Engineered microorganisms capable of producing target compounds under anaerobic conditions

Abstract

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.

1 . A recombinant microorganism comprising an engineered metabolic pathway for producing isobutanol under aerobic and anaerobic conditions, wherein said recombinant microorganism produces isobutanol under anaerobic conditions at a rate higher than a parental microorganism comprising a native or unmodified metabolic pathway. 
     
     
         2 . The recombinant microorganism of  claim 1 , wherein said engineered metabolic pathway comprises an overexpressed transhydrogenase that converts NADH to NADPH. 
     
     
         3 . The recombinant microorganism of  claim 2 , wherein said transhydrogenase is a membrane-bound transhydrogenase. 
     
     
         4 . The recombinant microorganism of  claim 3 , wherein said membrane-bound transhydrogenase is encoded by the  Escherichia coli  pntAB genes. 
     
     
         5 . The recombinant microorganism of  claim 1 , wherein said engineered metabolic pathway comprises an NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. 
     
     
         6 . The recombinant microorganism of  claim 5 , wherein said NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase is encoded by the  Clostridium acetobutylicum  gapC gene or the  Kluyveromyces lactis  GDP1 gene. 
     
     
         7 . The recombinant microorganism of  claim 1 , wherein said engineered metabolic pathway 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. 
     
     
         8 . The recombinant microorganism of  claim 7 , wherein said engineered metabolic pathway comprises genes encoding an NADH-dependent ketol-acid reductoisomerase (KARI) and an NADH-dependent alcohol dehydrogenase (ADH). 
     
     
         9 . The recombinant microorganism of  claim 8 , wherein said KARI and/or said ADH are identified in nature with increased activity using NADH as a cofactor as compared to the wild-type  E. coli  KARI llvC and a native  E. coli  ADH YqhD, respectively. 
     
     
         10 . The recombinant microorganism of  claim 9 , wherein said KARI and/or said ADH show at least a 10-fold higher catalytic efficiency using NADH as the cofactor as compared to the wild-type  E. coli  KARI llvC and a native  E. coli  ADH YqhD, respectively. 
     
     
         11 . The recombinant microorganism of  claim 8 , wherein said KARI and/or said ADH have been modified or mutated to have increased activity using NADH as a cofactor as compared to the wild-type  E. coli  KARI llvC and a native  E. coli  ADH YqhD, respectively. 
     
     
         12 . The recombinant microorganism of  claim 11 , wherein said KARI and/or said ADH show at least a 10-fold higher catalytic efficiency using NADH as the cofactor as compared to the wild-type  E. coli  KARI llvC and a native  E. coli  ADH YqhD, respectively. 
     
     
         13 . The recombinant microorganism of  claim 11 , wherein said KARI and/or said ADH have been modified or mutated to be NADH-dependent. 
     
     
         14 . The recombinant microorganism of  claim 8 , wherein said KARI enhances the recombinant microorganism's ability to convert acetolactate to 2,3-dihydroxyisovalerate under anaerobic conditions. 
     
     
         15 . The recombinant microorganism of  claim 8 , wherein said KARI enhances the recombinant microorganism's ability to utilize NADH for the conversion of acetolactate to 2,3-dihydroxyisovalerate. 
     
     
         16 . The recombinant microorganism of  claim 11 , wherein said KARI comprises two or more mutations or modifications 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. 
     
     
         17 . The recombinant microorganism of  claim 16 , wherein said alanine 71 residue of said KARI is replaced with a serine residue, said arginine 76 residue is replaced with an aspartic acid residue, said serine 78 residue is replaced with an aspartic acid residue, and said glutamine 110 residue is replaced with a valine residue. 
     
     
         18 . The recombinant microorganism of  claim 16 , wherein said KARI has at least about a 25% increased catalytic efficiency with NADH as compared to the wild-type KAR1. 
     
     
         19 . The recombinant microorganism of  claim 16 , wherein the catalytic efficiency of the KARI with NADH is at least about 25% of the catalytic efficiency with NADPH of the wild-type KAR1. 
     
     
         20 . The recombinant microorganism of  claim 16 , wherein the KARI preferentially utilizes NADH rather than NADPH. 
     
     
         21 . The recombinant microorganism of  claim 16 , wherein the KARI demonstrates a switch in cofactor preference from NADPH to NADH as compared to a corresponding wild-type KAR1. 
     
     
         22 . The recombinant microorganism of  claim 16 , wherein the KARI exhibits at least about a 1:1 ratio of catalytic efficiency (k cat /K M ) with NADH over catalytic efficiency with NADPH. 
     
     
         23 . The recombinant microorganism of  claim 16 , wherein the KARI exhibits at least about a 1:10 ratio of K M  for NADH over K M  for NADPH. 
     
     
         24 . The recombinant microorganism of  claim 16 , wherein the KARI is selected from the group consisting of  Escherichia coli  (GenBank No: NP — 418222, SEQ ID NO 13),  Saccharomyces cerevisiae  (GenBank No: NP — 013459, SEQ ID NO: 70),  Methanococcus maripaludis  (GenBank No: YP — 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: Q01292 and CAA40356, SEQ ID NO: 75),  Oryza sativa  (GenBank No: NP — 001056384, SEQ ID NO: 76)  Chlamydomonas reinhardtii  (GenBank No: XP — 001702649, SEQ ID NO: 77),  Neurospora crassa  (GenBank No: XP — 961335, SEQ ID NO: 78),  Schizosaccharomyces pombe  (GenBank No: NP — 001018845, SEQ ID NO: 79),  Laccaria bicolor  (GenBank No: XP — 001880867, SEQ ID NO: 80),  Ignicoccus hospitalis  (GenBank No: YP — 001435197, SEQ ID NO: 81),  Picrophilus torridus  (GenBank No: YP — 023851, SEQ ID NO: 82),  Acidiphilium cryptum  (GenBank No: YP — 001235669, SEQ ID NO: 83),  Cyanobacteria/Synechococcus  sp. (GenBank No: YP — 473733, SEQ ID NO: 84),  Zymomonas mobilis  (GenBank No: YP — 162876, SEQ ID NO: 85),  Bacteroides thetaiotaomicron  (GenBank No: NP — 810987, SEQ ID NO: 86),  Vibrio fischeri  (GenBank No: YP — 205911, SEQ ID NO: 87),  Shewanella  sp (GenBank No: YP — 732498, SEQ ID NO: 88),  Gramella forsetti  (GenBank No: YP — 862142, SEQ ID NO: 89),  Psychromonas ingrhamaii  (GenBank No: YP — 942294, SEQ ID NO: 90), and  Cytophaga hutchinsonii  (GenBank No: YP — 677763, SEQ ID NO: 91). 
     
     
         25 . The recombinant microorganism of  claim 16 , wherein the KARI is derived from a genus selected from the group consisting of  Escherichia, Zymomonas, Staphylococcus, Corynebacterium, Clostridium, Salmonella, Pseudomonas, Bacillus, Lactobacillus, Lactococcus, Enterobactor, Enterococcus, Klebsiella, Saccharomyces, Kluyveromyces, Pichia, Hansenula, Candida, Trichosporon, Yamadazyma, Schizosaccharomyces, Cryptococcus, Aspergillus, Neurospora, Piromyces, Orpinomyces , and  Neocallimastix, Piromyces, Buchnera, Spinacia, Oryza, Chlamydomonas, Neurospora, Schizosaccharomyces, Laccaria, Ignicoccus, Picrophilus, Acidiphilium, Cyanobacteria/Synechococcus, Zymomonas, Bacteroides, Methanococcus, Vibrio, Shewanella, Gramella, Psychromonas , and  Cytophaga.    
     
     
         26 . The recombinant microorganism of  claim 16 , wherein the KARI has further been codon optimized for expression in a host cell, and wherein said host cell is yeast. 
     
     
         27 . The recombinant microorganism of  claim 16 , wherein the KARI is selected from the 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. 
     
     
         28 . The recombinant microorganism of  claim 1 , 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. 
     
     
         29 . The recombinant microorganism of  claim 28 , wherein said first dehydrogenase is encoded by the  E. coli  gene maeA and the second dehydrogenase is encoded by the  E. coli  gene maeB or wherein said first dehydrogenase is encoded by the  E. coli  gene maeA and the second dehydrogenase is encoded by the  S. cerevisiae  gene MAE1. 
     
     
         30 . The recombinant microorganism of  claim 1 , 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. 
     
     
         31 . The recombinant microorganism of  claim 30 , wherein said engineered metabolic pathway further comprises the overexpression of the genes mdh and maeB or wherein said engineered metabolic pathway further comprises the overexpression of the  S. cerevisiae  genes MDH1 and MAE1. 
     
     
         32 . A recombinant microorganism according to  claim 1 , wherein said 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. 
     
     
         33 . The recombinant microorganism of  claim 1 , wherein said recombinant microorganism produces said isobutanol under anaerobic conditions at a yield which is at least about the same yield as under aerobic conditions. 
     
     
         34 . The recombinant microorganism of  claim 1 , wherein said recombinant microorganism produces isobutanol at substantially the same rate under anaerobic conditions as the parental microorganism produces under aerobic conditions. 
     
     
         35 . The recombinant microorganism of  claim 1 , wherein said engineered metabolic pathway is balanced with respect to NADH and NADPH as compared to a native or unmodified metabolic pathway from a corresponding parental microorganism, and wherein said native or unmodified metabolic pathway is not balanced with respect to NADH and NADPH. 
     
     
         36 . A method of producing isobutanol under anaerobic conditions, comprising:
 (a) providing a recombinant microorganism according to  claim 1 ;   (b) cultivating the recombinant microorganism under anaerobic conditions in a culture medium containing a feedstock providing the carbon source, until a recoverable quantity of isobutanol is produced; and   (c) recovering isobutanol.   
     
     
         37 . The method according to  claim 36 , wherein the recombinant microorganism is selected from:
 (i)  E. coli  that produces isobutanol at a yield of greater than 80% theoretical; and   (ii) Yeast that produces isobutanol at a yield of greater than 30% theoretical.   
     
     
         38 . The method according to  claim 36 , wherein isobutanol is produced under anaerobic conditions at a yield which is at least about the same yield as under aerobic conditions. 
     
     
         39 . A mutant ketol-acid reductoisomerase (KARI) comprising two or more mutations or modifications 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.