Preparation of adipic acid

The invention relates to a method for preparing adipic acid, comprising converting alpha-ketoglutaric acid (AKG) into alpha-ketoadipic acid (AKA), converting alpha-ketoadipic acid into alpha-ketopimelic acid (AKP), converting alpha-ketopimelic acid into 5-formylpentanoic acid (5-FVA), and converting 5-formylpentanoic acid into adipic acid, wherein at least one of these conversions is carried out using a heterologous biocatalyst.The invention further relates to a heterologous cell, comprising one or more heterologous nucleic acid sequences encoding one or more heterologous enzymes capable of catalysing at least one reaction step in said method.

Preparation of alpha-ketopimelic acid

The invention relates to a method for preparing alpha-ketopimelic acid, comprising converting alpha-ketoglutaric acid into alpha-ketoadipic acid and converting alpha-ketoadipic acid into alpha-ketopimelic acid, wherein at least one of these conversions is carried out using a heterologous biocatalyst. The invention further relates to a heterologous cell, comprising one or more heterologous nucleic acid sequences encoding one or more heterologous enzymes capable of catalysing at least one reaction step in the preparation of alpha-ketopimelic acid from alpha-ketoglutaric acid.

PREPARATION OF ALPHA-KETOPIMELIC ACID

The present invention relates to a method for preparing alpha-ketopimelic acid, comprising converting 2-hydroxyheptanedioic acid into alpha-ketopimelic acid, which conversion is catalysed using a biocatalyst. Further, the invention relates to a heterologous cell, comprising a nucleic acid sequence encoding an enzyme having catalytic activity in the conversion of 2-hydroxyheptanedioic acid into alpha-ketopimelic acid. Further, the invention relates to the use of a heterologous cell according to the invention in the preparation of caprolactam, diaminohexane or adipic acid. 1. Method for preparing alpha-ketopimelic acid , comprising converting 2-hydroxyheptanedioic acid into alpha-ketopimelic acid , which conversion is catalysed using a biocatalyst.2. Method according to claim 1 , wherein the biocatalyst comprises an enzyme selected from the group of ‘oxidoreductases acting on the CH—OH group of donors (EC 1.1)’ claim 1 , ‘oxidoreductases acting on the aldehyde or oxo group of donors (EC 1.2)’ claim 1 , enzymes with 2-hydroxypimelate dehydrogenase activity claim 1 , enzymes with 2-hydroxypimelate oxidase activity claim 1 , oxidoreductases classified under EC 1.97 claim 1 , and oxidoreductases classified under EC 1.98.3. Method according to claim 2 , wherein said enzyme is selected from the group ofoxidoreductases with oxygen as acceptor (EC 1.1.3), such as a lactate oxidase or another hydroxy acid oxidase;L-lactate dehydrogenases (EC 1.1.1.27);hydroxypyruvate reductases, beta-hydroxypyruvate reductases; NADH:hydropyruvate reductases and D-glycerate dehydrogenases (EC1.1.1.81);malate dehydrogenases [NADP+], NADP+-malic enzymes, NADP+-malic dehydrogenases (nicotinamide adenine dinucleotide phosphate); malate NADP dehydrogenases; NADP+ malate dehydrogenases; NADP+-linked malate dehydrogenase and malate dehydrogenases (NADP+) (EC 1.1.1.82);3-isopropylmalate dehydrogenases, beta-isopropylmalic enzymes; beta-isopropylmalate dehydrogenases; threo-Ds-3-isopropylmalate ...

HIGH EFFICIENCY, SMALL VOLUME NUCLEIC ACID SYNTHESIS

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes). 1. A multiwell plate for non-template directed synthesis of nucleic acid molecules , the plate comprising:(a) a magnetic bead located in each of a plurality of wells of the plate, and(b) an electrochemically generated acid being present in one or more well, wherein the bead is between 1.0 μm and 100 μm in diameter.2. The multiwell plate of claim 1 , wherein the number of wells in the plate is between 10 and 50 claim 1 ,000.3. The multiwell plate of claim 1 , wherein the total volume of each well is between 0.1 μl and 50 μl.4. The multiwell plate of claim 1 , wherein each well is operably connected to a pair of electrodes.5. The multiwell plate of claim 1 , wherein the wells of the plate are connected to microfluidic channels for the introduction and removal of reagents.6. A method for the generation of an assembled nucleic acid molecule claim 1 , the method comprising:(a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a well of a plate in an average amount of from about 0.001 nanomoles to about 1,000 nanomoles;(b) combining the nucleic acid molecules generated in (a) to produce a pool;(c) oining some or all of the nucleic acid molecules present in the pool formed in (b) to form a plurality of larger nucleic acid molecules;(d) eliminating nucleic acid molecules which contain sequence errors from the plurality of larger nucleic acid molecules formed in (c) to produce an error corrected nucleic acid molecule pool; and(e) assembling the nucleic acid molecules in the error corrected nucleic ...

PREPARATION OF 6-AMINOCAPROIC ACID FROM ALPHA-KETOPIMELIC ACID

The invention is directed to a method for preparing 6-aminocaproic acid, comprising decarboxylating alpha-aminopimelic acid, using at least one biocatalyst comprising an enzyme having alpha-aminopimelic acid decarboxylase activity. The invention is further directed to a method for preparing caprolactam from 6-aminocaproic acid prepared by said method, to a host cell suitable for use in a method according to the invention and to a polynucleotide encoding a decarboxylase that may be used in a method according to the invention. 1. Method for preparing 6-aminocaproic acid , comprising decarboxylating alpha-aminopimelic acid , using at least one biocatalyst comprising an enzyme having alpha-aminopimelic acid decarboxylase activity , wherein said enzyme comprises an amino acid sequence selected from the group of sequences represented by any of the SEQUENCE ID NO's: 2 , 5 , 8 and 11 and homologues of said sequences having alpha-aminopimelic acid decarboxylase activity.2. Method according to claim 1 , wherein said enzyme comprises a homologue having at least 40% claim 1 , preferably at least 60% claim 1 , in particular at least 80% claim 1 , more in particular at least 90% sequence identity with any of the SEQUENCE ID NO's: 2 claim 1 , 5 claim 1 , 8 and 11.3. Method according to claim 1 , comprising preparing alpha-aminopimelic acid from alpha-ketopimelic acid.4. Method according to claim 3 , wherein the preparation of alpha-aminopimelic acid is catalysed by a biocatalyst in the presence of an amino donor claim 3 , said biocatalyst having catalytic activity with respect to the transamination or the reductive amination of alpha-ketopimelic acid.5. Method according to claim 4 , wherein the biocatalyst comprises an enzyme having catalytic activity with respect to the transamination or the reductive amination of alpha-ketopimelic acid selected from the group of aminotransferases (E.C. 2.6.1) and amino acid dehydrogenases (E.C. 1.4.1).6. Method according to claim 5 , wherein the ...

METHODS FOR USING A THERMOSTABLE PHYTASE IN ETHANOL PRODUCTION

A method for using a thermostable phytase for eliminating or reducing phytic acid or salts of phytic acid in an alcohol production process is disclosed. The phytase can be added anywhere in the alcohol production process including a feedstock, a hammer mill, a slurry tank, a jet cooker, a liquefaction, a mash cooker, a fermentation, a beer, a distillation system, a whole stillage, a centrifuge, a thin stillage, an evaporator, a condensate, a syrup, a wet grain, a drum dryer, a distillers dried grain, distillers solubles, distillers wet grain, condensed distillers solubles distillers dried grains with solubles a molecular sieves, or any combination thereof. The alcohol production process can be in an ethanol production plant; a spirit or a drinkable alcohol production plant; or a fuel ethanol plant. 1. A method for eliminating or reducing phytic acid and salts of phytic acid in an alcohol production process comprising:(a) providing a polypeptide having phytase activity under high temperature conditions, and retains 0.1% to 100% phytase activity after exposure to temperatures from about 72 degrees C. to about 100 degrees C.;(b) providing a composition comprising a phytic acid or a salt of phytic acid;(c) contacting the phytase of (a) with the composition of (b), wherein the phytase can hydrolyze the phytic acid or salt of phytic acid.2. The method of claim 1 , wherein the phytase is added anywhere in the alcohol production process comprising: a feedstock claim 1 , a hammer mill claim 1 , a slurry tank claim 1 , a jet cooker claim 1 , a liquefaction claim 1 , a mash cooker claim 1 , a fermentation claim 1 , a beer claim 1 , a distillation system claim 1 , a whole stillage claim 1 , a centrifuge claim 1 , a thin stillage claim 1 , an evaporator claim 1 , a condensate claim 1 , a syrup claim 1 , a wet grain claim 1 , a drum dryer claim 1 , a distillers dried grain claim 1 , distillers solubles distillers wet grain claim 1 , condensed distillers solubles claim 1 , ...

HIGH EFFICIENCY, SMALL VOLUME NUCLEIC ACID SYNTHESIS

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes). 1. A method for removing a bead from a fluid-filled well of a microchip for synthesizing nucleic acid molecules , wherein a nucleic acid molecule is attached to the bead , the method comprising:providing a voltage between a first electrode that is arranged at a bottom of the fluid-filled well and a second electrode, wherein the voltage is sufficient to cause fluid in the fluid-filled well to undergo electrolysis producing one or more bubbles in the fluid to rise to a top of the fluid-filled well along with the bead or to lift the bead to the top of the fluid-filled well.2. The method of claim 1 , further comprising collecting the bead that has risen to the top of the fluid-filled well with a bead-collection device.3. The method of claim 2 , further comprising transferring the bead that was collected to a well of a first multiwell collection plate.4. The method of claim 1 , wherein the fluid comprises an aqueous or a non-aqueous buffer solution.5. The method of claim 1 , wherein the fluid comprises water claim 1 , methanol claim 1 , acetonitrile claim 1 , and Net4pTsO.6. The method of claim 1 , wherein the first electrode is composed of platinum and the voltage is about 0.1 to about 100 volts.7. The method of claim 1 , wherein each well of the microchip is individually addressable by a controller.8. The method of claim 1 , wherein the bead is composed of: a synthetic polymer claim 1 , a modified naturally occurring polymer claim 1 , glass claim 1 , controlled pore glass claim 1 , magnetic controlled pore glass claim 1 , magnetic beads claim 1 ...

PREPARATION OF 5-FORMYL VALERIC ACID FROM ALPHA-KETOPIMELIC ACID

The invention relates to an alpha-ketopimelic acid decarboxylase enzyme that is a homologue of SEQ ID NO:2, comprising at least one mutation selected from a group of substitutions listed in the specification, to a method for preparing 5-formyl valeric acid (hereinafter also referred to as ‘5-FVA’), to a method for preparing 6-aminocaproic acid (hereinafter also referred to as ‘6-ACA’), to a method for preparing ε-caprolactam (hereinafter referred to as ‘caprolactam’) from 6-ACA, to a method for the preparation of adipic acid, to a method for preparing diaminohexane. The invention further relates to a host cell which may be used in a method according to the invention and to a polynucleotide encoding an alpha-ketopimelic acid decarboxylase enzyme. 1. An alpha-ketopimelic acid decarboxylase enzyme having at least 50% sequence identity with SEQ ID NO:2 , wherein the enzyme comprises at least one mutation selected from the group of substitutions corresponding to 072L , 072M , 101D , 101E , 101F , 101L , 104D , 104Q , 104W , 111M , 166K , 166R , 240A , 240G , 241L , 241N , 241R , 258R , 261A , 261D , 261G , 261W , 261Y , 284C , 284I , 284S , 284V , 290E , 290F , 290N , 290Q , 290Y , 291S , 377A , 377I , 377L , 377M , 377T , 377V , 381H , 382A , 382C , 382E , 382I , 382K , 382N , 382R , 382S , 382V , 382Y , 461I , 461 L , 461M , 461T , 465C , 465F , 465L , 465M , 532C , 532T , 534G , 535A , 535C , 535G , 535Q , 535S , 538A , 538C , 538G , 538H , 538L , 538S , 538W , 539H , 539L , 539Q , 539R , 539T , 541N , 541V , 542A , 542C , 542D , 542E , 542G , 542H , 542I , 542K , 542L , 542M , 542N , 542Q , 542R , 542S , 542T , 542V , 542W , 545C , 545D , 545E , 545F , 545K , 545R , 545S , 545T , 545V , 545W , 546A , 546E , 546F , 546G , 546H , 546P , 546T , 546V , 546W , 546Y and 547P in SEQ ID NO:2 , with the provisio that if the enzyme has only one mutation compared to SEQ ID NO:2 , that mutation is not 4611 or 538W in SEQ ID NO:2.2. An alpha-ketopimelic acid decarboxylase enzyme ...

PREPARATION OF 6-AMINOCAPROIC ACID FROM 5-FORMYL VALERIC ACID

The invention relates to a method for preparing 6-aminocaproic acid (hereinafter also referred to as ‘6-ACA’) using a biocatalyst. The invention further relates to a method for preparing ε-caprolactam (hereafter referred to as ‘caprolactam’) by cyclising such 6-ACA. The invention further relates to a host cell, a micro-organism, or a polynucleotide which may be used in the preparation of 6-ACA or caprolactam.

PREPARATION OF 6-AMINOCAPROIC ACID FROM 5-FORMYL VALERI C ACID

The invention relates to a method for preparing 6-aminocaproic acid (hereinafter also referred to as ‘6-ACA’) using a biocatalyst. The invention further relates to a method for preparing e-caprolactam (hereafter referred to as ‘caprolactam’) by cyclising such 6-ACA. The invention further relates to a host cell, a micro-organism, or a polynucleotide which may be used in the preparation of 6-ACA or caprolactam. 1. Method for preparing 6-aminocaproic acid , wherein the 6-aminocaproic acid is prepared from α-ketopimelic acid , using at least one biocatalyst.2. Method for preparing 6-aminocaproic acid , wherein the 6-aminocaproic acid is prepared from 5-formylpentanoate , using at least one biocatalyst.3. Method according to claim 1 , wherein the biocatalyst comprises an enzyme capable of catalysing a transamination and/or a reductive amination.4. Method according to claim 3 , wherein the enzyme capable of catalysing a transamination and/or a reductive amination is selected from the group of aminotransferases (E.C. 2.6.1) and amino acid dehydrogenases (E.C.1.4.1)5. Method according to claim 4 , wherein the aminotransferase or amino acid dehydrogenase is selected from the group of β-aminoisobutyrate: α-ketoglutarate aminotransferases claim 4 , β-alanine aminotransferases claim 4 , aspartate aminotransferases claim 4 , 4-amino-butyrate aminotransferases (EC 2.6.1.19) claim 4 , L-lysine 6-aminotransferase (EC 2.6.1.36) claim 4 , 2-aminoadipate aminotransferases (EC 2.6.1.39) claim 4 , 5-aminovalerate aminotransferases (EC 2.6.1.48) claim 4 , 2-aminohexanoate aminotransferases (EC 2.6.1.67) claim 4 , lysine:pyruvate 6-aminotransferases (EC 2.6.1.71) claim 4 , and lysine-6-dehydrogenases (EC 1.4.1.18).6Vibrio; Pseudomonas; Bacillus; Mercurialis; Asplenium; CeratoniaNeurospora; Escherichia; Thermus; Saccharomyces; Brevibacterium; Corynebacterium; Proteus; Agrobacterium; Geobacillus; Acinetobacter; Ralstonia; Salmonella; RhodobacterStaphylococcusBacillus subtilis, Bacillus ...

DEVICES AND METHODS FOR PRODUCING NUCLEIC ACIDS AND PROTEINS

The present disclosure generally relates to devices, compositions and methods for designing and producing nucleic acid molecules and the production of encoded proteins using these nucleic acid molecules. In some aspect, the disclosure relates to automation for the in vitro generation of coding DNA molecules, the in vitro transcription of these DNA molecules to generate protein coding RNA molecules, and the in vitro translation of these protein coding RNA molecules to produce proteins. 164.-. (canceled)65. A method of producing a protein , the method comprising:(a) designing a codon optimized nucleic acid molecule, wherein the codon optimized nucleic acid molecule encodes the protein, and wherein codons of the nucleic acid molecule are optimized for translation in a yeast,(b) generating oligonucleotides encoding subportions of the codon optimized nucleic acid molecule,(c) assembling the oligonucleotides generated in (b) to produce the codon optimized nucleic acid molecule,(d) contacting the codon optimized nucleic acid molecule assembled in (c) with a coupled in vitro translation/transcription reaction mixture under conditions that result in the production of (1) mRNA encoding the protein encoded by the codon optimized nucleic acid molecule and (2) the protein encoded by the codon optimized nucleic acid molecule,wherein the codons of the nucleic acid molecule are select to not contain inverse complimentary repeats sufficient to form secondary structures at the RNA level when present in the coupled in vitro translation/transcription reaction mixture, andwherein the reaction mixture of (d) comprises mammalian cell components.66. The method of claim 65 , wherein the codon optimized nucleic acid molecule encodes a human protein.67. The method of claim 66 , wherein the difference in production of the protein encoded by the codon optimized nucleic acid molecule is at least 20% higher than the same protein encoded by a nucleic acid molecule that is not codon optimized when ...

ADIPATE (ESTER OR THIOESTER) SYNTHESIS

The present invention relates to a method for preparing an adipate ester or thioester. The invention further relates to a method for preparing adipic acid from said ester or thioester. Further the invention provides a number of methods for preparing an intermediate for said ester or thioester. Further the invention relates to a method for preparing 6-amino caproic acid (6-ACA), a method for preparing 5-formyl valeric acid (5-FVA), and a method for preparing caprolactam. Further, the invention relates to a host cell for use in a method according to the invention. 1. Method for preparing an adipate ester or adipate thioester , comprising converting a 2 ,3-dehydroadipate ester or 2 ,3-dehydroadipate thioester into the adipate ester or thioester in the presence of a biocatalyst.2. Method according to claim 1 , wherein the biocatalyst comprises an enzyme capable of catalysing the reduction of a carbon-carbon double bond of a 2 claim 1 ,3-enoate moiety or a 2-enoyl moiety.3. Method according to claim 2 , wherein the biocatalyst comprises an enzyme selected from the group of oxidoreductases acting on the HC—CH group of donors (EC 1.3.1. or 1.3.99) claim 2 , preferably from the group of oxidoreductases (EC 1.3.1 and EC 1.3.99) claim 2 , preferably from the group of enoyl-CoA reductases EC 1.3.1.8 claim 2 , EC 1.3.1.38 and EC 1.3.1.44 claim 2 , from the group of enoyl-[acyl-carrier-protein] reductases EC 1.3.1.9 claim 2 , EC 1.3.1.10 and EC 1.3.1.39 claim 2 , and from the group butyryl-CoA dehydrogenase (EC 1.3.99.2) claim 2 , acyl-CoA dehydrogenase (1.3.99.3) and long-chain-acyl-CoA dehydrogenase (EC 1.3.99.13).4. Method according to claim 1 , wherein the biocatalyst comprises an enzyme claim 1 , which enzyme comprises an amino acid sequence selected from the group of amino acid sequences represented by any of the SEQUENCE ID's 42-67 claim 1 , 94 claim 1 , 96 claim 1 , 98 claim 1 , 100 claim 1 , 102 claim 1 , 103 claim 1 , 105 claim 1 , 106 claim 1 , 107 claim 1 , 109 claim ...

ADIPATE (ESTER OR THIOESTER) SYNTHESIS

The present invention relates to a method for preparing an adipate ester or thioester. The invention further relates to a method for preparing adipic acid from said ester or thioester. Further the invention provides a number of methods for preparing an intermediate for said ester or thioester. Further the invention relates to a method for preparing 6-amino caproic acid (6-ACA), a method for preparing 5-formyl valeric acid (5-FVA), and a method for preparing caprolactam. Further, the invention relates to a host cell for use in a method according to the invention. 131-. (canceled)32. A non-naturally occurring microbial organism , comprising a microbial organism having an adipate thioester pathway comprising at least one exogenous nucleic acid encoding a set of adipate thioester pathway enzymes expressed in a sufficient amount to produce an adipate thioester , wherein said set of adipate thioester pathway enzymes convert a succinate thioester and an acetate thioester to a 3-oxoadipate thioester , a 3-oxoadipate thioester to a 3-hydroxyadipate thioester , a 3-hydroxyadipate thioester to a 5-carboxy-2-pentenoate thioester and a 5-carboxy-2-pentenoate thioester to an adipate thioester.33. The non-naturally occurring microbial organism of claim 32 , wherein said adipate thioester pathway is an adipyl-CoA pathway claim 32 , said set of adipate thioester pathway enzymes are a set of adipyl-CoA pathway enzymes claim 32 , said adipyl-CoA pathway enzymes are expressed in a sufficient amount to produce adipyl-CoA claim 32 , and said set of adipyl-CoA pathway enzymes convert a succinyl-CoA and an acetyl-CoA to a 3-oxoadipyl-CoA claim 32 , a 3-oxoadipyl-CoA to a 3-hydroxyadipyl-CoA claim 32 , a 3-hydroxyadipyl-CoA to a 5-carboxy-2-pentenoyl-CoA and a 5-carboxy-2-pentenoyl-CoA to an adipyl-CoA.34. The non-naturally occurring microbial organism of claim 33 , wherein said set adipyl-CoA pathway enzymes comprise a succinyl-CoA:acetyl-CoA acyl transferase claim 33 , a 3-hydroxyacyl-CoA ...

PREPARATION OF 6-AMINOCAPROIC ACID FROM 5-FORMYL VALERIC ACID

The invention relates to a method for preparing 6-aminocaproic acid (hereinafter also referred to as ‘6-ACA’) using a biocatalyst. The invention further relates to a method for preparing ϵ-caprolactam (hereafter referred to as ‘caprolactam’) by cyclising such 6-ACA. The invention further relates to a host cell, a micro-organism, or a polynucleotide which may be used in the preparation of 6-ACA or caprolactam. 1. (canceled)2. Method for preparing 6-aminocaproic acid , wherein the 6-aminocaproic acid is prepared from 5-formylpentanoate , using at least one biocatalyst.325-. (canceled) This application is a divisional of U.S. patent application Ser. No. 14,105,705, filed Dec. 13, 2013, which is a continuation of U.S. patent application Ser. No. 12/921,733, filed Dec. 21, 2010, now issued U.S. Pat. No. 8,673,599, issued Mar. 18, 2014, which is a U.S. National Stage Application under 35 U.S.C. § 371 of International Patent Application No. PCT/NL2009/050117, filed Mar. 11, 2009, which claims the benefit of European Patent Application No. 08152584.2, filed Mar. 11, 2008, the entire contents of which are each incorporated herein by reference.The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 17, 2017, is named Sequence_Listing_12956-416-999.txt and is 249,748 bytes in size.The invention relates to a method for preparing 6-aminocaproic acid (hereinafter also referred to as ‘6-ACA’). The invention further relates to a method for preparing ϵ-caprolactam (hereafter referred to as ‘caprolactam’) from 6-ACA. The invention further relates to a host cell which may be used in the preparation of 6-ACA or caprolactam.Caprolactam is a lactam which may be used for the production of polyamide, for instance nylon-6 or nylon-6,12 (a copolymer of caprolactam and laurolactam). Various manners of preparing caprolactam from bulk chemicals are known in ...

HIGH EFFICIENCY, SMALL VOLUME NUCLEIC ACID SYNTHESIS

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes). 17.-. (canceled)8. A multiwell plate for non-template directed synthesis of nucleic acid molecules , the plate comprising:(a) a magnetic bead located in each of a plurality of wells of the plate, and(b) an electrochemically generated acid being present in one or more well,wherein the bead is between 1.0 μm and 100 μm in diameter.91. The multiwell plate of claim , wherein the number of wells in the plate is between 10 and 50 ,000.101. The multiwell plate of claim , wherein the total volume of each well is between 0.1 μl and 50 μl.111. The multiwell plate of claim , wherein each well is operably connected to a pair of electrodes.121. The multiwell plate of claim , wherein the wells of the plate are connected to microfluidic channels for the introduction and removal of reagents.13. A system for generating assembled nucleic acid molecule , the system comprising:a processor; and a memory encoded with processor-executable instructions for:(a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a microquantity in the well of a plate;(b) combining the nucleic acid molecules generated in (a) to produce a pool;(c) joining some or all of the nucleic acid molecules present in the pool formed in (b) to form a plurality of larger nucleic acid molecules;(d) eliminating nucleic acid molecules which contain sequence errors from the plurality of larger nucleic acid molecules formed in (c) to produce an error corrected nucleic acid molecule pool; andassembling the nucleic acid molecules in the error corrected ...

HIGH EFFICIENCY, SMALL VOLUME NUCLEIC ACID SYNTHESIS

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes). 1. A method for assembling nucleic acid molecules , the method comprising: (1) one or more insert nucleic acid molecule, one or more acceptor nucleic acid molecule, a plurality of oligonucleotides, wherein each oligonucleotide shares sequence complementarity with (i) one terminus of the insert nucleic acid molecule and the insertion site of the acceptor nucleic acid molecule or (ii) one terminus of two different insert nucleic acid molecules and wherein the number of oligonucleotides is represented by the formula O=2+2I, where O is the number of oligonucleotides and I is the number of insert nucleic acid molecules,', '(2) a cell extract, and', '(3) a protein composition comprising an exonuclease and, optionally, a single-stranded binding protein, and, '(a) forming a reaction mixture of(b) incubating the reaction formed in (a) under conditions which allow for the introduction of the insert nucleic acid molecule into the acceptor nucleic acid molecule.2. The method of claim 1 , wherein the cell extract is obtained from a single cellular organism selected from the group consisting of:{'i': 'Escherichia coli;', '(a)'}{'i': 'Bacillus subtilis;', '(b)'}{'i': 'Schizosaccharomyces pombe;', '(c) and'}{'i': 'Saccharomyces cerevisiae.', '(d)'}3Escherichia coli. The method of claim 2 , wherein the cells do not express redET genes.4Escherichia coli. The method of claim 3 , wherein the cells are strain DH10B.5. The method of claim 1 , wherein the exonuclease activity is provided by a DNA polymerase.6. The method of claim 1 , wherein the single-stranded ...

PHYTASES, NUCLEIC ACIDS ENCODING THEM AND METHODS FOR MAKING AND USING THEM

This invention relates to phytases, polynucleotides encoding them, uses of the polynucleotides and polypeptides of the invention, as well as the production and isolation of such polynucleotides and polypeptides. In particular, the invention provides polypeptides having phytase activity under high temperature conditions, and phytases that retain activity after exposure to high temperatures. The phytases of the invention can be thermotolerant and/or thermostable at low temperatures, in addition to higher temperatures. The phytases of the invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients. The phytases of the invention can be formulated as foods or feeds or supplements for either to, e.g., aid in the digestion of phytate. The foods or feeds of the invention can be in the form of pellets, liquids, powders and the like. In one aspect, phytases of the invention are stabile against thermal denaturation during pelleting; and this decreases the cost of the phytase product while maintaining in vivo efficacy and detection of activity in feed. 142-. (canceled)43. A variant polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:2 and one single amino acid substitution to the amino acid sequence as set forth in SEQ ID NO:2 , wherein said one single amino acid substitution is selected from the group consisting of: C97E , C97V , E168R , and C226D , wherein said variant polypeptide has phytase activity.44. A protein preparation comprising the variant polypeptide as set forth in claim 43 , wherein the protein preparation comprises a liquid claim 43 , a slurry claim 43 , a powder claim 43 , a spray claim 43 , a suspension claim 43 , a lyophilized composition/formulation claim 43 , a solid claim 43 , geltab claim 43 , pill claim 43 , implant claim 43 , a gel; or a pharmaceutical formulation claim 43 , a food or a feed or a supplement thereof.45. A food or feed claim 43 , or a food or feed supplement claim 43 , for an animal or ...

HIGH EFFICIENCY, SMALL VOLUME NUCLEIC ACID SYNTHESIS

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes). 1. A method for removing a bead from a fluid-filled well of a microchip for synthesizing nucleic acid molecules , wherein a nucleic acid molecule is attached to the bead , the method comprising:providing a voltage between a first electrode that is arranged at a bottom of the fluid-filled well and a second electrode, wherein the voltage is sufficient to cause fluid in the fluid-filled well to undergo electrolysis producing one or more bubbles in the fluid to rise to a top of the fluid-filled well along with the bead or to lift the bead to the top of the fluid-filled well.2. The method of claim 1 , further comprising collecting the bead that has risen to the top of the fluid-filled well with a bead-collection device.3. The method of claim 2 , further comprising transferring the bead that was collected to a well of a first multiwell collection plate.4. The method of claim 1 , wherein the fluid comprises an aqueous or a non-aqueous buffer solution.5. The method of claim 1 , wherein the fluid comprises water claim 1 , methanol claim 1 , acetonitrile claim 1 , and Net4pTsO.6. The method of claim 1 , wherein the first electrode is composed of platinum and the voltage is about 0.1 to about 100 volts.7. The method of claim 1 , wherein each well of the microchip is individually addressable by a controller.8. The method of claim 1 , wherein the bead is composed of: a synthetic polymer claim 1 , a modified naturally occurring polymer claim 1 , glass claim 1 , controlled pore glass claim 1 , magnetic controlled pore glass claim 1 , magnetic beads claim 1 ...

HIGH EFFICIENCY, SMALL VOLUME NUCLEIC ACID SYNTHESIS

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes). 1. A multiwell plate for non-template directed synthesis of nucleic acid molecules , the plate comprising:(a) a magnetic bead located in each of a plurality of wells of the plate, and(b) an electrochemically generated acid being present in one or more well,wherein the bead is between 1.0 μm and 100 μm in diameter.23.-. (canceled)4. The multiwell plate of claim 1 , wherein each well is operably connected to a pair of electrodes.5. (canceled)6. A method for the generation of an assembled nucleic acid molecule claim 1 , the method comprising:(a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a well of a plate in an average amount of from about 0.001 nanomoles to about 1,000 nanomoles;(b) combining the nucleic acid molecules generated in (a) to produce a pool;(c) joining some or all of the nucleic acid molecules present in the pool formed in (b) to form a plurality of larger nucleic acid molecules;(d) eliminating nucleic acid molecules which contain sequence errors from the plurality of larger nucleic acid molecules formed in (c) to produce an error corrected nucleic acid molecule pool; and(e) assembling the nucleic acid molecules in the error corrected nucleic acid molecule pool to form the assembled nucleic acid molecule.7. The method of claim 6 , wherein the joining in (c) is mediated by polymerase chain reaction and/or ligases.841.-. (canceled)42. A method for producing a plurality of nucleic acid molecules claim 6 , the method comprising:(a) synthesizing the plurality of nucleic acid ...

ENZYMATIC SYNTHESIS OF NUCLEIC ACID SEQUENCES

The disclosure generally relates to compositions and methods for the enzymatic synthesis of nucleic acid sequences. In particular embodiments 3′ protected nucleotides are used in conjunction with a universal template to direct synthesis. 1. A method for the synthesis of an oligonucleotide comprising:a) providing on a solid support a single or double stranded template comprising a primer binding site at the 3′ end and a universal template,b) adding a primer complimentary to the primer binding site,c) adding a polymerase,d) adding a 3′ protected nucleotide such that the protected nucleotide is added to the primer or the oligonucleotide extending from the primer,e) removing the unreacted protected nucleotide,f) removing the protective group from the 3′ protected nucleotide; andg) repeating steps (d)-(f) and optionally step (c) until synthesis of the oligonucleotide has been completed.2. The method of claim 1 , wherein step e) further comprises removing the polymerase.3. The method of claim 1 , wherein the primer binding site is from 10 to 25 nucleotides in length.4. The method of claim 1 , wherein the universal template is from 10 to 200 nucleotides in length.5. The method of claim 1 , wherein the universal template comprises a universal base.6. The method of claim 5 , wherein the universal base is selected from the group consisting of inosine and PPT.7. The method of claim 1 , wherein the protective group of the 3′ protected molecule is selected from the group consisting of 3′-O-allyl claim 1 , 3′-O-methoxymethyl claim 1 , 3′-O-nitrobenzyl claim 1 , 3′-O-azidomethylene claim 1 , and 3′-O-aminoalkoxyl.8. A non-transitory computer-readable storage media encoded with instructions claim 1 , executable by a processor claim 1 , for generating synthesized nucleic acid molecules claim 1 , the instructions comprising instructions for:a) adding to a solid support comprising a single or double stranded template comprising a primer binding site at the 3′ end and a universal ...

ENZYMATIC SYNTHESIS OF NUCLEIC ACID SEQUENCES

The disclosure generally relates to compositions and methods for the enzymatic synthesis of nucleic acid sequences. In particular embodiments 3′ protected nucleotides are used in conjunction with a universal template to direct synthesis. 1. A method for the synthesis of an oligonucleotide comprising:a) providing on a solid support a single or double stranded template comprising a primer binding site at the 3′ end and a universal template,b) adding a primer complimentary to the primer binding site,c) adding a polymerase,d) adding a 3′ protected nucleotide such that the protected nucleotide is added to the primer or the oligonucleotide extending from the primer,e) removing the unreacted protected nucleotide,f) removing the protective group from the 3′ protected nucleotide; andg) repeating steps (d)-(f) and optionally step (c) until synthesis of the oligonucleotide has been completed.2. The method of claim 1 , wherein step e) further comprises removing the polymerase.3. The method of claim 1 , wherein the primer binding site is from 10 to 25 nucleotides in length.4. The method of claim 1 , wherein the universal template is from 10 to 200 nucleotides in length.5. The method of claim 1 , wherein the universal template comprises a universal base.6. The method of claim 5 , wherein the universal base is selected from the group consisting of inosine and PPT.7. The method of claim 1 , wherein the protective group of the 3′ protected molecule is selected from the group consisting of 3′-O-allyl claim 1 , 3′-O-methoxymethyl claim 1 , 3′-O-nitrobenzyl claim 1 , 3′-O-azidomethylene claim 1 , and 3′-O-aminoalkoxyl.8. A non-transitory computer-readable storage media encoded with instructions claim 1 , executable by a processor claim 1 , for generating synthesized nucleic acid molecules claim 1 , the instructions comprising instructions for:a) adding to a solid support comprising a single or double stranded template comprising a primer binding site at the 3′ end and a universal ...

PREPARATION OF 6-AMINOCAPROIC ACID FROM 5-FORMYL VALERIC ACID

The invention relates to a method for preparing 6-aminocaproic acid (hereinafter also referred to as ‘6-ACA’) using a biocatalyst. The invention further relates to a method for preparing E-caprolactam (hereafter referred to as ‘caprolactam’) by cyclising such 6-ACA. The invention further relates to a host cell, a micro-organism, or a polynucleotide which may be used in the preparation of 6-ACA or caprolactam. 1. (canceled)2. Method for preparing 6-aminocaproic acid , wherein the 6-aminocaproic acid is prepared from 5-formylpentanoate , using at least one biocatalyst.325-. (canceled) This application is a divisional of U.S. patent application Ser. No. 15/599,314, filed May 18, 2017, which is a divisional of U.S. patent application Ser. No. 14/105,705, filed Dec. 13, 2013, now issued U.S. Pat. No. 9,663,805, issued Jun. 8, 2017, which is a continuation of U.S. patent application Ser. No. 12/921,733, filed Dec. 21, 2010, now issued U.S. Pat. No. 8,673,599, issued Mar. 18, 2014, which is a U.S. National Stage Application under 35 U.S.C. § 371 of International Patent Application No. PCT/NL2009/050117, filed Mar. 11, 2009, which claims the benefit of European Patent Application No. 08152584.2, filed Mar. 11, 2008, the entire contents of which are each incorporated herein by reference.The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 20, 2018, is named 12956-454-999_Sequence_Listing and is 249,788 bytes in size.The invention relates to a method for preparing 6-aminocaproic acid (hereinafter also referred to as ‘6-ACA’). The invention further relates to a method for preparing ε-caprolactam (hereafter referred to as ‘caprolactam’) from 6-ACA. The invention further relates to a host cell which may be used in the preparation of 6-ACA or caprolactam.Caprolactam is a lactam which may be used for the production of polyamide, for instance ...

ADIPATE (ESTER OR THIOESTER) SYNTHESIS

The present invention relates to a method for preparing an adipate ester or thioester. The invention further relates to a method for preparing adipic acid from said ester or thioester. Further the invention provides a number of methods for preparing an intermediate for said ester or thioester. Further the invention relates to a method for preparing 6-amino caproic acid (6-ACA), a method for preparing 5-formyl valeric acid (5-FVA), and a method for preparing caprolactam. Further, the invention relates to a host cell for use in a method according to the invention. 1. Method for preparing an adipate ester or adipate thioester , comprising converting a 2 ,3-dehydroadipate ester or 2 ,3-dehydroadipate thioester into the adipate ester or thioester in the presence of a biocatalyst.2. Method according to claim 1 , wherein the biocatalyst comprises an enzyme capable of catalysing the reduction of a carbon-carbon double bond of a 2 claim 1 ,3-enoate moiety or a 2-enoyl moiety.3. Method according to claim 2 , wherein the biocatalyst comprises an enzyme selected from the group of oxidoreductases acting on the HC—CH group of donors (EC 1.3.1. or 1.3.99) claim 2 , preferably from the group of oxidoreductases (EC 1.3.1 and EC 1.3.99) claim 2 , preferably from the group of enoyl-CoA reductases EC 1.3.1.8 claim 2 , EC 1.3.1.38 and EC 1.3.1.44 claim 2 , from the group of enoyl-[acyl-carrier-protein] reductases EC 1.3.1.9 claim 2 , EC 1.3.1.10 and EC 1.3.1.39 claim 2 , and from the group butyryl-CoA dehydrogenase (EC 1.3.99.2) claim 2 , acyl-CoA dehydrogenase (1.3.99.3) and long-chain-acyl-CoA dehydrogenase (EC 1.3.99.13).4. Method according to claim 1 , wherein the biocatalyst comprises an enzyme claim 1 , which enzyme comprises an amino acid sequence selected from the group of amino acid sequences represented by any of the SEQUENCE ID's 42-67 claim 1 , 94 claim 1 , 96 claim 1 , 98 claim 1 , 100 claim 1 , 102 claim 1 , 103 claim 1 , 105 claim 1 , 106 claim 1 , 107 claim 1 , 109 claim ...

HIGH EFFICIENCY, SMALL VOLUME NUCLEIC ACID SYNTHESIS

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes). 1. A multiwell plate for non-template directed synthesis of nucleic acid molecules , the plate comprising:(a) a magnetic bead located in each of a plurality of wells of the plate, and(b) an electrochemically generated acid being present in one or more well, wherein the bead is between 1.0 μm and 100 μm in diameter.2. The multiwell plate of claim 1 , wherein the number of wells in the plate is between 10 and 50 claim 1 ,000.3. The multiwell plate of claim 1 , wherein the total volume of each well is between 0.1 μl and 50 μl.4. The multiwell plate of claim 1 , wherein each well is operably connected to a pair of electrodes.5. The multiwell plate of claim 1 , wherein the wells of the plate are connected to microfluidic channels for the introduction and removal of reagents.616.-. (canceled)17. A method for producing a product nucleic acid molecule claim 1 , the method comprising:(a) designing the product nucleic acid molecule of between 10 kilobases and 500 kilobases in size, wherein the product nucleic acid molecule is defined by nucleotide sequence;{'sup': 7', '9, '(b) synthesizing a plurality of individual nucleic acid molecules which differ in nucleotide sequence, wherein each individual nucleic acid molecule is synthesized to prepare a quantity of between 1.0×10and 1.0×10copies and wherein the individual nucleic acid molecules are capable of hybridizing with one or more of the other individual nucleic acid molecules;'}(c) combining the individual nucleic acid molecules synthesized in (b) under conditions which allow for hybridization of the ...

DEVICES AND METHODS FOR PRODUCING NUCLEIC ACIDS AND PROTEINS

The present disclosure generally relates to devices, compositions and methods for designing and producing nucleic acid molecules and the production of encoded proteins using these nucleic acid molecules. In some aspect, the disclosure relates to automation for the in vitro generation of coding DNA molecules, the in vitro transcription of these DNA molecules to generate protein coding RNA molecules, and the in vitro translation of these protein coding RNA molecules to produce proteins. 1. A method of producing a protein , the method comprising:(a) designing a nucleic acid molecule encoding the protein,(b) generating oligonucleotides encoding subportions of the nucleic acid molecule,(c) assembling the oligonucleotides to produce a population of nucleic acid molecules encoding the protein,(d) contacting the population of nucleic acid molecules encoding the protein with a first mixture suitable for the in vitro transcription and translation of members of the population of nucleic acid molecules encoding the protein to form a second mixture, and(e) incubating the second mixture of (d) under conditions suitable for the production of (1) mRNA encoding the protein and (2) the protein,wherein the first mixture suitable for the in vitro transcription and translation of members of the population of nucleic acid molecules comprises cellular components from cells of a first organism,wherein some or all of the codons of the nucleic acid molecules encoding the protein are optimized for translation in a second organism, andwherein the first organism and the second organism are of different species.2. The method of claim 1 , wherein the population of nucleic acid molecules encoding the protein are linear.3. The method of claim 2 , wherein the linear nucleic acid molecules comprise a promoter operable linked to the protein coding regions.45.-. (canceled)6. The method of claim 1 , wherein the second mixture of (d) contains transfer RNA molecules obtained from an organism other than ...

PREPARATION OF 6-AMINOCAPROIC ACID FROM 5-FORMYL VALERIC ACID

The invention relates to a method for preparing 6-aminocaproic acid (hereinafter also referred to as ‘6-ACA’) using a biocatalyst. The invention further relates to a method for preparing ε-caprolactam (hereafter referred to as ‘caprolactam’) by cyclising such 6-ACA. The invention further relates to a host cell, a micro-organism, or a polynucleotide which may be used in the preparation of 6-ACA or caprolactam.

HIGH EFFICIENCY, SMALL VOLUME NUCLEIC ACID SYNTHESIS

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes). 1. A multiwell plate for non-template directed synthesis of nucleic acid molecules , the plate comprising:(a) a magnetic bead located in each of a plurality of wells of the plate, and(b) an electrochemically generated acid being present in one or more well, wherein the bead is between 1.0 μm and 100 μm in diameter.2. The multiwell plate of claim 1 , wherein the number of wells in the plate is between 10 and 50 claim 1 ,000.3. The multiwell plate of claim 1 , wherein the total volume of each well is between 0.1 μl and 50 μl.4. The multiwell plate of claim 1 , wherein each well is operably connected to a pair of electrodes.5. The multiwell plate of claim 1 , wherein the wells of the plate are connected to microfluidic channels for the introduction and removal of reagents.6. A method for the generation of an assembled nucleic acid molecule claim 1 , the method comprising:(a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a well of a plate in an average amount of from about 0.001 nanomoles to about 1,000 nanomoles;(b) combining the nucleic acid molecules generated in (a) to produce a pool;(c) joining some or all of the nucleic acid molecules present in the pool formed in (b) to form a plurality of larger nucleic acid molecules;(d) eliminating nucleic acid molecules which contain sequence errors from the plurality of larger nucleic acid molecules formed in (c) to produce an error corrected nucleic acid molecule pool; and(e) assembling the nucleic acid molecules in the error corrected nucleic ...

HIGH EFFICIENCY, SMALL VOLUME NUCLEIC ACID SYNTHESIS

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes). 1. A method for assembling nucleic acid molecules , the method comprising: (1) one or more insert nucleic acid molecule, one or more acceptor nucleic acid molecule, a plurality of oligonucleotides, wherein each oligonucleotide shares sequence complementarity with (i) one terminus of the insert nucleic acid molecule and the insertion site of the acceptor nucleic acid molecule or (ii) one terminus of two different insert nucleic acid molecules and wherein the number of oligonucleotides is represented by the formula O=2+2I, where O is the number of oligonucleotides and I is the number of insert nucleic acid molecules,', '(2) a cell extract, and', '(3) a protein composition comprising an exonuclease and, optionally, a single-stranded binding protein, and, '(a) forming a reaction mixture of(b) incubating the reaction formed in (a) under conditions which allow for the introduction of the insert nucleic acid molecule into the acceptor nucleic acid molecule.2. The method of claim 1 , wherein the cell extract is obtained from a single cellular organism selected from the group consisting of:{'i': 'Escherichia coli;', '(a)'}{'i': 'Bacillus subtilis;', '(b)'}{'i': 'Schizosaccharomyces pombe', '(c) ; and'}{'i': 'Saccharomyces cerevisiae.', '(d)'}3Escherichia coli. The method of claim 2 , wherein the cells do not express redET genes.4Escherichia coli. The method of claim 3 , wherein the cells are strain DH10B.5. The method of claim 1 , wherein the exonuclease activity is provided by a DNA polymerase.6. The method of claim 1 , wherein the single-stranded ...

preparation of 6-aminocaproic acid from α-ketopimelic acid

Butanol production in a prokaryotic cell

The present invention relates to a prokaryotic cell which does not naturally produce butanol, but which has been made capable of producing butanol by genetic modification of said microorganism and to a process for the production of butanol by fermenting the prokaryotic cell in a suitable fermentation medium according to the present invention.

Phytases, nucleic acids that encode them and methods for their production and use

Un ácido nucleico aislado, sintético o recombinante que comprende (a) una secuencia de ácido nucleico que codifica un polipéptido que tiene una actividad fitasa y que tiene al menos 95%, 96%, 97%, 98% o 99% o más identidad de secuencia con SEC ID Nº: 1, y que tiene una modificación de nucleótidos donde los nucleótidos en las posiciones 1045 a 1047 de SEC ID Nº: 1 se cambian a TAT O TAC, donde el polipéptido tiene termoestabilidad mejorada sobre la fitasa de SEC ID Nº: 2; (b) un ácido nucleico que codifica un polipéptido que tiene una actividad fitasa y que tiene al menos 95%, 96%, 97%, 98% o 99% o más identidad de secuencia de SEC ID Nº: 2, y que tiene una modificación de aminoácidos donde el aminoácido treonina en la posición de aminoácido 349 de SEC ID Nº: 2 se reemplaza por una tirosina, donde el polipéptido tiene termoestabilidad mejorada sobre la fitasa de SEC ID Nº: 2; (c) la secuencia de ácido nucleico de (a) o (b) que codifica un polipéptido que tiene una actividad fitasa pero que carece de: una secuencia señal o secuencia de proproteína, o una secuencia promotora homóloga; (d) el ácido nucleico de cualquiera de (a) a (c) que codifica un polipéptido que tiene una actividad fitasa y que comprende además una secuencia de aminoácidos heteróloga, o el ácido nucleico de cualquiera de (a) a (c) que comprende además una secuencia de nucleótidos heteróloga; (e) el ácido nucleico de (d), donde la secuencia de aminoácidos heteróloga comprende, o consiste en una secuencia que codifica una secuencia señal heteróloga (líder), o un marcador o un epítopo, o la secuencia de nucleótidos heteróloga que comprende un promotor heterólogo o codifica una secuencia diana; o (f) una secuencia de ácido nucleico completamente complementaria de la secuencia de ácido nucleico de cualquiera de (a) a (e). An isolated, synthetic or recombinant nucleic acid comprising (a) a nucleic acid sequence encoding a polypeptide having a phytase activity and having at least 95%, 96%, 97%, 98% or 99% ...

High efficiency, small volume nucleic acid synthesis

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

Multiwell plate for high efficiency, small volume nucleic acid synthesis

High efficiency, small volume nucleic acid synthesis

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

Devices and methods for producing proteins

The present disclosure generally relates to devices, compositions and methods for designing and producing nucleic acid molecules and the production of encoded proteins using these nucleic acid molecules. In some aspect, the disclosure relates to automation for the in vitro generation of coding DNA molecules, the in vitro transcription of these DNA molecules to generate protein coding RNA molecules, and the in vitro translation of these protein coding RNA molecules to produce proteins.

Phytases, nucleic acids encoding them and methods for making and using them

Phytases, nucleic acids encoding them and method for making and using them

This invention relates to phytases, polynucleotides encoding them, uses of the poiynucleotides and polypeptides of the invention, as well as the production and isolation of such polynucleotides and polypeptides. In particular, the invention provides polypeptides having phytase activity under high temperature conditions, and phytases that retain activity after exposure to high temperatures. The phytases of the invention can be thermotolerant and/or thermostable at low temperatures, in addition to higher temperatures. The phytases of the invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients. The phytases of the invention can be formulated as foods or feeds or supplements for either to, e.g., aid in the digestion of phytate. The foods or feeds of the invention can be in the form of pellets, liquids, powders and the like. In one aspect, phytases of the invention are stabile against thermal denaturation during pelleting; and this decreases the cost of the phytase product while maintaining in vivo efficacy and detection of activity in feed. (Figure 6)

Preparation of epsilon-caprolactam from (Z)-6,7-dihydro-1H-azepin-2(5H)-one

preparation of alpha-ketopimelic acid 6-aminocaproic acid

preparação de ácido 6-aminocapróico de ácido alfa-cetopimélico. a invenção é dirigida a um método para a preparação de ácido 6-aminocapróico, compreendendo descarboxilação de ácido alfa-aminopimélico, usando pelo menos um bicatalisador que compreende uma enzima tendo atividade de descarboxilase de ácido alfa-aminopimélico. a invenção é ainda dirigida a um método para a preparação de caprolactama a partir de ácido 6-aminocapróico preparada pelo referido método, a uma célula hospedeira adequada para uso num método de acordo com a invenção e a um polinucleotídeo que codifica uma descarboxilase que pode ser usada num método de acordo a invenção. preparation of 6-aminocaproic acid from alpha-ketopimelic acid. The invention is directed to a method for the preparation of 6-aminocaproic acid comprising alpha-aminopimelic acid decarboxylation using at least one bicatalyst comprising an enzyme having alpha-aminopimelic acid decarboxylase activity. The invention is further directed to a method for preparing caprolactam from 6-aminocaproic acid prepared by said method, a host cell suitable for use in a method according to the invention and a polynucleotide encoding a decarboxylase which may be used in a method according to the invention.

High efficiency, small volume nucleic acid synthesis

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

Adipate (ester or thioester) synthesis

Phytases, nucleic acids encoding them and methods for making and using them

This invention relates to phytases, polynucleotides encoding them, uses of the polynucleotides and polypeptides of the invention, as well as the production and isolation of such polynucleotides and polypeptides. In particular, the invention provides polypeptides having phytase activity under high temperature conditions, and phytases that retain activity after exposure to high temperatures. The phytases of the invention can be thermotolerant and/or thermostable at low temperatures, in addition to higher temperatures. The phytases of the invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients. The phytases of the invention can be formulated as foods or feeds or supplements for either to, e.g., aid in the digestion of phytate. The foods or feeds of the invention can be in the form of pellets, liquids, powders and the like. In one aspect, phytases of the invention are stabile against thermal denaturation during pelleting; and this decreases the cost of the phytase product while maintaining in vivo efficacy and detection of activity in feed.

High efficiency, small volume nucleic acid synthesis

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

Phytases, nucleic acids encoding them and methods for making and using them

This invention relates to phytases, polynucleotides encoding them, uses of the polynucleotides and polypeptides of the invention, as well as the production and isolation of such polynucleotides and polypeptides. In particular, the invention provides polypeptides having phytase activity under high temperature conditions, and phytases that retain activity after exposure to high temperatures. The phytases of the invention can be thermotolerant and/or thermostable at low temperatures, in addition to higher temperatures. The phytases of the invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients. The phytases of the invention can be formulated as foods or feeds or supplements for either to, e.g., aid in the digestion of phytate. The foods or feeds of the invention can be in the form of pellets, liquids, powders and the like. In one aspect, phytases of the invention are stabile against thermal denaturation during pelleting; and this decreases the cost of the phytase product while maintaining in vivo efficacy and detection of activity in feed.

Phytases, nucleic acids encoding them and methods for making and using them

This invention relates to phytases, polynucleotides encoding them, uses of the polynucleotides and polypeptides of the invention, as well as the production and isolation of such polynucleotides and polypeptides. In particular, the invention provides polypeptides having phytase activity under high temperature conditions, and phytases that retain activity after exposure to high temperatures. The phytases of the invention can be thermotolerant and/or thermostable at low temperatures, in addition to higher temperatures. The phytases of the invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients. The phytases of the invention can be formulated as foods or feeds or supplements for either to, e.g., aid in the digestion of phytate. The foods or feeds of the invention can be in the form of pellets, liquids, powders and the like. In one aspect, phytases of the invention are stabile against thermal denaturation during pelleting; and this decreases the cost of the phytase product while maintaining in vivo efficacy and detection of activity in feed.

High efficiency, small volume nucleic acid synthesis

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

Phytases, nucleic acids encoding them and methods for making and using them

This invention relates to phytases, polynucleotides encoding them, uses of the polynucleotides and polypeptides of the invention, as well as the production and isolation of such polynucleotides and polypeptides. In particular, the invention provides polypeptides having phytase activity under high temperature conditions, and phytases that retain activity after exposure to high temperatures. The phytases of the invention can be thermotolerant and/or thermostable at low temperatures, in addition to higher temperatures. The phytases of the invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients. The phytases of the invention can be formulated as foods or feeds or supplements for either to, e.g., aid in the digestion of phytate. The foods or feeds of the invention can be in the form of pellets, liquids, powders and the like. In one aspect, phytases of the invention are stabile against thermal denaturation during pelleting; and this decreases the cost of the phytase product while maintaining in vivo efficacy and detection of activity in feed.

Avilamycin derivatives

The invention relates to avilamycin derivatives, genetic engineering, biosynthetic methods for the production thereof, medicaments containing said compounds and the utilization of said compounds for the production of a medicament against infectious diseases. The invention also relates to the nucleic acids, proteins and gene clusters and corresponding cells connected to the production of said avilamycin derivatives.

Phytases, nucleic acids encoding them and methods for making and using them

Phytases, nucleic acids encoding them and methods for making and using them

Preparation of epsilon-caprolactam from (Z)-6,7-dihydro -1H-azepin-2(5H)-one

High efficiency, small volume nucleic acid synthesis

Devices and methods for producing nucleic acids and proteins

The present disclosure generally relates to devices, compositions and methods for designing and producing nucleic acid molecules and the production of encoded proteins using these nucleic acid molecules. In some aspect, the disclosure relates to automation for the in vitro generation of coding DNA molecules, the in vitro transcription of these DNA molecules to generate protein coding RNA molecules, and the in vitro translation of these protein coding RNA molecules to produce proteins.

Preparation of alpha-ketopimelic acid

The present invention relates to a method for preparing alpha-ketopimelic acid, comprising converting 2-hydroxyheptanedioic acid into alpha-ketopimelic acid, which conversion is catalysed using a biocatalyst. Further, the invention relates to a heterologous cell, comprising a nucleic acid sequence encoding an enzyme having catalytic activity in the conversion of 2-hydroxyheptanedioic acid into alpha-ketopimelic acid. Further, the invention relates to the use of a heterologous cell according to the invention in the preparation of caprolactam, diaminohexane or adipic acid.

Preparation of alpha-ketopimelic acid

The invention relates to a method for preparing alpha-ketopimelic acid, comprising converting alpha-ketoglutaric acid into alpha-ketoadipic acid and converting alpha- ketoadipic acid into alpha-ketopimelic acid, wherein at least one of these conversions is carried out using a heterologous biocatalyst. The invention further relates to a heterologous cell, comprising one or more heterologous nucleic acid sequences encoding one or more heterologous enzymes capable of catalysing at least one reaction step in the preparation of alpha-ketopimelic acid from alpha-ketoglutaric acid.

Adipate ester or thioester synthesis

The present invention relates to a method for preparing an adipate ester or thioester. The invention further relates to a method for preparing adipic acid from said ester or thioester. Further the invention provides a number of methods for preparing an intermediate for said ester or thioester. Further the invention relates to a method for preparing 6-amino caproic acid(6-ACA), a method for preparing 5-formyl valeric acid (5- FVA), and a method for preparingcaprolactam. Further, the invention relates to a host cell for use in a method according to the invention.

preparation of alpha ketopimelic acid.

preparação de ácido alfa-cetopimélico - a presente invenção refere-se a um método para a preparação de ácido alfa-cetopiméçico, compreendendo a conversão de ácido 2 -hidroxiheptanedioico em ácido alfacetopimélico, que a conversão é catalisada utilizando um biocatalisador. além disso, a invenção refere-se a uma célula heterólogo, que compreende uma sequência de ácido nucleico que codifica uma enzima possuindo atividade catalítica na conversão de 2- hidroxiheptanedioic ácido em alfa- cetopimélico ácido. além disso, a invenção refere-se a utilização de uma célula heteróloga de acordo com a invenção na preparação de diaminohexano caprolactama, ou ácido adípico. Alpha-Ketopimelic Acid Preparation - The present invention relates to a method for the preparation of alpha-ketopimecic acid, comprising converting 2-hydroxyheptanedioic acid to alpha-ketopimelic acid, which conversion is catalyzed using a biocatalyst. furthermore, the invention relates to a heterologous cell comprising an nucleic acid sequence encoding an enzyme having catalytic activity in the conversion of 2-hydroxyheptanedioic acid to alpha-ketopimelic acid. furthermore, the invention relates to the use of a heterologous cell according to the invention in the preparation of diaminohexane caprolactam, or adipic acid.

High efficiency, small volume nucleic acid synthesis

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

Method of finding a biocatalyst having ammonia lyase activity

A method of finding a biocatalyst having ammonia lyase activity, in particular a lysine ammonia lyase, comprising - providing a library comprising a plurality of cells in one or more cell cultures, which cells are candidates for having an ammonia lyase activity, which one or more cultures comprise a culture medium containing at least one nitrogen source selected from the group of amines, including amino acids, and structural analogues thereof, as sole nitrogen source or sources for the cells; - selecting at least one candidate which grows in said culture medium; and - screening the selected candidate or candidates which grow in said culture for the ammonia lyase activity.

Preparation of alpha-amino-epsilon-caprolactam via lysine cyclisation

Preparation of alpha-amino-epsilon-caprolactam via lysine cyclisation

The present invention relates to a method for preparing α-amino-ε-caprolactam, comprising converting lysine to α-amino-ε-caprolactam, wherein the conversion is catalysed by a biocatalyst. Further, the invention relates to a host cell comprising at least one recombinant vector comprising a nucleic acid sequence encoding a biocatalyst with lysine cyclase activity. Further a method is provided wherein α-amino- ε-caprolactam is used for preparing ε-caprolactam.

Avilamycin derivatives

Preparation of alpha-ketopimelic acid

The present invention relates to a method for preparing alpha-ketopimelic acid, comprising converting 2-hydroxyheptanedioic acid into alpha-ketopimelic acid, which conversion is catalysed using a biocatalyst. Further, the invention relates to a heterologous cell, comprising a nucleic acid sequence encoding an enzyme having catalytic activity in the conversion of 2-hydroxyheptanedioic acid into alpha-ketopimelic acid. Further, the invention relates to the use of a heterologous cell according to the invention in the preparation of caprolactam, diaminohexane or adipic acid.

Preparation of alpha-amino-epsilon-caprolactam via lysine cyclisation

The present invention relates to a method for preparing α-amino-ε-caprolactam, comprising converting lysine to α-amino-ε-caprolactam, wherein the conversion is catalysed by a biocatalyst. Further, the invention relates to a host cell comprising at least one recombinant vector comprising a nucleic acid sequence encoding a biocatalyst with lysine cyclase activity. Further a method is provided wherein α-amino- ε-caprolactam is used for preparing ε-caprolactam.

Preparation of alpha-amino-epsilon-caprolactam via lysine cyclisation

The present invention relates to a method for preparing α-amino-ε-caprolactam, comprising converting lysine to α-amino-ε-caprolactam, wherein the conversion is catalysed by a biocatalyst. Further, the invention relates to a host cell comprising at least one recombinant vector comprising a nucleic acid sequence encoding a biocatalyst with lysine cyclase activity. Further a method is provided wherein α-amino- ε-caprolactam is used for preparing ε-caprolactam.

Preparation of 6-aminocaproic acid from 5-formyl valeri C acid

The invention relates to a method for preparing 6-aminocaproic acid (hereinafter also referred to as ‘6-ACA’) using a biocatalyst. The invention further relates to a method for preparing e-caprolactam (hereafter referred to as ‘caprolactam’) by cyclising such 6-ACA. The invention further relates to a host cell, a micro-organism, or a polynucleotide which may be used in the preparation of 6-ACA or caprolactam.