to the identification of a pathway-specific transcriptional repressor that silences the gene cluster under normal growth conditions. The successful implementation of this approach will allow future discovery of cryptic metabolites with useful bioactivities from Actinomycete bacteria. 3. (canceled)4. (canceled)5. (canceled)6. (canceled)7. A method for activating silent biosynthetic gene clusters in actinomycete bacteria , the method comprising the steps of:providing at least one actinomycete bacterial cell, wherein the bacterial cell contains at least one gene cluster that is silent or lowly-expressed, and wherein the bacterial cell contains at least one of a promoter from a targeted gene cluster fused to at least one reporter gene at a neutral site or the promoter fused to at least one reporter gene at a site within the targeted gene cluster;exposing the bacterial cell to test compounds from a small molecule library;measuring the expression of the at least one reporter gene at a first point in time;measuring the expression of the at least one reporter gene at a second point in time;identifying activation of gene clusters by determining whether the expression of the at least one reporter gene has increased by more threshold amount from the first point in time to the ...

ENGINEERING OF MULTI-CARBON SUBSTRATE UTILIZATION PATHWAYS IN METHANOTROPHIC BACTERIA

The present disclosure relates to genetically engineered methanotrophic bacteria with the capability of growing on a multi-carbon substrate (e.g., glycerol) as a primary or sole carbon source and methods for growing methanotrophic bacteria on the multi-carbon substrate. 1. A recombinant methanotrophic bacterium , comprising an exogenous polynucleotide encoding at least two glycerol utilization pathway components , wherein a first encoded glycerol utilization pathway component comprises a glycerol kinase and a second encoded glycerol utilization pathway component comprises a glycerol-3-phosphate dehydrogenase (G3PDH);wherein the recombinant methanotrophic bacterium expresses an amount of glycerol kinase and G3PDH sufficient to permit utilization of glycerol as a primary carbon source as compared to an unmodified parent methanotrophic bacterium; and{'i': Methylococcus capsulatus, Methylomonas', 'Methylosinus trichosporium, Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus, Methylomonas flagellata, Methylacidiphilum infernorum, Methylomicrobium alcaliphilum Methylocella silvestris, Methylocella palustris, Methylocella tundrae, Methylocystis daltona, Methylocystis bryophila', 'Methylocapsa aurea., 'wherein the methanotrophic bacterium is selected from sp. 16A, , or'}2Escherichia coli, Acinetobacter baumannii, Fusobacterium nucleatumvincentii, PantoeaPseudomonas aeruginosa, Shigella flexneri, Shewanella balticaActinobacillus pleuropneumoniasSalmonella entericaentericaYersinia bercovieri, Aeromonas veroniiPseudomonas fluorescens, SerratiaVibrio fischeriHaemophilus haemolyticus, Vibrio harveyi, Vibrio cholera, Pseudomonas putidaPectobacterium carotovorumcarotovorumPseudomonas syringae, Acinetobacter, Photobacterium profundumCitrobacter freundii, Klebsiella pneumoniae, EnterobacterEnterococcus casseliflavus, Enterococcus faecalis, Bacillus stearothermophilus, Bacillus subtilis, Streptococcus pyogenes, ...

MICROORGANISMS FOR PRODUCING 1,3-BUTANEDIOL AND METHODS RELATED THERETO

Provided herein is a non-naturally occurring microbial organism having a 1,3-butanediol (1,3-BDO) pathway and comprising at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme expressed in a sufficient amount to produce 1,3-BDO. In some embodiments, the pathway includes reducing equivalents from CO or hydrogen. In certain embodiments, a 1,3-BDO pathway proceeds by way of central metabolites pyruvate, succinate or alpha-ketoglutarate. Also provided herein is a method for producing 1,3-BDO, includes culturing such microbial organisms under conditions and for a sufficient period of time to produce 1,3-BDO. 2. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism further comprises an exogenous nucleic acid encoding an enzyme selected from the group consisting of a pyruvate:ferredoxin oxidoreductase claim 1 , an aconitase claim 1 , an isocitrate dehydrogenase claim 1 , a succinyl-CoA synthetase claim 1 , a succinyl-CoA transferase claim 1 , a fumarase claim 1 , a malate dehydrogenase claim 1 , an acetate kinase claim 1 , a phosphotransacetylase claim 1 , an acetyl-CoA synthetase claim 1 , an NAD(P)H:ferredoxin oxidoreductase claim 1 , a ferredoxin claim 1 , and combinations thereof.3. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism further comprises an exogenous nucleic acid encoding an enzyme selected from the group consisting of a succinyl-CoA synthetase claim 1 , a succinyl-CoA transferase claim 1 , a fumarase claim 1 , a malate dehydrogenase claim 1 , and combinations thereof.4. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises two claim 1 , three claim 1 , four claim 1 , five claim 1 , six claim 1 , seven claim 1 , eight or nine exogenous nucleic acids claim 1 , each encoding a 1 claim 1 ,3-BDO pathway enzyme.5. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises ...

ALTERATION OF PROTEOLYTIC CLEAVAGE OF BOTULINUM NEUROTOXINS

The present invention pertains to a polynucleotide encoding a modified neurotoxin polypeptide comprising a modified neurotoxin light chain and a heavy chain, said modified light chain having at least one modification conferring altered cleavage by calpain proteases. Further encompassed by the present invention are vectors and host cells comprising the polynucleotide of the invention as well as polypeptides encoded by the said polynucleotide. In addition, the invention relates to compositions comprising the polynucleotide, vector, host cell or polypeptide of the invention as a medicament. 116-. (canceled)17. A polynucleotide encoding a modified neurotoxin polypeptide comprising a modified neurotoxin light chain and a heavy chain , wherein the modified neurotoxin light chain exhibits at least one modification conferring altered cleavage by calpain proteases.18. The polynucleotide of claim 17 , wherein the at least one modification confers an increased cleavage by calpain proteases compared to a non-modified neurotoxin light chain.19. The polynucleotide of claim 18 , wherein the at least one modification is at least one calpain cleavage site which has been introduced into the neurotoxin light chain.20. The polynucleotide of claim 19 , wherein the modified neurotoxin light chain and the heavy chain are derived from BoNT/A claim 19 , BoNT/B claim 19 , BoNT/C1 claim 19 , BoNT/D claim 19 , BoNT/F claim 19 , or BoNT/G.21. The polynucleotide of claim 17 , wherein the modified neurotoxin polypeptide exhibits a shortened duration of biological activity.22. The polynucleotide of claim 17 , wherein the at least one modification confers a decreased cleavage by calpain proteases compared to a non-modified neurotoxin light chain.23. The polynucleotide of claim 22 , wherein the at least one modification is at least one substitution within a calpain cleavage site in the neurotoxin light chain.24. The polynucleotide of claim 23 , wherein the at least one substitution is a substitution ...

TRANSAMINASE BIOCATALYSTS

The present disclosure relates to polypeptides having transaminase activity, polynucleotides encoding the polypeptides, and methods of using the polypeptides. 1. A recombinant transaminase polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:110.2. The recombinant transaminase polypeptide of claim 1 , wherein said recombinant transaminase polypeptide is capable of converting 4-oxo-4-[3-(trifluoromethyl)-5 claim 1 ,6-dihydro[1 claim 1 ,2 claim 1 ,4]triazolo[4 claim 1 ,3-a]pyrazin-7(8H)-yl]-1-(2 claim 1 ,4 claim 1 ,5-trifluorophenyl)butan-2-one (“ketoamide substrate”) to (2R)-4-oxo-4-[3-(trifluoromethyl)-5 claim 1 ,6-dihydro[1 claim 1 ,2 claim 1 ,4]triazolo[4 claim 1 ,3-a]pyrazin-7(8H)-yl]-1-(2 claim 1 ,4 claim 1 ,5-trifluorophenyl)butan-2-amine (“product”) under a defined reaction condition in the presence of an amino group donor isopropylamine to levels of the product detectable by HPLC-UV at 210 nm claim 1 , where the reaction condition comprises about 2 g/L ketoamide substrate claim 1 , about 0.5 M isopropylamine claim 1 , about 22° C. claim 1 , about pH 7.5 claim 1 , about 5% DMSO claim 1 , about 100 μM pyridoxal phosphate claim 1 , and about 20 mg/mL of transaminase polypeptide.3. The recombinant transaminase polypeptide of claim 1 , wherein said recombinant transaminase polypeptide is capable of converting the ketoamide substrate to the product with an activity that is equal to or greater than the activity of the polypeptide of SEQ ID NO: 4 under the defined reaction condition.4. The recombinant transaminase polypeptide of claim 1 , wherein said recombinant transaminase polypeptide is capable of converting the ketoamide substrate to product in at least 90% enantiomeric excess.5. The recombinant transaminase polypeptide of claim 1 , wherein said recombinant transaminase polypeptide is capable of converting the ketoamide substrate to product in at least 99% enantiomeric excess.6. The recombinant transaminase polypeptide of ...