Microorganisms for the production of 1,4-butanediol

Abstract

The invention provides non-naturally occurring microbial organisms comprising a 1,4-butanediol (BDO) pathway comprising at least one exogenous nucleic acid encoding a BDO pathway enzyme expressed in a sufficient amount to produce BDO. The invention additionally provides methods of using such microbial organisms to produce BDO.

1 - 39 . (canceled) 
     
     
         40 . A recombinant microorganism adapted to biosynthesize 1,4-butanediol (“1,4-BDO”), the recombinant microorganism comprising one or more nucleic acid sequences encoding one or more of 1) a first polypeptide providing acetyl-CoA acetyltransferase activity, 2) a second polypeptide providing β-hydroxybutyryl-CoA dehydrogenase activity; 3) a third polypeptide providing crotonase activity; a 4) fourth polypeptide providing vinylacetyl-CoA-Δ-isomerase and 4-hydroxybutyryl-CoA dehydratase activities; 5) a fifth polypeptide providing 4-hydroxybutyrate-CoA-hydrolase activity, and 6) a sixth polypeptide providing 1,3-propanediol dehydrogenase activity, wherein the recombinant microorganism biosynthesizes 1,4-BDO utilizing said polypeptides. 
     
     
         41 . The recombinant microorganism of  claim 40 , wherein the one or more nucleic acid sequences comprise at least one of thiL, hbd, crt, abfD, abfT, and dhaT. 
     
     
         42 . The recombinant microorganism of  claim 40 , wherein the recombinant microorganism is adapted to biosynthesize 1,4-BDO by condensing two acetyl-CoA moieties into acetoacetyl-CoA. 
     
     
         43 . The recombinant microorganism of  claim 42 , comprising aldehyde dehydrogenase. 
     
     
         44 . (canceled) 
     
     
         45 . A recombinant microorganism adapted to biosynthesize 1,4-butanediol (“1,4-BDO”), the recombinant microorganism comprising one or more nucleic acid sequences encoding one or more of 1) a first polypeptide providing a-ketoglutarate decarboxylase activity, 2) a second polypeptide providing 4-hydroxybutyrate dehydrogenase activity, and 3) a third polypeptide providing 1,3-propanediol dehydrogenase activity, wherein the recombinant microorganism biosynthesizes 1,4-BDO utilizing said polypeptides. 
     
     
         46 . The recombinant microorganism of  claim 45 , wherein the one or more nucleic acid sequences comprise at least one of kgd, 4hbd, and dhaT. 
     
     
         47 . The recombinant microorganism of  claim 45  wherein the recombinant microorganism is adapted to biosynthesize 1,4-BDO from citrate, wherein the citrate is derived from oxaloacetate and acetyl-CoA. 
     
     
         48 . The recombinant microorganism of  claim 47 , comprising: aconitase, isocitrate dehydrogenase, aldehyde dehydrogenase, and methylcitrate synthase; or aconitase, isocitrate dehydrogenase, aldehyde dehydrogenase, and citrate synthase. 
     
     
         49 . (canceled) 
     
     
         50 . A recombinant microorganism adapted to biosynthesize 1,4-butanediol (“1,4-BDO”), the recombinant microorganism comprising one or more nucleic acid sequences encoding one or more of 1) a first polypeptide providing fumarase activity, 2) a second polypeptide providing fumarate reductase activity, 3) a third polypeptide providing succinate semialdehyde dehydrogenase activity, 4) a fourth polypeptide providing one or both of succinyl-CoA synthetase activity and succinate semialdehyde dehydrogenase activity, 5) a fifth polypeptide providing 4-hydroxybutyrate dehydrogenase activity, 6) a sixth polypeptide providing aldehyde dehydrogenase activity, and 7) a seventh polypeptide providing 1,3-propanediol dehydrogenase activity, wherein the recombinant microorganism biosynthesizes 1,4-BDO utilizing said polypeptides. 
     
     
         51 . The recombinant microorganism of  claim 50 , wherein the one or more nucleic acid sequences comprise at least one of fumA, fumB, fumC, frd, yneI, sucC, sucD, 4hbD, adh, and dhaT. 
     
     
         52 . The recombinant microorganism of  claim 50  wherein the recombinant microorganism is adapted to biosynthesize 1,4-BDO from malate, wherein the malate is derived from oxaloacetate and/or from pyruvate. 
     
     
         53 . The recombinant microorganism of  claim 52 , comprising fumarase, succinate semialdehyde dehydrogenase, and aldehyde dehydrogenase, or fumarase, succinyl-CoA synthetase, and aldehyde dehydrogenase. 
     
     
         54 - 56 . (canceled) 
     
     
         57 . A recombinant microorganism of  claim 40  wherein a nucleic acid sequence encoding one of the indicated polypeptides selectively hybridizes with one of the nucleic acid sequences encoding kgd, 4hbd, dhaT, thiL, crt, bcd, etfB, etfA, hbd, abfD, abfT and yneI. 
     
     
         58 - 59 . (canceled) 
     
     
         60 . The recombinant microorganism of  claim 40 , wherein at least one of the one or more nucleic acid sequences is heterologous. 
     
     
         61 . A method of biosynthesis of 1,4-BDO comprising providing in a bioreactor vessel a microorganism of  claim 40 , a carbon source, and a media, and conducting a bio-production event under suitable conditions and for a suitable time to obtain a measurable quantity of 1,4-BDO. 
     
     
         62 . A method of biosynthesis of any of the identified downstream products of 1,4-BDO comprising practicing the method of  claim 61  and thereafter practicing a method for conversion of 1,4-BDO to one of said identified downstream products. 
     
     
         63 . The method of  claim 62  wherein the method for conversion comprises esterification or ester exchange reaction, or dehydration under acidic conditions to form tetrahydrofuran. 
     
     
         64 . (canceled) 
     
     
         65 . A bio-production system comprising a bioreactor vessel, a microorganism of  claim 40 , a carbon source, and a media, wherein the system is adapted to conduct a bio-production event under suitable conditions and for a suitable time to produce a measurable quantity of 1,4-BDO. 
     
     
         66 . An industrial-scale microbial bioreactor system comprising: (a) a bioreactor vessel; (b) a carbon source; (c) a recombinant microorganism of  claim 40 ; and (d) a media. 
     
     
         67 - 68 . (canceled) 
     
     
         69 . The industrial-scale microbial bioreactor of  claim 66 , wherein the carbon source is a sugar. 
     
     
         70 . The industrial-scale microbial bioreactor of  claim 69 , wherein the sugar is sucrose, glucose, xylose, cellulose or hemixellulose.