Microorganisms and methods for the biosynthesis of adipate, hexamethylenediamine and 6-aminocaproic acid

Abstract

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

1 - 282 . (canceled) 
     
     
         283 . Method for preparing 6-aminocaproic acid, wherein the 6-aminocaproic acid is prepared from 2-oxoheptane-1,7-dioate (2-OHD), using at least one biocatalyst. 
     
     
         284 . Method for preparing 6-aminocaproic acid, wherein the 6-aminocaproic acid is prepared from adipate semialdehyde, using at least one biocatalyst. 
     
     
         285 . Method according to  claim 283 , wherein the biocatalyst comprises an enzyme capable of catalysing a transamination and/or a reductive amination. 
     
     
         286 . Method according to  claim 285 , wherein the enzyme capable of catalysing a transamination and/or a reductive amination is selected from the group of aminotransferases (E.C. 2.6.1) and amino acid dehydrogenases (E.C. 1.4.1). 
     
     
         287 . Method according to  claim 286 , wherein the aminotransferase or amino acid dehydrogenase is selected from the group of β-aminoisobutyrate: a-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). 
     
     
         288 . Method according to  claim 283 , wherein the enzyme is selected from the group of enzymes capable of catalysing a transamination and/or a reductive amination 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; Salmonella; Rhodobacter  and  Staphylococcus , in particular from an organism selected from the group of  Bacillus subtilis, Bacillus weihenstephanensis, Rhodobacter sphaeroides, Staphylococcus aureus, Legionella pneumophila, Nitrosomonas europaea, Neisseria gonorrhoeae, Pseudomonas syringae, Rhodopseudomonas palustris, Vibrio fluvialis  and  Pseudomonas aeruginosa.    
     
     
         289 . Method according to  claim 286 , wherein an aminotransferase is used comprising an amino acid sequence according to: an enzyme from  Vibrio fluvialis, Bacillus weihenstephanensis, Pseudomonas aeruginosa, Bacillus subtilis , or  Pseudomonas aeruginosa  that catalyses the conversion of adipate semialdehyde to 6-aminocaproic acid; an enzyme from  Vibrio fluvialis, Pseudomonas aeruginosa, Pseudomonas syringae, Bacillus subtilis, Rhodobaeter sphaeroides, Legionella pneumophila, Nitrosomonas europaea, Neisseria gonorrhoeae, Pseudomonas aeruginosa , or  Rhodopseudomonas palustris  that catalyses the conversion of 2-oxoheptane-1,7-dioate (2-OHD) to 2-aminoheptane-1,7-dioate (2-AHD); a gene product of gabT from  Escherichia coli , puuE from  Escherichia coli , abat from  Mus musculus , gabT from  Pseudomonas fluorescens , or abat from  Sus scrofa ; or a homologue of any of these sequences. 
     
     
         290 . Method according to  claim 283 , wherein the biocatalyst comprises an enzyme capable of catalysing the decarboxylation of an α-keto acid or an amino acid. 
     
     
         291 . Method according to  claim 290 , wherein the enzyme capable of catalysing the decarboxylation is a decarboxylase (E.G. 4.1.1). 
     
     
         292 . Method according to  claim 291 , wherein the decarboxylase is selected from the group of glutamate decarboxylases (EC 4.1.1.15), diaminopimelate decarboxylases (EC 4.1.1.20) aspartate 1-decarboxylases (EC 4.1.1.11), branched chain α-keto acid decarboxylases, α-ketoisovalerate decarboxylases, a-ketoglutarate decarboxylases, pyruvate decarboxylases (EC 4.1.1.1), and oxaloacetate decarboxylases (E.C. 4.1.1.3). 
     
     
         293 . Method according to  claim 290 , wherein the enzyme capable of catalysing the decarboxylation is enzyme from an organism or part thereof selected from the group of  Cucurbitaceae; Saccharomyces; Candida; Hansenula; Kluyveromyces; Rhizopus; Neurospora; Zymomonas; Escherichia; Mycobacterium; Clostridium; Lactobacillus; Streptococcus; Pseudomonas  and  Lactococcus.    
     
     
         294 . Method according to  claim 290 , wherein the enzyme capable of catalysing the decarboxylation comprises an amino acid sequence according to: an enzyme from  Escherichia coli, Saccharomyces cerevisiae, Zymomonas mobilis, Lactococcus lactis  or  Mycobacterium tuberculosis  that catalyses the conversion of 2-oxoheptane-1,7-dioate (2-OHD) to adipate semialdehyde or 2-aminoheptane-1,7-dioate (2-AHD) to 6-aminocaproic acid; a gene product of pdc from  Zymomonas mobilus , pdc1 from  Saccharomyces cerevisiae , pdc from  Acetobacter pasteurians , pdc1 from  Kluyveromyces lactis , mdlC from  Pseudomonas putida , mdlC from  Pseudomonas aeruginosa , dpgB from  Pseudomonas stutzeri , ilvB-1 from  Pseudomonas fluorescens , kgd from  Mycobacterium tuberculosis , kgd from  Bradyrhizobium japonicum , kgd from  Mesorhizobium loti , kdcA from  Lactococcus lactis , BCKDHB from  Homo sapiens , BCKDHA from  Homo sapiens , BCKDHB from  Bos taurus , BCKDHA from  Bos taurus , panD from  Escherichia coli  K12, panD from  Corynebacterium glutamicum  or panD from  Mycobacterium tuberculosis ; or a homologue of any of these sequences. 
     
     
         295 . Method according to  claim 290 , wherein 2-OHD is biocatalytically converted into adipate semialdehyde in the presence of a biocatalyst capable of catalysing the decarboxylation of an α-keto acid, and adipate semialdehyde is biocatalytically converted into 6-aminocaproic acid in the presence of at least one amino donor and at least one biocatalyst capable of catalysing a transamination and/or a reductive amination of adipate semialdehyde. 
     
     
         296 . Method according to  claim 283 , wherein 2-OHD is biocatalytically converted into 2-aminoheptane-1,7-dioate (2-AHD) in the presence of at least one amino donor and at least one biocatalyst capable of catalysing a transamination and/or a reductive amination of 2-OHD thereby forming 2-AHD, and 2-AHD is biocatalytically converted into 6-aminocaproic acid in the presence of a biocatalyst capable of catalysing the decarboxylation of an amino acid. 
     
     
         297 . Method according to  claim 283 , wherein the 2-OHD has been obtained from a natural source. 
     
     
         298 . Method for preparing caprolactam, comprising cyclising the 6-aminocaproic acid prepared by a method according to  claim 283 , thereby forming caprolactam. 
     
     
         299 . A recombinant host cell comprising a nucleic acid sequence encoding an enzyme with 2-oxoheptane-1,7-dioate (2-OHD) decarboxylase activity and/or a nucleic acid sequence encoding an enzyme with adipate semialdehyde aminotransferase activity. 
     
     
         300 . A recombinant host cell according to  claim 299 , comprising a nucleic acid sequence encoding an enzyme with adipate semialdehyde aminotransferase comprising an amino acid sequence according to: an enzyme from  Vibrio fluvialis, Bacillus weihenstephanensis, Pseudomonas aeruginosa, Bacillus subtilis , or  Pseudomonas aeruginosa  that catalyses the conversion of adipate semialdehyde to 6-aminocaproic acid; a gene product of gabT from  Escherichia coli , abat from  Mus musculus , gabT from  Pseudomonas fluorescens , abat from  Sus scrofa ; or a homologue thereof. 
     
     
         301 . A recombinant host cell according to  claim 299 , comprising a nucleic acid sequence encoding an enzyme with 2-OHD decarboxylase activity comprising an amino acid sequence according to: an enzyme from  Escherichia coli, Saccharomyces cerevisiae, Zymomonas mobilis, Lactococcus lactis  or  Mycobacterium tuberculosis  that catalyses the conversion of 2-oxoheptane-1,7-dioate (2-OHD) to adipate semialdehyde or 2-aminoheptane-1,7-dioate (2-AHD) to 6-aminocaproic acid; a gene product of pdc from  Zymomonas mobilus , pdc1 from  Saccharomyces cerevisiae , pdc from  Acetobacter pasteurians , pdc1 from  Kluyveromyces lactis , mdlC from  Pseudomonas putida , mdlC from  Pseudomonas aeruginosa , dpgB from  Pseudomonas stutzeri , ilvB-1 from  Pseudomonas fluorescens , kgd from  Mycobacterium tuberculosis , kgd from  Bradyrhizobium japonicum , kgd from  Mesorhizobium loti , kdcA from  Lactococcus  lactis, BCKDHB from  Homo sapiens , BCKDHA from  Homo sapiens , BCKDHB from  Bos taurus , BCKDHA from  Bos taurus , panD from  Escherichia coli  K12, panD from  Corynebacterium glutamicum  or panD from  Mycobacterium tuberculosis ; or a homologue of any of these sequences. 
     
     
         302 . A recombinant host cell comprising a nucleic acid sequence encoding an enzyme with 2-oxoheptane-1,7-dioate (2-OHD) aminotransferase activity or 2-OHD dehydrogenase activity and/or a nucleic acid sequence encoding an enzyme with 2-OHD decarboxylase activity. 
     
     
         303 . A recombinant host cell according to  claim 302 , wherein the biocatalyst comprises a nucleic acid sequence encoding an aminotransferase comprising an amino acid sequence according to: an enzyme from  Vibrio fluvialis, Pseudomonas aeruginosa, Pseudomonas syringae, Bacillus subtilis, Rhodobaeter sphaeroides, Legionella pneumophila, Nitrosomonas europaea, Neisseria gonorrhoeae, Pseudomonas aeruginosa , or  Rhodopseudomonas palustris  that catalyses the conversion of 2-oxoheptane-1,7-dioate (2-OHD) to 2-aminoheptane-1,7-dioate (2-AHD); a gene product of gabT from  Escherichia coli , puuE from  Escherichia coli , abat from  Mus musculus , gabT from  Pseudomonas fluorescens , or abat from  Sus scrofa , or a homologue thereof. 
     
     
         304 . A recombinant host cell according to  claim 299 , comprising one or more nucleic acid sequences encoding one or more biocatalysts capable of catalysing at least one reaction step in the preparation of 2-oxoheptane-1,7-dioate (2-OHD) from alpha-ketoglutarate. 
     
     
         305 . A recombinant host cell according to  claim 299 , wherein the host cell is selected from the group of  Aspergillus, Penicillium, Saccharomyces, Kluyveromyces, Pichia, Candida, Hansenula, Bacillus, Corynebacterium , and  Escherichia.    
     
     
         306 . A micro-organism according to  claim 299 , comprising DNA containing a nucleic acid sequence selected from the group of sequences represented by any sequence selected from the group of: a gene from  Vibrio fluvialis, Bacillus weihenstephanensis, Pseudomonas aeruginosa, Bacillus subtilis , or  Pseudomonas aeruginosa  that encodes an enzyme that catalyses the conversion of adipate semialdehyde to 6-aminocaproic acid or a codon optimized variant thereof; a gene from  Vibrio fluvialis, Pseudomonas aeruginosa, Pseudomonas syringae, Bacillus subtilis, Rhodobaeter sphaeroides, Legionella pneumophila, Nitrosomonas europaea, Neisseria gonorrhoeae, Pseudomonas aeruginosa , or  Rhodopseudomonas palustris  that encodes an enzyme that catalyses the conversion of 2-oxoheptane-1,7-dioate (2-OHD) to 2-aminoheptane-1,7-dioate (2-AHD) or a codon optimized variant thereof; a gabT gene from  Escherichia coli , a puuE gene from  Escherichia coli , a abat gene from  Mus musculus , a gabT gene from  Pseudomonas fluorescens , or a abat gene from  Sus scrofa ; a gene from  Escherichia coli, Saccharomyces cerevisiae, Zymomonas mobilis, Lactococcus lactis  or  Mycobacterium tuberculosis  that encodes an enzyme that catalyses the conversion of 2-oxoheptane-1,7-dioate (2-OHD) to adipate semialdehyde or 2-aminoheptane-1,7-dioate (2-AHD) to 6-aminocaproic acid or a codon optimized variant thereof; a pdc gene from  Zymomonas mobilus , a pdc1 gene from  Saccharomyces cerevisiae , a pdc gene from  Acetobacter pasteurians , a pdc1 gene from  Kluyveromyces lactis , a mdlC gene from  Pseudomonas putida , a mdlC gene from  Pseudomonas aeruginosa , a dpgB gene from  Pseudomonas stutzeri , an ilvB-1 gene from  Pseudomonas fluorescens , a kgd gene from  Mycobacterium tuberculosis , a kgd gene from  Bradyrhizobium japonicum , a kgd gene from  Mesorhizobium loti , a kdcA gene from  Lactococcus lactis , a BCKDHB gene from  Homo sapiens , a BCKDHA gene from  Homo sapiens , a BCKDHB gene from  Bos taurus , a BCKDHA gene from  Bos taurus , a panD gene from  Escherichia coli  K12, a panD gene from  Corynebacterium glutamicum  or a panD gene from  Mycobacterium tuberculosis ; and functional analogues thereof. 
     
     
         307 . Polynucleotide comprising a nucleic acid sequence selected from the group of sequences as identified in a codon optimized variant of a gene from  Vibrio fluvialis  or  Bacillus weihenstephanensis  that encodes an enzyme that catalyses the conversion of adipate semialdehyde to 6-aminocaproic acid; a codon optimized variant of a gene from  Vibrio fluvialis  or  Pseudomonas syringae  that encodes an enzyme that catalyses the conversion of 2-oxoheptane-1,7-dioate (2-OHD) to 2-aminoheptane-1,7-dioate (2-AHD); a codon optimized variant of a gene from  Escherichia coli, Saccharomyces cerevisiae, Zymomonas mobilis, Lactococcus lactis  or  Mycobacterium tuberculosis  that encodes an enzyme that catalyses the conversion of 2-oxoheptane-1,7-dioate (2-OHD) to adipate semialdehyde or 2-aminoheptane-1,7-dioate (2-AHD) to 6-aminocaproic acid; and functional analogues thereof.