WO2014025604A2 - Microorganisms for improved production of fuels, chemicals, and amino acids - Google Patents

Abstract

The present application relates to recombinant microorganisms comprising engineered biosynthetic pathways and methods of using said recombinant microorganisms to produce various beneficial metabolites. In various aspects of the invention, the recombinant microorganisms may be engineered to reduce the expression and/or activity of at least one endogenous lactate-cytochrome-c- reductase ("LCR").

Description

MICROORGANISMS FOR IMPROVED PRODUCTION OF FUELS, CHEMICALS,

AND AMINO ACIDS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application Serial No. 61 /680,511 , filed August 7, 2012, which is herein incorporated by reference in its entirety for all purposes. TECHNICAL FIELD [0002] Recombinant microorganisms and methods of producing such microorganisms are provided. Also provided are methods of producing beneficial metabolites including fuels and chemicals by contacting a suitable substrate with the recombinant microorganisms and enzymatic preparations therefrom. DESCRIPTION OF TEXT FILE SUBMITTED ELECTRONICALLY [0003] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: GEVO_067_01 WO_SeqList_ST25.txt, date recorded: July 30, 2013, file size: 1.42 megabytes). BACKGROUND

[0004] The ability of microorganisms to convert sugars to beneficial metabolites including fuels, chemicals, and amino acids has been widely described in the literature in recent years. See, e.g., Alper et al., 2009, Nature Microbiol. Rev. 7: 715- 723 and McCourt et al., 2006, Amino Acids 31 : 173-210. Recombinant engineering techniques have enabled the creation of microorganisms that express biosynthetic pathways capable of producing a number of useful products, including the commodity chemical, isobutanol.

[0005] Isobutanol, also a promising biofuel candidate, has been produced in recombinant microorganisms expressing a heterologous, five-step metabolic pathway (See, e.g., WO/2007/050671 to Donaldson et al., WO/2008/098227 to Liao et al., and WO/2009/103533 to Festel et al.). However, the microorganisms produced to date have fallen short of commercial relevance due to their low performance characteristics, including, for example low productivities, low titers, and low yields.

[0006] The present application results from the study of lactate-cytochrome-c- reductases and shows that the suppression of one or more of endogenous lactate- cytochrome-c-reductases can improve the yields, titers, and productivities of isobutanol as well as reduce the formation of the fermentation by-products 2,3- butanediol and isobutyrate. SUMMARY OF THE INVENTION

[0007] In a first aspect, the application relates to a recombinant microorganism comprising an engineered isobutanol producing metabolic pathway, said engineered isobutanol producing metabolic pathway comprising at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, and wherein said microorganism is engineered to reduce the expression and/or activity of at least one endogenous lactate-cytochrome-c-reductase (“LCR”). In one embodiment, the LCR is a L-lactate-cytochrome-c-reductase (“L-LCR”). In another embodiment, the LCR is a D-lactate-cytochrome-c-reductase (“D-LCR”). In a specific embodiment, the L-LCR is the S. cerevisiae protein CYB2 (SEQ ID NO: 2). In another specific embodiment, the D-LCR is an S. cerevisiae protein selected from DLD1 (SEQ ID NO: 4), DLD2 (SEQ ID NO: 6), or DLD3 (SEQ ID NO: 8). In another specific embodiment, the LCR is an endogenous protein from a yeast other than S. cerevisiae, wherein said LCR is a homolog or variant of the S. cerevisiae proteins CYB2, DLD1 , DLD2, or DLD3. In a further specific embodiment, the homolog or variant of the S. cerevisiae proteins CYB2, DLD1 , DLD2, or DLD3 is an endogenous protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia or Zygosaccharomyces. A representative listing of LCR yeast homologs is found in SEQ ID NOs: 9-307.

[0008] In one embodiment, the recombinant microorganism includes a mutation in at least one gene encoding for a lactate-cytochrome-c-reductase resulting in a reduction of lactate-cytochrome-c-reductase activity of a polypeptide encoded by said gene. In another embodiment, the recombinant microorganism includes a partial deletion of gene encoding for a lactate-cytochrome-c-reductase resulting in a reduction of lactate-cytochrome-c-reductase activity of a polypeptide encoded by the gene. In another embodiment, the recombinant microorganism comprises a complete deletion of a gene encoding for a lactate-cytochrome-c-reductase resulting in a reduction of lactate-cytochrome-c-reductase activity of a polypeptide encoded by the gene. In yet another embodiment, the recombinant microorganism includes a modification of the regulatory region associated with the gene encoding for a lactate- cytochrome-c-reductase resulting in a reduction of expression of a polypeptide encoded by said gene. In yet another embodiment, the recombinant microorganism comprises a modification of the transcriptional regulator resulting in a reduction of transcription of a gene encoding for a lactate-cytochrome-c-reductase. In yet another embodiment, the recombinant microorganism comprises mutations in all genes encoding for a lactate-cytochrome-c-reductase resulting in a reduction of activity of a polypeptide encoded by the gene(s). In one embodiment, the lactate- cytochrome-c-reductase activity or expression is reduced by at least about 50%. In another embodiment, the lactate-cytochrome-c-reductase activity or expression is reduced by at least about 60%, by at least about 65%, by at least about 70%, by at least about 75%, by at least about 80%, by at least about 85%, by at least about 90%, by at least about 95%, or by at least about 99% as compared to a recombinant microorganism not comprising a reduction of the lactate-cytochrome-c-reductase activity or expression.

[0009] In one embodiment, the isobutanol producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanol. In another embodiment, the isobutanol producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least three exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least four exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least five exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, all of the isobutanol producing metabolic pathway steps in the conversion of pyruvate to isobutanol are converted by exogenously encoded enzymes.

[0010] In one embodiment, one or more of the isobutanol pathway genes encodes an enzyme that is localized to the cytosol. In one embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol. In another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least two isobutanol pathway enzymes localized in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least three isobutanol pathway enzymes localized in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least four isobutanol pathway enzymes localized in the cytosol. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with five isobutanol pathway enzymes localized in the cytosol. In yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with all isobutanol pathway enzymes localized in the cytosol.

[0011] In various embodiments described herein, the isobutanol pathway genes may encode enzyme(s) selected from the group consisting of acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), 2- keto-acid decarboxylase, e.g., keto-isovalerate decarboxylase (KIVD), and alcohol dehydrogenase (ADH). In one embodiment, the KARI is an NADH-dependent KARI (NKR). In another embodiment, the ADH is an NADH-dependent ADH. In yet another embodiment, the KARI is an NADH-dependent KARI (NKR) and the ADH is an NADH-dependent ADH.

[0012] In various embodiments described herein, the recombinant microorganisms of the invention that comprise an isobutanol producing metabolic pathway may be further engineered to reduce or eliminate the expression or activity of one or more enzymes selected from a pyruvate decarboxylase (PDC), a glycerol- 3-phosphate dehydrogenase (GPD), a 3-keto acid reductase (3-KAR), or an aldehyde dehydrogenase (ALDH).

[0013] In one embodiment, the recombinant microorganisms of the application may be recombinant prokaryotic microorganisms. In another embodiment, the recombinant microorganisms may be recombinant eukaryotic microorganisms. In a further embodiment, the recombinant eukaryotic microorganisms may be recombinant yeast microorganisms.

[0014] In some embodiments, the recombinant yeast microorganisms may be members of the Saccharomyces clade, Saccharomyces sensu stricto microorganisms, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre- WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

[0015] In some embodiments, the recombinant microorganisms may be yeast recombinant microorganisms of the Saccharomyces clade.

[0016] In some embodiments, the recombinant microorganisms may be Saccharomyces sensu stricto microorganisms. In one embodiment, the Saccharomyces sensu stricto is selected from the group consisting of S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis and hybrids thereof.

[0017] In some embodiments, the recombinant microorganisms may be Crabtree- negative recombinant yeast microorganisms. In one embodiment, the Crabtree- negative yeast microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Issatchenkia, Hansenula, or Candida. In additional embodiments, the Crabtree-negative yeast microorganism is selected from Saccharomyces kluyveri, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Hansenula anomala, Candida utilis and Kluyveromyces waltii.

[0018] In some embodiments, the recombinant microorganisms may be Crabtree- positive recombinant yeast microorganisms. In one embodiment, the Crabtree- positive yeast microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Candida, Pichia and Schizosaccharomyces. In additional embodiments, the Crabtree-positive yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, Kluyveromyces thermotolerans, Candida glabrata, Z. bailli, Z. rouxii, Debaryomyces hansenii, Pichia pastorius, Schizosaccharomyces pombe, and Saccharomyces uvarum.

[0019] In some embodiments, the recombinant microorganisms may be post- WGD (whole genome duplication) yeast recombinant microorganisms. In one embodiment, the post-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces or Candida. In additional embodiments, the post-WGD yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, and Candida glabrata.

[0020] In some embodiments, the recombinant microorganisms may be pre-WGD (whole genome duplication) yeast recombinant microorganisms. In one embodiment, the pre-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula, Pachysolen, Yarrowia and Schizosaccharomyces. In additional embodiments, the pre-WGD yeast is selected from the group consisting of Saccharomyces kluyveri, Kluyveromyces thermotolerans, Kluyveromyces marxianus, Kluyveromyces waltii, Kluyveromyces lactis, Candida tropicalis, Pichia pastoris, Pichia anomala, Pichia stipitis, Issatchenkia orientalis, Issatchenkia occidentalis, Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophilis, Yarrowia lipolytica, and Schizosaccharomyces pombe.

[0021] In some embodiments, the recombinant microorganisms may be microorganisms that are non-fermenting yeast microorganisms, including, but not limited to those, classified into a genera selected from the group consisting of Tricosporon, Rhodotorula, Myxozyma, or Candida. In a specific embodiment, the non-fermenting yeast is C. xestobii.

[0022] In another aspect, the present invention provides methods of producing isobutanol using a recombinant microorganism as described herein. In one embodiment, the method includes cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source until the isobutanol is produced and optionally, recovering the isobutanol. In one embodiment, the microorganism produces isobutanol from a carbon source at a yield of at least about 5 percent theoretical. In another embodiment, the microorganism produces isobutanol at a yield of at least about 10 percent, at least about 15 percent, about least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 60 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97.5 percent theoretical.

[0023] In one embodiment, the recombinant microorganism converts the carbon source to isobutanol under aerobic conditions. In another embodiment, the recombinant microorganism converts the carbon source to isobutanol under microaerobic conditions. In yet another embodiment, the recombinant microorganism converts the carbon source to isobutanol under anaerobic conditions. BRIEF DESCRIPTION OF DRAWINGS [0024] Figure 1 illustrates an isobutanol pathway.

[0025] Figure 2 illustrates an NADH-dependent isobutanol pathway.

[0026] Figures 3A and 3B illustrate exemplary lactate-cytochrome-c-reductase reactions catalyzed by D-LCR and L-LCR enzymes, respectively.

[0027] Figure 4 illustrates exemplary reactions capable of being catalyzed by lactate-cytochrome-c-reductases.

[0028] Figures 5A and 5B illustrate cell density profiles for control strains and strains with one or more LCR genes disrupted.

[0029] Figures 6A and 6B illustrate the effective isobutanol production profiles of control strains and strains with one or more LCR genes disrupted.

[0030] Figures 7A and 7B illustrate the 2,3-butanediol production profiles of control strains and strains with one or more LCR genes disrupted.

[0031] Figure 8 illustrates the isobutyrate production profile of a control strain as compared to strains with an LCR gene disruption.

[0032] Figure 9 illustrates the 2,3-butanediol production profile of a control strain as compared to strains with an LCR gene disruption. DETAILED DESCRIPTION [0033] As used herein and in the appended claims, the singular forms "a,” "an,” and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the microorganism" includes reference to one or more microorganisms, and so forth.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

[0035] Any publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

[0036] The term "microorganism" includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms "microbial cells" and "microbes" are used interchangeably with the term microorganism.

[0037] The term "prokaryotes" is art recognized and refers to cells which contain no nucleus or other cell organelles. The prokaryotes are generally classified in one of two domains, the Bacteria and the Archaea. The definitive difference between organisms of the Archaea and Bacteria domains is based on fundamental differences in the nucleotide base sequence in the 16S ribosomal RNA.

[0038] The term "Archaea" refers to a categorization of organisms of the division Mendosicutes, typically found in unusual environments and distinguished from the rest of the prokaryotes by several criteria, including the number of ribosomal proteins and the lack of muramic acid in cell walls. On the basis of ssrRNA analysis, the Archaea consist of two phylogenetically-distinct groups: Crenarchaeota and Euryarchaeota. On the basis of their physiology, the Archaea can be organized into three types: methanogens (prokaryotes that produce methane); extreme halophiles (prokaryotes that live at very high concentrations of salt (NaCl); and extreme (hyper) thermophiles (prokaryotes that live at very high temperatures). Besides the unifying archaeal features that distinguish them from Bacteria (i.e., no murein in cell wall, ester-linked membrane lipids, etc.), these prokaryotes exhibit unique structural or biochemical attributes which adapt them to their particular habitats. The Crenarchaeota consist mainly of hyperthermophilic sulfur-dependent prokaryotes and the Euryarchaeota contain the methanogens and extreme halophiles.

[0039] "Bacteria", or "eubacteria", refers to a domain of prokaryotic organisms. Bacteria include at least eleven distinct groups as follows: (1 ) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1 ) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple photosynthetic +non-photosynthetic Gram-negative bacteria (includes most "common" Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non- sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant micrococci and relatives; (11 ) Thermotoga and Thermosipho thermophiles.

[0040] "Gram-negative bacteria" include cocci, nonenteric rods, and enteric rods. The genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0041] "Gram positive bacteria" include cocci, nonsporulating rods, and sporulating rods. The genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0042] The term“genus” is defined as a taxonomic group of related species according to the Taxonomic Outline of Bacteria and Archaea (Garrity, G.M., Lilburn, T.G., Cole, J.R., Harrison, S.H., Euzeby, J., and Tindall, B.J. (2007) The Taxonomic Outline of Bacteria and Archaea. TOBA Release 7.7, March 2007. Michigan State University Board of Trustees. [http://www.taxonomicoutline.org/]).

[0043] The term“species” is defined as a collection of closely related organisms with greater than 97% 16S ribosomal RNA sequence homology and greater than 70% genomic hybridization and sufficiently different from all other organisms so as to be recognized as a distinct unit.

[0044] The terms "recombinant microorganism,"“modified microorganism,” and "recombinant host cell" are used interchangeably herein and refer to microorganisms that have been genetically modified to express or to overexpress endogenous polynucleotides, to express heterologous polynucleotides, such as those included in a vector, in an integration construct, or which have an alteration in expression of an endogenous gene. By "alteration" it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the alteration. For example, the term "alter" can mean "inhibit," but the use of the word "alter" is not limited to this definition. It is understood that the terms "recombinant microorganism" and "recombinant host cell" refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0045] The term "expression" with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein results from transcription and translation of the open reading frame sequence. The level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the cell, or the amount of the desired product encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantitated by qRT-PCR or by Northern hybridization (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). Protein encoded by a selected sequence can be quantitated by various methods, e.g., by ELISA, by assaying for the biological activity of the protein, or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay, using antibodies that recognize and bind the protein. See Sambrook et al., 1989, supra. [0046] The term“overexpression” refers to an elevated level (e.g., aberrant level) of mRNAs encoding for a protein(s), and/or to elevated levels of protein(s) in cells as compared to similar corresponding unmodified cells expressing basal levels of mRNAs or having basal levels of proteins. In particular embodiments mRNA(s) or protein(s) may be overexpressed by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8- fold, 10-fold, 12-fold, 15-fold or more in microorganisms engineered to exhibit increased gene mRNA, protein, and/or activity.

[0047] As used herein and as would be understood by one of ordinary skill in the art,“reduced activity and/or expression” of a protein such as an enzyme can mean either a reduced specific catalytic activity of the protein (e.g. reduced activity) and/or decreased concentrations of the protein in the cell (e.g. reduced expression).

[0048] The term "wild-type microorganism" describes a cell that occurs in nature, i.e. a cell that has not been genetically modified. A wild-type microorganism can be genetically modified to express or overexpress a first target enzyme. This microorganism can act as a parental microorganism in the generation of a microorganism modified to express or overexpress a second target enzyme. In turn, the microorganism modified to express or overexpress a first and a second target enzyme can be modified to express or overexpress a third target enzyme.

[0049] Accordingly, a“parental microorganism” functions as a reference cell for successive genetic modification events. Each modification event can be accomplished by introducing a nucleic acid molecule in to the reference cell. The introduction facilitates the expression or overexpression of a target enzyme. It is understood that the term "facilitates" encompasses the activation of endogenous polynucleotides encoding a target enzyme through genetic modification of e.g., a promoter sequence in a parental microorganism. It is further understood that the term "facilitates" encompasses the introduction of heterologous polynucleotides encoding a target enzyme in to a parental microorganism

[0050] The term "engineer" refers to any manipulation of a microorganism that results in a detectable change in the microorganism, wherein the manipulation includes but is not limited to inserting a polynucleotide and/or polypeptide heterologous to the microorganism and mutating a polynucleotide and/or polypeptide native to the microorganism.

[0051] The term "mutation" as used herein indicates any modification of a nucleic acid and/or polypeptide which results in an altered nucleic acid or polypeptide. Mutations include, for example, point mutations, deletions, or insertions of single or multiple residues in a polynucleotide, which includes alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A genetic alteration may be a mutation of any type. For instance, the mutation may constitute a point mutation, a frame-shift mutation, a nonsense mutation, an insertion, or a deletion of part or all of a gene. In addition, in some embodiments of the modified microorganism, a portion of the microorganism genome has been replaced with a heterologous polynucleotide. In some embodiments, the mutations are naturally-occurring. In other embodiments, the mutations are identified and/or enriched through artificial selection pressure. In still other embodiments, the mutations in the microorganism genome are the result of genetic engineering.

[0052] The term "biosynthetic pathway", also referred to as "metabolic pathway", refers to a set of anabolic or catabolic biochemical reactions for converting one chemical species into another. Gene products belong to the same "metabolic pathway" if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and metabolite end product.

[0053] As used herein, the term“isobutanol producing metabolic pathway” refers to an enzyme pathway which produces isobutanol from pyruvate.

[0054] The term“NADH-dependent” as used herein with reference to an enzyme, e.g., KARI and/or ADH, refers to an enzyme that catalyzes the reduction of a substrate coupled to the oxidation of NADH with a catalytic efficiency that is greater than the reduction of the same substrate coupled to the oxidation of NADPH at equal substrate and cofactor concentrations.

[0055] The term“exogenous” as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are not normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.

[0056] On the other hand, the term“endogenous” or“native” as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature. [0057] The term“heterologous” as used herein in the context of a modified host cell refers to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., wherein at least one of the following is true: (a) the molecule(s) is/are foreign (“exogenous”) to (i.e., not naturally found in) the host cell; (b) the molecule(s) is/are naturally found in (e.g., is“endogenous to”) a given host microorganism or host cell but is either produced in an unnatural location or in an unnatural amount in the cell; and/or (c) the molecule(s) differ(s) in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid sequence(s) such that the molecule differing in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid as found endogenously is produced in an unnatural (e.g., greater than naturally found) amount in the cell.

[0058] The term “feedstock” is defined as a raw material or mixture of raw materials supplied to a microorganism or fermentation process from which other products can be made. For example, a carbon source, such as biomass or the carbon compounds derived from biomass are a feedstock for a microorganism that produces a biofuel in a fermentation process. However, a feedstock may contain nutrients other than a carbon source.

[0059] The term "substrate" or "suitable substrate" refers to any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme. The term includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate, or derivatives thereof. Further, the term "substrate" encompasses not only compounds that provide a carbon source suitable for use as a starting material, such as any biomass derived sugar, but also intermediate and end product metabolites used in a pathway associated with a recombinant microorganism as described herein.

[0060] The term“fermentation” or“fermentation process” is defined as a process in which a microorganism is cultivated in a culture medium containing raw materials, such as feedstock and nutrients, wherein the microorganism converts raw materials, such as a feedstock, into products.

[0061] The term“volumetric productivity” or“production rate” is defined as the amount of product formed per volume of medium per unit of time. Volumetric productivity is reported in gram per liter per hour (g/L/h).

[0062] The term“specific productivity” or“specific production rate” is defined as the amount of product formed per volume of medium per unit of time per amount of cells. Specific productivity is reported in gram (or milligram) per liter per gram cell dry weight per hour (g/g h).

[0063] The term“yield” is defined as the amount of product obtained per unit weight of raw material and may be expressed as g product per g substrate (g/g). Yield may be expressed as a percentage of the theoretical yield. “Theoretical yield” is defined as the maximum amount of product that can be generated per a given amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to isobutanol is 0.41 g/g. As such, a yield of isobutanol from glucose of 0.39 g/g would be expressed as 95% of theoretical or 95% theoretical yield.

[0064] The term“titer” is defined as the strength of a solution or the concentration of a substance in solution. For example, the titer of a biofuel in a fermentation broth is described as g of biofuel in solution per liter of fermentation broth (g/L).

[0065] “Aerobic conditions” are defined as conditions under which the oxygen concentration in the fermentation medium is sufficiently high for an aerobic or facultative anaerobic microorganism to use as a terminal electron acceptor.

[0066] In contrast,“anaerobic conditions” are defined as conditions under which the oxygen concentration in the fermentation medium is too low for the microorganism to use as a terminal electron acceptor. Anaerobic conditions may be achieved by sparging a fermentation medium with an inert gas such as nitrogen until oxygen is no longer available to the microorganism as a terminal electron acceptor. Alternatively, anaerobic conditions may be achieved by the microorganism consuming the available oxygen of the fermentation until oxygen is unavailable to the microorganism as a terminal electron acceptor. Methods for the production of isobutanol under anaerobic conditions are described in commonly owned and co- pending publication, US 2010/0143997, the disclosures of which are herein incorporated by reference in its entirety for all purposes.

[0067] “Aerobic metabolism” refers to a biochemical process in which oxygen is used as a terminal electron acceptor to make energy, typically in the form of ATP, from carbohydrates. Aerobic metabolism occurs e.g. via glycolysis and the TCA cycle, wherein a single glucose molecule is metabolized completely into carbon dioxide in the presence of oxygen. [0068] In contrast, “anaerobic metabolism” refers to a biochemical process in which oxygen is not the final acceptor of electrons contained in NADH. Anaerobic metabolism can be divided into anaerobic respiration, in which compounds other than oxygen serve as the terminal electron acceptor, and substrate level phosphorylation, in which the electrons from NADH are utilized to generate a reduced product via a“fermentative pathway.”

[0069] In“fermentative pathways”, NAD(P)H donates its electrons to a molecule produced by the same metabolic pathway that produced the electrons carried in NAD(P)H. For example, in one of the fermentative pathways of certain yeast strains, NAD(P)H generated through glycolysis transfers its electrons to pyruvate, yielding ethanol. Fermentative pathways are usually active under anaerobic conditions but may also occur under aerobic conditions, under conditions where NADH is not fully oxidized via the respiratory chain. For example, above certain glucose concentrations, Crabtree positive yeasts produce large amounts of ethanol under aerobic conditions.

[0070] The term“byproduct” or“by-product” means an undesired product related to the production of an amino acid, amino acid precursor, chemical, chemical precursor, biofuel, or biofuel precursor.

[0071] The term“substantially free” when used in reference to the presence or absence of a protein activity (LCR enzymatic activity, 3-KAR enzymatic activity, ALDH enzymatic activity, PDC enzymatic activity, GPD enzymatic activity, etc.) means the level of the protein is substantially less than that of the same protein in the wild-type host, wherein less than about 50% of the wild-type level is preferred and less than about 30% is more preferred. The activity may be less than about 20%, less than about 10%, less than about 5%, or less than about 1 % of wild-type activity. Microorganisms which are“substantially free” of a particular protein activity (LCR enzymatic activity, 3-KAR enzymatic activity, ALDH enzymatic activity, PDC enzymatic activity, GPD enzymatic activity, etc.) may be created through recombinant means or identified in nature.

[0072] The term “non-fermenting yeast” is a yeast species that fails to demonstrate an anaerobic metabolism in which the electrons from NADH are utilized to generate a reduced product via a fermentative pathway such as the production of ethanol and CO₂ from glucose. Non-fermentative yeast can be identified by the “Durham Tube Test” (J.A. Barnett, R.W. Payne, and D. Yarrow. 2000. Yeasts Characteristics and Identification. 3^rd edition. p. 28-29. Cambridge University Press, Cambridge, UK) or by monitoring the production of fermentation productions such as ethanol and CO_2.

[0073] The term“polynucleotide” is used herein interchangeably with the term “nucleic acid” and refers to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof, including but not limited to single stranded or double stranded, sense or antisense deoxyribonucleic acid (DNA) of any length and, where appropriate, single stranded or double stranded, sense or antisense ribonucleic acid (RNA) of any length, including siRNA. The term “nucleotide” refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or a pyrimidine base and to a phosphate group, and that are the basic structural units of nucleic acids. The term“nucleoside” refers to a compound (as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids. The term “nucleotide analog” or “nucleoside analog” refers, respectively, to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length, DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called nucleotidic oligomer or oligonucleotide.

[0074] It is understood that the polynucleotides described herein include "genes" and that the nucleic acid molecules described herein include "vectors" or "plasmids." Accordingly, the term "gene", also called a "structural gene" refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all or part of one or more proteins or enzymes, and may include regulatory (non- transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. The transcribed region of the gene may include untranslated regions, including introns, 5'-untranslated region (UTR), and 3'-UTR, as well as the coding sequence.

[0075] The term "operon" refers to two or more genes which are transcribed as a single transcriptional unit from a common promoter. In some embodiments, the genes comprising the operon are contiguous genes. It is understood that transcription of an entire operon can be modified (i.e., increased, decreased, or eliminated) by modifying the common promoter. Alternatively, any gene or combination of genes in an operon can be modified to alter the function or activity of the encoded polypeptide. The modification can result in an increase in the activity of the encoded polypeptide. Further, the modification can impart new activities on the encoded polypeptide. Exemplary new activities include the use of alternative substrates and/or the ability to function in alternative environmental conditions.

[0076] A "vector" is any means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components. Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes), and the like, that are "episomes," that is, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine -conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that are not episomal in nature, or it can be an organism which comprises one or more of the above polynucleotide constructs such as an agrobacterium or a bacterium.

[0077] "Transformation" refers to the process by which a vector is introduced into a host cell. Transformation (or transduction, or transfection), can be achieved by any one of a number of means including chemical transformation (e.g. lithium acetate transformation), electroporation, microinjection, biolistics (or particle bombardment- mediated delivery), or agrobacterium mediated transformation.

[0078] The term "enzyme" as used herein refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide, but can include enzymes composed of a different molecule including polynucleotides.

[0079] The term“protein,”“peptide,” or“polypeptide” as used herein indicates an organic polymer composed of two or more amino acidic monomers and/or analogs thereof. As used herein, the term“amino acid” or“amino acidic monomer” refers to any natural and/or synthetic amino acids including glycine and both D or L optical isomers. The term“amino acid analog” refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, or with a different functional group. Accordingly, the term polypeptide includes amino acidic polymer of any length including full length proteins, and peptides as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called a protein oligomer or oligopeptide

[0080] The term "homolog," used with respect to an original polynucleotide or polypeptide of a first family or species, refers to distinct polynucleotides or polypeptides of a second family or species which are determined by functional, structural or genomic analyses to be a polynucleotide or polypeptide of the second family or species which corresponds to the original polynucleotide or polypeptide of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of a polynucleotide or polypeptide can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes.

[0081] A polypeptide has "homology" or is "homologous" to a second polypeptide if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a polypeptide has homology to a second polypeptide if the two polypeptides have "similar" amino acid sequences. (Thus, the terms "homologous polypeptides" or“homologous proteins” are defined to mean that the two polypeptides have similar amino acid sequences).

[0082] The term“analog” or“analogous” refers to polynucleotide or polypeptide sequences that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure. Isobutanol Producing Recombinant Microorganisms

[0083] A variety of microorganisms convert sugars to produce pyruvate, which is then utilized in a number of pathways of cellular metabolism. In recent years, microorganisms, including yeast, have been engineered to produce a number of desirable products via pyruvate-driven biosynthetic pathways, including isobutanol, an important commodity chemical and biofuel candidate (See, e.g., commonly owned and co-pending patent publications, US 2009/0226991 , US 2010/0143997, US 2011 /0020889, US 2011 /0076733, and WO 2010/075504). [0084] As described herein, the present invention relates to recombinant microorganisms for producing isobutanol, wherein said recombinant microorganisms comprise an isobutanol producing metabolic pathway. In one embodiment, the isobutanol producing metabolic pathway to convert pyruvate to isobutanol can be comprised of the following reactions:

1. pyruvate ĺ acetolactate + CO₂

2. acetolactate + NAD(P)H ĺ 2,3-dihydroxyisovalerate + NAD(P)⁺ 3. 2,3-dihydroxyisovalerate ĺ alpha-ketoisovalerate

4. alpha-ketoisovalerate ĺ isobutyraldehyde + CO₂

5. isobutyraldehyde + NAD(P)H ĺ isobutanol + NAD(P)⁺

[0085] In one embodiment, these reactions are carried out by the enzymes 1 ) Acetolactate synthase (ALS), 2) Ketol-acid reductoisomerase (KARI), 3) Dihydroxy- acid dehydratase (DHAD), 4) 2-keto-acid decarboxylase, e.g., Keto-isovalerate decarboxylase (KIVD), and 5) an Alcohol dehydrogenase (ADH) (Figure 1). In some embodiments, the recombinant microorganism may be engineered to overexpress one or more of these enzymes. In an exemplary embodiment, the recombinant microorganism is engineered to overexpress all of these enzymes.

[0086] Alternative pathways for the production of isobutanol in yeast have been described in WO/2007/050671 and in Dickinson et al., 1998, J Biol Chem 273:25751 -6. These and other isobutanol producing metabolic pathways are within the scope of the present application. In one embodiment, the isobutanol producing metabolic pathway comprises five substrate to product reactions. In another embodiment, the isobutanol producing metabolic pathway comprises six substrate to product reactions. In yet another embodiment, the isobutanol producing metabolic pathway comprises seven substrate to product reactions.

[0087] In various embodiments described herein, the recombinant microorganism comprises an isobutanol producing metabolic pathway. In one embodiment, the isobutanol producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanol. In another embodiment, the isobutanol producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least three exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least four exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least five exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, all of the isobutanol producing metabolic pathway steps in the conversion of pyruvate to isobutanol are converted by exogenously encoded enzymes.

[0088] In one embodiment, one or more of the isobutanol pathway genes encodes an enzyme that is localized to the cytosol. In one embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol. In another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least two isobutanol pathway enzymes localized in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least three isobutanol pathway enzymes localized in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least four isobutanol pathway enzymes localized in the cytosol. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with five isobutanol pathway enzymes localized in the cytosol. In yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with all isobutanol pathway enzymes localized in the cytosol. Isobutanol producing metabolic pathways in which one or more genes are localized to the cytosol are described in commonly owned U.S. Patent No. 8,232,089, which is herein incorporated by reference in its entirety for all purposes.

[0089] As is understood in the art, a variety of organisms can serve as sources for the isobutanol pathway enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including Y. spp. stipitis, Torulaspora pretoriensis, Issatchenkia orientalis, Schizosaccharomyces spp., including S. pombe, Cryptococcus spp., Aspergillus spp., Neurospora spp., or Ustilago spp. Sources of genes from anaerobic fungi include, but not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp. Sources of prokaryotic enzymes that are useful include, but not limited to, Escherichia spp., Zymomonas spp., Staphylococcus spp., Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., Streptococcus spp., Salmonella spp., Slackia spp., Cryptobacterium spp., and Eggerthella spp.

[0090] In some embodiments, one or more of these enzymes can be encoded by native genes. Alternatively, one or more of these enzymes can be encoded by heterologous genes.

[0091] For example, acetolactate synthases capable of converting pyruvate to acetolactate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including B. subtilis (GenBank Accession No. Q04789.3), L. lactis (GenBank Accession No. NP_267340.1 ), S. mutans (GenBank Accession No. NP_721805.1 ), K. pneumoniae (GenBank Accession No. ZP_06014957.1 ), C. glutamicum (GenBank Accession No. P42463.1 ), E. cloacae (GenBank Accession No. YP_003613611.1 ), M. maripaludis (GenBank Accession No. ABX01060.1 ), M. grisea (GenBank Accession No. AAB81248.1 ), T. stipitatus (GenBank Accession No. XP_002485976.1 ), or S. cerevisiae ILV2 (GenBank Accession No. NP_013826.1 ). Additional acetolactate synthases capable of converting pyruvate to acetolactate are described in commonly owned and co-pending US Publication No. 2011 /0076733, which is herein incorporated by reference in its entirety. A review article characterizing the biosynthesis of acetolactate from pyruvate via the activity of acetolactate synthases is provided by Chipman et al., 1998, Biochimica et Biophysica Acta 1385: 401 -19, which is herein incorporated by reference in its entirety. Chipman et al. provide an alignment and consensus for the sequences of a representative number of acetolactate synthases. Motifs shared in common between the majority of acetolactate synthases include:

SGPG(A/C/V)(T/S)N (SEQ ID NO: 308),

GX(P/A)GX(V/A/T) (SEQ ID NO: 309),

GX(Q/G)(T/A)(L/M)G(Y/F/W)(A/G)X(P/G)(W/A)AX(G/T)(A/V) (SEQ ID NO: 310), and

GD(G/A)(G/S/C)F (SEQ ID NO: 311)

motifs at amino acid positions corresponding to the 163-169, 240-245, 521 -535, and 549-553 residues, respectively, of the S. cerevisiae ILV2. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit acetolactate synthase activity.

[0092] Ketol-acid reductoisomerases capable of converting acetolactate to 2,3- dihydroxyisovalerate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including E. coli (GenBank Accession No. EGB30597.1 ), L. lactis (GenBank Accession No. YP_003353710.1 ), S. exigua (GenBank Accession No. ZP_06160130.1 ), C. curtam (GenBank Accession No. YP_003151266.1 ), Shewanella sp. (GenBank Accession No. YP_732498.1 ), V. fischeri (GenBank Accession No. YP_205911.1 ), M. maripaludis (GenBank Accession No. YP_001097443.1 ), B. subtilis (GenBank Accession No. CAB14789), S. pombe (GenBank Accession No. NP_001018845), B. thetaiotamicron (GenBank Accession No. NP_810987), or S. cerevisiae ILV5 (GenBank Accession No. NP_013459.1 ). Additional ketol-acid reductoisomerases capable of converting acetolactate to 2,3- dihydroxyisovalerate are described in commonly owned and co-pending US Publication No. 2011 /0076733, which is herein incorporated by reference in its entirety. An alignment and consensus for the sequences of a representative number of ketol-acid reductoisomerases is provided in commonly owned and co-pending US Publication No. 2010/0143997, which is herein incorporated by reference in its entirety. Motifs shared in common between the majority of ketol-acid reductoisomerases include:

G(Y/C/W)GXQ(G/A) (SEQ ID NO: 312),

(F/Y/L)(S/A)HG(F/L) (SEQ ID NO: 313),

V(V/I/F)(M/L/A)(A/C)PK (SEQ ID NO: 314),

D(L/I)XGE(Q/R)XXLXG (SEQ ID NO: 315), and

S(D/N/T)TA(E/Q/R)XG (SEQ ID NO: 316)

motifs at amino acid positions corresponding to the 89-94, 175-179, 194-200, 262- 272, and 459-465 residues, respectively, of the E. coli ketol-acid reductoisomerase encoded by ilvC. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit ketol-acid reductoisomerase activity.

[0093] To date, all known, naturally existing ketol-acid reductoisomerases are known to use NADPH as a cofactor. In certain embodiments, a ketol-acid reductoisomerase which has been engineered to used NADH as a cofactor may be utilized to mediate the conversion of acetolactate to 2,3-dihydroxyisovalerate. Engineered NADH-dependent KARI enzymes (“NKRs”) and methods of generating such NKRs are disclosed in commonly owned and co-pending US Publication No. 2010/0143997.

[0094] In accordance with the invention, any number of mutations can be made to a KARI enzyme, and in a preferred aspect, multiple mutations can be made to a KARI enzyme to result in an increased ability to utilize NADH for the conversion of acetolactate to 2,3-dihydroxyisovalerate. Such mutations include point mutations, frame shift mutations, deletions, and insertions, with one or more (e.g., one, two, three, four, five or more, etc.) point mutations preferred.

[0095] Mutations may be introduced into naturally existing KARI enzymes to create NKRs using any methodology known to those skilled in the art. Mutations may be introduced randomly by, for example, conducting a PCR reaction in the presence of manganese as a divalent metal ion cofactor. Alternatively, oligonucleotide directed mutagenesis may be used to create the NKRs which allows for all possible classes of base pair changes at any determined site along the encoding DNA molecule. In general, this technique involves annealing an oligonucleotide complementary (except for one or more mismatches) to a single stranded nucleotide sequence coding for the KARI enzyme of interest. The mismatched oligonucleotide is then extended by DNA polymerase, generating a double-stranded DNA molecule which contains the desired change in sequence in one strand. The changes in sequence can, for example, result in the deletion, substitution, or insertion of an amino acid. The double-stranded polynucleotide can then be inserted into an appropriate expression vector, and a mutant or modified polypeptide can thus be produced. The above-described oligonucleotide directed mutagenesis can, for example, be carried out via PCR.

[0096] Dihydroxy acid dehydratases capable of converting 2,3- dihydroxyisovalerate to Į -ketoisovalerate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including E. coli (GenBank Accession No. YP_026248.1 ), L. lactis (GenBank Accession No. NP_267379.1 ), S. mutans (GenBank Accession No. NP_722414.1 ), M. stadtmanae (GenBank Accession No. YP_448586.1 ), M. tractuosa (GenBank Accession No. YP_004053736.1 ), Eubacterium SCB49 (GenBank Accession No. ZP_01890126.1 ), G. forsetti (GenBank Accession No. YP_862145.1 ), Y. lipolytica (GenBank Accession No. XP_502180.2), N. crassa (GenBank Accession No. XP_963045.1 ), or S. cerevisiae ILV3 (GenBank Accession No. NP_012550.1 ). Additional dihydroxy acid dehydratases capable of 2,3-dihydroxyisovalerate to Į -ketoisovalerate are described in commonly owned and co-pending US Publication No. 2011 /0076733. Motifs shared in common between the majority of dihydroxy acid dehydratases include:

SLXSRXXIA (SEQ ID NO: 317),

CDKXXPG (SEQ ID NO: 318),

GXCXGXXTAN (SEQ ID NO: 319),

GGSTN (SEQ ID NO: 320),

GPXGXPGMRXE (SEQ ID NO: 321 ),

ALXTDGRXSG (SEQ ID NO: 322), and

GHXXPEA (SEQ ID NO: 323)

motifs at amino acid positions corresponding to the 93-101 , 122-128, 193-202, 276- 280, 482-491 , 509-518, and 526-532 residues, respectively, of the E. coli dihydroxy acid dehydratase encoded by ilvD. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit dihydroxy acid dehydratase activity.

[0097] 2-keto-acid decarboxylases capable of converting Į -ketoisovalerate to isobutyraldehyde may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including L. lactis kivD (GenBank Accession No. YP_003353820.1 ), E. cloacae (GenBank Accession No. P23234.1 ), M. smegmatis (GenBank Accession No. A0R480.1 ), M. tuberculosis (GenBank Accession No. O53865.1 ), M. avium (GenBank Accession No. Q742Q2.1 , A. brasilense (GenBank Accession No. P51852.1 ), L. lactis kdcA (GenBank Accession No. AAS49166.1 ), S. epidermidis (GenBank Accession No. NP_765765.1 ), M. caseolyticus (GenBank Accession No. YP_002560734.1 ), B. megaterium (GenBank Accession No. YP_003561644.1 ), S. cerevisiae ARO10 (GenBank Accession No. NP_010668.1 ), or S. cerevisiae THI3 (GenBank Accession No. CAA98646.1 ). Additional 2-keto-acid decarboxylases capable of converting Į -ketoisovalerate to isobutyraldehyde are described in commonly owned and co-pending US Publication No. 2011 /0076733. Motifs shared in common between the majority of 2-keto-acid decarboxylases include:

FG(V/I)(P/S)G(D/E)(Y/F) (SEQ ID NO: 324),

(T/V)T(F/Y)G(V/A)G(E/A)(L/F)(S/N) (SEQ ID NO: 325),

N(G/A)(L/I/V)AG(S/A)(Y/F)AE (SEQ ID NO: 326),

(V/I)(L/I/V)XI(V/T/S)G (SEQ ID NO: 327), and

GDG(S/A)(L/F/A)Q(L/M)T (SEQ ID NO: 328) motifs at amino acid positions corresponding to the 21 -27, 70-78, 81 -89, 93-98, and 428-435 residues, respectively, of the L. lactis 2-keto-acid decarboxylase encoded by kivD. An additional“HH”-motif found at amino acids 112-113 in the L. lactis 2- keto-acid decarboxylase encoded by kivD is characteristic of thiamin diphosphate- dependent decarboxylases, a class of enzymes of which 2-keto acid decarboxylases belong. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit 2-keto-acid decarboxylase activity.

[0098] Alcohol dehydrogenases capable of converting isobutyraldehyde to isobutanol may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including L. lactis (GenBank Accession No. YP_003354381 ), B. cereus (GenBank Accession No. YP_001374103.1 ), N. meningitidis (GenBank Accession No. CBA03965.1 ), S. sanguinis (GenBank Accession No. YP_001035842.1 ), L. brevis (GenBank Accession No. YP_794451.1 ), B. thuringiensis (GenBank Accession No. ZP_04101989.1 ), P. acidilactici (GenBank Accession No. ZP_06197454.1 ), B. subtilis (GenBank Accession No. EHA31115.1 ), N. crassa (GenBank Accession No. CAB91241.1 ) or S. cerevisiae ADH6 (GenBank Accession No. NP_014051.1 ). Additional alcohol dehydrogenases capable of converting isobutyraldehyde to isobutanol are described in commonly owned and co-pending US Publication Nos. 2011 /0076733 and 2011 /0201072. Motifs shared in common between the majority of alcohol dehydrogenases include:

C(H/G)(T/S)D(L/I)H (SEQ ID NO: 329),

GHEXXGXV (SEQ ID NO: 330),

(L/V)(Q/K/E)(V/I/K)G(D/Q)(R/H)(V/A) (SEQ ID NO: 331 ),

CXXCXXC (SEQ ID NO: 332),

(C/A)(A/G/D)(G/A)XT(T/V) (SEQ ID NO: 333), and

G(L/A/C)G(G/P)(L/I/V)G (SEQ ID NO: 334) motifs at amino acid positions corresponding to the 39-44, 59-66, 76-82, 91 -97, 147- 152, and 171 -176 residues, respectively, of the L. lactis alcohol dehydrogenase encoded by adhA. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit alcohol dehydrogenase activity.

[0099] In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutanol. In one embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutyraldehyde. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to keto-isovalerate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to 2,3-dihydroxyisovalerate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to acetolactate.

[00100] Furthermore, any of the genes encoding the foregoing enzymes (or any others mentioned herein (or any of the regulatory elements that control or modulate expression thereof)) may be optimized by genetic/protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in yeast.

[00101] In an exemplary embodiment, pathway steps 2 and 5 of the isobutanol pathway may be carried out by KARI and ADH enzymes that utilize NADH (rather than NADPH) as a cofactor. The present inventors have found that utilization of NADH-dependent KARI (NKR) and ADH enzymes to catalyze pathway steps 2 and 5, respectively, surprisingly enables production of isobutanol at theoretical yield and/or under anaerobic conditions. An example of an NADH-dependent isobutanol pathway is illustrated in Figure 2. Thus, in one embodiment, the recombinant microorganisms of the present invention may use an NKR to catalyze the conversion of acetolactate to produce 2,3-dihydroxyisovalerate. In another embodiment, the recombinant microorganisms of the present invention may use an NADH-dependent ADH to catalyze the conversion of isobutyraldehyde to produce isobutanol. In yet another embodiment, the recombinant microorganisms of the present invention may use both an NKR to catalyze the conversion of acetolactate to produce 2,3- dihydroxyisovalerate, and an NADH-dependent ADH to catalyze the conversion of isobutyraldehyde to produce isobutanol.

[00102] As described herein, the present inventors have found that the suppression of one or more of endogenous lactate-cytochrome-c-reductases can improve the yields, titers, and productivities of isobutanol as well as reduce the formation of the fermentation by-products 2,3-butanediol and isobutyrate. Accordingly, in one aspect, the application relates to a recombinant microorganism comprising an engineered isobutanol producing metabolic pathway, said engineered isobutanol producing metabolic pathway comprising at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, and wherein said microorganism is engineered to reduce the expression and/or activity of at least one endogenous lactate-cytochrome-c-reductase (“LCR”).

[00103] L-lactate-cytochrome-c-reductases and D-lactate-cytochrome-c- reductases generate pyruvate from L-lactate and D-lactate, respectively. In S. cerevisiae, LCRs are encoded by CYB2, DLD1, DLD2, and DLD3. Except for Dld3p which is cytosolic, the other three enzymes are associated with the mitochondria. Specifically, Dld1 p, Dld2p, and Cyb2p are localized in the inner membrane, matrix, and intermembrane space of the mitochondrial compartment, respectively. These enzymes exhibit stereospecificity for their substrate lactate. Cyb2p is known to mainly oxidize L-lactate, whereas Dld1 p, Dld2p, and Dld3p oxidize D-lactate. An illustration of exemplary reactions capable of being catalyzed by LCRs is shown in Figures 3A and 3B, corresponding to reaction catalyzed by D-LCR and L-LCR enzymes, respectively. The oxidation reaction results in the transfer of 2e- to cytochrome C, which in turn donates these to the mitochondrial electron transport chain.

[00104] Without being bound by theory, it is believed that because of the structural similarity between lactate (i.e., D-lactate) and 2,3-dihydroxyisovalerate“DHIV” (i.e., R-DHIV), LCRs oxidizes DHIV to 3-hydroxy-3-methyl-2-oxobutanoate (i.e., HKIV), resulting in a loss of 2e- to the mitochondrial electron transport chain. It is then theorized that the conversion of HKIV back to DHIV by an NADH-dependent ketol- acid reductoisomerase in the isobutanol pathway and the associated consumption of additional NADH creates a futile cycle leading to the loss of NADH, which is essential for isobutanol production. The present inventors have found that the suppression of one or more of endogenous lactate-cytochrome-c-reductases can improve the yields, titers, and productivities of isobutanol. Without being bound by theory, it is hypothesized that this result is observed because of LCR involvement in the unwanted oxidation of DHIV– an isobutanol pathway intermediate– to HKIV.

[00105] As illustrated herein, suitable lactate-cytochrome-c-reductases may generally be found in the enzyme classification subgroup 1.1.2.3 and/or 1.1.2.4. In one embodiment, the endogenous LCR targeted for reduced expression and/or activity is a L-lactate-cytochrome-c-reductase (“L-LCR”). In another embodiment, the endogenous LCR targeted for reduced expression and/or activity is a D-lactate- cytochrome-c-reductase (“D-LCR”). In a specific embodiment, the L-LCR is the S. cerevisiae protein CYB2 (SEQ ID NO: 2). In another specific embodiment, the D- LCR is an S. cerevisiae protein selected from DLD1 (SEQ ID NO: 4), DLD2 (SEQ ID NO: 6), or DLD3 (SEQ ID NO: 8). In another specific embodiment, the LCR is an endogenous protein from a yeast other than S. cerevisiae, wherein said LCR is a homolog or variant of the S. cerevisiae proteins CYB2, DLD1 , DLD2, or DLD3. In a further specific embodiment, the homolog or variant of the S. cerevisiae proteins CYB2, DLD1 , DLD2, or DLD3 is an endogenous protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia or Zygosaccharomyces. A representative listing of LCR yeast homologs is found in SEQ ID NOs: 9-307.

[00106] In one embodiment, the recombinant microorganism comprising an engineered isobutanol producing metabolic pathway includes a mutation in at least one gene encoding for a lactate-cytochrome-c-reductase resulting in a reduction of lactate-cytochrome-c-reductase activity of a polypeptide encoded by said gene. In another embodiment, the recombinant microorganism comprising an isobutanol producing metabolic pathway includes a partial deletion of a gene encoding for a lactate-cytochrome-c-reductase gene resulting in a reduction of lactate-cytochrome- c-reductase activity of a polypeptide encoded by the gene. In another embodiment, the recombinant microorganism comprising an isobutanol producing metabolic pathway comprises a complete deletion of a gene encoding for a lactate- cytochrome-c-reductase resulting in a reduction of lactate-cytochrome-c-reductase activity of a polypeptide encoded by the gene. In yet another embodiment, the recombinant microorganism comprising an isobutanol producing metabolic pathway includes a modification of the regulatory region associated with the gene encoding for a lactate-cytochrome-c-reductase resulting in a reduction of expression of a polypeptide encoded by said gene. In yet another embodiment, the recombinant microorganism comprising an isobutanol producing metabolic pathway comprises a modification of the transcriptional regulator resulting in a reduction of transcription of gene encoding for a lactate-cytochrome-c-reductase. In yet another embodiment, the recombinant microorganism comprising an isobutanol producing metabolic pathway comprises mutations in all genes encoding for a lactate-cytochrome-c- reductase resulting in a reduction of activity of a polypeptide encoded by the gene(s). In one embodiment, said lactate-cytochrome-c-reductase gene is a gene selected from the group consisting of the S. cerevisiae CYB2, DLD1, DLD2, and DLD3 genes or homologs thereof. As would be understood in the art, naturally occurring homologs of CYB2, DLD1, DLD2, and DLD3 in yeast other than S. cerevisiae can similarly be inactivated using the methods of the present invention. CYB2, DLD1, DLD2, and DLD3 homologs and methods of identifying such CYB2, DLD1, DLD2, and DLD3 homologs are described herein. A representative listing of LCR yeast homologs from yeast genera including Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia, and Zygosaccharomyces may be found in SEQ ID NOs: 9-307.

[00107] As is understood by those skilled in the art, there are several additional mechanisms available for reducing or disrupting the activity of a protein such as a lactate-cytochrome-c-reductase, including, but not limited to, the use of a regulated promoter, use of a weak constitutive promoter, disruption of one of the two copies of the gene in a diploid yeast, disruption of both copies of the gene in a diploid yeast, expression of an anti-sense nucleic acid, expression of an siRNA, over expression of a negative regulator of the endogenous promoter, alteration of the activity of an endogenous or heterologous gene, use of a heterologous gene with lower specific activity, the like or combinations thereof.

[00108] In various embodiments described herein, the yield of isobutanol is increased in the recombinant microorganisms comprising a reduction of the activity and/or expression of one or more endogenous lactate-cytochrome-c-reductases. In one embodiment, the yield of isobutanol is increased by at least about 1 % as compared to a parental microorganism that does not comprise a reduction of the activity and/or expression of one or more endogenous lactate-cytochrome-c- reductases. In another embodiment, the yield of isobutanol is increased by at least about 5%, by at least about 10%, by at least about 25%, or by at least about 50% as compared to a corresponding parental microorganism that does not comprise a reduction of the activity and/or expression of one or more endogenous lactate- cytochrome-c-reductases. The Microorganism in General

[00109] As described herein, the recombinant microorganisms of the present invention can express a plurality of heterologous and/or native enzymes involved in pathways for the production of a beneficial metabolite such as isobutanol.

[00110] As described herein, "engineered" or "modified" microorganisms are produced via the introduction of genetic material into a host or parental microorganism of choice and/or by modification of the expression of native genes, thereby modifying or altering the cellular physiology and biochemistry of the microorganism. Through the introduction of genetic material and/or the modification of the expression of native genes the parental microorganism acquires new properties, e.g., the ability to produce a new, or greater quantities of, an intracellular and/or extracellular metabolite. As described herein, the introduction of genetic material into and/or the modification of the expression of native genes in a parental microorganism results in a new or modified ability to produce beneficial metabolites such as isobutanol from a suitable carbon source. The genetic material introduced into and/or the genes modified for expression in the parental microorganism contains gene(s), or parts of genes, coding for one or more of the enzymes involved in a biosynthetic pathway for the production of a beneficial metabolite such as isobutanol and may also include additional elements for the expression and/or regulation of expression of these genes, e.g., promoter sequences.

[00111] In addition to the introduction of a genetic material into a host or parental microorganism, an engineered or modified microorganism can also include the alteration, disruption, deletion or knocking-out of a gene or polynucleotide to alter the cellular physiology and biochemistry of the microorganism. Through the alteration, disruption, deletion or knocking-out of a gene or polynucleotide, the microorganism acquires new or improved properties (e.g., the ability to produce a new metabolite or greater quantities of an intracellular metabolite, to improve the flux of a metabolite down a desired pathway, and/or to reduce the production of by-products).

[00112] Recombinant microorganisms provided herein may also produce metabolites in quantities not available in the parental microorganism. A "metabolite" refers to any substance produced by metabolism or a substance necessary for or taking part in a particular metabolic process. A metabolite can be an organic compound that is a starting material (e.g., glucose or pyruvate), an intermediate (e.g., 2-ketoisovalerate), or an end product (e.g., isobutanol) of metabolism. Metabolites can be used to construct more complex molecules, or they can be broken down into simpler ones. Intermediate metabolites may be synthesized from other metabolites, perhaps used to make more complex substances, or broken down into simpler compounds, often with the release of chemical energy.

[00113] The disclosure identifies specific genes useful in the methods, compositions and organisms of the disclosure; however it will be recognized that absolute identity to such genes is not necessary. For example, changes in a particular gene or polynucleotide comprising a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically such changes comprise conservative mutations and silent mutations. Such modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art.

[00114] Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or functionally equivalent polypeptides can also be used to clone and express the polynucleotides encoding such enzymes.

[00115] As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low- usage codons. Codons can be substituted to reflect the preferred codon usage of the host, in a process sometimes called "codon optimization" or "controlling for species codon bias."

[00116] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (Murray et al., 1989, Nucl Acids Res. 17: 477-508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon (Dalphin et al., 1996, Nucl Acids Res. 24: 216-8).

[00117] Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA compounds differing in their nucleotide sequences can be used to encode a given enzyme of the disclosure. The native DNA sequence encoding the biosynthetic enzymes described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide. Furthermore, the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.

[00118] In addition, homologs of enzymes useful for generating metabolites are encompassed by the microorganisms and methods provided herein. As used herein, two proteins (or a region of the proteins) are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[00119] When "homologous" is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (See, e.g., Pearson W.R., 1994, Methods in Mol Biol 25: 365-89).

[00120] The following six groups each contain amino acids that are conservative substitutions for one another: 1 ) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[00121] It is understood that a range of microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of beneficial metabolites such as isobutanol. In various embodiments, microorganisms may be selected from yeast microorganisms. Yeast microorganisms for the production of a metabolite such as isobutanol are described in commonly-owned U.S. Patent Nos. 8,017,375, 8,017,376, 8,133,715, 8,153,415, 8,158,404, and 8,232,089. In alternative embodiments, the recombinant microorganisms may be derived from bacterial microorganisms. In various embodiments the recombinant microorganism may be selected from a genus of Citrobacter, Corynebacterium, Lactobacillus, Lactococcus, Salmonella, Enterobacter, Enterococcus, Erwinia, Pantoea, Morganella, Pectobacterium, Proteus, Serratia, Shigella, and Klebsiella. In one specific embodiment, the recombinant microorganism is a lactic acid bacteria such as, for example, a microorganism derived from the Lactobacillus or Lactococcus genus.

[00122] In one embodiment, the yeast microorganism has reduced or no pyruvate decarboxylase (PDC) activity. PDC catalyzes the decarboxylation of pyruvate to acetaldehyde, which is then reduced to ethanol by ADH via an oxidation of NADH to NAD+. Ethanol production is the main pathway to oxidize the NADH from glycolysis. Deletion, disruption, or mutation of this pathway increases the pyruvate and the reducing equivalents (NADH) available for a biosynthetic pathway which uses pyruvate as the starting material and/or as an intermediate. Accordingly, deletion, disruption, or mutation of one or more genes encoding for pyruvate decarboxylase and/or a positive transcriptional regulator thereof can further increase the yield of the desired pyruvate-derived metabolite (e.g., isobutanol). In one embodiment, said pyruvate decarboxylase gene targeted for disruption, deletion, or mutation is selected from the group consisting of PDC1, PDC5, and PDC6, or homologs or variants thereof. In another embodiment, all three of PDC1, PDC5, and PDC6 are targeted for disruption, deletion, or mutation. In yet another embodiment, a positive transcriptional regulator of the PDC1, PDC5, and/or PDC6 is targeted for disruption, deletion or mutation. In one embodiment, said positive transcriptional regulator is PDC2, or homologs or variants thereof.

[00123] As is understood by those skilled in the art, there are several additional mechanisms available for reducing or disrupting the activity of a protein encoded by PDC1, PDC5, PDC6, and/or PDC2, including, but not limited to, the use of a regulated promoter, use of a weak constitutive promoter, disruption of one of the two copies of the gene in a diploid yeast, disruption of both copies of the gene in a diploid yeast, expression of an anti-sense nucleic acid, expression of an siRNA, over expression of a negative regulator of the endogenous promoter, alteration of the activity of an endogenous or heterologous gene, use of a heterologous gene with lower specific activity, the like or combinations thereof. Yeast strains with reduced PDC activity are described in commonly owned U.S. Pat. No. 8.017,375, as well as commonly owned and co-pending US Patent Publication No. 2011 /0183392. [00124] In another embodiment, the microorganism has reduced glycerol-3- phosphate dehydrogenase (GPD) activity. GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD+. Glycerol is then produced from G3P by Glycerol-3- phosphatase (GPP). Glycerol production is a secondary pathway to oxidize excess NADH from glycolysis. Reduction or elimination of this pathway would increase the pyruvate and reducing equivalents (NADH) available for the production of a pyruvate-derived metabolite (e.g., isobutanol). Thus, disruption, deletion, or mutation of the genes encoding for glycerol-3-phosphate dehydrogenases can further increase the yield of the desired metabolite (e.g., isobutanol). Yeast strains with reduced GPD activity are described in commonly owned and co-pending US Patent Publication Nos. 2011 /0020889 and 2011 /0183392.

[00125] In yet another embodiment, the microorganism has reduced 3-keto acid reductase (3-KAR) activity. 3-KARs catalyze the conversion of 3-keto acids (e.g., acetolactate) to 3-hydroxyacids (e.g., DH2MB). Yeast strains with reduced 3-KAR activity are described in commonly owned U.S. Pat. Nos. 8,133,715, 8,153,415, and 8,158,404, which are herein incorporated by reference in their entireties.

[00126] In yet another embodiment, the microorganism has reduced aldehyde dehydrogenase (ALDH) activity. Aldehyde dehydrogenases catalyze the conversion of aldehydes (e.g., isobutyraldehyde) to acid by-products (e.g., isobutyrate). Yeast strains with reduced ALDH activity are described in commonly owned U.S. Pat. Nos. 8,133,715, 8,153,415, and 8,158,404, which are herein incorporated by reference in their entireties. Methods in General

[00127] Any method can be used to identify genes that encode for enzymes that are homologous to the genes described herein (e.g., lactate-cytochrome-c-reductase homologs). Generally, genes that are homologous or similar to the lactate- cytochrome-c-reductases described herein may be identified by functional, structural, and/or genetic analysis. In most cases, homologous or similar genes and/or homologous or similar enzymes will have functional, structural, or genetic similarities.

[00128] Techniques known to those skilled in the art may be suitable to identify additional homologous genes and homologous enzymes. Generally, analogous genes and/or analogous enzymes can be identified by functional analysis and will have functional similarities. Techniques known to those skilled in the art may be suitable to identify analogous genes and analogous enzymes. For example, to identify homologous or analogous genes, proteins, or enzymes, techniques may include, but not limited to, cloning a gene by PCR using primers based on a published sequence of a gene/enzyme or by degenerate PCR using degenerate primers designed to amplify a conserved region among ketol-acid reductoisomerase genes. Further, one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity. Techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity (e.g. as described herein or in Kiritani, K. Branched-Chain Amino Acids Methods Enzymology, 1970), then isolating the enzyme with said activity through purification, determining the protein sequence of the enzyme through techniques such as Edman degradation, design of PCR primers to the likely nucleic acid sequence, amplification of said DNA sequence through PCR, and cloning of said nucleic acid sequence. To identify homologous or similar genes and/or homologous or similar enzymes, analogous genes and/or analogous enzymes or proteins, techniques also include comparison of data concerning a candidate gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC. The candidate gene or enzyme may be identified within the above mentioned databases in accordance with the teachings herein.

[00129] Methods for gene insertion, gene deletion, and gene overexpression may be found in commonly-owned U.S. Patent Nos. 8,017,375, 8,017,376, 8,071 ,358, 8,097,440, 8,133,175, 8,153,415, 8,158,404, and 8,232,089, each of which is herein incorporated by reference in its entirety for all purposes. Methods of Using Recombinant Microorganisms for High-Yield Fermentations

[00130] In one aspect, the present application provides methods of producing a desired metabolite, e.g., isobutanol, using a recombinant microorganism described herein. In one embodiment, the recombinant microorganism comprises an engineered isobutanol producing metabolic pathway, said engineered isobutanol producing metabolic pathway comprising at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol. In an exemplary embodiment, the recombinant microorganism is further engineered to reduce the expression and/or activity of at least one endogenous lactate-cytochrome-c- reductase (“LCR”). In one embodiment, the endogenous LCR targeted for reduced expression and/or activity is a L-lactate-cytochrome-c-reductase (“L-LCR”). In another embodiment, the endogenous LCR targeted for reduced expression and/or activity is a D-lactate-cytochrome-c-reductase (“D-LCR”). In a specific embodiment, the L-LCR is the S. cerevisiae protein CYB2 (SEQ ID NO: 2). In another specific embodiment, the D-LCR is an S. cerevisiae protein selected from DLD1 (SEQ ID NO: 4), DLD2 (SEQ ID NO: 6), or DLD3 (SEQ ID NO: 8). In another specific embodiment, the LCR is an endogenous protein from a yeast other than S. cerevisiae, wherein said LCR is a homolog or variant of the S. cerevisiae proteins CYB2, DLD1 , DLD2, or DLD3. In a further specific embodiment, the homolog or variant of the S. cerevisiae proteins CYB2, DLD1 , DLD2, or DLD3 is an endogenous protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia or Zygosaccharomyces. A representative listing of LCR yeast homologs is found in SEQ ID NOs: 9-307.

[00131] In a method to produce a beneficial metabolite (e.g., isobutanol) from a carbon source, the recombinant microorganism is cultured in an appropriate culture medium containing a carbon source. In certain embodiments, the method further includes isolating the beneficial metabolite (e.g., isobutanol) from the culture medium. For example, a beneficial metabolite (e.g., isobutanol) may be isolated from the culture medium by any method known to those skilled in the art, such as distillation, pervaporation, or liquid-liquid extraction.

[00132] In one embodiment, the recombinant microorganism may produce the beneficial metabolite (e.g., isobutanol) from a carbon source at a yield of at least 5 percent theoretical. In another embodiment, the microorganism may produce the beneficial metabolite (e.g., isobutanol) from a carbon source at a yield of at least about 10 percent, at least about 15 percent, about least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 60 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97.5 percent theoretical. In a specific embodiment, the beneficial metabolite is isobutanol Distillers Dried Grains Comprising Spent Yeast Biocatalysts

[00133] In an economic fermentation process, as many of the products of the fermentation as possible, including the co-products that contain biocatalyst cell material, should have value. Insoluble material produced during fermentations using grain feedstocks, like corn, is frequently sold as protein and vitamin rich animal feed called distillers dried grains (DDG). See, e.g., commonly owned and co-pending U.S. Publication No. 2009/0215137, which is herein incorporated by reference in its entirety for all purposes. As used herein, the term“DDG” generally refers to the solids remaining after a fermentation, usually consisting of unconsumed feedstock solids, remaining nutrients, protein, fiber, and oil, as well as spent yeast biocatalysts or cell debris therefrom that are recovered by further processing from the fermentation, usually by a solids separation step such as centrifugation.

[00134] Distillers dried grains may also include soluble residual material from the fermentation, or syrup, and are then referred to as "distillers dried grains and solubles" (DDGS). Use of DDG or DDGS as animal feed is an economical use of the spent biocatalyst following an industrial scale fermentation process.

[00135] Accordingly, in one aspect, the present invention provides an animal feed product comprised of DDG derived from a fermentation process for the production of a beneficial metabolite (e.g., isobutanol), wherein said DDG comprise a spent yeast biocatalyst of the present invention. In an exemplary embodiment, said spent yeast biocatalyst has been engineered to comprise an isobutanol producing metabolic pathway comprising at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol. In a further exemplary embodiment, said spent yeast biocatalyst has been engineered reduce the expression and/or activity of at least one endogenous lactate-cytochrome-c-reductase (“LCR”). In one embodiment, the endogenous LCR targeted for reduced expression and/or activity is a L-lactate- cytochrome-c-reductase (“L-LCR”). In another embodiment, the endogenous LCR targeted for reduced expression and/or activity is a D-lactate-cytochrome-c- reductase (“D-LCR”). In a specific embodiment, the L-LCR is the S. cerevisiae protein CYB2 (SEQ ID NO: 2). In another specific embodiment, the D-LCR is an S. cerevisiae protein selected from DLD1 (SEQ ID NO: 4), DLD2 (SEQ ID NO: 6), or DLD3 (SEQ ID NO: 8). In another specific embodiment, the LCR is an endogenous protein from a yeast other than S. cerevisiae, wherein said LCR is a homolog or variant of the S. cerevisiae proteins CYB2, DLD1 , DLD2, or DLD3. In a further specific embodiment, the homolog or variant of the S. cerevisiae proteins CYB2, DLD1 , DLD2, or DLD3 is an endogenous protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia or Zygosaccharomyces. A representative listing of LCR yeast homologs is found in SEQ ID NOs: 9-307.

[00136] In certain additional embodiments, the DDG comprising a spent yeast biocatalyst of the present invention comprise at least one additional product selected from the group consisting of unconsumed feedstock solids, nutrients, proteins, fibers, and oils.

[00137] In another aspect, the present invention provides a method for producing DDG derived from a fermentation process using a yeast biocatalyst (e.g., a recombinant yeast microorganism of the present invention), said method comprising: (a) cultivating said yeast biocatalyst in a fermentation medium comprising at least one carbon source; (b) harvesting insoluble material derived from the fermentation process, said insoluble material comprising said yeast biocatalyst; and (c) drying said insoluble material comprising said yeast biocatalyst to produce the DDG.

[00138] In certain additional embodiments, the method further comprises step (d) of adding soluble residual material from the fermentation process to said DDG to produce DDGS. In some embodiments, said DDGS comprise at least one additional product selected from the group consisting of unconsumed feedstock solids, nutrients, proteins, fibers, and oils. [00139] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures and the Sequence Listings, are incorporated herein by reference for all purposes Example 1 : Effect of LCR Deletions in Yeast Under Low Aeration Growth Conditions

[00140] The purpose of this example is to show how disruption of the DLD1 and/or DLD3 genes improves cell yield and isobutanol titers as compared to a corresponding yeast microorganism which does not comprise a disruption of DLD1 and/or DLD3. Example 1 illustrates growth under relatively low aeration conditions (4 mmol/g-h) and isobutanol productivity under lowered aeration conditions (0.1 mmol/g-h).

[00141] In this example, transformants of GEVO8011 were created which harbored a DLD1 disruption (GEVO8471 ), a DLD1 and DLD3 disruption (GEVO8931 ), and a DLD1 and DLD2 disruption (GEVO8933). The parent strain, GEVO8011 , is an S. cerevisiae yeast strain comprising disruptions of ALD6, TMA29, PDC1, PDC5, PDC6, GPD1, and GPD2, and harboring an acetolactate synthase from B. subtilis, an engineered NADH-dependent KARI derived from E. coli (P2D1 -A1 ), a dihydroxyacid dehydratase from L. lactis, a 2-keto acid decarboxylase from L. lactis (KivD), and an engineered NADH-dependent ADH derived from L. lactis (AdhA^RE1). GEVO7529 was also sampled as a URA+ control.

[00142] To prepare inoculum, cultures were started 19.5 h prior to the start of the fermentation. 160 mL cultures in 1 L baffled flask were started for GEVO8931 and GEVO8933 (Batch 1 ), and later, 80 mL cultures in a 500 mL baffled flask each were started for GEVO8011 , GEVO8471 , and GEVO8931 (Batch 2). Table 1 shows cell density for these inoculums measured after incubation for 19.5 h at 30ºC. Table 1. Cell Density Achieved After 19.5 h of Incubation for Strains in Batch Fermentations.

[00143] Each batch experiment was conducted in four 2 L top drive motor DasGip vessels with a working volume of 0.9 L per vessel. Growth was conducted at 33ºC at pH 6.0 using a starting cell density of 0.5 at an OD600. During the growth phase, the oxygen transfer rate was 4.0 mM/h, agitation was fixed at 380-400 rpm, and airflow was 10 sL/h. During the production phase, the specific OTR was 0.10 mmol/g-h, agitation was fixed at 150-200 rpm, and the airflow was 10 sL/h.

[00144] The fermentation duration was 48 h. The aerobic growth phase lasted 24 h prior to switch to microaerobic production phase. Vessels were sampled every 4 h. At the switch to production, 3 x 10 mL samples were taken from each fermenter and cell dry weight measurements were conducted in triplicate. Samples were analyzed using gas chromatography (GC) and high performance liquid chromatography (HPLC).

[00145] Figures 5A and 5B illustrate the cell density profiles of Batch 1 and Batch 2, respectively. As these figures illustrate, disruption of the DLD1 and DLD3 genes improved cell yield over the control by approximately 20%. Further, strain GEVO8931 (DLD1¨ /DLD3¨ ) grew better than GEVO8933 (DLD1¨ /DLD2¨ ).

[00146] Figures 6A and 6B illustrate the effective isobutanol production profiles of the tested strains for Batches 1 and 2, respectively. In Batch 1 , GEVO8931 (DLD1¨ /DLD3¨ ) was compared directly to GEVO8933 (DLD1¨ /DLD2¨ ). The comparison showed that GEVO8931 achieved a higher effective isobutanol titer than GEVO8933. GEVO8931 achieved 7.8 g/L while GEVO8933 achieved 5.7 g/L isobutanol in a 48 h run (24 h production phase). The batch 2 run supports that the presence of DLD1 and DLD3 deletions in GEVO8931 results in the improved production of isobutanol (8.6 g/L) over both controls, GEVO7529 and GEVO8011 (6.8 g/L). GEVO8471 , containing just the DLD1 disruption, showed slight improvements in isobutanol titer over the control strains (7.5 g/L vs. 6.8 g/L).

[00147] Figures 7A and 7B illustrate the 2,3-butanediol production profile for each strain tested during the growth and production phases. Figure 7A shows that GEVO8931 produced 0.8 g/L of 2,3-butanediol, primarily during the production phase of the experiment. In contrast, GEVO8933 produced less than 0.1 g/L of 2,3- butanediol, which is surprising since DLD2 activity was not previously known to be associated with the production of 2,3-butanediol. Figure 7B shows that GEVO7529 (URA+ Control) exhibited similar 2,3-butanediol production as GEVO8471 and GEVO8931 , while GEVO8011 (URA- Control) produced less 2,3-butanediol than the other strains. Example 2: Effect of LCR Deletions in Yeast Under High Aeration Growth Conditions

[00148] The purpose of this example is to show the effect of DLD1, DLD2, DLD3, and CYB2 disruptions on cell yields and isobutanol titers. Example 2 illustrates growth under relatively high aeration conditions (25 mmol/g-h) and isobutanol productivity under lowered aeration conditions (0.1 mmol/g-h).

[00149] In this example, transformants of GEVO8011 were created which harbored a CYB2 disruption (GEVO8467, GEVO8468, and GEVO8469), a DLD1 disruption (GEVO8471 and GEVO8472), a DLD2 disruption (GEVO8474 and GEVO8475), or a DLD3 disruption (GEVO8477, GEVO8479, and GEVO8480). The control strain, GEVO8011 , was as described in Example 1.

[00150] To prepare inoculum, cultures were started 24 h prior to the start of the fermentation. 160 mL cultures in 1 L baffled flask were started for all strains. Table 2 shows cell density for these inoculums measured after incubation for 24 h at 33ºC.

Table 2. Cell Density Achieved After 24 h of Incubation for Strains in Batch Fermentations.

[00151] Batch experiments were conducted in 2 L top drive motor DasGip vessels with a working volume of 0.9 L per vessel. Growth was conducted at 33ºC at pH 6.0 using a starting cell density of 0.5 at an OD600. During the growth phase, the oxygen transfer rate was 25.0 mM/h, agitation was fixed at 700 rpm, and airflow was 10 sL/h. During the production phase, the specific OTR was 0.10 mmol/g-h, agitation was fixed at 185-200 rpm, and the airflow was 10 sL/h.

[00152] The fermentation duration was 48 h. The aerobic growth phase lasted 24 h prior to switch to microaerobic production phase. Vessels were sampled every 4 h. At the switch to production, 3 x 10 mL samples were taken from each fermenter and cell dry weight measurements were conducted in triplicate. Samples were analyzed using gas chromatography (GC) and high performance liquid chromatography (HPLC).

[00153] Table 3 illustrates the cell density, cell dry weight, and isobutanol profiles for the tested strains. In addition to the isobutanol measurements, production of the by-products isobutyrate and 2,3-butanediol was also evaluated. Figure 8 depicts the graph of isobutyrate production. The GEVO8011 control produced 1.1 g/L isobutyrate over the course of the fermentation. CYB2 disruption strains produced slightly less isobutyrate than the control while DLD3 disruption strains produced more. Figure 9 depicts the graph of 2,3-butanediol production. One DLD3 disruption strain, GEVO8479, produced more 2,3-butanediol than the control (1.1 g/L vs. 0.9 g/L). CYB2 and DLD1 disruption strains exhibited a reduction in 2,3- butanediol production compared to the control, while DLD2 disruption isolates produced similar amounts as the control.

cket No. GEVO-067/01 WO 31 0142-2394 P Deletion Isolates.

GEVO GEVO GEVO GEVO

8475 8477 8479 8480 2 dld3¨ 16.7 17.7 19.0 19.7 10.0 10.6 11.4 11.8 21.5 19.6 19.2 19.3 0.82 0.72 0.69 0.72 0.078 0.065 0.058 0.059

[00154] The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood there from as modifications will be obvious to those skilled in the art.

[00155] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

[00156] The disclosures, including the claims, figures and/or drawings, of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entireties.

Claims

WHAT IS CLAIMED IS: 1. A recombinant yeast microorganism comprising at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, wherein said recombinant microorganism is engineered to reduce the expression and/or activity of at least one endogenous lactate-cytochrome-c-reductase.

2. The recombinant yeast microorganism of claim 1 , wherein said lactate- cytochrome-c-reductase is a L-lactate-cytochrome-c-reductase.

3. The recombinant yeast microorganism of claim 1 , wherein said lactate- cytochrome-c-reductase is a D-lactate-cytochrome-c-reductase.

4. The recombinant yeast microorganism of claim 1 , wherein said lactate- cytochrome-c-reductase is selected from the S. cerevisiae proteins CYB2, DLD1 , DLD2, and DLD3.

5. The recombinant yeast microorganism of claim 1 , wherein the lactate- cytochrome-c-reductase is an endogenous lactate-cytochrome-c-reductase derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia, and Zygosaccharomyces.

6. The recombinant yeast microorganism of claim 1 , wherein said lactate- cytochrome-c-reductase is selected from SEQ ID NOs: 9-307.

7. The recombinant yeast microorganism of claim 1 , wherein said enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol is selected from the group consisting of acetolactate synthase, ketol-acid reductoisomerase, dihydroxyacid dehydratase, 2-keto acid decarboxylase, and alcohol dehydrogenase.

8. The recombinant yeast microorganism of claim 7, wherein said ketol-acid reductoisomerase is an NADH-dependent ketol-acid reductoisomerase.

9. The recombinant yeast microorganism of claim 7, wherein said alcohol dehydrogenase is an NADH-dependent alcohol dehydrogenase.

10. The recombinant yeast microorganism of claim 1 , wherein said recombinant yeast microorganism produces less 2,3-butanediol as compared to the corresponding yeast microorganism which has not engineered to reduce the expression and/or activity of at least one endogenous lactate-cytochrome-c- reductase.

11. The recombinant yeast microorganism of claim 1 , wherein said recombinant yeast microorganism produces less isobutyrate as compared to the corresponding yeast microorganism which has not engineered to reduce the expression and/or activity of at least one endogenous lactate-cytochrome-c-reductase.

12. The recombinant yeast microorganism of any of the preceding claims, wherein said recombinant microorganism is engineered to reduce pyruvate decarboxylase activity.

13. The recombinant yeast microorganism of any of the preceding claims, wherein said recombinant microorganism is engineered to reduce glycerol-3- phosphate dehydrogenase activity.

14. The recombinant yeast microorganism of any of the preceding claims, wherein said recombinant microorganism is engineered to reduce 3-keto acid reductase activity.

15. The recombinant yeast microorganism of any of the preceding claims, wherein said recombinant microorganism is engineered to reduce aldehyde dehydrogenase activity.

16. A method of producing isobutanol, comprising:

(a) providing a recombinant microorganism according to any of the preceding claims; and

(b) cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source, until the isobutanol is produced.