Methods for control of flux in metabolic pathways

Abstract

The invention pertains to a method for preparing cells that can be used as biocatalysts by inducing in them a growth-decoupled state, in which interferase inhibits the expression of genes except the ones that code for the pathway enzymes of interest. mRNAs that code for interferase-resistant products are overexpressed in the background of a metabolically-frozen cell. Enzymes that compete for a substrate or product of the pathway of interest may be altered such that the enzyme is sensitive to a site-specific protease, which protease is inducible in the host cell.

1 . A cell genetically manipulated to comprise:
 an interferase coding sequence, which interferase cleaves mRNA at a specific sequence motif;   coding sequences for at least two enzymes involved in a pathway of interest, wherein the coding sequences are modified to be free of motifs cleaved by said interferase.   
     
     
         2 . The cell of  claim 1 , wherein said interferase coding sequence is operably linked to an inducible promoter. 
     
     
         3 . The cell of  claim 1 , further comprising a coding sequence encoding a cognate antitoxin for said interferase. 
     
     
         4 . The cell of  claim 1 , wherein said at least two enzymes are rate-limiting enzymes in said pathway of interest. 
     
     
         5 . The cell of  claim 1  wherein at least one of said enzymes is directly involved in said pathway of interest. 
     
     
         6 . The cell of  claim 1 , wherein at least one of said enzymes is indirectly involved in said pathway of interest. 
     
     
         7 . The cell of  claim 1 , wherein all the enzymes directly involved in the pathway of interest are modified to be free of motifs cleaved by said interferase. 
     
     
         8 . The cell of  claim 1 , wherein said interferase coding sequence is present on either an episomal vector, or a chromosome. 
     
     
         9 . The cell of  claim 1 , wherein the coding sequences for at least two enzymes involved in a pathway of interest are operably linked to an inducible promoter. 
     
     
         10 . The cell of  claim 1 , wherein the interferase is selected from MazF, ChpBK and MqsR. 
     
     
         11 . The cell of  claim 1 , wherein said pathway of interest is synthesis of shikimate or derivatives thereof. 
     
     
         12 . A cell genetically manipulated to comprise:
 a protease coding sequence operably linked to an inducible promoter, which protease cleaves proteins at a specific sequence motif;   a coding sequence for at least one competing enzyme involved in a pathway of interest, wherein the coding sequence is modified to encode a motif cleaved by said protease.   
     
     
         13 . The cell of  claim 12 , wherein said protease coding sequence is present on either an episomal vector, or a chromosome. 
     
     
         14 . The cell of  claim 12 , wherein said pathway of interest is synthesis of shikimate or derivatives thereof. 
     
     
         15 . The cell of  claim 12 , further comprising:
 an interferase coding sequence operably linked to a second inducible promoter, which interferase cleaves mRNA at a specific motif;   coding sequences for at least two enzymes involved in a pathway of interest, wherein the coding sequences are modified to be free of motifs cleaved by said interferase, and wherein the protease coding sequence is modified to be free of motifs cleaved by said interferase.   
     
     
         16 . A method of producing a product of a pathway of interest, the method comprising:
 growing a cell according to  claim 1  to a desired population density;   inducing expression of one or both of said interferase and said protease;   providing said population of cells with substrates required for producing said product, wherein said enzymes in said pathway of interest catalyze production of said product.   
     
     
         17 . The method of  claim 16 , wherein said microbial cell is intact. 
     
     
         18 . The method of  claim 16 , wherein said microbial cell is lysed. 
     
     
         19 . A system for producing a product of a pathway of interest, the system comprising:
 a cell according to  claim 1 ; and such substrate, nutrients, cofactors, buffers, reducing agents, ATP generating systems as are required for production of said product.   
     
     
         20 . The system of  claim 19 , wherein the cell is lysed.