Microorganisms for the production of adipic acid and other compounds

Abstract

The invention provides a non-naturally occurring microbial organism having an adipate, 6-aminocaproic acid or caprolactam pathway. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in the respective adipate, 6-aminocaproic acid or caprolactam pathway. The invention additionally provides a method for producing adipate, 6-aminocaproic acid or caprolactam. The method can include culturing an adipate, 6-aminocaproic acid or caprolactam producing microbial organism, where the microbial organism expresses at least one exogenous nucleic acid encoding an adipate, 6-aminocaproic acid or caprolactam pathway enzyme in a sufficient amount to produce the respective product, under conditions and for a sufficient period of time to produce adipate, 6-aminocaproic acid or caprolactam.

1 - 112 . (canceled) 
     
     
         113 . Method for preparing an adipate ester or adipate thioester, comprising converting a 2,3-dehydroadipate ester or 2,3-dehydroadipate thioester into the adipate ester or thioester in the presence of a biocatalyst. 
     
     
         114 . Method according to  claim 113 , wherein the biocatalyst comprises an enzyme capable of catalysing the reduction of a carbon-carbon double bond of a 2,3-enoate moiety or a 2-enoyl moiety. 
     
     
         115 . Method according to  claim 114 , wherein the biocatalyst comprises an enzyme selected from the group of oxidoreductases acting on the HC—CH group of donors (EC 1.3.1. or 1.3.99), preferably from the group of oxidoreductases (EC 1.3.1 and EC 1.3.99), preferably from the group of enoyl-CoA reductases EC 1.3.1.8, EC 1.3.1.38 and EC 1.3.1.44, from the group of enoyl-[acyl-carrier-protein] reductases EC 1.3.1.9, EC 1.3.1.10 and EC 1.3.1.39, and from the group butyryl-CoA dehydrogenases (EC 1.399.2), acyl-CoA dehydrogenase (1.3.99.3) and long-chain-acyl-CoA dehydrogenase (EC 1.3.99.13). 
     
     
         116 . Method according to  claim 113 , wherein the biocatalyst comprises an enzyme, which enzyme comprises an amino acid sequence selected from the group of amino acid sequences represented by:
 an enzyme from  Penicillium chrysogenum, Bos Taurus, Cavia  sp.,  Candida tropicalis, Clostridium kluyveri, Euglena gracilis, Homo sapiens, Mus musculus, Rattus norvegicus, Saccharomyces cerevisiae, Microscilla marina, Clostridium beijerinckii, Aeromonas hydrophila  subsp.  hydrophila, Yarrowia lipolytica, Megasphaera elsdenii, Acinetobacter  sp.,  Deinococcus radiodurans , or  Arabidopsis thaliana  that catalyses the reduction of a carbon-carbon double bond of a 2,3-enoate moiety or a 2-enoyl moiety, an electron transfer flavoprotein from  Clostridium kluyveri  or  Homo sapiens , or a homolog thereof; or   a gene product of bcd from  Clostridium acetobutylicum , TER from  Euglena gracilis , TDE0597 from  Treponema denticola , etfA from  Clostridium acetobutylicum , etfB from  Clostridium acetobutylicum , or a homolog thereof.   
     
     
         117 . Method according to  claim 113 , wherein the biocatalyst comprises an enzyme of an organism selected from the group of  Escherichia  (in particular  E. coli ),  Vibrio, Bacillus  (in particular  B. subtilis ),  Clostridia  (in particular  C. kluyveri, C. acetobutylicum  and  C. perfringens ),  Streptomyces  (in particular  S. coelicolor  and  S. avermitilis ),  Pseudomonas  (in particular  P. putida  and  P. aeruginosa ),  Shewanella, Xanthomonas, Xylella, Yersinia, Treponema  (in particular  T. denticola ),  Eubacterium  (in particular  E. pyruvativorans ),  Micorscilla  (in particular  Micorscilla marina ),  Aeromonas  (in particular  Aeromonas hydrophila ),  Megasphera  (in particular  Megasphera elsdenii ),  Acinetobacter  sp.,  Deinococcus  (in particular  Deinococcus radiourans ),  Yarrowia  (in particular  Yarrowia lypolytica ),  Euglenozoa  (in particular  Euglena gracilis ),  Saccharomyces  (in particular  S. cerevisiae ),  Kluyveromyces  (in particular  K. lactis ),  Schizosaccharomyces  (in particular  S. pombe ),  Candida  (in particular  C. tropicalis ),  Aspergillus  (in particular  A. niger  and  A. nidulans ),  Penicillium  (in particular  P. chrysogenum ),  Arabidopsis  (in particular  A. thaliana ),  Homo sapiens, Rattus norvegicus, Bos Taurus, Cavia  sp.,  Caenorhabditis elegans , and  Drosophila melanogaster.    
     
     
         118 . Method according to  claim 113 , wherein 2,3-dehydroadipate ester or 2,3-dehydroadipate thioester is prepared by converting a 3-hydroxyadipate ester or 3-hydroxyadipate thioester. 
     
     
         119 . Method according to  claim 118 , wherein the 3-hydroxyadipate ester or thioester is biocatalytically converted in the presence of a biocatalyst capable of catalysing the dehydration of a 3-hydroxyacyl ester or 3-hydroxyacyl thioester to a 2-enoyl ester or thioester, preferably a biocatalyst comprising an enzyme selected from the group of hydrolyases (EC. 4.2.1), preferably from the group of enoyl-CoA hydratases (EC 4.2.1.17), 3-hydroxybutyryl-CoA dehydratases (EC 4.2.1.55) and long-chain-enoyl-CoA hydratases (EC 4.2.1.74). 
     
     
         120 . Method according to  claim 119 , wherein said biocatalyst comprises an enzyme of an organism selected from the group of  Acinetobacter  (in particular  Acinetobacter  sp. strain ADP1 and  A. calcoaceticus ),  Alicaligenes  (in particular  Alicaligenes  D2),  Aspergillus  (in particular  A. niger ),  Azoarcus  (in particular  A. evansii ),  Bacillus  (in particular  B. halodurans ),  Corynebacterium  (in particular  C. glutamicum  and  C. aurantiacum ),  E. coli, Flavobacterium, Neurospora  (in particular  N. crassa ),  Penicillium  (in particular  P. chrysogenum ),  Pseudomonas  (in particular  P. putida  and  P. fluorescens ),  Rhodopseudomonas  (in particular  R. palustris ),  Rhodococcus  (in particular  Rhodococcus  sp strain RHA1),  Aeromonas  (in particular  A. caviae ),  Clostridium  (in particular  C. acetobutylicumi  and  C. kluyveri ),  Gossypium  (in particular  G. hirsutum ),  Rhodospirillum  (in particular  R. rubrumi ), and  Ralstonia  (in particular  Ralstonia eutropha ),  Euglenozoa  (in particular  Euglena gracilis, Megasphera  (in particular  M. elsdenii ), and  Saccharomyces  (in particular  S. cerevisiae ), and mammals (in particular  Bos taurus, Homo sapiens, Rattus norvegicus , and  Sus scrofa ). 
     
     
         121 . Method according to  claim 118 , wherein the 3-hydroxyadipate ester or thioester is prepared by converting a 3-oxoadipate ester or 3-oxoadipate thioester. 
     
     
         122 . Method according to  claim 121 , comprising biocatalytically converting the 3-oxoadipate ester or 3-oxoadipate thioester, in the presence of a biocatalyst, in particular a biocatalyst capable of catalysing the reduction of a carbonyl group to an alcohol group or capable of catalysing the reduction of a 3-oxoacyl ester or 3-oxoacyl thioester to the corresponding 3-hydroxyacyl ester or thioester, selected from the group of dehydrogenases (E.C. 1.1.1), preferably from the group 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 and EC 1.1.1.36), 3-hydroxybutanoyl-CoA dehydrogenase (EC 1.1.1.157), 3-hydroxypimeloyl-CoA dehydrogenase (EC 1.1.1.259) and long-chain-3-hydroxyacyl-CoA dehydrogenases (EC 1.1.1.211). 
     
     
         123 . Method according to  claim 122 , wherein said biocatalyst comprises an enzyme of an organism selected from the group of  Acinetobacter  (in particular  Acinetobacter  sp. Strain ADP1 and  A. calcoaceticus ),  Alicaligenes  (in particular  Alicaligenes strain  D2 and  A. eutrophus ),  Arzoarcus  (in particular  A. evansii ),  Bacillus  (in particular  B. halodurans ),  Bordetella  (in particular  B. pertussis ),  Burkholderia  (in particular  B. pseudomallei  and  B. xenovorans ),  Corynebacterium  (in particular  C. glutamicum, C. aurantiacum  and  C. efficiens ),  Deinococcus  (in particular  D. radiodurans ),  E. coli, Flavobacterium, Klebsiella  (in particular  K. pneumonia ),  Pseudomonas  (in particular  P. putida  and  P. fluorescens ),  Rhodopseudomonas  (in particular  R. palustris ),  Rodococcus  (in particular  R. erythropolis, R. opacus , and  Rodococcus  sp strain RHA1),  Aspergillus  (in particular  A. niger ),  Neurospora  (in particular  N. crassa ),  Penicillium  (in particular  P. chrysogenum ),  Saccharomyces, Bos taurus, Rattus norvegicus, Sus scrofa, Homo sapiens. Clostridia  (in particular  C. acetobutylicum  and  C. kluyveri ),  Euglenozoa  (in particular)  Euglena gracilis, Megasphera  (in particular  Megasphera elsdenii ),  Ralstonia  (in particular  Ralstonia eutropha ), and  Zoogloea  (in particular  Zoogloea ramigera ). 
     
     
         124 . Method according to  claim 121 , wherein the 3-oxoadipate ester or 3-oxoadipate thioester is prepared by reacting a succinate ester or succinate thioester with an acetate ester or acetate thioester. 
     
     
         125 . Method according to  claim 124 , comprising biocatalytically reacting a succinate ester or thioester with a acetate ester or thioester, in the presence of a biocatalyst, preferably a biocatalyst comprising an enzyme capable of acetyl-group transfer selected from the group of acyltransferases (E.C. 2.3.1), in particular an acyltransferase from the group of acetyl-CoA:acetyl-CoA C-acetyltransferases (EC 23.1.9), acyl-CoA:acetyl-CoA C-acyltransferases (EC 2.3.1.16) and succinyl-CoA:acetyl-CoA C-succinyltransferases (EC 2.3.1.174), more in particular an enzyme comprising an amino acid sequence as identified in:
 an enzyme from  Penicillium chrysogenum, Acinetobacter  sp.,  Clostridium kluyveri, Corynebacterium glutamicum, Escherichia coli, Pseudomonas putida, Ralstonia eutropha , or  Rhodococcus  sp. that catalyses the formation of 3-oxoadipate ester or thioester from succinate ester or thioester and acetate ester or thioester, or a homolog thereof; or   a gene product of paaJ from  Escherichia coli , pcaF from  Pseudomonas knackmussii  (B13), phaD from  Pseudomonas putida , paaE from  Pseudomonas fluorescens ; paaJ from  Klebsiella pneumoniae , paaJ from  Serratia proteamaculans , paaJ from  Pseudomonas putida , pcaF from  Streptomyces  sp. 2065, pcaF from  Pseudomonas putida , pcaF from  Pseudomonas aeruginosa , atoB from  Escherichia coli , yqeF from  Escherichia coli , phaA from  Ralstonia eutropha , bktB from  Ralstonia eutropha , thiA from  Clostridium acetobutylicum , thiB from  Clostridium acetobutylicum , or a homolog thereof.   
     
     
         126 . Method according to  claim 124 , wherein said biocatalyst comprises an enzyme of an organism selected from the group of,  Acinetobacter  (in particular  Acinetobacter  sp. Strain ADP1 and  A. calcoaceticus ),  Agrobacterium  (in particular  A. tumefaciens ), Alicaligenes (in particular  Alicaligenes  strains D2 and  A. eutrophus ),  Arthrobacter, Arzoarcus  (in particular  A. evansii ),  Azomonas, Azotobacter, Bacillus  (in particular  B. halodurans ),  Beijerinckia, Bradyrhizobium, Burkholderia, Clostridia  (in particular  C. kluyveri ),  Comamonas, Corynebacterium  (in particular  C. glutamicum  and  C. aurantiacum ),  E. coli, Enterobacter, Flavobacterium, Megasphera  (in particular  M. elsdenii ),  Norcadia, Pseudomonas  (in particular  P. putida, P. aeruginosa  and  Pseudomonas  sp. strain B13),  Ralstonia  (in particular  R. eutropha ),  Rhizobium, Rhodopseudomonas  (in particular  R. palustris ),  Rodococcus  (in particular  R. ervthropolis, R. opacus , and  Rodococcus  sp strain RHA1),  Aspergillus  (in particular  A. niger ),  Euglenozoa  (in particular  Euglena gracilis ),  Neurospora  (in particular  N. crassa ),  Penicillium  (in particular  P. chrysogenum ),  Rhodotorula, Saccharomyces, Trichosporon  (in particular  T. cutaneum ). 
     
     
         127 . Method according to  claim 113 , wherein any of said esters is selected from the group of biological activating groups, in particular from the group of coenzyme A, phospho-pantetheine, which may be bound to an acyl or peptidyl carrier protein, N-acetyl-cysteamine, methyl-thio-glycolate, methyl-mercapto-propionate, ethyl-mercapto-propionate, methyl-mercapto-butyrate, methyl-mercapto-butyrate and mercaptopropionate. 
     
     
         128 . Method for preparing adipic acid comprising preparing adipate ester or adipate thioester in a method according to  claim 113 , and hydrolysing the adipate ester or adipate thioester obtained in a method according to any of the preceding claims, wherein the hydrolysis is preferably catalysed by a biocatalyst, in particular by a biocatalyst comprising an enzyme selected from the group of hydrolases (EC 3.1.2). 
     
     
         129 . Method for preparing adipic acid, comprising preparing adipate ester or adipate thioester in a method according to  claim 113 , and transferring the activating group of the adipate ester or adipate thioester obtained wherein the activating group transfer is catalysed by a biocatalyst, preferably a biocatalyst comprising an enzyme catalysing the transfer of sulphur-containing groups (EC 2.8), more preferably from the group of CoA transferases (EC 2.8.3). 
     
     
         130 . Method for preparing adipate semialdehyde comprising preparing adipate ester or adipate thioester in a method according to  claim 113 , or preparing adipic acid and converting the adipate ester, the adipate thioester or the adipic acid into adipate semialdehyde. 
     
     
         131 . Method according to  claim 130 , wherein the conversion into adipate semialdehyde is catalysed by a biocatalyst, preferably a biocatalyst comprising an enzyme selected from the group of oxidoreductases (EC 1.2.1), preferably from the group of aldehyde dehydrogenase (EC 1.2.1.3, EC 1.2.1.4 and EC malonate-semialdehyde dehydrogenase (EC 1.2.1.15), succinate-semialdehyde dehydrogenase (EC 1.2.1.16 and EC 1.2.1.24); glutarate-semialdehyde dehydrogenase (EC 1.2.1.20), aminoadipate semialdehyde dehydrogenase (EC 1.2.1.31), adipate semialdehyde dehydrogenase (EC 1.2.1.63) or from the group of aldehyde dehydrogenases (acetylating) (EC 1.2.1.10), fatty acyl-CoA reductases (EC 1.2.1.42), long-chain-fatty-acyl-CoA reductases (EC 1.2.1.50), butanal dehydrogenases (EC 1.2.1.57) and succinate semialdehyde dehydrogenases (acetylating). 
     
     
         132 . Method for preparing 6-amino caproic acid, comprising preparing adipate semialdehyde in a method according to  claim 130 , and converting the adipate semialdehyde into 6-amino caproic acid. 
     
     
         133 . Method according to  claim 132 , wherein the conversion is catalysed by a biocatalyst, in particular a biocatalyst capable of catalysing a transamination and/or a reductive amination, preferably a biocatalyst comprising an enzyme selected from the group of aminotransferases (EC 2.6.1) and amino acid dehydrogenases (EC 1.4.1), more preferably from the group of β-aminoisobutyrate:α-ketoglutarate aminotransferases, β-alanine aminotransferases, aspartate aminotransferases, 4-amino-butyrate aminotransferases (EC 2.6.1.19), L-lysine 6-aminotransferase (EC 2.6.1.36), 2-aminoadipate aminotransferases (EC 2.6.1.39), 5-aminovalerate aminotransferases (EC 2.6.1.48), 2-aminohexanoate aminotransferases (EC 2.6.1.67), lysine:pyruvate 6-aminotransferases (EC 2.6.1.71), and lysine-6-dehydrogenases (EC 1.4.1.18). 
     
     
         134 . Method according to  claim 132 , wherein an aminotransferase is used comprising an amino acid sequence according to:
 an aminotransferase from  Vibrio fluvialis, Bacillus weihenstephanensis, Pseudomonas aeruginosa , or  Bacillus subtilis , or a homolog of any of these sequences; or   a gene product of gabT from  Escherichia coli , abat from  Mus musculus , gabT from  Pseudomonas fluorescens , abat from  Sus scrofa , or a homolog of any of these sequences.   
     
     
         135 . Method according to  claim 132 , wherein the biocatalyst comprises an enzyme from an organism selected from the group of  Vibrio; Pseudomonas; Bacillus; Mercurialis; Asplenium; Ceratonia ; mammals;  Neurospora; Escherichia; Thermus; Saccharomyces; Brevibacterium; Corynebacterium; Proteus; Agrobacterium; Geobacillus; Acinetobacter; Ralstonia  and  Salmonella.    
     
     
         136 . Method for preparing caprolactam, comprising preparing 6-amino caproic acid in a method according to  claim 132 , and cyclising the 6-amino caproic acid, thereby forming caprolactam. 
     
     
         137 . A host cell comprising one or more nucleic acid sequences encoding an enzyme as defined in  claim 114 , preferably comprising at least two nucleic acid sequences each encoding a different enzyme. 
     
     
         138 . A host cell according to  claim 137 , comprising a nucleic acid sequence encoding a polypeptide as represented by:
 an enzyme from  Penicillium chrysogenum, Bos Taurus, Cavia  sp.,  Candida tropicalis, Clostridium kluyveri, Euglena gracilis, Homo sapiens, Mus musculus, Rattus norvegicus, Saccharomyces cerevisiae, Microscilla marina, Clostridium beijerinckii, Aeromonas hydrophila  subsp.  hydrophila, Yarrowia lipolytica, Megasphaera elsdenii, Acinetobacter  sp.,  Deinococcus radiodurans , or  Arabidopsis thaliana  that is capable of catalysing the reduction of a carbon-carbon double bond of a 2,3-enoate moiety or a 2-enoyl moiety, an electron transfer flavoprotein from  Clostridium kluyveri  or  Homo sapiens , an enzyme from  Clostridium kluyveri, Porphyromonas gingivalis, Propionibacterium freudenreichi , or  Clostridium acetobutylicum  that catalyses the formation of adipate semialdehyde from an adipate ester or an adipate thioester, or a homolog thereof; or   a gene product of bcd from  Clostridium acetobutylicum , TER from  Euglena gracilis , TDE0597 from  Treponema denticola , etfA from  Clostridium acetobutylicum , etfB from  Clostridium acetobutylicum , acr1 from  Acinetobacter calcoaceticus , acr1 from  Acinetobacter  sp. Strain M-1, sucD from  Clostridium kluyveri , or a homolog thereof.   
     
     
         139 . Method according to  claim 113 , comprising contacting cells comprising the biocatalyst—which biocatalyst may comprise a host cell or a different biocatalyst—with a fermentable carbon source, wherein the carbon source contains any of said compounds which are to be converted into the compound to be prepared or wherein the cells prepare the compound to be converted into the compound to be prepared from the carbon source. 
     
     
         140 . Method for preparing adipic acid from succinic acid or an ester or thioester of succinic acid thereof, optionally according to  claim 128 , via a plurality of reactions, wherein at least one of the reactions is catalysed by a biocatalyst. 
     
     
         141 . Method according to  claim 140 , comprising
 (1) providing a succinate ester or thioester and reacting said ester or thioester with an acetate ester or thioester, thereby forming a 3-oxoadipate ester or thioester;   (2) hydrogenating the 3-oxo group of the 3-oxoadipate ester or thioester thereby forming a 3-hydroxyadipate ester or thioester;   (3) dehydrating the 3-hydroxyadipate ester or thioester thereby forming a 2,3-dehydro adipate ester or thioester;   (4) hydrogenating of the C—C double bond of the 2,3-dehydro adipate ester or thioester, thereby forming an adipate ester or thioester; and   (5) hydrolysing the ester bond or thioester bond, thereby forming adipic acid.   
     
     
         142 . Polypeptide comprising an amino acid sequence according to:
 a reductase from  Penicillium chrysogenum  that is capable of catalysing the reduction of a carbon-carbon double bond of a 2,3-enoate moiety or a 2-enoyl, a hydrolase from  Penicillium chrysogenum  that is capable of catalyzing adipate ester or thioester to adipic acid, a dehydrogenase from  Propionibacterium freudenreichi  that is capable of catalysing the formation of adipate semialdehyde from an adipate ester or an adipate thioester, a CoA transferase from  Penicillium chrysogenum  that is capable of catalyzing adipate ester or thioester to adipic acid, or a homologue thereof; or   a gene product of bcd from  Clostridium acetobutylicum , TER from  Euglena gracilis , TDE0597 from  Treponema denticola , etfA from  Clostridium acetobutylicum , etfB from  Clostridium acetobutylicum , acr1 from  Acinetobacter calcoaceticus , acr1 from  Acinetobacter  sp. Strain M-1, sucD from  Clostridium kluyveri , tesB from  Escherichia coli , acot8 from  Homo sapiens , acot8 from  Rattus norvegicus , cat1 from  Clostridium kluyveri , cat2 from  Clostridium kluyveri , cat3 from  Clostridium kluyveri , or a homolog thereof.   
     
     
         143 . Polynucleotide, encoding a polypeptide according to  claim 142 .