BALANCED FOUR-STEP PATHWAYS TO RENEWABLE BUTANOLS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 61/344,589, filed August 27, 2010, which is hereby 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.

BACKGROUND

[0003] Biofuels have a long history ranging back to the beginning of the 20th century. As early as 1900, Rudolf Diesel demonstrated at the World Exhibition in Paris, France, an engine running on peanut oil. Soon thereafter, Henry Ford demonstrated his Model T running on ethanol derived from corn. Petroleum-derived fuels displaced biofuels in the 1930s and 1940s due to increased supply, and efficiency at a lower cost.

[0004] Market fluctuations in the 1970s coupled to the decrease in US oil production led to an increase in crude oil prices and a renewed interest in biofuels. Today, many interest groups, including policy makers, industry planners, aware citizens, and the financial community, are interested in substituting petroleum- derived fuels with biomass-derived biofuels. The leading motivations for developing biofuels are of economical, political, and environmental nature.

[0005] One is the threat of 'peak oil', the point at which the consumption rate of crude oil exceeds the supply rate, thus leading to significantly increased fuel cost results in an increased demand for alternative fuels. In addition, instability in the Middle East and other oil-rich regions has increased the demand for domestically produced biofuels. Also, environmental concerns relating to the possibility of carbon dioxide related climate change is an important social and ethical driving force which is starting to result in government regulations and policies such as caps on carbon dioxide emissions from automobiles, taxes on carbon dioxide emissions, and tax incentives for the use of biofuels.

[0006] Ethanol is the most abundant fermentatively produced fuel today but has several drawbacks when compared to gasoline. Butanol, in comparison, has several advantages over ethanol as a fuel: it can be made from the same feedstocks as ethanol but, unlike ethanol, it is compatible with gasoline at any ratio and can also be used as a pure fuel in existing combustion engines without modifications. Unlike ethanol, butanol does not absorb water and can thus be stored and distributed in the existing petrochemical infrastructure. Due to its higher energy content which is close to that of gasoline, the fuel economy (miles per gallon) is better than that of ethanol. Also, butanol-gasoline blends have lower vapor pressure than ethanol-gasoline blends, which is important in reducing evaporative hydrocarbon emissions.

[0007] Isobutanol has the same advantages as butanol with the additional advantage of having a higher octane number due to its branched carbon chain. Isobutanol is also useful as a commodity chemical and is also a precursor to MTBE. Isobutanol has been produced recombinantly in microorganisms expressing a 5-step heterologous metabolic pathway (See, e.g., WO/2007/050671 to Donaldson et al., and WO/2008/098227 to Liao et al.). However, these microorganisms fall short of commercial relevance due to their low performance characteristics, including low productivity, low titer, low yield, and the requirement for oxygen during the fermentation process. Thus, there is an existing need to identify additional metabolic processes catalyzing the conversion of pyruvate of isobutanol. The present inventors have addressed this need by identifying a previously unknown process for producing isobutanol from pyruvate which circumvents the need for the dihydroxyacid dehydratase (DHAD) and 2-keto-acid decarboxylase (KIVD) enzymes.

SUMMARY OF THE INVENTION

[0008] In a first aspect, the present application provides a recombinant microorganism having an engineered isobutanol biosynthetic pathway. The engineered microorganism may be used for the production of isobutanol. Accordingly, in one embodiment, the invention provides a recombinant microorganism comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from: (i) pyruvate to acetolactate (pathway step a),

(ii) acetolactate to 2,3-dihydroxyisovalerate (pathway step b),

(iii) 2,3-dihydroxyisovalerate to isobutyraldehyde (pathway step c), and

(iv) isobutyraldehyde to isobutanol; (pathway step d), wherein the at least one DNA molecule is heterologous to said microorganism and wherein said microorganism produces isobutanol.

[0009] In various embodiments described herein, the recombinant microorganisms can be engineered to express an isobutanol producing metabolic pathway comprising at least one exogenous gene that catalyzes a step in the conversion of pyruvate to isobutanol. In one embodiment, the recombinant microorganism may be engineered to express an isobutanol producing metabolic pathway comprising at least two exogenous genes. In another embodiment, the recombinant microorganism may be engineered to express an isobutanol producing metabolic pathway comprising at least three exogenous genes. In another embodiment, the recombinant microorganism may be engineered to express an isobutanol producing metabolic pathway comprising at least four exogenous genes.

[0010] In various embodiments described herein, the isobutanol pathway enzyme(s) is/are selected from the group consisting of acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI), mevalonate diphosphate decarboxylase (MDC), and alcohol dehydrogenase (ADH).

[0011] In various embodiments described herein, the isobutanol pathway enzyme(s) is/are selected from the group consisting of acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDC), and alcohol dehydrogenase (ADH).

[0012] In various embodiments described herein, the isobutanol pathway enzyme(s) is/are selected from the group consisting of acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI), mevalonate kinase (MK), phosphomevalonate kinase (PMK), mevalonate diphosphate decarboxylase (MDC), and alcohol dehydrogenase (ADH).

[0013] In various embodiments described herein, the isobutanol pathway enzyme(s) is/are selected from the group consisting of acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI), phosphomevalonate decarboxylase (PMDC), and alcohol dehydrogenase (ADH). [0014] In various embodiments described herein, the isobutanol pathway enzyme(s) is/are selected from the group consisting of acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI), mevalonate kinase (MK), phosphomevalonate decarboxylase (PMDC), and alcohol dehydrogenase (ADH).

[0015] 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, all of the isobutanol producing metabolic pathway steps in the conversion of pyruvate to isobutanol are converted by exogenously encoded enzymes.

[0016] 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 yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with all isobutanol pathway enzymes localized in the cytosol.

[0017] In various embodiments described herein, the recombinant microorganisms of the application that comprise an isobutanol producing metabolic pathway may be further engineered to reduce or eliminate the expression and/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).

[0018] 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.

[0019] 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.

[0020] In some embodiments, the recombinant microorganisms may be yeast recombinant microorganisms of the Saccharomyces clade.

[0021] 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.

[0022] 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.

[0023] 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, Kiuyveromyces thermotolerans, Candida glabrata, Z. bailli, Z. rouxii, Debaryomyces hansenii, Pichia pastorius, Schizosaccharomyces pombe, and Saccharomyces uvarum.

[0024] 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.

[0025] 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, Kiuyveromyces, 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, Kiuyveromyces thermotolerans, Kiuyveromyces marxianus, Kiuyveromyces waltii, Kiuyveromyces lactis, Candida tropicalis, Pichia pastoris, Pichia anomala, Pichia stipitis, Issatchenkia orientalis, Issatchenkia occidentalis, Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophilis, Yarrowia lipolytica, and Schizosaccharomyces pombe.

[0026] 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.

[0027] In another aspect, the present application 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.

[0028] 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.

[0029] In a second aspect, the present application provides a recombinant microorganism having an engineered 2-butanol biosynthetic pathway. The engineered microorganism may be used for the production of 2-butanol. Accordingly, in one embodiment, the invention provides a recombinant microorganism comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from:

(i) pyruvate to acetolactate (pathway step a),

(ii) acetolactate to 2,3-dihydroxy-2-methylbutanoic acid (pathway step b),

(iii) 2,3-dihydroxy-2-methylbutanoic acid to 2-butanone (pathway step c), and

(iv) 2-butanone to 2-butanol (pathway step d), wherein the at least one DNA molecule is heterologous to said microorganism and wherein said microorganism produces 2-butanol.

[0030] In various embodiments described herein, the recombinant microorganisms can be engineered to express an 2-butanol producing metabolic pathway comprising at least one exogenous gene that catalyzes a step in the conversion of pyruvate to 2-butanol. In one embodiment, the recombinant microorganism may be engineered to express an 2-butanol producing metabolic pathway comprising at least two exogenous genes. In another embodiment, the recombinant microorganism may be engineered to express an 2-butanol producing metabolic pathway comprising at least three exogenous genes. In another embodiment, the recombinant microorganism may be engineered to express an 2- butanol producing metabolic pathway comprising at least four exogenous genes.

[0031] In various embodiments described herein, the 2-butanol pathway enzyme(s) is/are selected from the group consisting of acetolactate synthase (ALS), 3-keto acid reductase (3-KAR), mevalonate diphosphate decarboxylase (MDC), and alcohol dehydrogenase (ADH).

[0032] In various embodiments described herein, the 2-butanol pathway enzyme(s) is/are selected from the group consisting of acetolactate synthase (ALS), 3-keto acid reductase (3-KAR), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDC), and alcohol dehydrogenase (ADH).

[0033] In various embodiments described herein, the 2-butanol pathway enzyme(s) is/are selected from the group consisting of acetolactate synthase (ALS), 3-keto acid reductase (3-KAR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), mevalonate diphosphate decarboxylase (MDC), and alcohol dehydrogenase (ADH).

[0034] In various embodiments described herein, the 2-butanol pathway enzyme(s) is/are selected from the group consisting of acetolactate synthase (ALS), 3-keto acid reductase (3-KAR), phosphomevalonate decarboxylase (PMDC), and alcohol dehydrogenase (ADH).

[0035] In various embodiments described herein, the 2-butanol pathway enzyme(s) is/are selected from the group consisting of acetolactate synthase (ALS), 3-keto acid reductase (3-KAR), mevalonate kinase (MK), phosphomevalonate decarboxylase (PMDC), and alcohol dehydrogenase (ADH).

[0036] In another aspect, the present application provides methods of producing 2-butanol 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 2-butanol is produced and optionally, recovering the 2-butanol. In one embodiment, the microorganism produces 2-butanol from a carbon source at a yield of at least about 5 percent theoretical. In another embodiment, the microorganism produces 2- butanol 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.

[0037] In one embodiment, the recombinant microorganism converts the carbon source to 2-butanol under aerobic conditions. In another embodiment, the recombinant microorganism converts the carbon source to 2-butanol under microaerobic conditions. In yet another embodiment, the recombinant microorganism converts the carbon source to 2-butanol under anaerobic conditions.

BRIEF DESCRIPTION OF DRAWINGS

[0038] Illustrative embodiments of the invention are illustrated in the drawings, in which:

[0039] Figure 1 illustrates the five-step isobutanol pathway.

[0040] Figure 2 illustrates the mevalonate biosynthesis pathway.

[0041] Figure 3 illustrates a four-step pathway by which pyruvate is converted to isobutanol via acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI), mevalonate diphosphate decarboxylase (MDC), and alcohol dehydrogenase (ADH). The pathway is redox-balanced and thus is capable of functioning anaerobically.

[0042] Figure 4 illustrates a four-step pathway by which pyruvate is converted to 2-butanol via acetolactate synthase (ALS), 3-ketoacid reductase (3-KAR), mevalonate diphosphate decarboxylase (MDC), and alcohol dehydrogenase (ADH). The pathway is redox-balanced and thus is capable of functioning anaerobically.

DETAILED DESCRIPTION

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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 (NaCI); 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.

[0049] "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; (1 1 ) Thermotoga and Thermosipho thermophiles.

[0050] "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.

[0051] "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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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). [0058] 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.

[0059] 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

[0060] 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.

[0061] 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, 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 the results of artificial selection pressure. In still other embodiments, the mutations in the microorganism genome are the result of genetic engineering.

[0062] 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.

[0063] As used herein, the term "isobutanol producing metabolic pathway" refers to an enzyme pathway which produces isobutanol from pyruvate.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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).

[0071] 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).

[0072] 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 also be expressed as mol product per mol substrate (mol/mol). Yield may be also 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. Yield may also be expressed as "carbon yield." The carbon yield of a fermentation product is defined as the amount of carbon in said fermentation product divided by the amount of carbon consumed from the carbon source. As an example, if 1 mol of glucose (C6H12O6) is consumed and 1 mol of pyruvate (C3H3O3) is produced, then the pyruvate carbon yield is 0.5 of 50%.

[0073] 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).

[0074] "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.

[0075] 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 copending publication, US 2010/0143997, the disclosures of which are herein incorporated by reference in its entirety for all purposes.

[0076] "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.

[0077] 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."

[0078] 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.

[0079] The term "byproduct" or "by-product" means an undesired product related to the production of a biofuel or biofuel precursor. Byproducts are generally disposed as waste, adding cost to a production process.

[0080] The term "substantially free" when used in reference to the presence or absence of a protein 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 (3-KAR enzymatic activity, ALDH enzymatic activity, PDC enzymatic activity, GPD enzymatic activity, etc.) may be created through recombinant means or identified in nature.

[0081] 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 CO2.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] "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.

[0087] 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.

[0088] 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

[0089] The term "homolog," used with respect to an original enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of an enzyme or gene 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. [0090] A protein has "homology" or is "homologous" to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences. (Thus, the term "homologous proteins" is defined to mean that the two proteins have similar amino acid sequences).

[0091] The term "analog" or "analogous" refers to nucleic acid or protein sequences or protein structures 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 Biosynthetic Pathways

[0092] The metabolite isobutanol can be produced by a recombinant microorganism which expresses or over-expresses a metabolic pathway that converts pyruvate to isobutanol. Recombinant microorganisms expressing five-step metabolic pathways that convert pyruvate to isobutanol are disclosed in the art, See, e.g., WO/2007/050671 , WO/2008/098227, and Atsumi et ai, Nature, 2008 Jan 3; 451 (7174):86-9. These five-step metabolic pathways which convert pyruvate to isobutanol may be comprised of an acetolactate synthase (ALS), a ketol-acid reductoisomerase (KARI), a dihyroxy-acid dehydratase (DHAD), a 2-keto-acid decarboxylase (KIVD), and an alcohol dehydrogenase (ADH).

[0093] The present inventors have discovered a process for producing isobutanol from pyruvate using a four-step metabolic process which circumvents the need for the dihydroxyacid dehydratase (DHAD) and 2-keto-acid decarboxylase (KIVD) enzymes.

[0094] The four-step isobutanol pathway comprises the following substrate to product conversions:

(i) pyruvate to acetolactate, as catalyzed by an acetolactate synthase (ALS) (pathway step a),

(ii) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed by a ketol-acid reductoisomerase (KARI) (pathway step b), (iii) 2,3-dihydroxyisovalerate to isobutyraldehyde, as catalyzed by a mevalonate diphosphate decarboxylase (MDC) (pathway step c), and

(iv) isobutyraldehyde to isobutanol, as catalyzed by an alcohol dehydrogenase (ADH) (pathway step d).

[0095] Accordingly, provided herein are recombinant microorganisms that produce isobutanol and in some aspects may include the expression or elevated expression of target enzymes such as ALS, KARI, MDC, and ADH.

[0096] In one embodiment, the recombinant microorganism is engineered to overexpress these enzymes. For example, these enzymes can be encoded by native genes. Alternatively, these enzymes can be encoded by heterologous genes.

[0097] Alternatively, the four-step isobutanol pathway comprises the following substrate to product conversions:

(i) pyruvate to acetolactate, as catalyzed by an acetolactate synthase (ALS) (pathway step a),

(ii) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed by a ketol-acid reductoisomerase (KARI) (pathway step b),

(iii) 2,3-dihydroxyisovalerate to isobutyraldehyde, as catalyzed by a phosphomevalonate decarboxylase (PMDC) (pathway step c), and

(iv) isobutyraldehyde to isobutanol, as catalyzed by an alcohol dehydrogenase (ADH) (pathway step d).

[0098] Accordingly, provided herein are recombinant microorganisms that produce isobutanol and in some aspects may include the expression or elevated expression of target enzymes such as ALS, KARI, PMDC, and ADH.

[0099] In one embodiment, the recombinant microorganism is engineered to overexpress these enzymes. For example, these enzymes can be encoded by native genes. Alternatively, these enzymes can be encoded by heterologous genes.

[00100] Alternatively, the four-step isobutanol pathway comprises the following substrate to product conversions:

(i) pyruvate to acetolactate, as catalyzed by an acetolactate synthase (ALS) (pathway step a),

(ii) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed by a ketol-acid reductoisomerase (KARI) (pathway step b),

(iii) 2,3-dihydroxyisovalerate to isobutyraldehyde, as catalyzed by one or more enzymes of the mevalonate biosynthesis pathway, including a mevalonate kinase (MK), a phosphomevalonate kinase (PMK), and a mevalonate diphosphate decarboxylase (MDC) (pathway step c), and

(iv) isobutyraldehyde to isobutanol, as catalyzed by an alcohol dehydrogenase (ADH) (pathway step d).

[00101] Accordingly, provided herein are recombinant microorganisms that produce isobutanol and in some aspects may include the expression or elevated expression of target enzymes such as ALS, KARI, MK, PMK, MDC, and ADH.

[00102] Alternatively, the four-step isobutanol pathway comprises the following substrate to product conversions:

(i) pyruvate to acetolactate, as catalyzed by an acetolactate synthase (ALS) (pathway step a),

(ii) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed by a ketol-acid reductoisomerase (KARI) (pathway step b),

(iii) 2,3-dihydroxyisovalerate to isobutyraldehyde, as catalyzed by one or more enzymes of the mevalonate biosynthesis pathway, including a mevalonate kinase (MK), and a phosphomevalonate decarboxylase (PMDC) (pathway step c), and

(iv) isobutyraldehyde to isobutanol, as catalyzed by an alcohol dehydrogenase (ADH) (pathway step d).

[00103] Accordingly, provided herein are recombinant microorganisms that produce isobutanol and in some aspects may include the expression or elevated expression of target enzymes such as ALS, KARI, MK, PMDC, and ADH.

[00104] In one embodiment, the recombinant microorganism is engineered to overexpress these enzymes. For example, these enzymes can be encoded by native genes. Alternatively, these enzymes can be encoded by heterologous genes.

[00105] As used herein, the terms "acetolactate synthase" or "ALS" are used interchangeably to refer to an enzyme that catalyzes the conversion of pyruvate to acetolactate. Acetolactate synthase enzymes are available from a number of sources, including, but not limited to, Bacillus subtilis, Lactobacillus lactis, Klebsiella pneumoniae. Examples of ALS enzymes capable of converting pyruvate to acetolactate are described in commonly owned and co-pending US Application 201 1/0076733. In exemplary embodiment, the ALS is derived from B. subtilis.

[00106] As used herein, the terms "ketol-acid reductoisomerase" or "KARI" are used interchangeably to refer to an enzyme that catalyzes the conversion of acetolactate to 2,3-dihydroxyisovalerate. Ketol-acid reductoisomerase are available from a number of sources, including, but not limited to, E. coli, B. subtilis, and L. lactis. Examples of KARI enzymes capable of converting pyruvate to acetolactate are described in co-pending and commonly owned application US 201 1/0076733. There are two distinct classes of KARI enzymes known in the art: (1 ) KARI enzymes which are dependent upon the use of NADPH as a cofactor, categorized into enzyme classification (EC) number 1 .1 .1 .86; and (2) KARI enzymes which are dependent upon the use of NADH as a cofactor (i.e., an NADH-dependent KARI or "NKR".) In a preferred embodiment, pathway step b of the isobutanol producing metabolic pathway described herein is carried out by a KARI enzyme that utilizes NADH (rather than NADPH) as a cofactor. Such enzymes are described in commonly owned and co-pending US application 2010/0143997.

[00107] As used herein in the context of isobutanol production, the terms "mevalonate diphosphate decarboxylase" or "MDC" are used interchangeably to refer to an enzyme of the mevalonate biosynthesis pathway (Figure 2) that catalyzes the conversion of 2-hydroxy-3-diphospho-isovalerate to isobutyraldehyde Alternatively, MDC catalyzes the conversion of 2-hydroxy-3-phospho-isovalerate to isobutyraldehyde. Alternatively, MDC catalyzes the conversion of dihydroxyisovalerate to isobutyraldehyde (Figure 3). In mevalonate biosynthesis, the diphosphorylated form of mevalonate is decarboxylated by mevalonate diphosphate decarboxylase (MDC, EC 4.1 .1 .33). MDC is an enzyme involved in the biosynthesis of cholesterol. This enzyme has been isolated from various organisms, such as, animals, fungi, yeasts, and certain bacteria. It may also be expressed by some plants. Numerous genes specifying this enzyme have been cloned and sequenced. These enzymes generally consist of 300-400 amino acids, and they involve ATP as a cosubstrate which is transformed during the reaction to ADP and inorganic phosphate. The phosphate group is transferred from the ATP molecule to the tertiary alcohol of mevalonate diphosphate by releasing ADP. The reaction intermediate which is phosphorylated on the 3-hydroxyl group then undergoes an elimination of the phosphate group, releasing isopentenyl pyrophosphate. The three-dimensional structure of several enzymes of this family has been solved. WO 2010/001078 shows that certain MDC enzymes also act non-phosphorylated forms of the following 3-hydroxyacids: 3-hydroxyisovalerate, 3-hydroxybutyrate, and 3-hydroxyrpopionate. The difference between the 3-hydroxyisovalerate and 2,3-dihydroxyisovalerate (DHIV) is an additional 2-hydroxyl group in DHIV.

[00108] MDC thus acts to replace DHAD and KIVD, which are required to convert steps 3 and 4, respectively, in the five-step isobutanol metabolic pathways disclosed in WO/2007/050671 and WO/2008/098227. Mevalonate diphosphate decarboxylase enzymes are available from a number of sources, including, but not limited to, S. cerevisiae (GenBank Accession Nos. CAA66158.1 and CAY82238.1 ), Kluyveromyces lactis (GenBank Accession No. CAG98256.1 ), Candida glabrata (GenBank Accession No. CAG58241 .1 ), Debaryomyces hansenii (GenBank Accession No. CAG84889.2), Staphylococcus aureus (GenBank Accession No. AAG02425.1 ), and Arabidopsis thaliana (GenBank Accession No. CAA74700.1 ). In an exemplary embodiment, the MDC is derived from S. cerevisiae.

[00109] In some embodiments, the MDC is engineered to increase the enzyme's specificity for DHIV as compared to mevalonate. In some embodiments, the MDC is engineered to increase the enzyme's specificity for 2-hydroxy-3-diphospho- isovalerate as compared to mevalonate diphosphate. In some embodiments, the MDC is engineered to increase the enzyme's specificity for 2-hydroxy-3-phospho- isovalerate as compared to mevalonate-5-phosphate.

[00110] As used herein in the context of isobutanol production, the terms "phosphomevalonate decarboxylase" or "PMDC" are used interchangeably to refer to an enzyme that catalyzes the conversion 2-hydroxy-3-phospho-isovalerate to isobutyraldehyde. Alternatively, PMDC catalyzes the conversion of dihydroxyisovalerate to isobutyraldehyde.

[00111] Certain archaeal enzymes have been described that likely decarboxylate the monophosphorylated form of mevalonate, Mevalonate-5-phosphate. Such mevalonate-5-phosphate decarboxylases, or PMDC enzymes, may be suited for catalyzing the conversion of DHIV. Based on work with the methanogen Methanocaidococcus jannaschii, the archaeal mevalonate pathway is believed to be somewhat different from the pathway found in other organisms, differing in the order of action of the two enzymes that catalyze the conversion of mevaionate-5- phosphate to isopentenyi diphosphate . !n the bacterla!/eukaryotic pathway mevalonate-5-phosphate is first phosphorylated by a kinase, resulting in the intermediate mevaionate-diphosphate , which is then decarboxyiated to isopentenyi diphosphate . At least in some archaea, the decarboxylase acts first, generating isopenteny! phosphate . which is then phosphory!ated to isopentenyi diphosphate by a isopentenyi phosphate kinase (Grochowski et ai. Journal of Bacteriology, 2008. 188(9): 3192).

[00112] This enzyme thus has acts to replace DHAD and KIVD, which are required to convert steps 3 and 4, respectively, in the five-step isobutanol metabolic pathways disclosed in WO/2007/050671 and WO/2008/098227. In some embodiments, the MDC is engineered to increase the enzyme's specificity for DHIV as compared to mevalonate. In some embodiments, the MDC is engineered to increase the enzyme's specificity for 2-hydroxy-3-diphospho-isovalerate as compared to mevalonate diphosphate. In some embodiments, the MDC is engineered to increase the enzyme's specificity for 2-hydroxy-3-phospho-isovalerate as compared to mevalonate-5-phosphate.

[00113] As used herein in the context of isobutanol production, the terms "alcohol dehydrogenase" or "ADH" are used interchangeably to refer to an enzyme that catalyzes the conversion of isobutyraldehyde to isobutanol. Examples of ADH enzymes capable of converting isobutyraldehyde to isobutanol are described in commonly owned and co-pending US Application 201 1/0076733. In one embodiment, pathway step d in the production of isobutanol may be carried out by an ADH that utilizes NADH (rather than NADPH) as a cofactor. Such enzymes are described in commonly owned and co-pending US application 2010/0143997. In an exemplary embodiment, the ADH may be derived from L. lactis or D. melanogaster.

2-Butanol Biosynthetic Pathways

[00114] The metabolite 2-butanol can be produced by a recombinant microorganism which expresses or overexpresses a metabolic pathway that converts pyruvate to 2-butanol.

[00115] The present inventors have discovered a process for producing 2-butanol from pyruvate using a four-step metabolic process.

[00116] In one embodiment, the four-step 2-butanol pathway comprises the following substrate to product conversions:

(i) pyruvate to acetolactate, as catalyzed by an acetolactate synthase (ALS) (pathway step a),

(ii) acetolactate to DH2MB, as catalyzed by a 3-keto acid reductase (3- KAR) (pathway step b), (iii) DH2MB to 2-butanone, as catalyzed by a mevalonate diphosphate decarboxylase (MDC) (pathway step c), and

(iv) 2-butanone to 2-butanol, as catalyzed by an alcohol dehydrogenase (ADH) (pathway step d).

[00117] Accordingly, provided herein are recombinant microorganisms that produce 2-butanol and in some aspects may include the expression or elevated expression of target enzymes such as ALS, KARI, MDC, and ADH.

[00118] In one embodiment, the recombinant microorganism is engineered to overexpress these enzymes. For example, these enzymes can be encoded by native genes. Alternatively, these enzymes can be encoded by heterologous genes.

[00119] Alternatively, the four-step 2-butanol pathway comprises the following substrate to product conversions:

(i) pyruvate to acetolactate, as catalyzed by an acetolactate synthase (ALS) (pathway step a),

(ii) acetolactate to DH2MB, as catalyzed by a 3-keto acid reductase (3- KAR) (pathway step b),

(iii) DH2MB to 2-butanone, as catalyzed by a phosphomevalonate decarboxylase (PMDC) (pathway step c), and

(iv) 2-butanone to 2-butanol, as catalyzed by an alcohol dehydrogenase (ADH) (pathway step d).

[00120] Accordingly, provided herein are recombinant microorganisms that produce 2-butanol and in some aspects may include the expression or elevated expression of target enzymes such as ALS, KARI, PMDC, and ADH.

[00121] In one embodiment, the recombinant microorganism is engineered to overexpress these enzymes. For example, these enzymes can be encoded by native genes. Alternatively, these enzymes can be encoded by heterologous genes.

[00122] Alternatively, the four-step 2-butanol pathway comprises the following substrate to product conversions:

(i) pyruvate to acetolactate, as catalyzed by an acetolactate synthase (ALS) (pathway step a),

(ii) acetolactate to DH2MB, as catalyzed by a 3-keto acid reductase (3- KAR) (pathway step b),

(iii) DH2MB to 2-butanone, as catalyzed by one or more enzymes of the mevalonate biosynthesis pathway, including a mevalonate kinase (MK), a phosphomevalonate kinase (PMK), and a mevalonate diphosphate decarboxylase (MDC) (pathway step c), and

(iv) 2-butanone to 2-butanol, as catalyzed by an alcohol dehydrogenase (ADH) (pathway step d).

[00123] Accordingly, provided herein are recombinant microorganisms that produce 2-butanol and in some aspects may include the expression or elevated expression of target enzymes such as ALS, KARI, MK, PMK, MDC, and ADH.

[00124] Alternatively, the four-step 2-butanol pathway comprises the following substrate to product conversions:

(i) pyruvate to acetolactate, as catalyzed by an acetolactate synthase (ALS) (pathway step a),

(ii) acetolactate to DH2MB, as catalyzed by a 3-keto acid reductase (3- KAR) (pathway step b),

(iii) DH2MB to 2-butanone, as catalyzed by one or more enzymes of the mevalonate biosynthesis pathway, including a mevalonate kinase (MK), and a phosphomevalonate decarboxylase (PMDC) (pathway step c), and

(iv) 2-butanone to 2-butanol, as catalyzed by an alcohol dehydrogenase (ADH) (pathway step d).

[00125] Accordingly, provided herein are recombinant microorganisms that produce 2-butanol and in some aspects may include the expression or elevated expression of target enzymes such as ALS, KARI, MK, PMDC, and ADH.

[00126] In one embodiment, the recombinant microorganism is engineered to overexpress these enzymes. For example, these enzymes can be encoded by native genes. Alternatively, these enzymes can be encoded by heterologous genes.

[00127] As used herein, the terms "3-keto acid reductase" or "3-KAR" are used interchangeably to refer to an enzyme that catalyzes the reaction of acetolactate to 2,3-Dihydroxy-2-Methylbutanoic Acid (DH2MB). 3-keto acid reductases are available from a number of sources, including, but not limited to, S. cerevisiae and C. albicans. Examples of 3-keto acid reductase enzymes capable of converting acetolactate to DH2MB are described in co-pending and commonly owned US Application 201 1/0201090. In one embodiment, the 3-keto acid reductase is TMA29 or homologs thereof. In an exemplary embodiment, the TMA29 is derived from S. cerevisiae. In a specific embodiment, the TMA29 derived from S. cerevisiae comprises SEQ ID NO: 1 . [00128] As used herein in the context of 2-butanol production, the terms "mevalonate diphosphate decarboxylase" or "MDC" are used interchangeably to refer to an enzyme of the mevalonate biosynthesis pathway (Figure 2) that catalyzes the conversion of 2-hydroxy-2-methyl-3-diphosphobutanoate to 2-butanone Alternatively, MDC catalyzes the conversion of 2-hydroxy-2-methyl-3- phosphobutanoate to 2-butanone. Alternatively, MDC catalyzes the conversion of DH2MB to 2-butanone (Figure 4). In mevalonate biosynthesis, the diphosphorylated form of mevalonate is decarboxylated by mevalonate diphosphate decarboxylase (MDC, EC 4.1 .1 .33). MDC is an enzyme involved in the biosynthesis of cholesterol. This enzyme has been isolated from various organisms, such as, animals, fungi, yeasts, and certain bacteria. It may also be expressed by some plants. Numerous genes specifying this enzyme have been cloned and sequenced. These enzymes generally consist of 300-400 amino acids, and they involve ATP as a cosubstrate which is transformed during the reaction to ADP and inorganic phosphate. The phosphate group is transferred from the ATP molecule to the tertiary alcohol of mevalonate diphosphate by releasing ADP. The reaction intermediate which is phosphorylated on the 3-hydroxyl group then undergoes an elimination of the phosphate group, releasing isopentenyl pyrophosphate. The three-dimensional structure of several enzymes of this family has been solved. WO 2010/001078 shows that certain MDC enzymes also act non-phosphorylated forms of the following 3-hydroxyacids: 3-hydroxyisovalerate, 3-hydroxybutyrate, and 3-hydroxyrpopionate. The difference between the 3-hydroxybutyrate and DH2MB are an additional 2- hydroxyl group and an additional 2-methyl group in DH2MB.

[00129] As described above, mevalonate diphosphate decarboxylase enzymes are available from a number of sources, including, but not limited to, S. cerevisiae (GenBank Accession Nos. CAA66158.1 and CAY82238.1 ), Kluyveromyces lactis (GenBank Accession No. CAG98256.1 ), Candida glabrata (GenBank Accession No. CAG58241 .1 ), Debaryomyces hansenii (GenBank Accession No. CAG84889.2), Staphylococcus aureus (GenBank Accession No. AAG02425.1 ), and Arabidopsis thaliana (GenBank Accession No. CAA74700.1 ). In an exemplary embodiment, the MDC is derived from S. cerevisiae.

[00130] In some embodiments, the MDC is engineered to increase the enzyme's specificity for DH2MB as compared to mevalonate. In some embodiments, the MDC is engineered to increase the enzyme's specificity for 2-hydroxy-2-methyl-3- diphosphobutanoate as compared to mevalonate diphosphate. In some embodiments, the MDC is engineered to increase the enzyme's specificity for 2- hydroxy-2-methyl-3-phosphobutanoate as compared to mevalonate-5-phosphate. As used herein in the context of 2-butanol production, the terms "alcohol dehydrogenase" or "ADH" are used interchangeably to refer to an enzyme that catalyzes the conversion of 2-butanone to 2-butanol. Examples of ADH enzymes capable of converting 2-butanone to 2-butanol are described in commonly owned and co-pending US Application 201 1/0076733. In one embodiment, pathway step d in the production of isobutanol may be carried out by an ADH that utilizes NADH (rather than NADPH) as a cofactor. Such enzymes are described in commonly owned and co-pending US application 2010/0143997. In an exemplary embodiment, the ADH may be derived from L. lactis or D. melanogaster.

The Microorganism in General

[00131] 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 or 2- butanol.

[00132] 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 or 2-butanol 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 isobutanol or 2- butanol and may also include additional elements for the expression and/or regulation of expression of these genes, e.g., promoter sequences. [00133] 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).

[00134] 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.

[00135] 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.

[00136] 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.

[00137] 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."

[00138] 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 ai, 1996, Nucl Acids Res. 24: 216-8). Methodology for optimizing a nucleotide sequence for expression in a plant is provided, for example, in U.S. Pat. No. 6,015,891 , and the references cited therein.

[00139] 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.

[00140] In addition, homologs of enzymes useful for generating metabolites are encompassed by the microorganisms and methods provided herein.

[00141] 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 nonhomologous 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.

[00142] 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).

[00143] 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). [00144] Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See commonly owned and co-pending application US 2009/0226991 . A typical algorithm used comparing a molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST. When searching a database containing sequences from a large number of different organisms, it is typical to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms described in commonly owned and co-pending application US 2009/0226991 .

[00145] It is understood that a range of microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of beneficial metabolites from biosynthetic pathways requiring the use of DHIV as an intermediate. In various embodiments, microorganisms may be selected from yeast microorganisms. Yeast microorganisms for the production of a metabolite such as isobutanol or 2-butanol may be selected based on certain characteristics:

[00146] One characteristic may include the property that the microorganism is selected to convert various carbon sources into beneficial metabolites such as isobutanol or 2-butanol. The term "carbon source" generally refers to a substance suitable to be used as a source of carbon for prokaryotic or eukaryotic cell growth. Examples of suitable carbon sources are described in commonly owned and copending application US 2009/0226991 . Accordingly, in one embodiment, the recombinant microorganism herein disclosed can convert a variety of carbon sources to products, including but not limited to glucose, galactose, mannose, xylose, arabinose, lactose, sucrose, and mixtures thereof.

[00147] The recombinant microorganism may thus further include a pathway for the production of isobutanol or 2-butanol from five-carbon (pentose) sugars including xylose. Most yeast species metabolize xylose via a complex route, in which xylose is first reduced to xylitol via a xylose reductase (XR) enzyme. The xylitol is then oxidized to xylulose via a xylitol dehydrogenase (XDH) enzyme. The xylulose is then phosphorylated via a xylulokinase (XK) enzyme. This pathway operates inefficiently in yeast species because it introduces a redox imbalance in the cell. The xylose-to- xylitol step uses primarily NADPH as a cofactor (generating NADP+), whereas the xylitol-to-xylulose step uses NAD+ as a cofactor (generating NADH). Other processes must operate to restore the redox imbalance within the cell. This often means that the organism cannot grow anaerobically on xylose or other pentose sugars. Accordingly, a yeast species that can efficiently ferment xylose and other pentose sugars into a desired fermentation product is therefore very desirable.

[00148] Thus, in one aspect, the recombinant microorganism is engineered to express a functional exogenous xylose isomerase. Exogenous xylose isomerases (XI) functional in yeast are known in the art. See, e.g., Rajgarhia et ai, US2006/0234364, which is herein incorporated by reference in its entirety. In an embodiment according to this aspect, the exogenous XI gene is operatively linked to promoter and terminator sequences that are functional in the yeast cell. In a preferred embodiment, the recombinant microorganism further has a deletion or disruption of a native gene that encodes for an enzyme {e.g., XR and/or XDH) that catalyzes the conversion of xylose to xylitol. In a further preferred embodiment, the recombinant microorganism also contains a functional, exogenous xylulokinase (XK) gene operatively linked to promoter and terminator sequences that are functional in the yeast cell. In one embodiment, the xylulokinase (XK) gene is overexpressed.

[00149] In one embodiment, the 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 of this pathway increases the pyruvate and the reducing equivalents (NADH) available for the isobutanol producing metabolic pathway. Accordingly, deletion of genes encoding for pyruvate decarboxylases can further increase the yield of desired metabolites.

[00150] In another embodiment, the microorganism has reduced or no 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 isobutanol producing metabolic pathway. Thus, deletion of genes encoding for glycerol-3-phosphate dehydrogenases can further increase the yield of desired metabolites, including isobutanol. [00151] In yet another embodiment, the microorganism has reduced or no PDC activity and reduced or no GPD activity. PDC-minus, GPD-minus yeast production strains are described in commonly owned and co-pending publications, US 2009/0226991 and US 201 1/0020889, both of which are herein incorporated by reference in their entireties for all purposes.

[00152] In yet another embodiment relating to recombinant microorganisms comprising a four-step isobutanol pathway, the microorganism has reduced or no 3- keto acid reductase (3-KAR) activity. 3-keto acid reductase catalyzes the conversion of 3-keto acids {e.g., acetolactate) to 3-hydroxyacids {e.g., DH2MB). 3-KAR-minus yeast production strains are described in commonly owned and co-pending application US 201 1/0201090, which is herein incorporated by reference in its entirety for all purposes.

[00153] In yet another embodiment, the microorganism has reduced or no aldehyde dehydrogenase (ALDH) activity. Aldehyde dehydrogenases catalyze the conversion of aldehydes {e.g., isobutyraldehyde) to acid by-products {e.g., isobutyrate). ALDH-minus yeast production strains are described in commonly owned and co-pending application US 201 1/0201090, which is herein incorporated by reference in its entirety for all purposes.

[00154] In one embodiment, the yeast microorganisms may be selected from the "Saccharomyces Yeast Clade", as described in commonly owned and co-pending application US 2009/0226991 .

[00155] The term "Saccharomyces sensu stricto" taxonomy group is a cluster of yeast species that are highly related to S. cerevisiae (Rainieri et al., 2003, J. Biosci Bioengin 96: 1 -9). Saccharomyces sensu stricto yeast species include but are not limited to S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis and hybrids derived from these species (Masneuf et al., 1998, Yeast 7: 61 - 72).

[00156] An ancient whole genome duplication (WGD) event occurred during the evolution of the hemiascomycete yeast and was discovered using comparative genomic tools (Kellis et al., 2004, Nature 428: 617-24; Dujon et al., 2004, Nature 430:35-44; Langkjaer et al., 2003, Nature 428: 848-52; Wolfe et al., 1997, Nature 387: 708-13). Using this major evolutionary event, yeast can be divided into species that diverged from a common ancestor following the WGD event (termed "post-WGD yeast" herein) and species that diverged from the yeast lineage prior to the WGD event (termed "pre-WGD yeast" herein).

[00157] Accordingly, in one embodiment, the yeast microorganism may be selected from a post-WGD yeast genus, including but not limited to Saccharomyces and Candida. The favored post-WGD yeast species include: S. cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S. castelli, and C. glabrata.

[00158] In another embodiment, the yeast microorganism may be selected from a pre-whole genome duplication (pre-WGD) yeast genus including but not limited to Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula, Yarrowia and, Schizosaccharomyces. Representative pre-WGD yeast species include: S. kluyveri, K. thermotolerans, K. marxianus, K. waltii, K. lactis, C. tropicalis, P. pastoris, P. anomala, P. stipitis, I. orientalis, I. occidentalis, I. scutulata, D. hansenii, H. anomala, Y. lipolytica, and S. pombe.

[00159] A yeast microorganism may be either Crabtree-negative or Crabtree- positive as described in described in commonly owned and co-pending application US 2009/0226991 . In one embodiment the yeast microorganism may be selected from yeast with a Crabtree-negative phenotype including but not limited to the following genera: Saccharomyces, Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida. Crabtree-negative species include but are not limited to: S. kluyveri, K. lactis, K. marxianus, P. anomala, P. stipitis, I. orientalis, I. occidentalis, I. scutulata, H. anomala, and C. utilis. In another embodiment, the yeast microorganism may be selected from yeast with a Crabtree-positive phenotype, including but not limited to Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Pichia and Schizosaccharomyces. Crabtree-positive yeast species include but are not limited to: S. cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S. castelli, K. thermotolerans, C. glabrata, Z. bailli, Z. rouxii, D. hansenii, P. pastorius, and S. pombe.

[00160] Another characteristic may include the property that the microorganism is that it is non-fermenting. In other words, it cannot metabolize a carbon source anaerobically while the yeast is able to metabolize a carbon source in the presence of oxygen. Nonfermenting yeast refers to both naturally occurring yeasts as well as genetically modified yeast. During anaerobic fermentation with fermentative yeast, the main pathway to oxidize the NADH from glycolysis is through the production of ethanol. Ethanol is produced by alcohol dehydrogenase (ADH) via the reduction of acetaldehyde, which is generated from pyruvate by pyruvate decarboxylase (PDC). In one embodiment, a fermentative yeast can be engineered to be non-fermentative by the reduction or elimination of the native PDC activity. Thus, most of the pyruvate produced by glycolysis is not consumed by PDC and is available for the isobutanol pathway. Deletion of this pathway increases the pyruvate and the reducing equivalents available for the biosynthetic pathway. Fermentative pathways contribute to low yield and low productivity of desired metabolites such as isobutanol. Accordingly, deletion of PDC genes may increase yield and productivity of desired metabolites such as isobutanol or 2-butanol.

[00161] 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.

[00162] 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.

Methods in General

Identification of Homologous Four-Step Isobutanol and 2-Butanol Pathway Enzymes

[00163] Any method can be used to identify genes that encode for enzymes that are homologous to the pathway enzymes described herein. Generally, genes that are homologous or similar to the pathway enzymes 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.

[00164] 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.

Genetic Insertions and Deletions

[00165] Any method can be used to introduce a nucleic acid molecule into yeast and many such methods are well known. For example, transformation and electroporation are common methods for introducing nucleic acid into yeast cells. See, e.g., Gietz et al., 1992, Nuc Acids Res. 27: 69-74; Ito et al., 1983, J. Bacteriol. 153: 163-8; and Becker et al., 1991 , Methods in Enzymology 194: 182-7.

[00166] In an embodiment, the integration of a gene of interest into a DNA fragment or target gene of a yeast microorganism occurs according to the principle of homologous recombination. According to this embodiment, an integration cassette containing a module comprising at least one yeast marker gene and/or the gene to be integrated (internal module) is flanked on either side by DNA fragments homologous to those of the ends of the targeted integration site (recombinogenic sequences). After transforming the yeast with the cassette by appropriate methods, a homologous recombination between the recombinogenic sequences may result in the internal module replacing the chromosomal region in between the two sites of the genome corresponding to the recombinogenic sequences of the integration cassette. (Orr-Weaver et al., 1981 , PNAS USA 78: 6354-58).

[00167] In an embodiment, the integration cassette for integration of a gene of interest into a yeast microorganism includes the heterologous gene under the control of an appropriate promoter and terminator together with the selectable marker flanked by recombinogenic sequences for integration of a heterologous gene into the yeast chromosome. In an embodiment, the heterologous gene includes an appropriate native gene desired to increase the copy number of a native gene(s). The selectable marker gene can be any marker gene used in yeast, including but not limited to, HIS3, TRP1, LEU2, URA3, bar, ble, hph, and kan. The recombinogenic sequences can be chosen at will, depending on the desired integration site suitable for the desired application.

[00168] In another embodiment, integration of a gene into the chromosome of the yeast microorganism may occur via random integration (Kooistra et al., 2004, Yeast 21 : 781 -792).

[00169] Additionally, in an embodiment, certain introduced marker genes are removed from the genome using techniques well known to those skilled in the art. For example, URA3 marker loss can be obtained by plating URA3 containing cells in FOA (5-fluoro-orotic acid) containing medium and selecting for FOA resistant colonies (Boeke et al., 1984, Mol. Gen. Genet 197: 345-47).

[00170] The exogenous nucleic acid molecule contained within a yeast cell of the disclosure can be maintained within that cell in any form. For example, exogenous nucleic acid molecules can be integrated into the genome of the cell or maintained in an episomal state that can stably be passed on ("inherited") to daughter cells. Such extra-chromosomal genetic elements (such as plasmids, mitochondrial genome, etc.) can additionally contain selection markers that ensure the presence of such genetic elements in daughter cells. Moreover, the yeast cells can be stably or transiently transformed. In addition, the yeast cells described herein can contain a single copy, or multiple copies of a particular exogenous nucleic acid molecule as described above. Reduction of Enzymatic Activity

[00171] Yeast microorganisms within the scope of the invention may have reduced enzymatic activity such as reduced 3-KAR, ALDH, PDC, or GPD activity. The term "reduced" as used herein with respect to a particular polypeptide activity refers to a lower level of polypeptide activity than that measured in a comparable yeast cell of the same species. The term reduced also refers to the elimination of polypeptide activity as compared to a comparable yeast cell of the same species. Thus, yeast cells lacking activity for an endogenous 3-KAR, ALDH, PDC, or GPD are considered to have reduced activity for 3-KAR, ALDH, PDC, or GPD since most, if not all, comparable yeast strains have at least some activity for 3-KAR, ALDH, PDC, or GPD. Such reduced 3-KAR, ALDH, PDC, or GPD activities can be the result of lower 3-KAR, ALDH, PDC, or GPD concentration {e.g., via reduced expression), lower specific activity of the 3-KAR, ALDH, PDC, or GPD, or a combination thereof. Many different methods can be used to make yeast having reduced 3-KAR, ALDH, PDC, or GPD activity. For example, a yeast cell can be engineered to have a disrupted 3-KAR-, ALDH-, PDC-, or GPD-encoding locus using common mutagenesis or knock-out technology. See, e.g., Methods in Yeast Genetics (1997 edition), Adams, Gottschling, Kaiser, and Stems, Cold Spring Harbor Press (1998). In addition, a yeast cell can be engineered to partially or completely remove the coding sequence for a particular 3-KAR, ALDH, PDC, or GPD. Furthermore, the promoter sequence and/or associated regulatory elements can be mutated, disrupted, or deleted to reduce the expression of a 3-KAR, ALDH, PDC, or GPD. Moreover, certain point-mutation(s) can be introduced which results in a 3-KAR, ALDH, PDC, or GPD with reduced activity. Also included within the scope of this invention are yeast strains which when found in nature, are substantially free of one or more 3-KAR, ALDH, PDC, or GPD activities.

[00172] Alternatively, antisense technology can be used to reduce 3-KAR, ALDH, PDC, or GPD activity. For example, yeasts can be engineered to contain a cDNA that encodes an antisense molecule that prevents a 3-KAR, ALDH, PDC, or GPD from being made. The term "antisense molecule" as used herein encompasses any nucleic acid molecule that contains sequences that correspond to the coding strand of an endogenous polypeptide. An antisense molecule also can have flanking sequences (e.g., regulatory sequences). Thus antisense molecules can be ribozymes or antisense oligonucleotides. A ribozyme can have any general structure including, without limitation, hairpin, hammerhead, or axhead structures, provided the molecule cleaves RNA.

Overexpression of Genes

[00173] Methods for overexpressing a polypeptide from a native or heterologous nucleic acid molecule are well known. Such methods include, without limitation, constructing a nucleic acid sequence such that a regulatory element promotes the expression of a nucleic acid sequence that encodes the desired polypeptide. Typically, regulatory elements are DNA sequences that regulate the expression of other DNA sequences at the level of transcription. Thus, regulatory elements include, without limitation, promoters, enhancers, and the like. For example, the exogenous genes can be under the control of an inducible promoter or a constitutive promoter. Moreover, methods for expressing a polypeptide from an exogenous nucleic acid molecule in yeast are well known. For example, nucleic acid constructs that are used for the expression of exogenous polypeptides within Kluyveromyces and Saccharomyces are well known (see, e.g., U.S. Pat. Nos. 4,859,596 and 4,943,529, for Kluyveromyces and, e.g., Gellissen et al., Gene 190(1 ):87-97 (1997) for Saccharomyces). Yeast plasmids have a selectable marker and an origin of replication. In addition certain plasmids may also contain a centromeric sequence. These centromeric plasmids are generally a single or low copy plasmid. Plasmids without a centromeric sequence and utilizing either a 2 micron (S. cerevisiae) or 1 .6 micron (K. lactis) replication origin are high copy plasmids. The selectable marker can be either prototrophic, such as HIS3, TRP1, LEU2, URA3 or AD E2, or antibiotic resistance, such as, bar, ble, hph, or kan.

[00174] In another embodiment, heterologous control elements can be used to activate or repress expression of endogenous genes. Additionally, when expression is to be repressed or eliminated, the gene for the relevant enzyme, protein or RNA can be eliminated by known deletion techniques.

[00175] As described herein, any yeast within the scope of the disclosure can be identified by selection techniques specific to the particular polypeptide (e.g. an isobutanol pathway enzyme) being expressed, over-expressed or repressed. Methods of identifying the strains with the desired phenotype are well known to those skilled in the art. Such methods include, without limitation, PCR, RT-PCR, and nucleic acid hybridization techniques such as Northern and Southern analysis, altered growth capabilities on a particular substrate or in the presence of a particular substrate, a chemical compound, a selection agent and the like. In some cases, immunohistochemistry and biochemical techniques can be used to determine if a cell contains a particular nucleic acid by detecting the expression of the encoded polypeptide. For example, an antibody having specificity for an encoded enzyme can be used to determine whether or not a particular yeast cell contains that encoded enzyme. Further, biochemical techniques can be used to determine if a cell contains a particular nucleic acid molecule encoding an enzymatic polypeptide by detecting a product produced as a result of the expression of the enzymatic polypeptide. For example, transforming a cell with a vector encoding acetolactate synthase and detecting increased acetolactate concentrations compared to a cell without the vector indicates that the vector is both present and that the gene product is active. Methods for detecting specific enzymatic activities or the presence of particular products are well known to those skilled in the art. For example, the presence of acetolactate can be determined as described by Hugenholtz and Starrenburg, 1992, Appl. Micro. Biot. 38:17-22.

Increase of Enzymatic Activity

[00176] Microorganisms of the invention may be further engineered to have increased activity of enzymes {e.g., increased activity of enzymes involved in an isobutanol producing metabolic pathway). The term "increased" as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured in a comparable yeast cell of the same species. For example, overexpression of a specific enzyme can lead to an increased level of activity in the cells for that enzyme. Increased activities for enzymes involved in glycolysis or the isobutanol pathway would result in increased productivity and yield of isobutanol.

[00177] Methods to increase enzymatic activity are known to those skilled in the art. Such techniques may include increasing the expression of the enzyme by increased copy number and/or use of a strong promoter, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the K _M for the substrate, or by directed evolution. See, e.g., Methods in Molecular Biology (vol. 231 ), ed. Arnold and Georgiou, Humana Press (2003). Methods of Using Recombinant Microorganisms for High-Yield Fermentations

[00178] For a biocatalyst to produce a beneficial metabolite such as isobutanol or

2-butanol most economically, it is desirable to produce said metabolite at a high yield. Preferably, the only product produced is the desired metabolite, as extra products (i.e. by-products) lead to a reduction in the yield of the desired metabolite and an increase in capital and operating costs, particularly if the extra products have little or no value. These extra products also require additional capital and operating costs to separate these products from the desired metabolite.

[00179] In one aspect, the present invention provides a method of producing isobutanol derived from a recombinant microorganism comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from:

(i) pyruvate to acetolactate (pathway step a),

(ii) acetolactate to 2,3-dihydroxyisovalerate (pathway step b),

(iii) 2,3-dihydroxyisovalerate to isobutyraldehyde (pathway step c), and

(iv) isobutyraldehyde to isobutanol; (pathway step d), wherein the at least one DNA molecule is heterologous to said microorganism.

[00180] In another aspect, the present invention provides a method of producing 2- butanol derived from a recombinant microorganism comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from:

(i) pyruvate to acetolactate (pathway step a),

(ii) acetolactate to 2,3-dihydroxy-2-methylbutanoic acid (pathway step b),

(iii) 2,3-dihydroxy-2-methylbutanoic acid to 2-butanone (pathway step c), and

(iv) 2-butanone to 2-butanol (pathway step d), wherein the at least one DNA molecule is heterologous to said microorganism.

[00181] In a method to produce a beneficial metabolite such as isobutanol or 2- butanol 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 from the culture medium. For example, 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.

[00182] In one embodiment, the recombinant microorganism may produce the beneficial metabolite such as isobutanol or 2-butanol from a carbon source at a yield of at least 5 percent theoretical. In another embodiment, the microorganism may produce the beneficial metabolite such as isobutanol or 2-butanol 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% theoretical. In a specific embodiment, the beneficial metabolite is isobutanol.

[00183] 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.

[00184] 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.

[00185] 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.