Recombinant Host Cells Comprising Phosphoketalase

This application is related to and claims the benefit of priority of U.S. Provisional Patent Application No. 61/356,379, filed on Jun. 18, 2010, the entirety of which is herein incorporated by reference.

The invention relates generally to the field of industrial microbiology. The invention relates to recombinant host cells comprising (i) a modification in an endogenous gene encoding a polypeptide that converts pyruvate to acetyl-CoA, acetaldehyde or acetyl-phosphate and (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. The invention also relates to recombinant host cells comprising (i) a modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase (PDC) activity, or a modification in an endogenous polypeptide having PDC activity, and (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. The invention also relates to recombinant host cells further comprising (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. Additionally, the invention relates to methods of making and using such recombinant host cells including, for example, methods of increasing cell growth, methods of reducing or eliminating the requirement of an exogenous carbon substrate for cell growth, methods of increasing glucose consumption and methods of increasing the production of a product of a pyruvate-utilizing pathway.

Global demand for liquid transportation fuel is projected to strain the ability to meet certain environmentally driven goals, for example, the conservation of oil reserves and limitation of green house gas emissions. Such demand has driven the development of technology which allows utilization of renewable resources to mitigate the depletion of oil reserves and to minimize green house gas emissions.

Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a food grade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase in the future.

Methods for the chemical synthesis of isobutanol are known, such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCH Verlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) and Guerbet condensation of methanol with n-propanol (Carlini et al., J. Molec. Catal. A: Chem. 220:215-220, 2004). These processes use starting materials derived from petrochemicals, are generally expensive, and are not environmentally friendly. The production of isobutanol from plant-derived raw materials would minimize green house gas emissions and would represent an advance in the art.

2-Butanone, also referred to as methyl ethyl ketone (MEK), is a widely used solvent and is the most important commercially produced ketone, after acetone. It is used as a solvent for paints, resins, and adhesives, as well as a selective extractant, activator of oxidative reactions, and it can be chemically converted to 2-butanol by reacting with hydrogen in the presence of a catalyst (Nystrom, R. F. and Brown, W. G. (J. Am. Chem. Soc. (1947) 69:1198). 2,3-butanediol can be used in the chemical synthesis of butene and butadiene, important industrial chemicals currently obtained from cracked petroleum, and esters of 2,3-butanediol may be used as plasticizers (Voloch et al., “Fermentation Derived 2,3-Butanediol,” in Comprehensive Biotechnology, Pergamon Press Ltd., England Vol. 2, Section 3:933-947 (1986)).

Microorganisms can be engineered for the expression of biosynthetic pathways that initiate with cellular pyruvate to produce, for example, 2,3-butanediol, 2-butanone, 2-butanol and isobutanol. U.S. Pat. No. 7,851,188 discloses the engineering of recombinant microorganisms for production of isobutanol. U.S. Patent Application Publication Nos. US 20070259410 A1 and US 20070292927 A1 disclose the engineering of recombinant microorganisms for production of 2-butanone or 2-butanol. Multiple pathways are disclosed for biosynthesis of isobutanol and 2-butanol, all of which initiate with cellular pyruvate. Butanediol is an intermediate in the 2-butanol pathway disclosed in U.S. Patent Application Publication No. US 20070292927 A1.

The disruption of the enzyme pyruvate decarboxylase (PDC) in recombinant host cells engineered to express a pyruvate-utilizing biosynthetic pathway has been used to increase the availability of pyruvate for product formation via the biosynthetic pathway. For example, U.S. Application Publication No. US 20070031950 A1 discloses a yeast strain with a disruption of one or more pyruvate decarboxylase genes (a PDC knock-out or PDC-KO) and expression of a D-lactate dehydrogenase gene, which is used for production of D-lactic acid. U.S. Application Publication No. US 20050059136 A1 discloses glucose tolerant two-carbon source-independent (GCSI) yeast strains with no PDC activity, which may have an exogenous lactate dehydrogenase gene. Nevoigt and Stahl (Yeast 12:1331-1337 (1996)) describe the impact of reduced PDC and increased NAD-dependent glycerol-3-phosphate dehydrogenase in Saccharomyces cerevisiae on glycerol yield. U.S. Application Publication No. 20090305363 A1 discloses increased conversion of pyruvate to acetolactate by engineering yeast for expression of a cytosol-localized acetolactate synthase and substantial elimination of PDC activity.

While PDC-KO recombinant host cells can be used to produce the products of pyruvate-utilizing biosynthetic pathways, PDC-KO recombinant host cells require exogenous carbon substrate supplementation (e.g., ethanol or acetate) for their growth (Flikweert et al. 1999. FEMS Microbiol. Lett. 174(1):73-79 “Growth requirements of pyruvate-decarboxylase-negative Saccharomyces cerevisiae”). A similar auxotrophy is observed in Escherichia coli strains carrying a mutation of one or more genes encoding pyruvate dehydrogenase (Langley and Guest, 1977, J. Gen. Microbiol. 99:263-276).

In commercial applications, addition of exogenous carbon substrate in addition to the substrate converted to a desired product can lead to increased costs. There remains a need in the art for recombinant host cells with reduced or eliminated need for exogenous carbon substrate supplementation.

One aspect of the invention relates to a recombinant host cell comprising (i) at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide that converts pyruvate to acetaldehyde, acetyl-phosphate, or acetyl-CoA; and ii) a heterologous polynuclotide encoding a polypeptide having phosphoketolase activity. Another aspect of the invention relates to such a recombinant host cell further comprising (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. In embodiments, the polypeptide that converts pyruvate to acetaldehyde, acetyl-phosphate, or acetyl-CoA is pyruvate decarboxylase, pyruvate-formate lyase, pyruvate dehydrogenase, pyruvate oxidase, or pyruvate:ferredoxin oxidoreductase.

One aspect of the invention relates to a recombinant host cell comprising (i) a modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity or in an endogenous polypeptide having pyruvate decarboxylase activity; and (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. Another aspect of the invention relates to such a recombinant host cell further comprising (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

One aspect of the invention relates to a recombinant host cell comprising (i) at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity; and (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. Another aspect of the invention relates to a recombinant host cell further comprising: (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. Another aspect of invention relates to a reduced or eliminated requirement of such cells for an exogenous two-carbon substrate for its growth in culture compared to a recombinant eukaryotic host cell comprising (i) and not (ii) or (iii). Another aspect of the invention relates to the growth of such host cells in culture media that is not supplemented with an exogenous two-carbon substrate, for example, at a growth rate substantially equivalent to, or greater than, the growth rate of a host cell comprising (i) and not (ii) or (iii) in culture media supplemented with an exogenous two-carbon substrate.

In one aspect of the invention, the recombinant host cell is a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, or Saccharomyces. In another aspect of the invention, the recombinant host cell is S. cerevisiae

In another aspect of the invention, the recombinant host cell expresses a pyruvate-utilizing biosynthetic pathway including, for example, a biosynthetic pathway for a product such as 2,3-butanediol, isobutanol, 2-butanol, 2-butanone, valine, leucine, alanine, lactic acid, malic acid, fumaric acid, succinic acid, or isoamyl alcohol. Another aspect of the invention relates to expression of an isobutanol biosynthetic pathway in the recombinant host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of (i) pyruvate to acetolactate; (ii) acetolactate to 2,3-dihydroxyisovalerate; (iii) 2,3-dihydroxyisovalerate to 2-ketoisovalerate; (iv) 2-ketoisovalerate to isobutyraldehyde; and (v) isobutyraldehyde to isobutanol. Another aspect of the invention relates to expression of a 2-butanone biosynthetic pathway in the recombinant host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of (i) pyruvate to acetolactate; (ii) acetolactate to acetoin; (iii) acetoin to 2,3-butanediol; and (iv) 2,3-butanediol to 2-butanone.

Another aspect of the invention relates to expression of a 2-butanol biosynthetic pathway in the recombinant host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of (i) pyruvate to acetolactate; (ii) acetolactate to acetoin; (iii) acetoin to 2,3-butanediol; (iv) 2,3-butanediol to 2-butanone; and (v) 2-butanone to 2-butanol.

One aspect of the invention relates to methods for the production of a product selected from the group consisting of 2,3-butanediol, isobutanol, 2-butanol, 2-butanone, valine, leucine, alanine, lactic acid, malic acid, fumaric acid, succinic acid and isoamyl alcohol comprising growing the recombinant host cells described herein under conditions wherein the product is produced and optionally recovering the product. Another aspect of the invention relates to methods of producing a recombinant host cell comprising transforming a host cell comprising at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity with (i) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and optionally (ii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

Another aspect of the invention relates to methods of improving the growth of a recombinant host cell comprising at least one deletion, mutation or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, comprising (i) transforming the recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and optionally (ii) transforming the recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. In embodiments, the methods further comprise growing the recombinant host cell in media containing limited carbon substrate.

Another aspect of the invention relates to methods of reducing the requirement for an exogenous two-carbon substrate for the growth of a recombinant host cell comprising at least one deletion, mutation or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, comprising (i) transforming the host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and optionally (ii) transforming the host cell with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

Another aspect of the invention relates to methods of eliminating the requirement for an exogenous two-carbon substrate for the growth of a recombinant host cell comprising at least one deletion, mutation or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, comprising (i) transforming the host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and optionally (ii) transforming the host cell with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

Still another aspect of the invention relates to methods for increasing the activity of the phosphoketolase pathway in a recombinant host cell comprising (i) providing a recombinant host cell of the invention; and (ii) growing the recombinant host cell under conditions whereby the activity of the phosphoketolase pathway in the recombinant host cell is increased.

In another aspect, the recombinant host cells comprise a phosphoketolase that matches the Profile HMM given in Table 6 with an E value of less than 7.5E-242. In another aspect, the phosphoketolase has at least about 40% identity to at least one of SEQ ID NO: 355, 379, 381, 388, 481, 486, 468, or 504. In another aspect, the phosphoketolase has at least about 90% identity to at least one of SEQ ID NO: 355, 379, 381, 388, 481, 486, 468, or 504. In another aspect, the phosphoketolase matches the Profile HMMs given in Tables 6, 7, 8, and 9 with E values of less than 7.5E-242, 1.1E-124, 2.1E-49, 7.8E-37, respectively. In another aspect, the recombinant host cells further comprise a phosphotransacetylase which matches the Profile HMM given in Table 14 with an E value of less than 5E-34. In another aspect, the phosphotransacetylase has at least about 40% identity to SEQ ID NO: 1475, 1472, 1453, 1422, 1277, 1275, 1206, 1200, 1159, or 1129. In another aspect, the phosphotransacetylase has at least about 90% identity to SEQ ID NO: 1475, 1472, 1453, 1422, 1277, 1275, 1206, 1200, 1159, or 1129

Tables 6, 7, 8, 9, and 14 are tables of the Profile HMMs described herein. Table 6, 7, 8, 9, and 14 are submitted herewith electronically and are incorporated herein by reference.

The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions which form a part of this application.

The sequence listing provided herewith is herein incorporated by reference and conforms with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and is consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (2009) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822. The content of the electronically submitted sequence listing Name: 20110615_CL4871USNA_SeqList.txt; Size: 6.67 MB; and Date of Creation/Modification: Jun. 9, 2011/Jun. 15, 2011 is incorporated herein by reference in its entirety.

SEQ ID NOs: 1-20 are sequences of PDC target gene coding regions and proteins.

SEQ ID NOs: 21-638 are phosphoketolase target gene coding regions and proteins.

SEQ ID NOs: 762-1885 are phosphotransacetylase target gene coding regions and proteins.

SEQ ID NOs: 1893-1897 are hybrid promoter sequences.

SEQ ID NOs: 639-642, 644-654, 656-660, 662-701-714, 725-726, 729-740, 742-748, and 750-761 are primers.

SEQ ID NO: 643 is the vector pRS426::GPD-xpk1+ADH1-eutD.

SEQ ID NO: 655 is the TEF1p-kan-TEF1t gene.

SEQ ID NO: 661 is vector pLA54.

SEQ ID NO: 715 is vector pRS423::pGAL1-cre.

SEQ ID NO: 716 is the vector pLH468-sadB.

SEQ ID NOs: 717 and 718 are the amino acid and nucleic acid sequences for sadB from Achromobacter xylosoxidans.

SEQ ID NO: 719 is the kivD coding region from L. lactis.

SEQ ID NO: 720 is the plasmid pRS425::GPM-sadB.

SEQ ID NO: 721 is the GPM promoter.

SEQ ID NO: 722 is the ADH1 terminator.

SEQ ID NO: 723 is the GPM-sadB-ADHt segment.

SEQ ID NO: 724 is the pUC19-URA3 plasmid.

SEQ ID NO: 741 is the ilvD-FBA1t segment.

SEQ ID NO: 749 is URA3r2 template DNA.

SEQ ID NO: 1886 is the ilvD coding region from S. mutans.

SEQ ID NO: 1888 is vector pLH468.

SEQ ID NO: 1898 is pUC19-URA3::pdc1::GPD-xpk1+ADH1-eutD.

SEQ ID NOs: 1899-1906 are the sequences of modified S. cerevisiae loci.

SEQ ID NO: 1907 is the sequence of pLH702.

SEQ ID NO: 1908 is the sequence of pYZ067DkivDDhADH

SEQ ID NO: 1909 is the amino acid sequence of ALD6.

SEQ ID NO: 1910 is the amino acid sequence of K9D3.

SEQ ID NO: 1911 is the amino acid sequence of K9G9.

SEQ ID NO: 1912 is the amino acid sequence of YMR226c.

SEQ ID NOs: 1913 and 1914 are the nucleic acid and amino acid sequences of AFT1.

SEQ ID NOs: 1915 and 1916 are the nucleic acid and amino acid sequences of AFT2.

SEQ ID NOs: 1917 and 1918 are the nucleic acid and amino acid sequences of FRA2.

SEQ ID NOs: 1919 and 1920 are the nucleic acid and amino acid sequences of GRX3.

SEQ ID NOs: 1921 and 1922 are the nucleic acid and amino acid sequences of CCC1.

SEQ ID NO: 1923 is the amino acid sequence of an alcohol dehydrogenase from Beijerinkia indica.

Applicants have solved the stated problem by reducing or eliminating the need for providing two substrates, one of which is converted to a desired product, the other fully or partly into acetyl-CoA by recombinant host cells requiring such supplementation for growth comprising the expression of enzymes of the phosphoketolase pathway in such cells. One such enzyme, phosphoketolase (Enzyme Commission Number EC 4.1.2.9), catalyzes the conversion of xylulose 5-phosphate into glyceraldehyde 3-phosphate and acetyl-phosphate (Heath et al., J. Biol. Chem. 231: 1009-29; 1958). Another such enzyme is phosphotransacetylase (Enzyme Commission Number EC 2.3.1.8) which converts acetyl-phosphate into acetyl-CoA.

Applicants have provided PDC-KO recombinant host cells comprising a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity, and optionally a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. Such cells exhibit a reduced or eliminated requirement for exogenous two-carbon substrate supplementation for their growth compared to PDC-KO cells. Applicants have also provided methods of making and using such recombinant host cells including, for example, methods of increasing cell growth, methods of reducing or eliminating the requirement of an exogenous two-carbon substrate for cell growth, methods of increasing glucose consumption and methods of increasing the production of a product of a pyruvate-utilizing pathway.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference, unless only specific sections of patents or patent publications are indicated to be incorporated by reference.

Although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention, suitable methods and materials are described below. The materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description and from the claims.

In order to further define this invention, the following terms, abbreviations and definitions are provided.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” or “containing,” or any other variation thereof, are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances, i.e., occurrences of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the application.

As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or to carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.

The term “butanol” as used herein, refers to 2-butanol, 1-butanol, isobutanol, or mixtures thereof.

The term “pyruvate-utilizing biosynthetic pathway” refers to an enzyme pathway to produce a biosynthetic product from pyruvate.

The term “isobutanol biosynthetic pathway” refers to an enzyme pathway to produce isobutanol from pyruvate.

The term “2-butanol biosynthetic pathway” refers to an enzyme pathway to produce 2-butanol from pyruvate.

The term “2-butanone biosynthetic pathway” refers to an enzyme pathway to produce 2-butanone from pyruvate.

The terms “pdc-,” “PDC knock-out,” or “PDC-KO” as used herein refer to a cell that has a genetic modification to inactivate or reduce expression of at least one gene encoding pyruvate decarboxylase (PDC) so that the cell substantially or completely lacks pyruvate decarboxylase enzyme activity. If the cell has more than one expressed (active) PDC gene, then each of the active PDC genes may be inactivated or have minimal expression thereby producing a pdc-cell.

The term “carbon substrate” refers to a carbon source capable of being metabolized by the recombinant host cells disclosed herein. Non-limiting examples of carbon substrates are provided herein and include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides, ethanol, lactate, succinate, glycerol, carbon dioxide, methanol, glucose, fructose, sucrose, xylose, arabinose, dextrose, or mixtures thereof.

The term “exogenous two-carbon substrate” refers to the carbon source provided to be metabolized into acetyl-CoA by a host cell that lacks the ability to convert pyruvic acid into acetyl-CoA. The term is used to distinguish from the carbon substrate which is converted into a pyruvate-derived product by a pyruvate-utilizing biosynthetic pathway, herein also referred to as the “pathway substrate” which includes, for example, glucose.

The term “polynucleotide” is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to a nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide can contain the nucleotide sequence of the full-length cDNA sequence, or a fragment thereof, including the untranslated 5′ and 3′ sequences and the coding sequences. The polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. “Polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.

A polynucleotide sequence may be referred to as “isolated,” in which it has been removed from its native environment. For example, a heterologous polynucleotide encoding a polypeptide or polypeptide fragment having dihydroxy-acid dehydratase activity contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. An isolated polynucleotide fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

The term “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “heterologous gene” refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. “Heterologous gene” includes a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding region that is a portion of a chimeric gene including non-native regulatory regions that is reintroduced into the native host. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.

As used herein the term “coding region” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.

As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

By an “isolated” polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

As used herein, the term “variant” refers to a polypeptide differing from a specifically recited polypeptide of the invention by amino acid insertions, deletions, mutations, and substitutions, created using, e.g., recombinant DNA techniques, such as mutagenesis. Guidance in determining which amino acid residues may be replaced, added, or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous polypeptides, e.g., yeast or bacterial, and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequences.

Alternatively, recombinant polynucleotide variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code. Various codon substitutions, such as silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector for expression. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide.

Amino acid “substitutions” may be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements, or they may be the result of replacing one amino acid with an amino acid having different structural and/or chemical properties, i.e., non-conservative amino acid replacements. “Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Alternatively, “non-conservative” amino acid substitutions may be made by selecting the differences in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of any of these amino acids. “Insertions” or “deletions” may be within the range of variation as structurally or functionally tolerated by the recombinant proteins. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.

The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. For example, it will be understood that “FBA1 promoter” can be used to refer to a fragment derived from the promoter region of the FBA1 gene.

The term “terminator” as used herein refers to DNA sequences located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The 3′ region can influence the transcription, RNA processing or stability, or translation of the associated coding sequence. It is recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical terminator activity. For example, it will be understood that “CYC1 terminator” can be used to refer to a fragment derived from the terminator region of the CYC1 gene.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression,” as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.

The term “overexpression,” as used herein, refers to expression that is higher than endogenous expression of the same or related gene. A heterologous gene is overexpressed if its expression is higher than that of a comparable endogenous gene.

As used herein the term “transformation” refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid” and “vector” as used herein, refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.

As used herein the term “codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

The term “codon-optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.

Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The “genetic code” which shows which codons encode which amino acids is reproduced herein as Table 1. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.

Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference, or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at http://www.kazusa.or.jp/codon/ (visited Mar. 20, 2008), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. Nucl. Acids Res. 28:292 (2000). Codon usage tables for yeast, calculated from GenBank Release 128.0 [15 Feb. 2002], are reproduced below as Table 2. This table uses mRNA nomenclature, and so instead of thymine (T) which is found in DNA, the tables use uracil (U) which is found in RNA. Table 2 has been adapted so that frequencies are calculated for each amino acid, rather than for all 64 codons.

By utilizing this or similar tables, one of ordinary skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide, but which uses codons optimal for a given species.

Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence, can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly. Additionally, various algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the “EditSeq” function in the Lasergene Package, available from DNAstar, Inc., Madison, Wis., the backtranslation function in the VectorNTI Suite, available from InforMax, Inc., Bethesda, Md., and the “backtranslate” function in the GCG-Wisconsin Package, available from Accelrys, Inc., San Diego, Calif. In addition, various resources are publicly available to codon-optimize coding region sequences, e.g., the “backtranslation” function at http://www.entelechon.com/bioinformatics/backtranslation.php?lang=eng (visited Apr. 15, 2008) and the “backtranseq” function available at http://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html (visited Jul. 9, 2002). Constructing a rudimentary algorithm to assign codons based on a given frequency can also easily be accomplished with basic mathematical functions by one of ordinary skill in the art.

Codon-optimized coding regions can be designed by various methods known to those skilled in the art including software packages such as “synthetic gene designer” (http://phenotype.biosci.umbc.edu/codon/sgd/index.php).

A polynucleotide or nucleic acid fragment is “hybridizable” to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^nded., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding particular proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.

The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the “Clustal method of alignment” which encompasses several varieties of the algorithm including the “Clustal V method of alignment” corresponding to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program. Additionally the “Clustal W method of alignment” is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191 (1992)) and found in the MegAlign™ v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of the sequences using the Clustal W program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.

It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, such as from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100% may be useful in describing the present invention, such as 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.

The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Additional methods used here are in Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.). Other molecular tools and techniques are known in the art and include splicing by overlapping extension polymerase chain reaction (PCR) (Yu, et al. (2004) Fungal Genet. Biol. 41:973-981), positive selection for mutations at the URA3 locus of Saccharomyces cerevisiae (Boeke, J. D. et al. (1984) Mol. Gen. Genet. 197, 345-346; M A Romanos, et al. Nucleic Acids Res. 1991 Jan. 11; 19(1): 187), the cre-lox site-specific recombination system as well as mutant lox sites and FLP substrate mutations (Sauer, B. (1987) Mol Cell Biol 7: 2087-2096; Senecoff, et al. (1988) Journal of Molecular Biology, Volume 201, Issue 2, Pages 405-421; Albert, et al. (1995) The Plant Journal. Volume 7, Issue 4, pages 649-659), “seamless” gene deletion (Akada, et al. (2006) Yeast; 23(5):399-405), and gap repair methodology (Ma et al., Genetics 58:201-216; 1981). Applicants have discovered that activation of the phosphoketolase pathway in a recombinant host cell comprising a modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity or a modification in an endogenous polypeptide having pyruvate decarboxylase activity, reduces or eliminates the need for an exogenous carbon substrate for the growth of such a cell. In embodiments, the recombinant host cells comprise (i) at least one deletion, mutation and/or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity); (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and optionally (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

The genetic manipulations of the host cells described herein can be performed using standard genetic techniques and screening and can be made in any host cell that is suitable to genetic manipulation (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). In embodiments, the recombinant host cells disclosed herein can be any bacteria, yeast or fungi host useful for genetic modification and recombinant gene expression. In other embodiments, a recombinant host cell can be a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, Issatchenkia, or Saccharomyces. In other embodiments, the host cell can be Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces thermotolerans, Kluyveromyces marxianus, Candida glabrata, Candida albicans, Pichia stipitis, Yarrowia lipolytica, E. coli, or L. plantarum. In still other embodiments, the host cell is a yeast host cell. In some embodiments, the host cell is a member of the genera Saccharomyces. In some embodiments, the host cell is Kluyveromyces lactis, Candida glabrata or Schizosaccharomyces pombe. In some embodiments, the host cell is Saccharomyces cerevisiae. S. cerevisiae yeast are known in the art and are available from a variety of sources, including, but not limited to, American Type Culture Collection (Rockville, Md.), Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex, and Lallemand. S. cerevisiae include, but are not limited to, BY4741, CEN.PK 113-7D, Ethanol Red® yeast, Ferm Pro™ yeast, Bio-Ferm® XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMax™ Green yeast, FerMax™ Gold yeast, Thermosacc® yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

Acetyl-CoA is a major cellular building block, required for the synthesis of fatty acids, sterols, and lysine. Pyruvate is often a major contributor to the acetyl-CoA pool. Pyruvate dehydrogenase catalyzes the direct conversion of pyruvate to acetyl-CoA (E.C. 1.2.4.1, E.C. 1.2.1.51) or acetate (E.C. 1.2.2.2) and is almost ubiquitous in nature. Other enzymes involved in conversion of pyruvate to acetyl-CoA, acetyl-phosphate or acetate include pyruvate-formate lyase (E.C. 2.3.1.54), pyruvate oxidase (E.C. 1.2.3.3, E.C. 1.2.3.6), pyruvate-ferredoxin oxidoreductase (E.C. 1.2.7.1), and pyruvate decarboxylase (E.C. 4.1.1.1). Genetic modifications made to a host cell to conserve the pyruvate pool for a product of interest may include those that restrict conversion to acetyl-CoA, leading to decreased growth in the absence of an exogenously supplied two-carbon substrate, a carbon substrate that can be readily converted to acetyl-CoA independent of pyruvate (e.g. ethanol or acetate). An example is the documented auxotrophy observed in pyruvate decarboxylase deficient Saccharomyces cerevisiae (Flikweert et al. 1999, supra). Another example is the documented auxotrophy observed in pyruvate dehydrogenase deficient Escherichia coli when grown aerobically on glucose (Langley and Guest, 1977, J. Gen. Microbiol. 99:2630276).

In embodiments, the recombinant host cells disclosed herein comprise a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase (PDC) or a modification in an endogenous polypeptide having PDC activity. In embodiments, the recombinant host cells disclosed herein can have a modification or disruption of one or more polynucleotides, genes or polypeptides encoding PDC. In embodiments, the recombinant host cell comprises at least one deletion, mutation, and/or substitution in one or more endogenous polynucleotides or genes encoding a polypeptide having PDC activity, or in one or more endogenous polypeptides having PDC activity. Such modifications, disruptions, deletions, mutations, and/or substitutions can result in PDC activity that is reduced or eliminated, resulting in a PDC knock-out (PDC-KO) phenotype.

In embodiments, the endogenous pyruvate decarboxylase activity of the recombinant host cells disclosed herein converts pyruvate to acetaldehyde, which can then be converted to ethanol or to acetyl-CoA via acetate.

In embodiments, the recombinant host cell is Kluyveromyces lactis containing one gene encoding pyruvate decarboxylase, Candida glabrata containing one gene encoding pyruvate decarboxylase, or Schizosaccharomyces pombe containing one gene encoding pyruvate decarboxylase.

In other embodiments, the recombinant host cell is Saccharomyces cerevisiae containing three isozymes of pyruvate decarboxylase encoded by the pdc1, pdc5, and pdc6 genes, as well as a pyruvate decarboxylase regulatory gene, pdc2. In a non-limiting example in S. cerevisiae, the pdc1 and pdc5 genes, or all three genes, are disrupted. In another non-limiting example in S. cerevisiae, pyruvate decarboxylase activity may be reduced by disrupting the pdc2 regulatory gene. In another non-limiting example in S. cerevisiae, polynucleotides or genes encoding pyruvate decarboxylase proteins such as those having about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to pdc1 or pdc5 can be disrupted.

In embodiments, the polypeptide having PDC activity or the polynucleotide or gene encoding a polypeptide having PDC activity is associated with Enzyme Commission Number EC 4.1.1.1. In other embodiments, a PDC gene of the recombinant host cells disclosed herein is not active under the fermentation conditions used, and therefore such a gene would not need to be modified or inactivated.

Examples of recombinant host cells with reduced pyruvate decarboxylase activity due to disruption of pyruvate decarboxylase encoding genes have been reported, such as for Saccharomyces in Flikweert et al. (Yeast (1996) 12:247-257), for Kluyveromyces in Bianchi et al. (Mol. Microbiol. (1996) 19(1):27-36), and disruption of the regulatory gene in Hohmann (Mol. Gen. Genet. (1993) 241:657-666). Saccharomyces strains having no pyruvate decarboxylase activity are available from the ATCC with Accession #200027 and #200028.

Examples of PDC polynucleotides, genes and polypeptides that can be targeted for modification or inactivation in the recombinant host cells disclosed herein include, but are not limited to, those of the following table.

Other examples of PDC polynucleotides, genes and polypeptides that can be targeted for modification or inactivation in the recombinant host cells disclosed herein include, but are not limited to, PDC polynucleotides, genes and/or polypeptides having at least about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any one of the sequences of Table 3.

In embodiments, the sequences of other PDC polynucleotides, genes and/or polypeptides can be identified in the literature and in bioinformatics databases well known to the skilled person using sequences disclosed herein and available in the art. For example, such sequences can be identified through BLAST (as described above) searching of publicly available databases with known PDC encoding polynucleotide or polypeptide sequences. In such a method, identities can be based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Additionally, the PDC polynucleotide or polypeptide sequences described herein or known the art can be used to identify other PDC homologs in nature. For example, each of the PDC encoding nucleic acid fragments described herein can be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to (1) methods of nucleic acid hybridization; (2) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and (3) methods of library construction and screening by complementation.

In embodiments, PDC polynucleotides, genes and/or polypeptides related to the recombinant host cells described herein can be modified or disrupted. Many methods for genetic modification and disruption of target genes to reduce or eliminate expression are known to one of ordinary skill in the art and can be used to create the recombinant host cells described herein. Modifications that can be used include, but are not limited to, deletion of the entire gene or a portion of the gene encoding a PDC protein, inserting a DNA fragment into the encoding gene (in either the promoter or coding region) so that the protein is not expressed or expressed at lower levels, introducing a mutation into the coding region which adds a stop codon or frame shift such that a functional protein is not expressed, and introducing one or more mutations into the coding region to alter amino acids so that a non-functional or a less active protein is expressed. In other embodiments, expression of a target gene can be blocked by expression of an antisense RNA or an interfering RNA, and constructs can be introduced that result in cosuppression. In other embodiments, the synthesis or stability of the transcript can be lessened by mutation. In embodiments, the efficiency by which a protein is translated from mRNA can be modulated by mutation. All of these methods can be readily practiced by one skilled in the art making use of the known or identified sequences encoding target proteins.

In other embodiments, DNA sequences surrounding a target PDC coding sequence are also useful in some modification procedures and are available, for example, for yeasts such as Saccharomyces cerevisiae in the complete genome sequence coordinated by Genome Project ID9518 of Genome Projects coordinated by NCBI (National Center for Biotechnology Information) with identifying GOPID #13838. An additional non-limiting example of yeast genomic sequences is that of Candida albicans, which is included in GPID #10771, #10701 and #16373. Other yeast genomic sequences can be readily found by one of skill in the art in publicly available databases.

In other embodiments, DNA sequences surrounding a target PDC coding sequence can be useful for modification methods using homologous recombination. In a non-limiting example of this method, PDC gene flanking sequences can be placed bounding a selectable marker gene to mediate homologous recombination whereby the marker gene replaces the PDC gene. In another non-limiting example, partial PDC gene sequences and PDC gene flanking sequences bounding a selectable marker gene can be used to mediate homologous recombination whereby the marker gene replaces a portion of the target PDC gene. In embodiments, the selectable marker can be bounded by site-specific recombination sites, so that following expression of the corresponding site-specific recombinase, the resistance gene is excised from the PDC gene without reactivating the latter. In embodiments, the site-specific recombination leaves behind a recombination site which disrupts expression of the PDC protein. In other embodiments, the homologous recombination vector can be constructed to also leave a deletion in the PDC gene following excision of the selectable marker, as is well known to one skilled in the art.

In other embodiments, deletions can be made to a PDC target gene using mitotic recombination as described in Wach et al. (Yeast, 10:1793-1808; 1994). Such a method can involve preparing a DNA fragment that contains a selectable marker between genomic regions that can be as short as 20 bp, and which bound a target DNA sequence. In other embodiments, this DNA fragment can be prepared by PCR amplification of the selectable marker gene using as primers oligonucleotides that hybridize to the ends of the marker gene and that include the genomic regions that can recombine with the yeast genome. In embodiments, the linear DNA fragment can be efficiently transformed into yeast and recombined into the genome resulting in gene replacement including with deletion of the target DNA sequence ((as described, for example, in Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.)).

Moreover, promoter replacement methods can be used to exchange the endogenous transcriptional control elements allowing another means to modulate expression such as described in Mnaimneh et al. ((2004) Cell 118(1):31-44).

In other embodiments, the PDC target gene encoded activity can be disrupted using random mutagenesis, which can then be followed by screening to identify strains with dependency on carbon substrates for growth. In this type of method, the DNA sequence of the target gene encoding region, or any other region of the genome affecting carbon substrate dependency for growth, need not be known. In embodiments, a screen for cells with reduced PDC activity and/or two-carbon substrate dependency, or other mutants having reduced PDC activity and a reduced or eliminated dependency for exogenous two-carbon substrate for growth, can be useful as recombinant host cells of the invention.

Methods for creating genetic mutations are common and well known in the art and can be applied to the exercise of creating mutants. Commonly used random genetic modification methods (reviewed in Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) include spontaneous mutagenesis, mutagenesis caused by mutator genes, chemical mutagenesis, irradiation with UV or X-rays, or transposon mutagenesis.

Chemical mutagenesis of host cells can involve, but is not limited to, treatment with one of the following DNA mutagens: ethyl methanesulfonate (EMS), nitrous acid, diethyl sulfate, or N-methyl-N′-nitro-N-nitroso-guanidine (MNNG). Such methods of mutagenesis have been reviewed in Spencer et al. (Mutagenesis in Yeast, 1996, Yeast Protocols: Methods in Cell and Molecular Biology. Humana Press, Totowa, N.J.). In embodiments, chemical mutagenesis with EMS can be performed as described in Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Irradiation with ultraviolet (UV) light or X-rays can also be used to produce random mutagenesis in yeast cells. The primary effect of mutagenesis by UV irradiation is the formation of pyrimidine dimers which disrupt the fidelity of DNA replication. Protocols for UV-mutagenesis of yeast can be found in Spencer et al. (Mutagenesis in Yeast, 1996, Yeast Protocols: Methods in Cell and Molecular Biology. Humana Press, Totowa, N.J.). In embodiments, the introduction of a mutator phenotype can also be used to generate random chromosomal mutations in host cells. In embodiments, common mutator phenotypes can be obtained through disruption of one or more of the following genes: PMS1, MAG1, RAD18 or RAD51. In other embodiments, restoration of the non-mutator phenotype can be obtained by insertion of the wildtype allele. In other embodiments, collections of modified cells produced from any of these or other known random mutagenesis processes may be screened for reduced or eliminated PDC activity.

Genomes have been completely sequenced and annotated and are publicly available for the following yeast strains: Ashbya gossypii ATCC 10895, Candida glabrata CBS 138, Kluyveromyces lactis NRRL Y-1140, Pichia stipitis CBS 6054, Saccharomyces cerevisiae S288c, Schizosaccharomyces pombe 972h-, and Yarrowia lipolytica CLIB122. Typically BLAST (described above) searching of publicly available databases with known PDC polynucleotide or polypeptide sequences, such as those provided herein, is used to identify PDC-encoding sequences of other host cells, such as yeast cells.

Accordingly, it is within the scope of the invention to provide pyruvate decarboxylase polynucleotides and polypeptides having at least about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any of the PDC polypeptides or polypeptides disclosed herein (SEQ ID NOs: 1-20). Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

The modification of PDC in the host cells disclosed herein to reduce or eliminate PDC activity can be confirmed using methods known in the art. For example, PCR methods well known in the art can be used to confirm deletion of PDC. Other suitable methods will be known to those of skill in the art and include, but are not limited to, lack of growth on yeast extract peptone-dextrose medium (YPD).

Applicants have found that expression of enzymes associated with the phosphoketolase pathway (e.g., phosphoketolase and/or phosphotransacetylase) results in a reduced or eliminated requirement for exogenous two-carbon substrate supplementation for growth of PDC-KO cells. Phosphoketolases and/or phosphotransacetylases identified as described herein, can be expressed in such cells using methods described herein.

Enzymes of the phosphoketolase pathway include phosphoketolase and phosphotransacetylase (FIG. 1). Phosphoketolase (Enzyme Commission Number EC 4.1.2.9) catalyzes the conversion of xylulose 5-phosphate into glyceraldehyde 3-phosphate and acetyl-phosphate (Heath et al., J. Biol. Chem. 231: 1009-29; 1958). Phosphoketolase activity has been identified in several yeast strains growing with xylose as the sole carbon source but not in yeast strains grown with glucose (Evans and Ratledge, Arch. Microbiol. 139: 48-52; 1984). Inhibitors of phosphoketolase include, but are not limited to, erythrose 4-phosphate and glyceraldehyde 3-phosphate. Phosphotransacetylase (Enzyme Commission Number EC 2.3.1.8) converts acetyl-phosphate into acetyl-CoA.

In embodiments, the phosphoketolase pathway is activated in the recombinant host cells disclosed herein by engineering the cells to express polynucleotides and/or polypeptides encoding phosphoketolase and, optionally, phosphotransacetylase. In embodiments, the recombinant host cells disclosed herein comprise a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. In other embodiments, the recombinant host cells disclosed herein comprise a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity and a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. In other embodiments, the heterologous polynucleotide encoding a polypeptide having phosphoketolase activity is overexpressed, or expressed at a level that is higher than endogenous expression of the same or related endogenous gene, if any. In still other embodiments, the heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity is overexpressed, or expressed at a level that is higher than endogenous expression of the same or related endogenous gene, if any.

In embodiments, a polypeptide having phosphoketolase activity catalyzes the conversion of xylulose 5-phosphate into glyceraldehyde-3-phosphate and acetyl-phosphate and/or the conversion of fructose-6-phosphate into erythrose-4-phosphate and acetyl-phosphate. In embodiments, the activity of a polypeptide having phosphoketolase activity is inhibited by erythrose 4-phosphate and/or glyceraldehyde 3-phosphate. In other embodiments, a polypeptide having phosphotransacetylase activity catalyzes the conversion of acetyl-phosphate into acetyl-CoA.

Numerous examples of polynucleotides, genes and polypeptides encoding phosphoketolase activity are known in the art and can be used in the recombinant host cells disclosed herein. In embodiments, such a polynucleotide, gene and/or polypeptide can be the xylulose 5-phosphateketolase (XpkA) of Lactobacillus pentosus MD363 (Posthuma et al., Appl. Environ. Microbiol. 68: 831-7; 2002). XpkA is the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and exhibits a specific activity of 4.455 μmol/min/mg (Posthuma et al., Appl. Environ. Microbiol. 68: 831-7; 2002). In other embodiments, such a polynucleotide, gene and/or polypeptide can be the phosphoketolase of Leuconostoc mesenteroides which exhibits a specific activity of 9.9 μmol/min/mg and is stable at pH above 4.5 (Goldberg et al., Methods Enzymol. 9: 515-520; 1966). This phosphoketolase exhibits a Km of 4.7 mM for D-xylulose 5-phosphate and a Km of 29 mM for fructose 6-phosphate (Goldberg et al., Methods Enzymol. 9: 515-520; 1966). In other embodiments, such a polynucleotide, gene and/or polypeptide can be the D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase gene xfp from B. lactis, as described, for example, in a pentose-metabolizing S. cerevisiae strain by Sonderegger et al. (Appl. Environ. Microbiol. 70: 2892-7; 2004).

In embodiments, a polynucleotide, gene and/or polypeptide encoding phosphoketolase corresponds to the Enzyme Commission Number EC 4.1.2.9.

In embodiments, host cells comprise a polypeptide having at least about 80%, at least about 85%, at least about 90%, or 100% identity to a polypeptide of Table 4 or an active fragment thereof or a polynucleotide encoding such a polypeptide. In other embodiments, a polynucleotide, gene and/or polypeptide encoding phosphoketolase can include, but is not limited to, a sequence provided in the following tables 4 or 5.

In other embodiments, a polynucleotide, gene and/or polypeptide encoding phosphoketolase can have at least about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to that of any one of the sequences of Table 4, wherein the polynucleotide, gene and/or polypeptide encodes a polypeptide having phosphoketolase activity.

In other embodiments, a polynucleotide, gene and/or polypeptide encoding phosphoketolase can be used to identify other phosphoketolase polynucleotide, gene and/or polypeptide sequences or to identify phosphoketolase homologs in other cells, as described above for PDC. Such phosphoketolase encoding sequences can be identified, for example, in the literature and/or in bioinformatics databases well known to the skilled person. For example, the identification of phosphoketolase encoding sequences in other cell types using bioinformatics can be accomplished through BLAST (as described above) searching of publicly available databases with known phosphoketolase encoding DNA and polypeptide sequences, such as those provided herein. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Additional phosphoketolase target gene coding regions were identified using diversity search, clustering, experimentally verified xylulose-5-phosphate/fructose-6-phosphate phosphoketolases and domain architecture. Briefly, a BLAST search with the experimentally verified sequences with an Evalue cut-off of 0.01 resulted in 595 sequence matches. Clustering with the CD-HIT program at 95% sequence identity and 90% length overlap reduced the number to 436. CD-HIT is a program for clustering large protein database at high sequence identity threshold. The program removes redundant sequences and generates a database of only the representatives. (Clustering of highly homologous sequences to reduce the size of large protein database, Weizhong Li, Lukasz Jaroszewski & Adam Godzik Bioinformatics, (2001) 17:282-283)

Xylulose-5-phosphate/fructose-6-phosphate phosphoketolases have three Pfam domains: XFP_N; XFP; XFP_C. Although each of these domains may be present in several domain architectures, e.g. XFP_N is found in eight architectures. The architecture of interest was determined to be XFP_N; XFP; XFP_C. The cumulative length of the three domains is 760 amino acids.

A structure/function characterization of the phosphoketolases was performed using the HMMER software package. The following information based on the HMMER software user guide gives some description of the way that the hmmbuild program prepares a Profile HMM. A Profile HMM is capable of modeling gapped alignments, e.g. including insertions and deletions, which lets the software describe a complete conserved domain (rather than just a small ungapped motif). Insertions and deletions are modeled using insertion (I) states and deletion (D) states. All columns that contain more than a certain fraction x of gap characters will be assigned as an insert column. By default, x is set to 0.5. Each match state has an I and a D state associated with it. HMMER calls a group of three states (M/D/I) at the same consensus position in the alignment a “node”. These states are interconnected with arrows called state transition probabilities. M and I states are emitters, while D states are silent. The transitions are arranged so that at each node, either the M state is used (and a residue is aligned and scored) or the D state is used (and no residue is aligned, resulting in a deletion-gap character, ‘-’). Insertions occur between nodes, and I states have a self-transition, allowing one or more inserted residues to occur between consensus columns.

The scores of residues in a match state (i.e. match state emission scores), or in an insert state (i.e. insert state emission scores) are proportional to Log_2 (p_x)/(null_x). Where p_x is the probability of an amino acid residue, at a particular position in the alignment, according to the Profile HMM and null_x is the probability according to the Null model. The Null model is a simple one state probabilistic model with pre-calculated set of emission probabilities for each of the 20 amino acids derived from the distribution of amino acids in the SWISSPROT release 24. State transition scores are also calculated as log odds parameters and are propotional to Log_2 (t_x). Where t_x is the probability of transiting to an emitter or non-emitter state.

Using a multiple sequence alignment of experimentally verified sequences containing the architecture of interest XFP_N; XFP; XFP_C, a profile Hidden Markov Model (HMM) was created for representing members of the xylulose-5-phosphate/fructose-6-phosphate phosphoketolases (XPK-XFP). As stated in the user guide, Profile HMMs are statistical models of multiple sequence alignments. They capture position-specific information about how conserved each column of the alignment is, and which amino acid residues are most likely to occur at each position. Thus HMMs have a formal probabilistic basis. Profile HMMs for a large number of protein families are publicly available in the PFAM database (Janelia Farm Research Campus, Ashburn, Va.), see ftp://ftp.sanger.ac.uk/pub/databases/Pfam/releases/Pfam24.0/.

Eight xylulose-5-phosphate/fructose-6-phosphate phosphoketolases sequences with experimentally verified function were identified in the BRENDA database:

1. CBF76492.1 from Aspergillus nidulans FGSC A4 (SEQ ID NO: 355)
2. AAR98787.1 from Bifidobacterium longum (SEQ ID NO: 379)
3. ZP_03646196.1 from Bifidobacterium bifidum NCIMB 41171 (SEQ ID NO: 381)
4. ZP_02962870.1 from Bifidobacterium animalis subsp. lactis HN019 (SEQ ID NO: 388)
5. NP_786060.1 from Lactobacillus plantarum WCFS1 (SEQ ID NO: 481)
6. ZP_03940142.1 from Lactobacillus brevis subsp. gravesensis ATCC 27305 (SEQ ID NO: 486)
7. ZP_03073172.1 from Lactobacillus reuteri 100-23 (SEQ ID NO 468)
8. YP_818922.1 from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 (SEQ ID NO: 504)
The BRENDA database is a freely available information system containing biochemical and molecular information on all classified enzymes as well as software tools for querying the database and calculating molecular properties. The database covers information on classification and nomenclature, reaction and specificity, functional parameters, occurrence, enzyme structure and stability, mutants and enzyme engineering, preparation and isolation, the application of enzymes, and ligand-related data. (BRENDA, AMENDA and FRENDA the enzyme information system: new content and tools in 2009. Nucleic Acids Res. 2009 January; 37 (Database issue):D588-92. Epub 2008 Nov. 4. Chang A, Scheer M, Grote A, Schomburg I, Schomburg D.) The eight sequences were used to build a profile HMM which is provided herein as Table 6.

To further identify the proteins of interest, the 436 sequences were searched with four profile HMMs: the generated XPK_XFP_HMM profile HMM provided in Table 6 as well as the three published profiles for the three domains XFP_N; XFP; XFP_C (PFAM DATABASE) described in Tables 7, 8, and 9, respectively. 309 protein sequences which lengths were between 650 amino acids and 900 amino acids, and contained the three domains were retained.

All 309 sequences are at least 40% identical to an experimentally verified phosphoketolase, with exception of 12 sequences that are within 35% identity distance. However, all 309 sequences have a highly significant match to all 4 profile HMMs. The least significant matches have Evalues of 7.5E-242, 1.1E-124, 2.1E-49, 7.8E-37 to XFP_XPK HMM, XFP_N, XFP, and XFP_C profile HMMs respectively. The 309 sequences are provided in Table 5, however, it is understood that any xylulose-5-phosphate/fructose-6-phosphate phosphoketolase identifiable by the method described may be expressed in host cells as described herein. Where accession information is given as “complement (NN_NNNNN.N:X . . . Y)”, it should be understood to mean the reverse complement of nucleotides X to Y of the sequence with Accession number NN_NNNNN.N. Where accession information is given as “join (NNNNNN.N:X..Y, NNNNNN.N:Z..Q)”, it should be understood to mean the sequence resulting from joining nucleotides X to Y of NNNNNN.N to nucleotides Z to Q of NNNNNN.N.

Numerous examples of polynucleotides, genes and/or polypeptides encoding phosphotransacetylase are known in the art and can be used in relation to the recombinant host cells disclosed herein. In embodiments, the phosphotransacetylase can be EutD from Lactobacillus plantarum. In embodiments, the phosphotransacetylase can be the phosphotransacetylase from Bacillus subtilis. This phosphotransacetylase has a specific activity of 1371 μmol/min/mg and a Km 0.06 mM for acetyl-CoA (Rado and Hoch, Biochim. Biophys. Acta. 321: 114-25; 1973). In addition, the equilibrium constant (Keq) of this reaction was found to be 154±14 in favor of the formation of acetyl-CoA according to the following formula:

[acetyl-CoA][Pi][CoA][acetyl-P]=Keq

In embodiments, host cells comprise a polypeptide having at least about 80%, at least about 85%, at least about 90%, or at least about 99% identity to a polypeptide of Table 10 or an active fragment thereof or a polynucleotide encoding such a polypeptide. In embodiments, the phosphotransacetylase can be a polypeptide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to SEQ ID NO: 1472 or an active fragment thereof. In other embodiments, a polynucleotide, gene and/or polypeptide encoding phosphotransacetylase can include, but is not limited to, a sequence provided in the following tables 10 or 12.

Additional suitable phosphotransacetylase target gene coding regions and proteins were identified by diversity searching and clustering. A blast search of the non redundant GenBank protein database (NR) was performed with the L. plantarum EutD protein as a query sequence. A blast cut-off (Evalue) of 0.01 was applied. This search resulted in 2124 sequence matches. Redundancy reduction was achieved by clustering proteins with the CD-HIT program with parameters set at 95% sequence identity and 90% length overlap. The longest seed sequence, representative of each cluster, is retained for further analysis. Clustering reduced the number of protein sequences to 1336. Further clean-up of the sequences by removing sequences <280 amino acids and sequences >795 amino acids resulted in 1231 seqs.

The Brenda database was queried for experimentally verified phosphate acetyltransferases. Thirteen were found in the following organisms: S. enterica, E. coli K12, V. Parvula, C. Kluyveri, C. Acetobutylicum, C. Thermocellum, M thermophila, S. pyogenes, B. subtilis, L. fermentum, L. plantarum, L. sanfranciscensis, B. subtilis, L. fermentum, L. plantarum, L. sanfranciscensis, R. palustris, E. coli.

Experimentally verified phosphate acetyltransferases (EC 2.3.1.8) belong to the PTA_PTB pfam family. However, the PTA_PTB domain is present in 13 distinct architectures (http://pfam.janelia.org/family/PF01515, Pfam database version 24). The motivation for investigating the domain architecture is to determine which of the proteins, that were identified by BLAST search, are likely to be phosphate acetyltransferases.

Experimentally verified sequences extracted from the BRENDA database as well as sequences retained after the CD-HIT clustering and clean-up, were searched against the Pfam database to determine their domain architecture. Pfam is a collection of multiple sequence alignments and profile hidden Markov models (HMMs). Each Pfam HMM represents a protein family or domain. By searching a protein sequence against the Pfam library of HMMs, it is possible to determine which domains it carries i.e. its domain architecture. (The Pfam protein families database: R. D. Finn, J. Tate, J. Mistry, P. C. Coggill, J. S. Sammut, H. R. Hotz, G. Ceric, K. Forslund, S. R. Eddy, E. L. Sonnhammer and A. Bateman Nucleic Acids Research (2008) Database Issue 36:D281-D288)

Twelve of the experimentally verified proteins only contained the PTA_PTB domain. Two sequences, from R. palustris and E. coli, contained two domains PTA_PTB and DRTGG, a domain of unknown function. Therefore, from the CD-HIT clustering results, proteins that contained either the PTA_PTB domain only (Group 1: 549 sequences) or a combination of PTA_PTB+DRTGG domains (Group 2: 201 sequences) were chosen.

Furthermore, the PTA_PTB domain, as the name indicates, is actually not specific to phosphate acetyltransferases. The domain is also a signature for phosphate butyryltransferases (EC 2.3.1.19). Two methods to distinguish between the two subfamilies: acetyltransferases and butyryltransferases were employed and are as follows:

To further characterize the relationship among the sequences, multiple sequence alignment MSA), phylogenetic analysis, profile HMMs and GroupSim analysis were performed. For this set of analyses, the phosphate acetyltransferases are split in two groups. Group 1 contains phosphate acetyltransferases with the PTA_PTB domain only, while Group 2 contains phosphate acetyltransferases with PTA_PTB+DRTGG. The motivation here is to generate groups with similar lengths.

Clustal X, version 2.0 was used for sequence alignments with default parameters. (Thompson J D, et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. (1997) 25:4876-4882.)

Alignment results were utilized to compute % sequence identities to a reference sequence. If the sequence from L. plantarum is taken as a reference, % IDs range from as low as 10.5% to 75.6% for the closest sequence. Alignment results also provided the basis for re-constructing phylogenetic trees. The Neighbor Joining method, available in the Jalview package version 2.3, was used to produce the trees, and computed trees were visualized in MEGA 4 (Tamura, Dudley, Nei, and Kumar 2007). The Neighbor Joining method is a method for re-constructing phylogenetic trees, and computing the lengths of the branches of this tree. In each stage, the two nearest nodes of the tree (the term “nearest nodes” will be defined in the following paragraphs) are chosen and defined as neighbors in our tree. This is done recursively until all of the nodes are paired together. “The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987 July; 4(4):406-25. Saitou N, Nei M.” Jalview Version 2 is a system for interactive editing, analysis and annotation of multiple sequence alignments (Waterhouse, A. M., Procter, J. B., Martin, D. M. A, Clamp, M. and Barton, G. J. (2009) “Jalview Version 2—a multiple sequence alignment editor and analysis workbench” Bioinformatics 25 (9) 1189-1191). The MEGA software provides tools for exploring, discovering, and analyzing DNA and protein sequences from an evolutionary perspective. MEGA4 enables the integration of sequence acquisition with evolutionary analysis. It contains an array of input data and multiple results explorers for visual representation; the handling and editing of sequence data, sequence alignments, inferred phylogenetic trees; and estimated evolutionary distances. The results explorers allow users to browse, edit, summarize, export, and generate publication-quality captions for their results. MEGA 4 also includes distance matrix and phylogeny explorers as well as advanced graphical modules for the visual representation of input data and output results (Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599).

Taken together, % IDs and the generated tree (FIG. 4) indicated that potential phosphate acetyltransferases (PTA_PTB domain only) are divided in two major subfamilies. Subfamily 1 from 10.5% ID to ˜20% ID (176 sequences) and Subfamily 2 from ˜20% ID to 75.6% ID (361 sequences). The third Subfamily, of 12 sequences, has % ID ranging from 17% ID to 25% ID with respect to the L. plantarum sequence.

Based on experimentally verified sequences contained within each of the Subfamilies, Subfamily 1 and Subfamily 2 were determined to represent phosphate butyryltransferases (PTB) and phosphate acetlytransferases (PTA) respectively.

Discrimination between Subfamily 1 members and Subfamily 2 members was also performed by GroupSim analysis (Capra and Singh (2008) Bioinformatics 24: 1473-1480). The GroupSim method identifies amino acid residues that determine a protein's functional specificity. In a multiple sequence alignment (MSA) of a protein family whose sequences are divided into multiple Subfamilies, amino acid residues that distinguish between the functional Subfamilies of sequences can be identified. The method takes a multiple sequence alignment (MSA) and known specificity groupings as input, and assigns a score to each amino acid position in the MSA. Higher scores indicate a greater likelihood that an amino acid position is a specificity determining position (SDP).

GroupSim analysis performed on the MSA of 537 sequences (divided into Subfamily 1 and Subfamily 2 by the phylogenetic analysis above) identified highly discriminating positions. Listed in Table 11 are positions (Pos) having scores greater than to 0.7, where a perfect score of 1.0 would indicate that all proteins within the Subfamily have the listed amino acid in the specified position and between Subfamilies the amino acid would always be different. The “Pattern” columns give the amino acid(s) in single letter code. Numbers between parentheses indicate frequency of occurrence of each amino acid at the particular position. The amino acid position number in column 1 is for the PTA protein sequence from Lactobacillus plantarum, the representative protein of group 2 with a GI#28377658 (SEQ ID NO: 1472).

An alternative structure/function characterization of the PTA and PTB subfamilies of enzymes was performed using the HMMER software package (the theory behind profile HMMs is described in R. Durbin, S. Eddy, A. Krogh, and G. Mitchison, Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998; Krogh et al., 1994; J. Mol. Biol. 235:1501-1531), following the user guide which is available from HMMER (Janelia Farm Research Campus, Ashburn, Va.).

Using a multiple sequence alignment of the experimentally verified sequences (containing the PTA_PTB domain only) in Subfamily 2, a profile Hidden Markov Model (HMM) was created for representing Subfamily 2 members. The sequences were:

1. BAB19267.1 from Lactobacillus sanfranciscensis (SEQ ID NO: 1475)
2. NP_784550.1 from Lactobacillus plantarum WCFS1 (SEQ ID NO: 1472)
3. ZP_03944466.1 from Lactobacillus fermentum ATCC 14931 (SEQ ID NO: 1453)
4. NP_391646.1 from Bacillus subtilis subsp. subtilis str. 168 (SEQ ID NO: 1422)
5. AAA72041.1 from Methanosarcina thermophila (SEQ ID NO: 1277)
6. ZP_03152606.1 from Clostridium thermocellum DSM 4150 (SEQ ID NO: 1275)
7. NP_348368.1 from Clostridium acetobutylicum ATCC 824 (SEQ ID NO: 1206)
8. YP_001394780.1 from Clostridium kluyveri DSM 555 (SEQ ID NO: 1200)
9. ZP_03855267.1 from Veillonella parvula DSM 2008 (SEQ ID NO: 1159)
10. YP_149725.1 from Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150 (SEQ ID NO: 1129)

The Profile HMM was built as follows: The 10 seed sequences (sequences representing experimentally verified function) that are in Subfamily 2 were aligned using Clustal X (interface to Clustal W) with default parameters. The hmmbuild program was run on each set of the aligned sequences using default parameters. hmmbuild reads the multiple sequence alignment file, builds a new Profile HMM, and saves the Profile HMM to file. Using this program an un-calibrated profile was generated from the multiple alignment for each set of subunit sequences described above.

The Profile HMM was read using hmmcalibrate which scores a large number of synthesized random sequences with the Profile (the default number of synthetic sequences used is 5,000), fits an extreme value distribution (EVD) to the histogram of those scores, and re-saves the HMM file now including the EVD parameters. These EVD parameters (μ and λ) are used to calculate the E-values of bit scores when the profile is searched against a protein sequence database. hmmcalibrate writes two parameters into the HMM file on a line labeled “EVD”: these parameters are the μ (location) and λ (scale) parameters of an extreme value distribution (EVD) that best fits a histogram of scores calculated on randomly generated sequences of about the same length and residue composition as SWISS-PROT. This calibration was done once for the Profile HMM.

The calibrated pofile HMM for the Subfamily 2 set is provided as Table 14. The Profile HMM table gives the probability of each amino acid occurring at each position in the amino acid sequence. The amino acids are represented by the one letter code. The first line for each position reports the match emission scores: probability for each amino acid to be in that state (highest score is highlighted). The second line reports the insert emission scores, and the third line reports on state transition scores: M→M, M→I, M→D; I→M, I→I; D→M, D→D; B→M; M→E. Table 14 shows that in the Subfamily 2 profile HMM, methionine has a 3792 ans 4481 probability of being in the first two positions.

The Subfamily 2 profile HMM was evaluated using hmmsearch, with the Z parameter set to one billion, for the ability to discriminate Subfamily 1 members from those of Subfamily 2. The hmmsearch program takes the hmm file for the Subfamily 2 profile HMM and all the sequences from both Subfamilies and assigns an E-value score to each sequence. This E-value score is a measure of fit to the Profile HMM, with a lower score being a better fit. The Profile HMM distinguished Subfamily 2 members from Subfamily 1 members and there was a large margin of E-value difference between the worst scoring Subfamily 2 member (5e-34) and the best scoring Subfamily 1 member (4.3e-07). This analysis shows that the Profile HMM prepared for Subfamily 2 phosphate acetyltransferases (PTA) distinguishes PTA sequences from phosphate butyryltransferase PTB protein sequences.

Based on these analyses, 361 phosphate acetyltransferase sequences (PTA_PTB domain only) were identified and are provided in Table 12a.