Novel Gene Sms 37

Abstract

The present invention relates to newly identified genes that encode proteins that are involved in the synthesis of L-ascorbic acid (hereinafter also referred to as Vitamin C). The invention also features polynucleotides comprising the full-length polynucleotide sequences of the novel genes and fragments thereof, the novel polypeptides encoded by the polynucleotides and fragments thereof, as well as their functional equivalents. The present invention also relates to the use of said polynucleotides and polypeptides as biotechnological tools in the production of Vitamin C from microorganisms, whereby a modification of said polynucleotides and/or encoded polypeptides has a direct or indirect impact on yield, production, and/or efficiency of production of the fermentation product in said microorganism. Also included are methods/processes of using the polynucleotides and modified polynucleotide sequences to transform host microorganisms. The invention also relates to genetically engineered microorganisms and their use for the direct production of Vitamin C.

1 . A polynucleotide selected from the group consisting of:
 (a) polynucleotides encoding a polypeptide comprising the amino acid sequence according to SEQ ID NO:2;   (b) polynucleotides comprising the nucleotide sequence according to SEQ ID NO:1;   (c) polynucleotides comprising a nucleotide sequence obtainable by nucleic acid amplification such as polymerase chain reaction, using genomic DNA from a microorganism as a template and a primer set according to SEQ ID NO:3 and SEQ ID NO:4;   (d) polynucleotides comprising a nucleotide sequence encoding a fragment or derivative of a polypeptide encoded by a polynucleotide of any of (a) to (c) wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said fragment or derivative has the activity of a sugar exporter and/or sugar alcohol exporter;   (e) polynucleotides the complementary strand of which hybridizes under stringent conditions to a polynucleotide as defined in any one of (a) to (d) and which encode a sugar exporter and/or sugar alcohol exporter; and   (f) polynucleotides which are at least 60%, such as 70, 85, 90 or 95% identical to a polynucleotide as defined in any one of (a) to (d) and which encode a sugar exporter and/or sugar alcohol exporter   or   the complementary strand of such a polynucleotide.   
     
     
         2 . A vector containing the polynucleotide according to  claim 1 . 
     
     
         3 . The vector of  claim 2  in which the polynucleotide is operatively linked to expression control sequences allowing the expression in prokaryotic or eukaryotic host cells. 
     
     
         4 . A microorganism genetically engineered with a polynucleotide according to  claim 1  or with the vector of  claim 2  or  3 . 
     
     
         5 . A microorganism according to  claim 4  capable of directly producing Vitamin C from D-sorbitol in quantities of 300 mg/l or more when measured in a resting cell method after an incubation period of 20 hours. 
     
     
         6 . A microorganism according to  claim 5  capable of directly producing Vitamin C from L-sorbose in quantities of 800 mg/l or more. 
     
     
         7 . A polypeptide encoded by a polynucleotide according to  claim 1 . 
     
     
         8 . Process for producing cells capable of expressing a polypeptide according to  claim 7 , comprising the step of genetically engineering cells with the vector of  claim 2  or  3  or with a polynucleotide according to  claim 1 . 
     
     
         9 . Use of a polynucleotide according to  claim 1  or a vector according to  claims 2  or  3  for the production of Vitamin C. 
     
     
         10 . Use according to  claim 9 , wherein the polynucleotide is operatively linked to expression control sequences and transferred into a microorganism. 
     
     
         11 . Use according to  claim 10 , wherein the expression control sequences comprise a regulation-, and/or promoter-, and/or terminator sequence and wherein at least one of these sequences is altered in such a way that it leads to an improved yield and/or efficiency of production of Vitamin C produced by said microorganism. 
     
     
         12 . Use according to  claim 11 , wherein the expression control sequences comprise a regulation-, and/or promoter-, and/or terminator sequence and wherein at least one of these sequences is altered in such a way that it leads to an increased and/or improved sugar exporter and/or sugar alcohol exporter activity. 
     
     
         13 . A microorganism according to  claim 4  or a microorganism containing an endogenous gene comprising a polynucleotide according to  claim 1 , said microorganism being genetically altered in such a way that it leads to an improved yield and/or efficiency of production of Vitamin C produced by said microorganism. 
     
     
         14 . A microorganism according to  claim 13  producing a polypeptide according to  claim 7  with increased and/or improved a sugar exporter and/or sugar alcohol exporter activity. 
     
     
         15 . A microorganism according to any one of  claims 4  to  6 ,  13  or  14  wherein the polynucleotide according to  claim 1  is overexpressed. 
     
     
         16 . A microorganism according to any one of  claim 4  to  6  or  13  to  15  selected from the group consisting of  Pseudomonas, Pantoea, Escherichia, Ketogulonicigenium  and acetic acid bacteria like e.g.,  Gluconobacter, Acetobacter  or  Gluconacetobacter , preferably  Acetobacter  sp.,  Acetobacter aceti, Gluconobacter frateurii, Gluconobacter cerinus, Gluconobacter thailandicus, Gluconobacter oxydans , preferably  Gluconobacter oxydans , more preferably  Gluconobacter oxydans  DSM 17078. 
     
     
         17 . Process for the production of an enhanced endogenous sugar exporter and/or sugar alcohol exporter, gene in a microorganism, said microorganism comprising a polynucleotide according to  claim 1 , said process comprising the step of altering said polynucleotide in such a way that it leads to an improved yield and/or efficiency of production of Vitamin C produced by said microorganism. 
     
     
         18 . Process for the production of a microorganism capable of producing Vitamin C, comprising the step of altering said microorganism so that the microorganism produces a polypeptide with increased and/or improved sugar exporter and/or sugar alcohol exporter activity leading to an improved yield and/or efficiency of production of Vitamin C produced by said microorganism. 
     
     
         19 . Process for the production of a microorganism containing an endogenous gene comprising a polynucleotide according to  claim 1 , comprising the step of altering said microorganism so that the endogenous gene is overexpressed, leading to an improved yield and/or efficiency of production of Vitamin C produced by said microorganism. 
     
     
         20 . Process according to  claim 18  or  19  for the production of a microorganism according to any one of  claims 13  to  16 . 
     
     
         21 . Process for the production of Vitamin C with a microorganism according to any one of  claims 13  to  16  or  claims 4  to  6  wherein said microorganism is incubated in a aqueous medium under conditions that allow the direct production of Vitamin C from D-sorbitol or L-sorbose and wherein optionally Vitamin C is isolated as the fermentation product.