Metabolically Engineered Bacterial Strains Having Non-Functional Endogenous Gluconate Transporters

Abstract

The present invention relates to engineering metabolic pathways in bacterial host cells which results in enhanced carbon flow for the production of ascorbic acid (ASA) intermediates. In particular, the invention relates to increasing the production of ASA intermediates in bacterial cells by enhancing the availability of gluconate resulting from the inactivation of endogenous gluconate transporter genes.

1 . A method for enhancing the level of production of an ascorbic acid (ASA) intermediate comprising
 a) obtaining an altered bacterial host cell which is capable of producing an ASA intermediate, wherein an endogenous gluconate transporter gene has been rendered non-functional;   b) culturing the altered bacterial host cell under suitable culture conditions in the presence of a carbon source to allow the production of an ASA intermediate; and   c) obtaining the ASA intermediate,   
       wherein the level of production of the ASA intermediate in the altered bacterial host cell is enhanced compared to the level of production of the ASA intermediate in a corresponding unaltered bacterial cell grown under essentially the same culture conditions. 
     
     
         2 . The method according to  claim 1  further comprising isolating the ASA intermediate. 
     
     
         3 . The method according to  claim 1 , wherein the ASA intermediate is selected from the group consisting of gluconate, 2-keto-D-gluconate (2-KDG), 2,5-diketo-D-gluconate (2,5-DKG), 2-keto-L-gulonic acid (2-KLG), 5-keto-D-gluconate (5-KDG), and L-idonic acid (IA). 
     
     
         4 . The method according to  claim 3 , wherein the ASA intermediate is 2-KDG. 
     
     
         5 . The method according to  claim 3 , wherein the ASA intermediate is 2-KLG. 
     
     
         6 . The method according to  claim 1 , further comprising converting the ASA intermediate to erythorbic acid. 
     
     
         7 . The method according to  claim 1 , wherein the endogenous gluconate transporter gene encodes a gluconate transporter having the amino acid sequence of a) SEQ ID NO: 2, b) a sequence having at least 40% sequence identity to SEQ ID NO: 2, c) SEQ ID NO: 4 or d) a sequence having at least 80% identity to SEQ ID NO: 4. 
     
     
         8 . The method according to  claim 1 , wherein two endogenous gluconate transporter genes are inactivated, wherein a first gluconate transporter gene encodes a gluconate transporter having at least 90% sequence identity to SEQ ID NO: 2 and a second gluconate transporter gene encodes a gluconate transporter having at least 90% sequence identity to SEQ ID NO: 4. 
     
     
         9 . The method according to  claim 1 , wherein the altered bacterial host cell is a  Pantoea  cell or an  E. coli  cell. 
     
     
         10 . The method according to  claim 9 , wherein the  Pantoea  cell is a  P. citrea  cell. 
     
     
         11 . The altered bacterial host cell obtained according to  claim 1 . 
     
     
         12 . The method according to  claim 9 , wherein the endogenous gluconate transporter gene encodes a gluconate transporter having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or a sequence having at least 80% identity to either SEQ ID NO: 2 or SEQ ID NO: 4. 
     
     
         13 . The method according to  claim 1 , wherein the carbon source is selected from the group consisting of glucose, gluconate, fructose, galactose and combinations thereof. 
     
     
         14 . The method according to  claim 1 , wherein the endogenous gluconate transporter gene has been rendered non-functional by deletion of the gene. 
     
     
         15 . The method according to  claim 1 , wherein the altered bacterial host cell further comprises an inactivated endogenous glucokinase gene. 
     
     
         16 . A method for obtaining an improved bacterial strain for the production of an ascorbic acid (ASA) intermediate comprising
 a) obtaining a bacterial host cell which is capable of producing an ASA intermediate;   b) inactivating at least one endogenous gluconate transporter gene, wherein the endogenous gluconate transporter gene encodes a gluconate transporter having the amino acid sequence of at least 40% sequence identity with SEQ ID NO: 2 or at least 80% sequence identity with SEQ ID NO: 4; and   c) culturing the bacterial host cell under suitable culture conditions in the presence of a carbon source to allow for the production of ASA intermediates.   
     
     
         17 . The method according to  claim 16 , wherein the endogenous gluconate transporter gene has been deleted. 
     
     
         18 . The improved bacterial strain obtained according to the method of  claim 16 . 
     
     
         19 . The improved bacterial strain obtained according to the method of  claim 17 . 
     
     
         20 . The improved bacterial strain of  claim 18 , wherein said strain is a  Pantoea  strain. 
     
     
         21 . An altered Enterobacteriaceae strain comprising one or more inactivated endogenous gluconate transporter proteins, wherein the one or more inactivated endogenous gluconate transporter proteins are selected from the group consisting of the amino acid sequence of SEQ ID NO: 2, an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4 and an amino acid sequence having at least 80% identity with SEQ ID NO: 4. 
     
     
         22 . The altered Enterobacteriaceae strain of  claim 21 , wherein the one or more inactivated endogenous gluconate transporter proteins include two inactivated gluconate transporter proteins. 
     
     
         23 . The altered Enterobacteriaceae strain of  claim 21 , wherein the one or more inactivated endogenous gluconate transporter proteins is rendered nonfunctional by deletion of the genes encoding the one or more endogenous gluconate transporter proteins. 
     
     
         24 . The altered Enterobacteriaceae strain of  claim 21 , wherein said Enterobacteriaceae strain is selected from the group of  Pantoea, Gluconobacter, Erwinia, Klebsiella , and  Escherichia  strains. 
     
     
         25 . The altered Enterobacteriaceae strain of  claim 24  further comprising an inactivated endogenous glucokinase gene, said inactivation preventing the expression of an active glucokinase. 
     
     
         26 . A recombinant  Pantoea  strain comprising
 a) a first inactivated endogenous gluconate transporter gene, wherein the first gluconate transporter gene prior to inactivation encoded a gluconate transporter having at least 90% sequence identity with SEQ ID NO: 2,   b) a second inactivated endogenous gluconate transporter gene, wherein the second gluconate transporter gene prior to inactivation encoded a gluconate transporter having at least 90% sequence identity with SEQ ID NO: 4, and   c) an inactivated endogenous glucokinase gene.   
     
     
         27 . The recombinant  Pantoea  strain of  claim 26 , wherein the first inactivated endogenous gluconate transporter gene and the second inactivated endogenous gluconate transporter gene have been deleted. 
     
     
         28 . A method for increasing the availability of gluconate for oxidative pathway production of an ascorbic acid (ASA) intermediate comprising
 a) obtaining an altered bacterial host cell, wherein an endogenous gluconate transporter gene has been rendered non-functional;   b) culturing the altered bacterial host cell under suitable culture conditions in the presence of a carbon source to allow the production of an ASA intermediate; and   c) obtaining the ASA intermediate,   
       wherein the ASA intermediate is selected from the group consisting of 2-keto-D-gluconate (2-KDG), 2,5-diketo-D-gluconate (2,5-DKG), 2-keto-L-gulonic acid (2-KLG), 5-keto-D-gluconate (5-KDG) and L-idonic acid (IA) and the endogenous gluconate transporter gene encodes a gluconate transporter having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or a sequence having at least 80% identity to either SEQ ID NO: 2 or SEQ ID NO: 4. 
     
     
         29 . The method according to  claim 28 , wherein the altered bacterial host cell is a  Pantoea  cell or an  E. coli  cell. 
     
     
         30 . An isolated polynucleotide encoding a gluconate transporter having the amino acid sequence of
 a) SEQ ID NO: 2,   b) a sequence having at least 40% sequence identity with SEQ ID NO: 2,   c) SEQ ID NO: 4 or   d) a sequence having at least 80% sequence identity with SEQ ID NO: 4.   
     
     
         31 . The isolated polynucleotide of  claim 30  having the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3 or a sequence having at least 80% identity to either SEQ ID NO: 1 or SEQ ID NO: 3. 
     
     
         32 . The isolated polynucleotide of  claim 30  encoding the gluconate transporter having at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4. 
     
     
         33 . A gluconate transporter having the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4 or an amino acid sequence having at least 80% identity to either SEQ ID NO: 2 or SEQ ID NO: 4.