Engineering microbes and metabolic pathways for the production of ethylene glycol

Abstract

The invention relates to recombinant cells and their use in the production of ethylene glycol.

What is claimed is: 
     
         1 . A cell engineered to produce ethylene glycol, optionally wherein the cell has reduced or eliminated activity or reduced or eliminated expression of aldehyde dehydrogenase relative to a wild type cell, optionally wherein the aldehyde dehydrogenase is aldehyde dehydrogenase A and optionally wherein the cell is engineered to produce ethylene glycol from sugar, a pentose, xylose, L-arabinose, glucose, glycerol, serine, or a metabolite comprising glycolaldehyde. 
     
     
         2 . (canceled) 
     
     
         3 . The cell of  claim 1 , wherein the aldehyde dehydrogenase A is encoded by an aldA gene, and wherein the cell comprises a deletion of the aldA gene (ΔaldA). 
     
     
         4 . The cell of  claim 1 , wherein the cell has reduced or eliminated activity or reduced or eliminated expression of xylulokinase relative to a wild type cell, and optionally wherein the xylulokinase is encoded by a xylB gene and the cell comprises a deletion of the xylB gene (ΔxylB). 
     
     
         5 . (canceled) 
     
     
         6 . The cell of  claim 1 , wherein the cell has reduced or eliminated activity or reduced or eliminated expression of L-ribulokinase relative to a wild type cell, and optionally wherein the L-ribulokinase is encoded by a araB gene and the cell comprises a deletion of the araB gene (ΔaraB). 
     
     
         7 . (canceled) 
     
     
         8 . The cell of  claim 1 , wherein the cell recombinantly expresses an enzyme that interconverts D-xylulose and D-ribulose and/or interconverts L-ribulose and L-xylulose. 
     
     
         9 . The cell of  claim 8 , wherein the enzyme that interconverts D-xylulose and D-ribulose and/or interconverts L-ribulose and L-xylulose is D-tagatose 3-epimerase (DTE), and optionally wherein DTE is encoded by a dte gene, and optionally wherein the dte gene is from  Pseudomonas cichorii.    
     
     
         10 - 11 . (canceled) 
     
     
         12 . The cell of  claim 9 , wherein the cell overexpresses a dte gene. 
     
     
         13 . The cell of  claim 1 , wherein the cell recombinantly expresses D-ribulokinase, D-ribulose-phosphate aldolase and glycolaldehyde reductase, and optionally wherein the D-ribulokinase is encoded by a fucK gene and/or wherein the D-ribulose-phosphate aldolase is encoded by a fucA gene and/or wherein the glycolaldehyde reductase is encoded by a fucO gene. 
     
     
         14 . (canceled) 
     
     
         15 . The cell of  claim 13 , wherein the cell overexpresses the fucK gene and/or the fucA gene and/or the fucO gene. 
     
     
         16 . The cell of  claim 13 , wherein the fucK gene, the fucA gene and the fucO gene are expressed as part of an operon in conjunction with a dte gene, and optionally wherein the order of the genes in the operon is dte-fucA-fucO-fucK. 
     
     
         17 . (canceled) 
     
     
         18 . The cell of  claim 1 , wherein the cell recombinantly expresses ATP:L-xylulose 1-phosphotransferase, L-xylulose-1-phosphate aldolase, and glycolaldehyde reductase, optionally wherein the ATP:L-xylulose 1-phosphotransferase is encoded by a rhaB gene and/or the L-xylulose-1-phosphate aldolase is encoded by a rhaD gene and/or the glycolaldehyde reductase is encoded by a fucO gene, and optionally wherein the cell overexpresses the rhaB gene and/or the rhaD gene and/or the fucO gene. 
     
     
         19 - 20 . (canceled) 
     
     
         21 . The cell of  claim 18 , wherein the rhaB gene, the rhaD gene and the fucO gene are expressed as part of an operon in conjunction with a dte gene, and optionally wherein the order of the genes in the operon is dte-rhaB-rhaD fucO. 
     
     
         22 . (canceled) 
     
     
         23 . The cell of  claim 1 , wherein the cell expresses a subunit of the E1 component of 2-oxoglutarate dehydrogenase within ΔaldA ΔxylB, optionally wherein the subunit of the E1 component of 2-oxoglutarate dehydrogenase is encoded by a sucA gene, and optionally wherein the cell overexpresses a sucA gene. 
     
     
         24 - 25 . (canceled) 
     
     
         26 . The cell of  claim 1 , wherein the cell is a bacterial cell, a fungal cell (including a yeast cell), a plant cell, an insect cell or an animal cell. 
     
     
         27 - 28 . (canceled) 
     
     
         29 . The cell of  claim 1 , wherein the cell endogenously expresses the gene encoding D-ribulokinase, D-ribulose-phosphate aldolase, glycolaldehyde reductase, ATP:L-xylulose 1-phosphotransferase, L-xylulose-1-phosphate aldolase, and/or subunit of the E1 component of 2-oxoglutarate dehydrogenase, and wherein endogenous expression of the gene encoding D-ribulokinase, D-ribulose-phosphate aldolase, glycolaldehyde reductase, ATP:L-xylulose 1-phosphotransferase, L-xylulose-1-phosphate aldolase, and/or subunit of the E1 component of 2-oxoglutarate dehydrogenase is increased through modification of the gene(s) and/or their promoter(s) and/or their ribosome binding sites (RBSs). 
     
     
         30 - 31 . (canceled) 
     
     
         32 . The cell of  claim 1 , wherein the cell recombinantly expresses a 3-phosphoglycerate dehydrogenase, a glycerate kinase, a 3-phosphohydroxypyruvate phosphatase, a serine:pyruvate aminotransferase (SPT), an alanine:glyoxylate aminotransferase (AGT), a serine decarboxylase, and/or an ethanolamine oxidase. 
     
     
         33 . The cell of  claim 32 , wherein the 3-phosphoglycerate dehydrogenase is a mutant resistant to inhibition by serine and/or is encoded by a serA gene of  E. coli.    
     
     
         34 - 35 . (canceled) 
     
     
         36 . The cell of  claim 32 , wherein the glycerate kinase is a glycerate kinase II encoded by a glxK gene of  E. coli , or a glycerate kinase I encoded by a garK gene of  E. coli.    
     
     
         37 . The cell of  claim 1 , wherein the expression or activity in the cell of one or more phosphoglycerate mutases and/or the expression or activity in the cell of enolase is attenuated, thereby increasing the amount of 3-phosphoglycerate in the cell by reducing flux to 2-phosphoglycerate and/or increasing the amount of 2-phosphoglycerate in the cell by reducing flux to phosphoenolpyruvate, and optionally wherein the one or more phosphoglycerate mutases is encoded by gpmA, gpmB, and gpmM genes of  E. coli  and/or the enolase is encoded by an eno gene from  E. coli.    
     
     
         38 . (canceled) 
     
     
         39 . The cell of  claim 1 , wherein the cell recombinantly expresses a 3-phosphoserine aminotransferase to convert 3-phosphohydroxypyruvate to 3-phospho-L-serine and optionally further recombinantly expresses a phosphoserine phosphatase to convert 3-phospho-L-serine to L-serine, and optionally wherein the 3-phosphoserine aminotransferase and phosphoserine phosphatase are encoded by serC and serB genes of  E. coli , respectively. 
     
     
         40 . (canceled) 
     
     
         41 . The cell of  claim 1 , wherein the expression or activity in the cell of one or more serine deaminases is attenuated, and optionally wherein the one or more serine deaminases is encoded by sdaA, sdaB, tdcB, and tdcG genes of  E. coli.    
     
     
         42 - 43 . (canceled) 
     
     
         44 . The cell of  claim 32 , wherein the serine decarboxylase is encoded by a gene from  Arabidopsis thaliana , and/or the ethanolamine oxidase is encoded by a gene from  Arthrobacter  sp., and optionally wherein the gene from  Arabidopsis thaliana  is sdc, and/or the gene from  Arthrobacter  sp. is aao. 
     
     
         45 . (canceled) 
     
     
         46 . The cell of  claim 44 , wherein the serine decarboxylase gene from  A. thaliana  is truncated, and optionally wherein the truncated gene from  A. thaliana  is t-sdc. 
     
     
         47 - 48 . (canceled) 
     
     
         49 . The cell of  claim 32 , wherein the 3-phosphohydroxypyruvate phosphatase is encoded by a yeaB gene of  E. coli  or by GPP2 of  S. cerevisiae.    
     
     
         50 . (canceled) 
     
     
         51 . The cell of  claim 32 , wherein the SPT and/or the AGT is encoded by a gene of  Arabidopsis thaliana, Drosophila melanogaster, Canis lupus familiaris, Homo sapiens  or  Rattus norvegicus.    
     
     
         52 . A method for producing ethylene glycol, comprising culturing the cell of  claim 1  to produce ethylene glycol. 
     
     
         53 - 62 . (canceled) 
     
     
         63 . A cell culture produced by culturing the cell of  claim 1 . 
     
     
         64 - 65 . (canceled) 
     
     
         66 . A supernatant of a cell culture produced by culturing the cell of  claim 1 . 
     
     
         67 - 68 . (canceled) 
     
     
         69 . A method for producing ethylene glycol in a cell, comprising:
 reducing or eliminating the activity or expression of aldehyde dehydrogenase A and/or xylulokinase in the cell, relative to a wild type cell;   increasing the expression of an enzyme that interconverts D-xylulose and D-ribulose, a D-ribulokinase, D-ribulose-phosphate aldolase, glycolaldehyde reductase and/or subunit of the E1 component of 2-oxoglutarate dehydrogenase in the cell, relative to a wild type cell; and   culturing the cell.   
     
     
         70 - 110 . (canceled) 
     
     
         111 . A method for producing ethylene glycol in a cell, comprising:
 reducing or eliminating the activity or expression of aldehyde dehydrogenase A and/or L-ribulokinase in the cell, relative to a wild type cell;   increasing the expression of an enzyme that interconverts L-ribulose and L-xylulose, an ATP:L-xylulose 1-phosphotransferase, L-xylulose-1-phosphate aldolase, and glycolaldehyde reductase in the cell, relative to a wild type cell; and   culturing the cell.   
     
     
         112 - 158 . (canceled)