Methods And Microorganisms For The Production Of 1,3-Butanediol

Abstract

A non-naturally occurring microorganism having a 1,3-BDO pathway is provided. The microorganism expresses at least one of the following 1,3-BDO pathway enzymes: an aldolase that catalyzes condensation of two acetaldehydes to produce 3-hydroxybutanal; and an aldo-ketoreductase, oxidoreductase, aldehyde reductase or alcohol dehydrogenase that reduces 3-hydroxybutanal to 1,3-BDO. The organism may further express one or more enzymes for producing acetaldehyde. A biosynthetic process involves condensing two acetaldehyde molecules to 3-hydroxybutanal using an enzyme from class aldolases; and selectively reducing 3-hydroxybutanal to 1,3-BDO using an enzyme belonging to the class aldo-ketoreductase, oxidoreductase, aldehyde reductase or alcohol dehydrogenase. The process can further include producing acetaldehyde by a biosynthetic method.

1 - 25 . (canceled) 
     
     
         26 . A non-naturally occurring microorganism that produces 1,3-butanediol (1,3-BDO) from 3-hydroxybutanal, the microorganism being engineered to express an exogenous polynucleotide encoding an aldo-keto reductase (AKR) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 25, wherein the AKR catalyzes reduction of the 3-hydroxybutanal to 1,3-BDO. 
     
     
         27 . The non-naturally occurring microorganism of  claim 26 , wherein the AKR comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 25. 
     
     
         28 . The non-naturally occurring microorganism of  claim 26 , wherein the AKR comprises residues R214, R227, R281, Q285, G279, and R208, with respect to the amino acid numbering of SEQ ID NO: 25. 
     
     
         29 . The non-naturally occurring microorganism of  claim 26 , which is further engineered to express at least one further exogenous polynucleotide encoding a decarboxylase that catalyzes the decarboxylation of pyruvate to yield acetaldehyde and carbon dioxide. 
     
     
         30 . The non-naturally occurring microorganism of  claim 29 , wherein the decarboxylase is pyruvate decarboxylase (PDC), benzoylformate decarboxylase (BFD), or alpha-ketoacid decarboxylase (KDC). 
     
     
         31 . The non-naturally occurring microorganism of  claim 29 , which is further engineered to express at least one further exogenous polynucleotide encoding an aldolase that catalyzes condensation of two acetaldehydes to produce 3-hydroxybutanal. 
     
     
         32 . The non-naturally occurring microorganism of  claim 31 , wherein the aldolase is a deoxyribose-5-phosphate aldolase (DERA). 
     
     
         33 . The non-naturally occurring microorganism of  claim 26 , which is further engineered to delete or disrupt an enzyme that utilizes pyruvate, as compared to a corresponding non-engineered microorganism. 
     
     
         34 . The non-naturally occurring microorganism of  claim 26 , which is further engineered to increase acetaldehyde production, as compared to a corresponding non-engineered microorganism. 
     
     
         35 . A non-naturally occurring microorganism that produces 1,3-BDO from 3-hydroxybutanal, the microorganism being engineered to express an exogenous polynucleotide encoding an enzyme that catalyzes reduction of the 3-hydroxybutanal to 1,3-BDO, wherein said enzyme is a variant of a  Pseudomonas aeruginosa  AKR, said  Pseudomonas aeruginosa  AKR comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 25. 
     
     
         36 . The non-naturally occurring microorganism of  claim 35 , wherein said variant  Pseudomonas aeruginosa  AKR enzyme comprises residues R214, R227, R281, Q285, G279, and R208, with respect to the amino acid numbering of SEQ ID NO: 25. 
     
     
         37 . The non-naturally occurring microorganism of  claim 35 , which is further engineered to express at least one further exogenous polynucleotide encoding a decarboxylase that catalyzes the decarboxylation of pyruvate to yield acetaldehyde and carbon dioxide. 
     
     
         38 . The non-naturally occurring microorganism of  claim 35 , wherein the decarboxylase is pyruvate decarboxylase (PDC), benzoylformate decarboxylase (BFD), or alpha-ketoacid decarboxylase (KDC). 
     
     
         39 . The non-naturally occurring microorganism of  claim 35 , which is further engineered to express at least one further exogenous polynucleotide encoding an aldolase that catalyzes condensation of two acetaldehydes to produce 3-hydroxybutanal. 
     
     
         40 . The non-naturally occurring microorganism of  claim 39 , wherein the aldolase is a deoxyribose-5-phosphate aldolase (DERA). 
     
     
         41 . The non-naturally occurring microorganism of  claim 35 , which is further engineered to delete or disrupt an enzyme that utilizes pyruvate, as compared to a corresponding non-engineered microorganism. 
     
     
         42 . The non-naturally occurring microorganism of  claim 35 , which is further engineered to increase acetaldehyde production, as compared to a corresponding non-engineered microorganism. 
     
     
         43 . A method for producing 1,3-BDO from 3-hydroxybutanal, the method comprising: (a) culturing the non-naturally occurring microorganism as defined in  claim 26  under conditions and for a sufficient period of time to produce 1,3-BDO; and (b) isolating the 1,3-BDO produced. 
     
     
         44 . A method for producing 1,3-BDO from 3-hydroxybutanal, the method comprising: (a) culturing the non-naturally occurring microorganism as defined in  claim 35  under conditions and for a sufficient period of time to produce 1,3-BDO; and (b) isolating the 1,3-BDO produced. 
     
     
         45 . A method for producing 1,3-BDO from 3-hydroxybutanal, the method comprising contacting 3-hydroxybutanal with an aldo-keto reductase (AKR) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 25, wherein the AKR catalyzes reduction of the 3-hydroxybutanal to 1,3-BDO.