The present invention is generally provides recombinant microorganisms comprising engineered metabolic pathways capable of producing C3-C5 alcohols under aerobic and anaerobic conditions. The invention further provides ketol-acid reductoisomerase enzymes which have been mutated or modified to increase their NADH-dependent activity or to switch the cofactor preference from NADPH to NADH and are expressed in the modified microorganisms. In addition, the invention provides isobutyraldehyde dehydrogenase enzymes expressed in modified microorganisms. Also provided are methods of producing beneficial metabolites under aerobic and anaerobic conditions by contacting a suitable substrate with the modified microorganisms of the present invention.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention provides recombinant microorganisms comprising an engineered metabolic pathway capable of producing one or more C3-C5 alcohols under aerobic and anaerobic conditions. In a preferred embodiment, the recombinant microorganism produces the C3-C5 alcohol under anaerobic conditions at a rate higher than a parental microorganism comprising a native or unmodified metabolic pathway. In another preferred embodiment, the recombinant microorganism produces the C3-C5 alcohol under anaerobic conditions at a rate of at least about 2-fold higher than a parental microorganism comprising a native or unmodified metabolic pathway. In another preferred embodiment, the recombinant microorganism produces the C3-C5 alcohol under anaerobic conditions at a rate of at least about 10-fold, of at least about 50-fold, or of at least about 100-fold higher than a parental microorganism comprising a native or unmodified metabolic pathway.

In various embodiments described herein, the C3-C5 alcohol may be selected from 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol, and 1-pentanol. In a preferred embodiment, the C3-C5 alcohol is isobutanol. In another preferred embodiment, isobutanol is produced at a specific productivity of at least about 0.025 g l.sup.-1 h.sup.-1 OD.sup.-1.

In one aspect, there are provided recombinant microorganisms comprising an engineered metabolic pathway for producing one or more C3-C5 alcohols under anaerobic and aerobic conditions that comprises an overexpressed transhydrogenase that converts NADH to NADPH. In one embodiment, the transhydrogenase is a membrane-bound transhydrogenase. In a specific embodiment, the membrane-bound transhydrogenase is encoded by the E. coli pntAB genes or homologues thereof.

In another aspect, there are provided recombinant microorganisms comprising an engineered metabolic pathway for producing one or more C3-C5 alcohols under anaerobic and aerobic conditions that comprises an NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. In one embodiment, the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase is encoded by the Clostridium acetobutylicum gapC gene. In another embodiment, the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase is encoded by the Kluyveromyces lactis GDP1 gene.

In yet another aspect, there are provided recombinant microorganisms comprising an engineered metabolic pathway for producing one or more C3-C5 alcohols under anaerobic and aerobic conditions that comprises one or more enzymes catalyzing conversions in said engineered metabolic pathway that are not catalyzed by glyceraldehyde-3-phosphate dehydrogenase, and wherein said one or more enzymes have increased activity using NADH as a cofactor. In one embodiment, said one or more enzymes are selected from an NADH-dependent ketol-acid reductoisomerase (KARI) and an NADH-dependent alcohol dehydrogenase (ADH). In various embodiments described herein, the KARI and/or ADH enzymes may be engineered to have increased activity with NADH as the cofactor as compared to the wild-type E. coli KARI llvC and a native E. coli ADH YqhD, respectively. In some embodiments, the KARI and/or the ADH are modified or mutated to be NADH-dependent. In other embodiments, the KARI and/or ADH enzymes are identified in nature with increased activity with NADH as the cofactor as compared to the wild-type E. coli KARI llvC and a native E. coli ADH YqhD, respectively.

In various embodiments described herein, the KARI and/or ADH may show at least a 10-fold higher catalytic efficiency using NADH as a cofactor as compared to the wild-type E. coli KARI llvC and the native ADH YqhD, respectively. In a preferred embodiment, the KARI enhances the recombinant microorganism's ability to convert acetolactate to 2,3-dihydroxyisovalerate under anaerobic conditions. In another embodiment, the KARI enhances the recombinant microorganism's ability to utilize NADH from the conversion of acetolactate to 2,3-dihydroxyisovalerate.

The present invention also provides modified or mutated KARI enzymes that preferentially utilize NADH rather than NADPH, and recombinant microorganisms comprising said modified or mutated KARI enzymes. In general, these modified or mutated KARI enzymes may enhance the cell's ability to produce beneficial metabolites such as isobutanol and enable the production of beneficial metabolites such as isobutanol under anaerobic conditions.

In certain aspects, the invention includes KARIs which have been modified or mutated to increase the ability to utilize NADH. Examples of such KARIs include enzymes having one or more modifications or mutations at positions corresponding to amino acids selected from the group consisting of: (a) alanine 71 of the wild-type E. coli llvC (SEQ ID NO: 13); (b) arginine 76 of the wild-type E. coli llvC; (c) serine 78 of the wild-type E. coli llvC; and (d) glutamine 110 of the wild-type E. coli llvC, wherein llvC (SEQ ID NO: 13) is encoded by codon optimized E. coli ketol-acid reductoisomerase (KARI) genes Ec_ilvC_coEc (SEQ ID NO: 11) or Ec_ilvC_coSc (SEQ ID NO: 12).

In one embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 71 of the wild-type E. coli llvC (SEQ ID NO: 13). In another embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 76 of the wild-type E. coli llvC (SEQ ID NO: 13). In yet another embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 78 of the wild-type E. coli llvC (SEQ ID NO: 13). In yet another embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 110 of the wild-type E. coli llvC (SEQ ID NO: 13).

In one embodiment, the KARI enzyme contains two or more modifications or mutations at the amino acids corresponding to the positions described above. In another embodiment, the KARI enzyme contains three or more modifications or mutations at the amino acids corresponding to the positions described above. In yet another embodiment, the KARI enzyme contains four modifications or mutations at the amino acids corresponding to the positions described above.

In one specific embodiment, the invention is directed to KARI enzymes wherein the alanine at position 71 is replaced with serine. In another specific embodiment, the invention is directed to KARI enzymes wherein the arginine at position 76 is replaced with aspartic acid. In yet another specific embodiment, the invention is directed to KARI enzymes wherein the serine at position 78 is replaced with aspartic acid. In yet another specific embodiment, the invention is directed to KARI enzymes wherein the glutamine at position 110 is replaced with valine. In yet another specific embodiment, the invention is directed to KARI enzymes wherein the glutamine at position 110 is replaced with alanine. In certain embodiments, the KARI enzyme contains two or more modifications or mutations at the amino acids corresponding to the positions described in these specific embodiments. In certain other embodiments, the KARI enzyme contains three or more modifications or mutations at the amino acids corresponding to the positions described in these specific embodiments. In an exemplary embodiment, the KARI enzyme contains four modifications or mutations at the amino acids corresponding to the positions described in these specific embodiments. In additional embodiments described herein, the KARI may further comprise an amino acid substitution at position 68 of the wild-type E. coli llvC (SEQ ID NO: 13).

In one embodiment, the modified or mutated KARI is selected from group consisting of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 44.

Further included within the scope of the invention are KARI enzymes, other than the E. coli llvC (SEQ ID NO: 13), which contain alterations corresponding to those set out above. Such KARI enzymes may include, but are not limited to, the KARI enzymes encoded by the S. cerevisiae ILV5 gene, the KARI enzyme encoded by the E. coli ilvC gene and the KARI enzymes from Piromyces sp., Buchnera aphidicola, Spinacia oleracea, Oryza sativa, Chlamydomonas reinhardtii, Neurospora crassa, Schizosaccharomyces pombe, Laccaria bicolor, Ignicoccus hospitalis, Picrophilus torridus, Acidiphilium cryptum, Cyanobacteria/Synechococcus sp., Zymomonas mobilis, Bacteroides thetaiotaomicron, Methanococcus maripaludis, Vibrio fischeri, Shewanella sp, Gramella forsetti, Psychromonas ingrhamaii, and Cytophaga hutchinsonii.

In certain exemplary embodiments, the KARI to be modified or mutated is a KARI selected from the group consisting of Escherichia coli (GenBank No: NP.sub.--418222, SEQ ID NO 13), Saccharomyces cerevisiae (GenBank No: NP.sub.--013459, SEQ ID NO: 70), Methanococcus maripaludis (GenBank No: YP.sub.--001097443, SEQ ID NO: 71), Bacillus subtilis (GenBank Nos: CAB14789, SEQ ID NO: 72), Piromyces sp (GenBank No: CAA76356, SEQ ID NO: 73), Buchnera aphidicola (GenBank No: AAF13807, SEQ ID NO: 74), Spinacia oleracea (GenBank Nos: .quadrature.01292 and CAA40356, SEQ ID NO: 75), Oryza sativa (GenBank No: NP.sub.--001056384, SEQ ID NO: 76) Chlamydomonas reinhardtii (GenBank No: XP.sub.--001702649, SEQ ID NO: 77), Neurospora crassa (GenBank No: XP.sub.--961335, SEQ ID NO: 78), Schizosaccharomyces pombe (GenBank No: NP.sub.--001018845, SEQ ID NO: 79), Laccaria bicolor (GenBank No: XP.sub.--001880867, SEQ ID NO: 80), Ignicoccus hospitalis (GenBank No: YP.sub.--001435197, SEQ ID NO: 81), Picrophilus torridus (GenBank No: YP.sub.--023851, SEQ ID NO: 82), Acidiphilium cryptum (GenBank No: YP.sub.--001235669, SEQ ID NO: 83), Cyanobacteria/Synechococcus sp. (GenBank No: YP.sub.--473733, SEQ ID NO: 84), Zymomonas mobilis (GenBank No: YP.sub.--162876, SEQ ID NO: 85), Bacteroides thetaiotaomicron (GenBank No: NP.sub.--810987, SEQ ID NO: 86), Vibrio fischeri (GenBank No: YP.sub.--205911, SEQ ID NO: 87), Shewanella sp (GenBank No: YP.sub.--732498, SEQ ID NO: 88), Gramella forsetti (GenBank No: YP.sub.--862142, SEQ ID NO: 89), Psychromonas ingrhamaii (GenBank No: YP.sub.--942294, SEQ ID NO: 90), and Cytophaga hutchinsonii (GenBank No: YP.sub.--677763, SEQ ID NO: 91).

In various embodiments described herein, the modified or mutated KARI may exhibit an increased catalytic efficiency with NADH as compared to the wild-type KARI. In one embodiment, the KARI has at least about a 5% increased catalytic efficiency with NADH as compared to the wild-type KARI. In another embodiment, the KARI has at least about a 25%, at least about a 50%, at least about a 75%, or at least about a 100% increased catalytic efficiency with NADH as compared to the wild-type KARI.

In some embodiments described herein, the catalytic efficiency of the modified or mutated KARI with NADH is increased with respect to the catalytic efficiency with NADPH of the wild-type KARI. In one embodiment, the catalytic efficiency of said KARI with NADH is at least about 10% of the catalytic efficiency with NADPH of the wild-type KARI. In another embodiment, the catalytic efficiency of said KARI with NADH is at least about 25%, at least about 50%, or at least about 75% of the catalytic efficiency with NADPH of the wild-type KARI. In some embodiments, the modified or mutated KARI preferentially utilizes NADH rather than NADPH.

In one embodiments, the invention is directed to modified or mutated KARI enzymes that demonstrate a switch in cofactor preference from NADPH to NADH. In one embodiment, the modified or mutated KARI has at least about a 2:1 ratio of k.sub.cat with NADH over k.sub.cat with NADPH. In an exemplary embodiment, the modified or mutated KARI has at least about a 10:1 ratio of k.sub.cat with NADH over k.sub.cat with NADPH.

In one embodiments, the invention is directed to a modified or mutated KARI enzyme that exhibits at least about a 1:10 ratio of catalytic efficiency (k.sub.cat/K.sub.M) with NADH over catalytic efficiency with NADPH. In another embodiment, the modified or mutated KARI enzyme exhibits at least about a 1:1 ratio of catalytic efficiency (k.sub.cat/K.sub.M) with NADH over catalytic efficiency with NADPH. In yet another embodiment, the modified or mutated KARI enzyme exhibits at least about a ratio of catalytic efficiency (k.sub.cat/K.sub.M) with NADH over catalytic efficiency with NADPH. In an exemplary embodiment, the modified or mutated KARI enzyme exhibits at least about a 100:1 ratio of catalytic efficiency (k.sub.cat/K.sub.M) with NADH over catalytic efficiency with NADPH.

In some embodiments, the modified or mutated KARI has been modified to be NADH-dependent. In one embodiment, the KARI exhibits at least about a 1:10 ratio of K.sub.M for NADH over K.sub.M for NADPH.

In additional embodiments, the invention is directed to modified or mutated KARI enzymes that have been codon optimized for expression in certain desirable host organisms, such as yeast and E. coli. In other aspects, the present invention is directed to recombinant host cells (e.g. recombinant microorganisms) comprising a modified or mutated KARI enzyme of the invention. According to this aspect, the present invention is also directed to methods of using the modified or mutated KARI enzymes in any fermentation process where the conversion of acetolactate to 2,3-dihydroxyisovalerate is desired. In one embodiment according to this aspect, the modified or mutated KARI enzymes may be suitable for enhancing a host cell's ability to produce isobutanol and enable the production of isobutanol under anaerobic conditions. In another embodiment according to this aspect, the modified or mutated KARI enzymes may be suitable for enhancing a host cell's ability to produce 3-methyl-1-butanol.

According to this aspect, the present invention is also directed to methods of using the modified or mutated KARI enzymes in any fermentation process where the conversion of 2-aceto-2-hydroxy-butyrate to 2,3-dihydroxy-3-methylvalerate is desired. In one embodiment according to this aspect, the modified or mutated KARI enzymes may be suitable for enhancing a host cell's ability to produce 2-methyl-1-butanol and enable the production of 2-methyl-1-butanol under anaerobic conditions.

In another aspect, there are provided recombinant microorganisms comprising an engineered metabolic pathway for producing one or more C3-C5 alcohols under anaerobic conditions, wherein said engineered metabolic pathway comprises a first dehydrogenase and a second dehydrogenase that catalyze the same reaction, and wherein the first dehydrogenase is NADH-dependent and wherein the second dehydrogenase is NADPH dependent. In an exemplary embodiment, the first dehydrogenase is encoded by the E. coli gene maeA and the second dehydrogenase is encoded by the E. coli gene maeB.

In another aspect, there are provided recombinant microorganisms comprising an engineered metabolic pathway for producing one or more C3-C5 alcohols under anaerobic conditions, wherein said engineered metabolic pathway comprises a replacement of a gene encoding for pyk or homologs thereof with a gene encoding for ppc or pck or homologs thereof. In another embodiment, the engineered metabolic pathway may further comprise the overexpression of the genes mdh and maeB.

In various embodiments described herein, the recombinant microorganisms may further 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. In another embodiment, the recombinant microorganism may be engineered to express an isobutanol producing metabolic pathway comprising five exogenous genes.

In various embodiments described herein, the isobutanol pathway enzyme(s) may be selected from the group consisting of acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), 2-keto-acid decarboxylase (KIVD), and alcohol dehydrogenase (ADH).

In another embodiment, the recombinant microorganism further comprises a pathway for the fermentation of isobutanol from a pentose sugar. In one embodiment, the pentose sugar is xylose. In one embodiment, the recombinant microorganism is engineered to express a functional xylose isomerase (XI). In another embodiment, the recombinant microorganism further comprises a deletion or disruption of a native gene encoding for an enzyme that catalyzes the conversion of xylose to xylitol. In one embodiment, the native gene is xylose reductase (XR). In another embodiment, the native gene is xylitol dehydrogenase (XDH). In yet another embodiment, both native genes are deleted or disrupted. In yet another embodiment, the recombinant microorganism is engineered to express a xylulose kinase enzyme.

In another embodiment, the recombinant microorganisms of the present invention may further be engineered to include reduced pyruvate decarboxylase (PDC) activity as compared to a parental microorganism. In one embodiment, PDC activity is eliminated. PDC catalyzes the decarboxylation of pyruvate to acetaldehyde, which is reduced to ethanol by alcohol dehydrogenases via the oxidation of NADH to NAD+. In one embodiment, the recombinant microorganism includes a mutation in at least one PDC gene resulting in a reduction of PDC activity of a polypeptide encoded by said gene. In another embodiment, the recombinant microorganism includes a partial deletion of a PDC gene resulting in a reduction of PDC activity of a polypeptide encoded by said gene. In another embodiment, the recombinant microorganism comprises a complete deletion of a PDC gene resulting in a reduction of PDC activity of a polypeptide encoded by said gene. In yet another embodiment, the recombinant microorganism includes a modification of the regulatory region associated with at least one PDC gene resulting in a reduction of PDC activity of a polypeptide encoded by said gene. In yet another embodiment, the recombinant microorganism comprises a modification of the transcriptional regulator resulting in a reduction of PDC gene transcription. In yet another embodiment, the recombinant microorganism comprises mutations in all PDC genes resulting in a reduction of PDC activity of the polypeptides encoded by said genes.

In another embodiment, the recombinant microorganisms of the present invention may further be engineered to include reduced glycerol-3-phosphate dehydrogenase (GPD) activity as compared to a parental microorganism. In one embodiment, GPD activity is eliminated. GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD.sup.+. Glycerol is produced from G3P by Glycerol-3-phosphatase (GPP). In one embodiment, the recombinant microorganism includes a mutation in at least one GPD gene resulting in a reduction of GPD activity of a polypeptide encoded by said gene. In another embodiment, the recombinant microorganism includes a partial deletion of a GPD gene resulting in a reduction of GPD activity of a polypeptide encoded by the gene. In another embodiment, the recombinant microorganism comprises a complete deletion of a GPD gene resulting in a reduction of GPD activity of a polypeptide encoded by the gene. In yet another embodiment, the recombinant microorganism includes a modification of the regulatory region associated with at least one GPD gene resulting in a reduction of GPD activity of a polypeptide encoded by said gene. In yet another embodiment, the recombinant microorganism comprises a modification of the transcriptional regulator resulting in a reduction of GPD gene transcription. In yet another embodiment, the recombinant microorganism comprises mutations in all GPD genes resulting in a reduction of GPD activity of a polypeptide encoded by the gene.

In various embodiments described herein, the recombinant microorganisms of the invention may produce one or more C3-C5 alcohols under anaerobic conditions at a yield which is at least about the same yield as under aerobic conditions. In additional embodiments described herein, the recombinant microorganisms of the invention may produce one or more C3-C5 alcohols at substantially the same rate under anaerobic conditions as the parental microorganism produces under aerobic conditions. In the various embodiments described herein, the engineered metabolic pathway may be balanced with respect to NADH and NADPH as compared to a native or unmodified metabolic pathway from a corresponding parental microorganism, wherein the native or unmodified metabolic pathway is not balanced with respect to NADH and NADPH.

In another aspect, the present invention provides a method of producing a C3-C5 alcohol, comprising (a) providing a recombinant microorganism comprising an engineered metabolic pathway capable of producing one or more C3-C5 alcohols under aerobic and anaerobic conditions; (b) cultivating the recombinant microorganism in a culture medium containing a feedstock providing the carbon source, until a recoverable quantity of the C3-C5 alcohol is produced; and (c) recovering the C3-C5 alcohol. In one embodiment, the recombinant microorganism is cultured under anaerobic conditions. In a preferred embodiment, the C3-C5 alcohol is produced under anaerobic conditions at a yield which is at least about the same yield as under aerobic conditions.

In various embodiments described herein, a preferred C3-C5 alcohol is 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 is selected to produce 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 theoretical, at least about 85 percent theoretical, at least about 90 percent theoretical, or at least about 95 percent theoretical. In one embodiment, the C3-C5 alcohol, such as isobutanol, is produced under anaerobic conditions at about the same yield as under aerobic conditions.

In another aspect, the present invention provides a recombinant microorganism comprising a metabolic pathway for producing a C3-C5 alcohol from a carbon source, wherein said recombinant microorganism comprises a modification that leads to the regeneration of redox co-factors within said recombinant microorganism. In one embodiment according to this aspect, the modification increases the production of a C3-C5 alcohol under anaerobic conditions as compared to the parental or wild-type microorganism. In a preferred embodiment, the fermentation product is isobutanol. In one embodiment, the re-oxidation or re-reduction of said redox co-factors does not require the pentose phosphate pathway, the TCA cycle, or the generation of additional fermentation products. In another embodiment, the re-oxidation or re-reduction of said redox co-factors does not require the production of byproducts or co-products. In yet another embodiment, additional fermentation products are not required for the regeneration of said redox co-factors.

In another aspect, the present invention provides a method of producing a C3-C5 alcohol, comprising providing a recombinant microorganism comprising a metabolic pathway for producing a C3-C5 alcohol, wherein said recombinant microorganism comprises a modification that leads to the regeneration of redox co-factors within said recombinant microorganism; cultivating the microorganism in a culture medium containing a feedstock providing the carbon source, until a recoverable quantity of said C3-C5 alcohol is produced; and optionally, recovering the C3-C5 alcohol. In one embodiment, said microorganism is cultivated under anaerobic conditions. In another embodiment, the C3-C5 alcohol is produced under anaerobic conditions at about the same yield as under aerobic conditions. In a preferred embodiment, the C3-C5 alcohol is isobutanol.

In various embodiments described herein, the recombinant microorganisms may be microorganisms 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.

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

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.

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 Kluyveromyces, Pichia, Hansenula, or Candida. In additional embodiments, the Crabtree-negative yeast microorganism is selected from Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Hansenula anomala, Candida utilis, Issatchenkia orientalis and Kluyveromyces waltii.

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, Saccharomyces kluyveri, Kluyveromyces thermotolerans, Candida glabrata, Z. baiffi, Z. rouxii, Debaryomyces hansenii, Pichia pastorius, Schizosaccharomyces pombe, and Saccharomyces uvarum.

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 casteffi, and Candida glabrata.

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

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, or Myxozyma.

In certain specific embodiments, there are provided recombinant microorganisms comprising an engineered metabolic pathway for producing one or more C3-C5 alcohols under anaerobic conditions, wherein the recombinant microorganism is selected from GEVO1846, GEVO1886, GEVO1993, GEVO2158, GEVO2302, GEVO1803, GEVO2107, GEVO2710, GEVO2711, GEVO2712, GEVO2799, GEVO2847, GEVO2848, GEVO2849, GEVO2851, GEVO2852, GEVO2854, GEVO2855 and GEVO2856. In another specific embodiment, the present invention provides a plasmid is selected from the group consisting of pGV1698 (SEQ ID NO: 112), pGV1720 (SEQ ID NO: 115), pGV1745 (SEQ ID NO: 117), pGV1655 (SEQ ID NO: 109), pGV1609 (SEQ ID NO: 108), pGV1685 (SEQ ID NO: 111), and pGV1490 (SEQ ID NO: 104).

In yet another aspect, the present invention provides methods for the conversion of an aldehyde with three to five carbon atoms to the corresponding alcohol is provided. The method includes providing a microorganism comprising a heterologous polynucleotide encoding a polypeptide having NADH-dependent aldehyde reduction activity that is greater than its NADPH-dependent aldehyde reduction activity and having NADH-dependent aldehyde reduction activity that is greater than the endogenous NADPH-dependent aldehyde reduction activity of the microorganism; and contacting the microorganism with the aldehyde.

In another embodiment, a method for the conversion of an aldehyde derived from the conversion of a 2-ketoacid by a 2-ketoacid decarboxylase is provided. The method includes providing a microorganism comprising a heterologous polynucleotide encoding a polypeptide having NADH-dependent aldehyde reduction activity that is greater than its NADPH-dependent aldehyde reduction activity and having NADH-dependent aldehyde reduction activity that is greater than the endogenous NADPH-dependent aldehyde reduction activity of the microorganism; and contacting the microorganism with the aldehyde. In various embodiments described herein, the aldehyde may be selected from 1-propanal, 1-butanal, isobutyraldehyde, 2-methyl-1-butanal, or 3-methyl-1-butanal. In a preferred embodiment, the aldehyde is isobutyraldehyde.

In another embodiment, an microorganism include a heterologous polynucleotide encoding a polypeptide having NADH-dependent aldehyde reduction activity that is greater than its NADPH-dependent aldehyde reduction activity and having NADH-dependent aldehyde reduction activity that is greater than the endogenous NADPH-dependent aldehyde reduction activity of the microorganism is provided. The microorganism converts an aldehyde comprising three to five carbon atoms to the corresponding alcohol.

In another embodiment, an isolated microorganism is provided. The microorganism includes a heterologous polynucleotide encoding a polypeptide having NADH-dependent aldehyde reduction activity that is greater than its NADPH-dependent aldehyde reduction activity and having NADH-dependent aldehyde reduction activity that is greater than the endogenous NADPH-dependent aldehyde reduction activity of the microorganism. The microorganism converts an aldehyde derived from a 2-ketoacid by a 2-ketoacid decarboxylase. In one embodiment, the polypeptide is encoded by the Drosophila melanogaster ADH gene or homologs thereof. In a preferred embodiment, the Drosophila melanogaster ADH gene is set forth in SEQ ID NO: 60. In an alternative embodiment, the Drosophila melanogaster alcohol dehydrogenase is set forth in SEQ ID NO: 61. In another embodiment, the polypeptide possesses 1,2 propanediol dehydrogenase activity and is encoded by a 1,2 propanediol dehydrogenase gene. In a preferred embodiment, the 1,2-propanediol dehydrogenase gene is the Klebsiella pneumoniae dhaT gene as set forth in SEQ ID NO: 62. In an alternative embodiment, the 1,2-propanediol dehydrogenase is set forth in SEQ ID NO: 63. In another embodiment, the polypeptide possesses is encoded by a 1,3-propanediol dehydrogenase gene. In a preferred embodiment, the 1,3-propanediol dehydrogenase gene is the Escherichia coli fucO gene as set forth in SEQ ID NO: 64. In an alternative embodiment, the 1,3-propanediol dehydrogenase is set forth in SEQ ID NO: 65.

In yet another aspect, the present invention provides a recombinant microorganism producing isobutanol, wherein said recombinant microorganism i) does not overexpress an alcohol dehydrogenase; and ii) produces isobutanol at a higher rate, titer, and productivity as compared to recombinant microorganism expressing the S. cerevisiae alcohol dehydrogenase ADH2.

DETAILED DESCRIPTION

The Microorganism in General

Microorganism Characterized by Producing C3-C5 Alcohols from Pyruvate Via an Overexpressed Metabolic Pathway

Native producers of butanol, and more specifically 1-butaanol, such as Clostridium acetobutylicum, are known, but these organisms generate byproducts such as acetone, ethanol, and butyrate during fermentations. Furthermore, these microorganisms are relatively difficult to manipulate, with significantly fewer tools available than in more commonly used production hosts such as E. coli. Additionally, the physiology and metabolic regulation of these native producers are much less well understood, impeding rapid progress towards high-efficiency production. Furthermore, no native microorganisms have been identified that can metabolize glucose into isobutanol in industrially relevant quantities or yields.

The production of isobutanol and other fusel alcohols by various yeast species, including Saccharomyces cerevisiae is of special interest to the distillers of alcoholic beverages, for whom fusel alcohols constitute often undesirable off-notes. Production of isobutanol in wild-type yeasts has been documented on various growth media, ranging from grape must from winemaking (Romano, et al., Metabolic diversity of Saccharomyces cerevisiae strains from spontaneously fermented grape musts, 19:311-315, 2003), in which 12-219 mg/L isobutanol were produced, supplemented to minimal media (Oliviera, et al. (2005) World Journal of Microbiology and Biotechnology 21:1569-1576), producing 16-34 mg/L isobutanol. Work from Dickinson, et al. (J Biol. Chem. 272(43):26871-8, 1997) has identified the enzymatic steps utilized in an endogenous S. cerevisiae pathway converting branch-chain amino acids (e.g., valine or leucine) to isobutanol.

A number of recent publications have described methods for the production of industrial chemicals such as C3-C5 alcohols such as isobutanol using engineered microorganisms. See, e.g., WO/2007/050671 to Donaldson et al., and WO/2008/098227 to Liao et al., which are herein incorporated by reference in their entireties. These publications disclose recombinant microorganisms that utilize a series of heterologously expressed enzymes to convert sugars into isobutanol. However, the production of isobutanol using these microorganisms is feasible only under aerobic conditions and the maximum yield that can be achieved is limited.

Recombinant microorganisms provided herein can express a plurality of target enzymes involved in pathways for the production isobutanol from a suitable carbon source under anaerobic conditions.

Accordingly, "engineered" or "modified" microorganisms are produced via the introduction of genetic material into a host or parental microorganism of choice thereby modifying or altering the cellular physiology and biochemistry of the microorganism. Through the introduction of genetic material the parental microorganism acquires new properties, e.g. the ability to produce a new, or greater quantities of, an intracellular metabolite under anaerobic conditions. As described herein, the introduction of genetic material into a parental microorganism results in a new or modified ability to produce isobutanol under anaerobic conditions. The genetic material introduced into 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 under anaerobic conditions and may also include additional elements for the expression and/or regulation of expression of these genes, e.g. promoter sequences.

An engineered or modified microorganism can also include in the alternative or in addition to the introduction of a genetic material into a host or parental microorganism, the disruption, deletion or knocking out of a gene or polynucleotide to alter the cellular physiology and biochemistry of the microorganism. Through the reduction, disruption 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, improve the flux of a metabolite down a desired pathway, and/or reduce the production of undesirable by-products).

Microorganisms provided herein are modified to produce under anaerobic conditions 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.

Exemplary metabolites include glucose, pyruvate, and C3-C5 alcohols, including isobutanol. The metabolite isobutanol can be produced by a recombinant microorganism engineered to express or over-express metabolic pathway that converts pyruvate to isobutanol. An exemplary metabolic pathway that converts pyruvate to isobutanol may be comprised of a acetohydroxy acid synthase (ALS) enzyme encoded by, for example, alsS from B. subtilis, a ketolacid reductoisomerase (KARI) encoded by, for example ilvC from E. coli, a dihyroxy-acid dehydratase (DHAD), encoded by, for example ilvD from E. coli, a 2-keto-acid decarboxylase (KIVD) encoded by, for example kivd from L. lactis, and an alcohol dehydrogenase (ADH), encoded by, for example, by a native E. coli alcohol dehydrogenase gene, like Ec_yqhD.

Accordingly, provided herein are recombinant microorganisms that produce isobutanol and in some aspects may include the elevated expression of target enzymes such as ALS (encoded e.g. by the ilvIH operon from E. coli or by alsS from Bacillus subtilis), KARI (encoded e.g. by ilvC from E. coli), DHAD (encoded, e.g. by ilvD from E. coli, or by ILV3 from S. cerevisiae, and KIVD (encoded, e.g. by, AR010 from S. cerevisiae, THI3 from S. cerevisiae, kivd from L. lactis).

The recombinant microorganism may further include the deletion or reduction of the activity of enzymes that (a) directly consume a precursor of the product, e.g. an isobutanol precursor, (b) indirectly consume a precursor of the product, e.g. of isobutanol, or (c) repress the expression or function of a pathway that supplies a precursor of the product, e.g. of isobutanol. These enzymes include pyruvate decarboxylase (encoded, e.g. by PDC1, PDC2, PDC3, PDC5, or PDC6 of S. cerevisiae), glycerol-3-phosphate dehydrogenase (encoded, e.g. by GPD1 or GPD2 of S. cerevisiae) an alcohol dehydrogenase (encoded, e.g., by adhE of E. coli or ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, or ADH7 of S. cerevisiae), lacate dehydrogenase (encoded, e.g., by IdhA of E. coli), fumarate reductase (encoded, e.g., by frdB, frdC or frdBC of E. coli), FNR (encoded, e.g. by fnr of E. coli), 2-isopropylmalate synthase (encoded, e.g. by leuA of E. coli or by LEU4 or LEU9 of S. cerevisiae), valine transaminase (encoded, e.g. by ilvE of E. coli or by BAT1 or BAT2 of S. cerevisiae), pyruvate oxidase (e.g. encoded by poxB of E. coli), Threonine deaminase (encoded, e.g. by i/vA of E. coli or CHA1 or ILV1 of S. cerevisiae), pyruvate-formate-lyase (encoded, e.g. by pflB of E. coli), or phosphate acetyltransferase (encoded, e.g. by pta of E. coli), or any combination thereof, to increase the availability of pyruvate or reduce enzymes that compete for a metabolite in a desired biosynthetic pathway.

In yeast microorganisms, pyruvate decarboxylase (PDC) is a major competitor for pyruvate. During anaerobic fermentation, 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). Thus, most of the pyruvate produced by glycolysis is consumed by PDC and is not available for the isobutanol pathway. Another pathway for NADH oxidation is through the production of glycerol. Dihydroxyacetone-phospate, an intermediate of glycolysis is reduced to glycerol 3-phosphate by glycerol 3-phosphate dehydrogenase (GPD). Glycerol 3-phosphatase (GPP) converts glycerol 3-phosphate to glycerol. This pathway consumes carbon from glucose as well as reducing equivalents (NADH) resulting in less pyruvate and reducing equivalents available for the isobutanol pathway. These pathways contribute to low yield and low productivity of C3-C5 alcohols, including isobutanol. Accordingly, deletion or reduction of the activity of PDC and GPD may increase yield and productivity of C3-C5 alcohols, including isobutanol.

Reduction of PDC activity can be accomplished by 1) mutation or deletion of a positive transcriptional regulator for the structural genes encoding for PDC or 2) mutation or deletion of all PDC genes in a given organism. The term "transcriptional regulator" can specify a protein or nucleic acid that works in trans to increase or to decrease the transcription of a different locus in the genome. For example, in S. cerevisiae, the PDC2 gene, which encodes for a positive transcriptional regulator of PDC1,5,6 genes can be deleted; a S. cerevisiae in which the PDC2 gene is deleted is reported to have only .about.10% of wildtype PDC activity (Hohmann, Mol Gen Genet, 241:657-666 (1993)). Alternatively, for example, all structural genes for PDC (e.g. in S. cerevisiae, PDC1, PDC5, and PDC6, or in K. lactis, PDC1) are deleted.

Crabtree-positive yeast strains such as Saccharomyces. cerevisiae strain that contains disruptions in all three of the PDC alleles no longer produce ethanol by fermentation. However, a downstream product of the reaction catalyzed by PDC, acetyl-CoA, is needed for anabolic production of necessary molecules. Therefore, the Pdc-mutant is unable to grow solely on glucose, and requires a two-carbon carbon source, either ethanol or acetate, to synthesize acetyl-CoA. (Flikweert M T, de Swaaf M, van Dijken J P, Pronk J T. FEMS Microbiol Lett. 1999 May 1; 174(1):73-9. PMID:10234824 and van Maris A J, Geertman J M, Vermeulen A, Groothuizen M K, Winkler A A, Piper M D, van Dijken J P, Pronk J T. Appl Environ Microbiol. 2004 January; 70(1):159-66. PMID: 14711638).

Thus, in an embodiment, such a Crabtree-positive yeast strain may be evolved to generate variants of the PDC mutant yeast that do not have the requirement for a two-carbon molecule and has a growth rate similar to wild type on glucose. Any method, including chemostat evolution or serial dilution may be utilized to generate variants of strains with deletion of three PDC alleles that can grow on glucose as the sole carbon source at a rate similar to wild type (van Maris et al., Directed Evolution of Pyruvate Decarboxylase-Negative Saccharomyces cerevisiae, Yielding a C2-Independent, Glucose-Tolerant, and Pyruvate-Hyperproducing Yeast, Applied and Environmental Microbiology, 2004, 70(1), 159-166).

Another byproduct that would decrease yield of isobutanol is glycerol. Glycerol is produced by 1) the reduction of the glycolysis intermediate, dihydroxyacetone phosphate (DHAP), to glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD.sup.+ by Glycerol-3-phosphate dehydrogenase (GPD) followed by 2) the dephosphorylation of glycerol-3-phophate to glycerol by glycerol-3-phosphatase (GPP). Production of glycerol results in loss of carbons as well as reducing equivalents. Reduction of GPD activity would increase yield of isobutanol. Reduction of GPD activity in addition to PDC activity would further increase yield of isobutanol. Reduction of glycerol production has been reported to increase yield of ethanol production (Nissen et al., Anaerobic and aerobic batch cultivation of Saccharomyces cerevisiae mutants impaired in glycerol synthesis, Yeast, 2000, 16, 463-474; Nevoigt et al., Method of modifying a yeast cell for the production of ethanol, WO 2009/056984). Disruption of this pathway has also been reported to increase yield of lactate in a yeast engineered to produce lactate instead of ethanol (Dundon et al., Yeast cells having disrupted pathway from dihydroxyacetone phosphate to glycerol, US 2009/0053782).

In one embodiment, the microorganism is a crab-tree positive yeast with reduced or no GPD activity. In another embodiment, the microorganism is a crab-tree positive yeast with reduced or no GPD activity, and expresses an isobutanol biosynthetic pathway and produces isobutanol. In yet another embodiment, the microorganism is a crab-tree positive yeast with reduced or no GPD activity and with reduced or no PDC activity. In another embodiment, the microorganism is a crab-tree positive yeast with reduced or no GPD activity, with reduced or no PDC activity, and expresses an isobutanol biosynthetic pathway and produces isobutanol.

In another embodiment, the microorganism is a crab-tree negative yeast with reduced or no GPD activity. In another embodiment, the microorganism is a crab-tree negative yeast with reduced or no GPD activity, expresses the isobutanol biosynthetic pathway and produces isobutanol. In yet another embodiment, the microorganism is a crab-tree negative yeast with reduced or no GPD activity and with reduced or no PDC activity. In another embodiment, the microorganism is a crab-tree negative yeast with reduced or no GPD activity, with reduced or no PDC activity, expresses an an isobutanol biosynthetic pathway and produces isobutanol.

Any method can be used to identify genes that encode for enzymes with pyruvate decarboxylase (PDC) activity. PDC catalyzes the decarboxylation of pyruvate to form acetaldehyde. Generally, homologous or similar PDC genes and/or homologous or similar PDC enzymes can be identified by functional, structural, and/or genetic analysis. In most cases, homologous or similar PDC genes and/or homologous or similar PDC enzymes will have functional, structural, or genetic similarities. Techniques known to those skilled in the art may be suitable to identify 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 PDC 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 PDC 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, 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. Furthermore, PDC activity can be determined phenotypically. For example, ethanol production under fermentative conditions can be assessed. A lack of ethanol production may be indicative of a yeast microorganism with no PDC activity.

Any method can be used to identify genes that encode for enzymes with glycerol-3-phosphate dehydrogenase (GPD) activity. GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) with the corresponding oxidation of NADH to NAD+. Generally, homologous or similar GPD genes and/or homologous or similar GPD enzymes can be identified by functional, structural, and/or genetic analysis. In most cases, homologous or similar GPD genes and/or homologous or similar GPD enzymes will have functional, structural, or genetic similarities. Techniques known to those skilled in the art may be suitable to identify 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 GPD 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 GPD 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, 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. Furthermore, GPD activity can be determined phenotypically. For example, glycerol production under fermentative conditions can be assessed. A lack of glycerol production may be indicative of a yeast microorganism with no GPD activity.

The recombinant microorganism may further include metabolic pathways for the fermentation of a C3-C5 alcohols 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 an 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 NADH as a cofactor, whereas the xylitol-to-xylulose step uses NADPH as a cofactor. 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 sugar. Accordingly, a yeast species that can efficiently ferment xylose and other pentose sugars into a desired fermentation product is therefore very desirable.

Thus, in one aspect, the recombinant microorganism is engineered to express a functional exogenous xylose isomerase. Exogenous xylose isomerases functional in yeast are known in the art. See, e.g., Rajgarhia et al, US20060234364, which is herein incorporated by reference in its entirety. In an embodiment according to this aspect, the exogenous xylose isomerase 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.

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 mutation 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.

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

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, a process sometimes called "codon optimization" or "controlling for species codon bias."

Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl. Acids Res. 17:477-508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon (Dalphin et al. (1996) Nucl. Acids Res. 24: 216-218). 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.

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 they 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.

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

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

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 et al., 1994, hereby incorporated herein by reference).

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), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1.

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 (Altschul, S. F., et al. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410; Gish, W. and States, D. J. (1993) "Identification of protein coding regions by database similarity search." Nature Genet. 3:266-272; Madden, T. L., et al. (1996) "Applications of network BLAST server" Meth. Enzymol. 266:131-141; Altschul, S. F., et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389-3402; Zhang, J. and Madden, T. L. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation." Genome Res. 7:649-656), especially blastp or tblastn (Altschul, S. F., et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389-3402). Typical parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.

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 other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, W. R. (1990) "Rapid and Sensitive Sequence Comparison with FASTP and FASTA" Meth. Enzymol. 183:63-98). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, hereby incorporated herein by reference.

It is understood that a range of microorganisms can be modified to include recombinant metabolic pathways suitable for the production of C3-C5 alcohols, including isobutanol. In various embodiments, microorganisms may be selected from bacterial or yeast microorganisms. Microorganisms for the production of C3-C5 alcohols, including isobutanol may be selected based on certain characteristics:

One characteristic may include the ability to metabolize a carbon source in the presence of a C3-C5 alcohol, including isobutanol. A microorganism capable of metabolizing a carbon source at a high isobutanol concentration is more suitable as a production microorganism compared to a microorganism capable of metabolizing a carbon source at a low isobutanol concentration. Another characteristic may include the property that the microorganism is selected to convert various carbon sources into C3-C5 alcohols, including isobutanol. 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.

Another characteristic specific to a yeast microorganism may include the property that the microorganism is able to metabolize a carbon source in the absence of pyruvate decarboxylase (PDC). In an embodiment, it is preferable that the yeast microorganism is able to metabolize 5- and 6-carbon sugar in the absence of PDC. In one embodiment, it is even more preferred that a yeast microorganism is able to grow on 5- and 6-carbon sugars in the absence of PDC.

Another characteristic may include the property that the wild-type or parental microorganism 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. Non-fermenting 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).

Thus, 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 isobutanol pathway. Fermentative pathways contribute to low yield and low productivity of isobutanol. Accordingly, deletion of PDC may increase yield and productivity of isobutanol. In one embodiment, the yeast microorganisms may be selected from the "Saccharomyces Yeast Clade", defined as an ascomycetous yeast taxonomic class by Kurtzman and Robnett in 1998 ("Identification and phylogeny of ascomycetous yeast from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences." Antonie van Leeuwenhoek 73: 331-371, see FIG. 2 (see Original Patent) of Leeuwenhook reference). They were able to determine the relatedness of yeast of approximately 500 yeast species by comparing the nucleotide sequence of the D1/D2 domain at the 5' end of the gene encoding the large ribosomal subunit 26S. In pair-wise comparisons of the D1/D2 nucleotide sequence of S. cerevisiae and the two most distant yeast in the Saccharomyces clade: K. lactis and K. marxianus, yeast from this clade share greater than 80% identity.

An ancient whole genome duplication (WGD) event occurred during the evolution of hemiascomycete yeast was discovered using comparative genomics tools (Kellis et al 2004 "Proof and evolutionary analysis of ancient genome duplication in the yeast S. cerevisiae." Nature 428:617-624. Dujon et al 2004 "Genome evolution in yeasts." Nature 430:35-44. Langkjaer et al 2003 "Yeast genome duplication was followed by asynchronous differentiation of duplicated genes." Nature 428:848-852. Wolfe and Shields 1997 "Molecular evidence for an ancient duplication of the entire yeast genome." Nature 387:708-713.) 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).

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. casteffi, and C. glabrata.

In another embodiment, a method provided herein includes a recombinant organism that is a Saccharomyces sensu stricto yeast microorganism. In one aspect, a Saccharomyces sensu stricto yeast microorganism is selected from one of the species: S. cerevisiae, S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis or hybrids thereof.

In another embodiment, the yeast microorganism may be selected from a pre-whole genome duplication (pre-WBD) yeast genus including but not limited to Saccharomyces, Kluyveromyces, Issatchenkia, Candida, Pichia, Debaryomyces, Hansenula, Pachysolen, 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, D. hansenii, H. anomala, P. tannophilis, I. orientalis, Y. lipolytica, and S. pombe.

A yeast microorganism may be either Crabtree-negative or Crabtree-positive. A yeast cell having a Crabtree-negative phenotype is any yeast cell that does not exhibit the Crabtree effect. The term "Crabtree-negative" refers to both naturally occurring and genetically modified organisms. Briefly, the Crabtree effect is defined as the inhibition of oxygen consumption by a microorganism when cultured under aerobic conditions due to the presence of a high concentration of glucose (e.g., 50 g-glucose L.sup.-1). In other words, a yeast cell having a Crabtree-positive phenotype continues to ferment irrespective of oxygen availability due to the presence of glucose, while a yeast cell having a Crabtree-negative phenotype does not exhibit glucose mediated inhibition of oxygen consumption.

Accordingly, in one embodiment the yeast microorgnanism may be selected from a yeast with a Crabtree-negative phenotype including but not limited to the following genera: Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida. Crabtree-negative species include but are not limited to: K. lactis, K. marxianus, P. anomala, P. stipitis, H. anomala, I. orientalis, and C. utilis.

In another embodiment, the yeast microorganism may be selected from a 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. casteffi, S. kluyveri, K. thermotolerans, C. glabrata, Z. bailli, Z. rouxii, D. hansenii, P. pastorius, and S. pombe.

Bacterial Microorganisms may be selected from a number of genera, including but not limited to Arthrobacter, Bacillus, Brevibacterium, Clostridium, Corynebacterium, Cyanobacterium, Escherichia, Gluconobacter, Lactobacillus, Nocardia, Pseudomonas, Rhodococcus, Saccharomyces, Shewanella, Streptomyces, Xanthomonas, and Zymomonas. In another embodiment, such hosts are Corynebacterium, Cyanobacterium, E. coli or Pseudomonas. In another embodiment, such hosts are E. coli W3110, E. coli B, Pseudomonas oleovorans, Pseudomonas fluorescens, or Pseudomonas putida.

One exemplary metabolic pathway for the conversion of a carbon source to a C3-C5 alcohol via pyruvate begins with the conversion of glucose to pyruvate via glycolysis. Glycolysis also produces 2 moles of NADH and 2 moles of ATP. Two moles of pyruvate are then used to produce one mole of isobutanol (PCT/US2006/041602, PCT/US2008/053514). Alternative isobutanol pathways have been described in International Patent Application No PCT/US2006/041602 and in Dickinson et al., Journal of Biological Chemistry 273:25751-15756 (1998).

Accordingly, the engineered isobutanol pathway to convert pyruvate to isobutanol can be, but is not limited to, the following reactions: 2 pyruvate.fwdarw.acetolactate+CO.sub.2 1. acetolactate+NADPH.fwdarw.2,3-dihydroxyisovalerate+NADP+ 2. 2,3-dihydroxyisovalerate.fwdarw.alpha-ketoisovalerate 3. alpha-ketoisovalerate.fwdarw.isobutyraldehyde+CO.sub.2 4. isobutyraldehyde+NADPH.fwdarw.isobutanol+NADP.sup.+ 5.

These reactions are carried out by the enzymes 1) Acetolactate Synthase (ALS), 2) Ketol-acid Reducto-Isomerase (KARI), 3) Dihydroxy-acid dehydratase (DHAD), 4) Keto-isovalerate decarboxylase (KIVD), and 5) an Alcohol Dehydrogenase (ADH).

In another embodiment, the microorganism is engineered to overexpress these enzymes. For example, ALS can be encoded by the aisS gene of B. subtilis, aisS of L. lactis, or the ilvK gene of K. pneumonia. For example, KARI can be encoded by the ilvC genes of E. coli, C. glutamicum, M. maripaludis, or Piromyces sp E2. For example, DHAD can be encoded by the ilvD genes of E. coli, L. lactis, or C. glutamicum, or by the ILV3 gene from S. cerevisiae. KIVD can be encoded by the kivd gene of L. lactis. ADH can be encoded by ADH2, ADH6, or ADH7 of S. cerevisiae, by the adhA gene product of L. lactis, or by an ADH from D. melanogaster.

The microorganism of the invention may be engineered to have increased ability to convert pyruvate to a C3-C5 alcohol, including isobutanol. In one embodiment, the microorganism may be engineered to have increased ability to convert pyruvate to isobutyraldehyde. In another embodiment, the microorganism may be engineered to have increased ability to convert pyruvate to keto-isovalerate. In another embodiment, the microorganism may be engineered to have increased ability to convert pyruvate to 2,3-dihydroxyisovalerate. In another embodiment, the microorganism may be engineered to have increased ability to convert pyruvate to acetolactate.

Furthermore, any of the genes encoding the foregoing enzymes (or any others mentioned herein (or any of the regulatory elements that control or modulate expression thereof)) may be optimized by genetic/protein engineering techniques, such as directed evolution or rational mutagenesis.

It is understood that various microorganisms can act as "sources" for genetic material encoding target enzymes suitable for use in a recombinant microorganism provided herein. For example, In addition, genes encoding these enzymes can be identified from other fungal and bacterial species and can be expressed for the modulation of this pathway. A variety of eukaryotic organisms could serve as sources for these enzymes, including, but not limited to, Drosophila spp., including D. melanogaster, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including Y. stipitis, Torulaspora pretoriensis, Schizosaccharomyces spp., including S. pombe, Cryptococcus spp., Aspergillus spp., Neurospora spp., or Ustilago spp. Sources of genes from anaerobic fungi include, but not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp. Sources of prokaryotic enzymes that are useful include, but not limited to, Escherichia. coli, Klebsiella spp., including K. pneumoniae, Zymomonas mobilis, Staphylococcus aureus, Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., and Salmonella spp.

Claim 1 of 19 Claims

1. An isolated nucleic acid molecule encoding an NADH-dependent ketol-acid reductoisomerase, wherein said NADH-dependent ketol-acid reductoisomerase comprises SEQ ID NO: 44.

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