Microbial production of 3,4-dihydroxybutyrate (3,4-dhba), 2,3- dihydroxybutyrate (2,3-dhba) and 3-hydroxybutyrolactone (3-hbl)

Abstract

The invention relates to recombinant cells and their use in the production of 3,4-dihydroxybutyrate, 2,3-dihydroxybutyrate and 3-hydroxybutyrolactone.

What is claimed is: 
     
         1 .- 31 . (canceled) 
     
     
         32 . A method for producing 3,4-dihydroxybutyrate (3,4-DHBA), 2,3-dihydroxybutyrate (2,3-DHBA) and/or 3-hydroxybutyrolactone (3-HBL) in a cell, comprising:
 increasing the expression of a ycdW gene, an aceA gene, and an aceK gene in the cell, relative to a wild type cell, through an overexpression system;   expressing a pct gene, at least one of a phaA, thil, atoB or bktB gene, and at least one of a phaB or hbd gene in the cell, through a heterologous expression system; and   culturing the cell in minimal medium supplemented with a carbon source.   
     
     
         33 . The method of  claim 32 , wherein the cell further recombinantly expresses a tesB gene. 
     
     
         34 . The method of  claim 32 , wherein the cell is a bacterial cell, a fungal cell (including a yeast cell), a plant cell, an insect cell or an animal cell. 
     
     
         35 . The method of  claim 34 , wherein the cell is a bacterial cell. 
     
     
         36 . The method of  claim 35 , wherein the bacterial cell is an  Escherichia coli  cell. 
     
     
         37 . The method of  claim 32 , wherein the cell endogenously expresses ycdW, aceA, and/or aceK, and wherein endogenous expression of ycdW, aceA, and/or aceK is increased through modification of the gene(s) and/or their promoter(s) and/or their ribosome binding sites (RBSs). 
     
     
         38 . The method of  claim 32 , wherein one or more of ycdW, aceA, aceK, pct, phaA, thil, atoB, bktB, phaB and hbd are expressed from a plasmid, or wherein one or more copies of ycdW, aceA, aceK, pct, phaA, thil, atoB, bktB, phaB and hbd are integrated into the genome of the cell. 
     
     
         39 .- 41 . (canceled) 
     
     
         42 . The method of  claim 32 , wherein the ycdW, aceA and/or aceK gene(s) is an  Escherichia coli  gene. 
     
     
         43 . (canceled) 
     
     
         44 . The method of  claim 32 , wherein the phaA, bktB and/or phaB gene(s) is a  Ralstonia eutropha  gene. 
     
     
         45 . (canceled) 
     
     
         46 . The method of  claim 32 , wherein the pct gene is a  Megasphaera elsdenii  gene. 
     
     
         47 . (canceled) 
     
     
         48 . The method of  claim 33 , wherein the tesB gene is an  Escherichia coli  gene. 
     
     
         49 . (canceled) 
     
     
         50 . The method of  claim 32 , wherein the carbon source comprises or consists of glucose. 
     
     
         51 . The method of  claim 32 , wherein the carbon source comprises or consists of glycerol. 
     
     
         52 . The method of  claim 32 , further comprising recovering the 3,4-DHBA, 2,3-DHBA and/or 3-HBL from the cell culture. 
     
     
         53 . A cell culture produced by the method of  claim 32 . 
     
     
         54 . The cell culture of  claim 53 , wherein the cell culture contains at least 0.1 g L −1  DHBA, at least 10 mg L −1 3-HBL or at least 0.1 g L-1 2,3-DHBA. 
     
     
         55 . (canceled) 
     
     
         56 . A supernatant of a cell culture produced by the method of  claim 32 . 
     
     
         57 . The supernatant of  claim 56 , wherein the supernatant is subjected to lactonization, optionally wherein lactonization is achieved through acidification to reduce the pH of the supernatant. 
     
     
         58 . (canceled) 
     
     
         59 . (canceled) 
     
     
         60 . A method comprising:
 recombinantly expressing a pct gene, at least one of a phaA, thil, atoB or bktB gene, and at least one of a phaB or hbd gene in a cell that overexpresses a ycdW gene, an aceA gene, and an aceK gene.   
     
     
         61 . The method of  claim 32 , wherein the cell has decreased or eliminated expression of an iclR gene, an aceB gene and a gcl gene, relative to a wild type cell.