Methods and organisms for utilizing synthesis gas or other gaseous carbon sources and methanol

Abstract

The invention provides a non-naturally occurring microbial organism having an acetyl-CoA pathway and the capability of utilizing syngas or syngas and methanol. In one embodiment, the invention provides a non-naturally occurring microorganism, comprising one or more exogenous proteins conferring to the microorganism a pathway to convert CO, CO 2 and/or H 2 to acetyl-coenzyme A (acetyl-CoA), methyl tetrahydrofolate (methyl-THF) or other desired products, wherein the microorganism lacks the ability to convert CO or CO 2 and H 2 to acetyl-CoA or methyl-THF in the absence of the one or more exogenous proteins. For example, the microbial organism can contain at least one exogenous nucleic acid encoding an enzyme or protein in an acetyl-CoA pathway. The microbial organism is capable of utilizing synthesis gases comprising CO, CO 2 and/or H 2 , alone or in combination with methanol, to produce acetyl-CoA. The invention additionally provides a method for producing acetyl-CoA, for example, by culturing an acetyl-CoA producing microbial organism, where the microbial organism expresses at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme or protein in a sufficient amount to produce acetyl-CoA, under conditions and for a sufficient period of time to produce acetyl-CoA.

1 - 191 . (canceled) 
     
     
         192 . 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. 
     
     
         193 . The recombinant microorganism of  claim 192 , wherein the one or more nucleic acid sequences comprise at least one of thiL, hbd, crt, abfD, abfT, and dhaT. 
     
     
         194 . The recombinant microorganism of  claim 192 , wherein the recombinant microorganism is adapted to biosynthesize 1,4-BDO by condensing two acetyl-CoA moieties into acetoacetyl-CoA. 
     
     
         195 . The recombinant microorganism of  claim 194 , comprising aldehyde dehydrogenase. 
     
     
         196 . (canceled) 
     
     
         197 . 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. 
     
     
         198 . The recombinant microorganism of  claim 197 , wherein the one or more nucleic acid sequences comprise at least one of kgd, 4hbd, and dhaT. 
     
     
         199 . The recombinant microorganism of  claim 197  wherein the recombinant microorganism is adapted to biosynthesize 1,4-BDO from citrate, wherein the citrate is derived from oxaloacetate and acetyl-CoA. 
     
     
         200 . The recombinant microorganism of  claim 199 , comprising: aconitase, isocitrate dehydrogenase, aldehyde dehydrogenase, and methylcitrate synthase; or aconitase, isocitrate dehydrogenase, aldehyde dehydrogenase, and citrate synthase. 
     
     
         201 . (canceled) 
     
     
         202 . 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. 
     
     
         203 . The recombinant microorganism of  claim 202 , wherein the one or more nucleic acid sequences comprise at least one of fumA, fumB, fumC, frd, yneI, sucC, sucD, 4hbD, adh, and dhaT. 
     
     
         204 . The recombinant microorganism of  claim 202  wherein the recombinant microorganism is adapted to biosynthesize 1,4-BDO from malate, wherein the malate is derived from oxaloacetate and/or from pyruvate. 
     
     
         205 . The recombinant microorganism of  claim 204 , comprising fumarase, succinate semialdehyde dehydrogenase, and aldehyde dehydrogenase, or fumarase, succinyl-CoA synthetase, and aldehyde dehydrogenase. 
     
     
         206 - 208 . (canceled) 
     
     
         209 . A recombinant microorganism of  claim 192  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. 
     
     
         210 - 211 . (canceled) 
     
     
         212 . The recombinant microorganism of  claim 192 , wherein at least one of the one or more nucleic acid sequences is heterologous. 
     
     
         213 . A method of biosynthesis of 1,4-BDO comprising providing in a bioreactor vessel a microorganism of  claim 192 , 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. 
     
     
         214 . A method of biosynthesis of any of the identified downstream products of 1,4-BDO comprising practicing the method of  claim 213  and thereafter practicing a method for conversion of 1,4-BDO to one of said identified downstream products. 
     
     
         215 . The method of  claim 214  wherein the method for conversion comprises esterification or ester exchange reaction, or dehydration under acidic conditions to form tetrahydrofuran. 
     
     
         216 . (canceled) 
     
     
         217 . A bio-production system comprising a bioreactor vessel, a microorganism of  claim 192 , 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. 
     
     
         218 . An industrial-scale microbial bioreactor system comprising: (a) a bioreactor vessel; (b) a carbon source; (c) a recombinant microorganism of  claim 192 ; and (d) a media. 
     
     
         219 - 220 . (canceled) 
     
     
         221 . The industrial-scale microbial bioreactor of  claim 218 , wherein the carbon source is a sugar. 
     
     
         222 . The industrial-scale microbial bioreactor of  claim 221 , wherein the sugar is sucrose, glucose, xylose, cellulose or hemicellulose.