Methods and organisms for the growth-coupled production of 1,4-butanediol

Abstract

The invention provides a non-naturally occurring microorganism comprising one or more gene disruptions, the one or more gene disruptions occurring in genes encoding an enzyme obligatory to coupling 1,4-butanediol production to growth of the microorganism when the gene disruption reduces an activity of the enzyme, whereby the one or more gene disruptions confers stable growth-coupled production of 1,4-butanediol onto the non-naturally occurring microorganism. The microorganism can further comprise a gene encoding an enzyme in a 1,4-butanediol (BDO) biosynthetic pathway. The invention additionally relates to methods of using microorganisms to produce BDO.

1 . A non-naturally occurring microorganism comprising one or more gene disruptions, said one or more gene disruptions occurring in genes encoding an enzyme obligatory to coupling 1,4-butanediol production to growth of said microorganism when said gene disruption reduces an activity of said enzyme, whereby said one or more gene disruptions confers stable growth-coupled production of 1,4-butanediol onto said non-naturally occurring microorganism. 
     
     
         2 . The non-naturally occurring microorganism of  claim 1 , wherein said one or more gene disruptions comprise a metabolic modification listed in Table 6 or 7. 
     
     
         3 . The non-naturally occurring microorganism of  claim 1 , wherein said one or more gene disruptions comprise a deletion of said one or more genes. 
     
     
         4 . The non-naturally occurring microorganism of  claim 1 , wherein said non-naturally occurring microorganism is selected from the group of microorganisms having a metabolic modification listed in Table 6 or 7. 
     
     
         5 . A non-naturally occurring microorganism comprising a set of metabolic modifications obligatory to coupling 1,4-butanediol production to growth of said microorganism, said set of metabolic modifications comprising disruption of one or more genes, or an ortholog thereof, wherein said set of metabolic modifications comprises disruption of adhE and ldhA, wherein said microorganism exhibits stable growth-coupled production of 1,4-butanediol. 
     
     
         6 . The non-naturally occurring microorganism of  claim 5 , wherein said set of metabolic modifications further comprise disruption of mdh; adhE, ldhA, pflAB, mdh, and aspA;
 or adhE, ldhA, pflAB, mdh, and aspA.   
     
     
         7 . The non-naturally occurring microorganism of  claim 6 , wherein said set of metabolic modifications further comprise disruption of one or more genes selected from the set of genes comprising mqo, aspA, sfcA, maeB, pntAB, and gdhA; or pykA, pykF, dhaKLM, deoC, edd, yiaE, ycdW, prpC, and gsk. 
     
     
         8 . The non-naturally occurring microorganism of  claim 7 , wherein said set of metabolic modifications comprise disruption of sfcA and maeB; or pykA, pykF, dhaKLM, deoC, edd, yiaE and ycdW. 
     
     
         9 . The non-naturally occurring microorganism of  claim 8 , wherein said set of metabolic modifications further comprise disruption of prpC and gsk. 
     
     
         10 . The non-naturally occurring microorganism of  claim 6 , wherein said set of metabolic modifications further comprise disruption of pflAB. 
     
     
         11 . The non-naturally occurring microorganism of  claim 7 , wherein said set of metabolic modifications further comprise disruption of pflAB. 
     
     
         12 . The non-naturally occurring microorganism of  claim 8 , wherein said set of metabolic modifications further comprise disruption of pflAB. 
     
     
         13 . The non-naturally occurring microorganism of  claim 9 , wherein said set of metabolic modifications further comprise disruption of pflAB. 
     
     
         14 . The non-naturally occurring microorganism of  claim 5 , wherein said microorganism further comprises a 1,4-butanediol (BDO) biosynthetic pathway, said pathway comprising at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, CoA-independent succinic semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase, glutamate: succinic semialdehyde transaminase, glutamate decarboxylase, CoA-independent aldehyde dehydrogenase, CoA-dependent aldehyde dehydrogenase or alcohol dehydrogenase, wherein said exogenous nucleic acid is expressed in sufficient amounts to produce 1,4-butanediol (BDO). 
     
     
         15 . A method of producing a non-naturally occurring microorganism having stable growth-coupled production of 1,4-butanediol, comprising: (a) identifying in silico a set of metabolic modifications requiring 1,4-butanediol production during exponential growth, and (b) genetically modifying a microorganism to contain said set of metabolic modifications requiring 1,4-butanediol production. 
     
     
         16 - 23 . (canceled) 
     
     
         24 . A method of producing 1,4-butanediol coupled to the growth of a microorganism, comprising: (a) culturing under exponential growth phase in a sufficient amount of nutrients and media the non-naturally occurring microorganism of  claim 8 ; and (b) isolating 1,4-butanediol produced from said non-naturally occurring microorganism. 
     
     
         25 - 31 . (canceled) 
     
     
         32 . The method of  claim 24 , wherein said non-naturally occurring microorganism further comprises a 1,4-butanediol (BDO) biosynthetic pathway, said pathway comprising at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, CoA-independent succinic semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase, glutamate: succinic semialdehyde transaminase, glutamate decarboxylase, CoA-independent aldehyde dehydrogenase, CoA-dependent aldehyde dehydrogenase or alcohol dehydrogenase, wherein said exogenous nucleic acid is expressed in sufficient amounts to produce 1,4-butanediol (BDO).