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 - 10 . (canceled) 
     
     
         11 . A recombinant microorganism comprising nucleic acid sequences encoding polypeptides that convert 4-hydroxybutyrate (4-HB) to 4-hydroxybutanal and 4-hydroxybutanal to 1,4-butanediol (1,4-BDO), thereby biosynthesizing 1,4-BDO from carbon sources provided to the recombinant microorganism. 
     
     
         12 . The recombinant microorganism of  claim 11  comprising nucleic acid sequences encoding polypeptides for at least one of the following pathways:
 (a) a pathway that converts citrate to cis-aconitate to D-isocitrate to alpha ketoglutarate to succinate semialdehyde to the 4-HB; 
 (b) a pathway that converts acetyl-CoA to acetoacetyl-CoA to 3-hydroxybutryryl-CoA to crotonyl-CoA to vinylacetyl-CoA to 4-hydroxybutyryl-CoA to the 4-HB; or 
 (c) a pathway that converts succinate to succinate semialdehyde (optionally including via succinyl-CoA) to the 4-HB; thereby providing 4-HB. 
 
     
     
         13 . A recombinant microorganism comprising a biosynthetic pathway with aconitase, isocitrate dehydrogenase, and alpha ketoglutarate decarboxylase enzymatic activities. 
     
     
         14 . The recombinant microorganism of  claim 13  additionally comprising 4-hydroxybutyrate dehydrogenase, aldehyde dehydrogenase, and 1,3 propanediol dehydrogenase enzymatic activities. 
     
     
         15 . The recombinant microorganism of  claim 13 , additionally comprising a biosynthetic pathway with acetyl-CoA acetyltransferase, beta-hydroxybutyryl-CoA dehydrogenase, crotonase, vinylacetyl-CoA-isomerase, 4-hydroxybutyryl-CoA dehydratase, and 4-hydroxybutyrate CoA transferase enzymatic activities. 
     
     
         16 . The recombinant microorganism of  claim 13 , additionally comprising a biosynthetic pathway with succinate semialdehyde dehydrogenase, and optionally with succinyl-CoA synthetase, enzymatic activities. 
     
     
         17 . A recombinant microorganism comprising a biosynthetic pathway with acetyl-CoA acetyltransferase, beta-hydroxybutyryl-CoA dehydrogenase, crotonase, vinylacetyl-CoA-isomerase, 4-hydroxybutyryl-CoA dehydratase, and 4-hydroxybutyrate CoA transferase enzymatic activities. 
     
     
         18 . The recombinant microorganism of  claim 17 , additionally comprising 4-hydroxybutyrate dehydrogenase, aldehyde dehydrogenase, and 1,3 propanediol dehydrogenase enzymatic activities. 
     
     
         19 . A recombinant microorganism of  claim 17 , additionally comprising a biosynthetic pathway with succinate semialdehyde dehydrogenase, and optionally with succinyl-CoA synthetase, enzymatic activities. 
     
     
         20 . A recombinant microorganism of  claim 19 , comprising modifications in nucleic acids encoding respective polypeptides of the biosynthetic pathways, and resulting in biosynthesis of 3-hydroxybutyryl-CoA. 
     
     
         21 . A recombinant microorganism comprising a biosynthetic pathway with succinate semialdehyde dehydrogenase, and optionally with succinyl-CoA synthetase, enzymatic activities. 
     
     
         22 . The recombinant microorganism of  claim 21  additionally comprising 4-hydroxybutyrate dehydrogenase, aldehyde dehydrogenase, and 1,3 propanediol dehydrogenase enzymatic activities. 
     
     
         23 . A method of biosynthesis of a polymer chemistry intermediate comprising providing in a bioreactor vessel a recombinant microorganism of  claim 13 , 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 the intermediate. 
     
     
         24 . The method of  claim 23 , wherein the intermediate is 1,4-BDO. 
     
     
         25 . A method of making a polyester, a polyurethane, butyrolactone, γ-butyrolactone, or tetrahydrofuran comprising converting the 1,4-BDO of  claim 24  to a polyester, a polyurethane, butyrolactone, γ-butyrolactone, or tetrahydrofuran. 
     
     
         26 . A method of biosynthesis of a polymer chemistry intermediate comprising providing in a bioreactor vessel a recombinant microorganism of  claim 17 , 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 the intermediate. 
     
     
         27 . The method of  claim 26 , wherein the intermediate is 1,4-BDO. 
     
     
         28 . A method of making a polyester, a polyurethane, butyrolactone, γ-butyrolactone, or tetrahydrofuran comprising converting the 1,4-BDO of  claim 27  to a polyester, a polyurethane, butyrolactone, γ-butyrolactone, or tetrahydrofuran. 
     
     
         29 . A method of biosynthesis of a polymer chemistry intermediate comprising providing in a bioreactor vessel a recombinant microorganism of  claim 21 , 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 the intermediate. 
     
     
         30 . The method of  claim 29 , wherein the intermediate is 1,4-BDO. 
     
     
         31 . A method of making a polyester, a polyurethane, butyrolactone, γ-butyrolactone, or tetrahydrofuran comprising converting the 1,4-BDO of  claim 30  to a polyester, a polyurethane, butyrolactone, γ-butyrolactone, or tetrahydrofuran. 
     
     
         32 . An industrial-scale microbial bioreactor system comprising:
 (a) a bioreactor vessel;   (b) a carbon source;   (c) a recombinant microorganism of  claim 11 ; and   (d) a media; wherein the carbon source is optionally selected from the group consisting of sucrose, glucose, xylose, cellulose or hemicelluloses; and wherein the media is optionally a minimal media; and further wherein said minimal media optionally comprises M9 minimal media, potassium sulfate minimal media, yeast synthetic minimal media and variations thereof.   
     
     
         33 . The industrial-scale microbial bioreactor system of  claim 32 , wherein the recombinant microorganism of  claim 32  (step (c)) additionally comprises nucleic acid sequences encoding polypeptides for at least one of the following pathways:
 (a) a pathway that converts citrate to cis-aconitate to D-isocitrate to alpha ketoglutarate to succinate semialdehyde to the 4-HB; 
 (b) a pathway that converts acetyl-CoA to acetoacetyl-CoA to 3-hydroxybutryryl-CoA to crotonyl-CoA to vinylacetyl-CoA to 4-hydroxybutyryl-CoA to the 4-HB; or 
 (c) a pathway that converts succinate to succinate semialdehyde (optionally including via succinyl-CoA) to the 4-HB; thereby providing 4-HB in the recombinant microorganism.