Compositions and methods for rapid and dynamic flux control using synthetic metabolic valves

Abstract

This invention relates to metabolically engineered microorganisms, such as bacterial and or fungal strains, and bioprocesses utilizing such strains. These strains enable the dynamic control of metabolic pathways, which can be used to optimize production. Dynamic control over metabolism is accomplished via a combination of methodologies including but not limited to transcriptional silencing and controlled enzyme proteolysis. These microbial strains are utilized in a multi-stage bioprocess encompassing at least two stages, the first stage in which organisms are grown and metabolism can be optimized for microbial growth and at least one other stage in which growth can be slowed or stopped, and dynamic changes can be made to metabolism to improve the production of desired product, such as a chemical or fuel.

1 .- 77 . (canceled) 
     
     
         78 . A genetically modified microorganism comprising:
 a production pathway comprising at least one enzyme for the production of a product, and   at least one synthetic metabolic valve comprising controlled transcriptional gene silencing of a gene encoding an enzyme that is one of: enoyl-ACP reductase (fabI), citrate synthase (gltA), soluble transhydrogenase (udhA), glucose-6-phosphate-l-dehydrogenase (zwf), lipoamide dehydrogenase (lpd), or combinations thereof;   wherein transcriptional gene silencing comprises expression of small guide RNAs specific for the gene;   wherein depletion of the limiting nutrient from a growth media in which the genetically modified microorganism is growing will inducing a stationary or non-dividing cellular state;   wherein the synthetic metabolic valve of the microorganism may be conditionally activated.   
     
     
         79 . The genetically modified microorganism of  claim 78 , wherein transcriptional gene silencing further comprises expression of a gene encoding a dCas9 protein. 
     
     
         80 . The genetically modified microorganism of  claim 78 , wherein a single plasmid expresses small guide RNAs for two or more genes. 
     
     
         81 . The genetically modified microorganism of  claim 78 , wherein the synthetic metabolic valve directed to controlled transcriptional gene silencing further comprises conditional expression of a proteolysis chaperone protein. 
     
     
         82 . The genetically modified microorganism of  claim 81 , wherein the chaperone protein is a sspB protein. 
     
     
         83 . The genetically modified microorganism of  claim 78  wherein the genetically modified microorganism is characterized by disruption or deletion of a gene naturally occurring in the genetically modified microorganism, the naturally occurring gene one of a gene encoding lactate dehydrogenase (ldhA), phosphate acetyltransferase (pta), pyruvate oxidase (poxB), pyruvate-formate lyase (pflB), the methylglyoxal synthase (mgsA), acetate kinase (ackA), alcohol dehydrogenase (adhE), ATP-dependent Lon protease (ion), outer membrane protease (ompT), arcA transcriptional dual regulator (arcA), iclR transcriptional regulator (icR), cas3, sspB or combinations thereof. 
     
     
         84 . The genetically modified microorganism of  claim 81  further comprising a synthetic metabolic valve comprising controlled proteolysis of an enzyme that is one of: enoyl-ACP reductase (fabI), citrate synthase (gltA), soluble transhydrogenase (udhA), glucose-6-phosphate-l-dehydrogenase (zwf), lipoamide dehydrogenase (lpd), or combinations thereof. 
     
     
         85 . A bioprocess for production of a product from the genetically modified microorganism of  claim 78 , the bioprocess comprising:
 in a first stage, growing the genetically modified microorganism in a medium and   in a second stage, reducing the genetically modified microorganism growth of the first stage and expressing a heterologous enzyme of the product production pathway,   wherein, a transition from the first stage to the second stage is at least partially controlled by depletion of a level of a limiting nutrient from the media and, as the limiting nutrient is depleted from the media growth of the genetically modified microorganism is stopped, and the transition comprising activation of the synthetic metabolic valve.   
     
     
         86 . The bioprocess of  claim 85 , wherein the product is one of an alcohol, a diol, a polyol, an organic acid, an amino acid, a fatty acid, a fatty acid derivative, an ester, an alkane, or an alkene. 
     
     
         87 . The bioprocess of  claim 85 , wherein the limiting nutrient comprises inorganic phosphate. 
     
     
         88 . The bioprocess of  claim 85 , wherein the genetically modified microorganism is characterized by disruption or deletion of a gene naturally occurring in the genetically modified microorganism, the naturally occurring gene one of a gene encoding lactate dehydrogenase (idhA), phosphate acetyltransferase (pta), pyruvate oxidase (poxB), pyruvate-formate lyase (pflB), the methylglyoxal synthase (mgsA), acetate kinase (ackA), alcohol dehydrogenase (adhE), ATP-dependent Lon protease (Ion), outer membrane protease (ompT), arcA transcriptional dual regulator (arcA), iclR transcriptional regulator (icR), or combinations thereof. 
     
     
         89 . The bioprocess of  claim 85 , further comprising a second synthetic metabolic valve controlling at least one enzyme essential for growth of the genetically modified microorganism that is one of: sucD, aceA, pfkA, Ion, rpoS, tktA or tktB.