Nucleic acid encoding a cobalamin-dependent methionine synthase polypeptide

Abstract

The present invention relates to nulceotide sequences encoding enzymatically active cobalamin-methionine synthase and functional fragments thereof being modified in comparison to the respective wild-type enzyme such that said enzymes show reduced product inhibition by methionine. The present invention also relates to polypeptides being encoded by such nucleotide sequences and host cells comprising such nucleotide sequences. Furthermore, the present invention relates to methods for producing methionine in host organisms by making use of such nucleotide sequences.

1 - 27 . (canceled) 
     
     
         28 . An isolated polynucleotide which encodes a polypeptide comprising SEQ ID NO:2 with a mutation at position 86 wherein the polypeptide exhibits cobalamin-dependent methionine synthase activity. 
     
     
         29 . The isolated polynucleotide of  claim 28 , wherein the polypeptide is SEQ ID NO:1 with a mutation at position 86. 
     
     
         30 . The isolated polynucleotide of  claim 28 , wherein the polypeptide has a mutation in its homocysteine-binding domain. 
     
     
         31 . The isolated polynucleotide of  claim 28 , wherein the polypeptide exhibits reduced product inhibition by methionine. 
     
     
         32 . The isolated polynucleotide of  claim 28 , wherein the mutation is Phenylalanine at position 86 is replaced by Leucine. 
     
     
         33 . The isolated polynucleotide of  claim 28 , wherein the polypeptide is selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22. 
     
     
         34 . An expression vector comprising the polynucleotide of  claim 28 . 
     
     
         35 . A host cell which comprises the polynucleotide of  claim 28 . 
     
     
         36 . The host cell of  claim 35 , wherein the host cell is a microorganism selected from the group consisting of:  Corynebacterium glutamicum, Escherichia coli, Streptomyces coelicolor  and  Thermotoga maritima.    
     
     
         37 . The host cell of  claim 35 , wherein one or more of the endogenous genes of the host cell which encode cobalamin-dependent methionine synthetase is deleted or functionally disrupted. 
     
     
         38 . The host cell of  claim 35 , wherein the amount and/or activity of at least one of the following nucleotide sequences selected from the group consisting of:
 nucleotide sequence coding for aspartate kinase lysC,   nucleotide sequence coding for glycerine aldehyde-3-phosphate dehydrogenase gap,   nucleotide sequence coding for 3-phosphoglycerate kinase pgk,   nucleotide sequence coding for pyruvatecarboxylase pyc,   nucleotide sequence coding for triosephosphate isomerase tpi,   nucleotide sequence coding for homoserin-O-acetyltransferase metA,   nucleotide sequence coding for cystathione-gamma-synthase metB,   nucleotide sequence coding for cystathione-gamma-lyase metC,   nucleotide sequence coding for serin-hydroxymethyl transferase glyA,   nucleotide sequence coding for O-acetylhomoserine-sulfhydrylase metY,   nucleotide sequence coding for phosphoserine aminotransferase serC,   nucleotide sequence coding for phosphoserine-phosphatase serB,   nucleotide sequence coding for serine acetyltransferase cysE,   nucleotide sequence coding for homoserine-dehydrogenase horn,   nucleotide sequence coding for methionine synthase metE,   nucleotide sequence coding for phosphoadenosine-phosphosulfate-reductase cysH,   nucleotide sequence coding for sulfate adenylyl transferase-subunit I,   nucleotide sequence coding for CysN-sulfate adenylyl transferase-subunit 2,   nucleotide sequence coding for ferredoxin-NADP-reductase,   nucleotide sequence coding for ferredoxin,   nucleotide sequence coding for glucose-6-phosphate-dehydrogenase, and   nucleotide sequence coding for fructose-1-6-bisphosphatase is increased in comparison to the corresponding parent strain.   
     
     
         39 . The host cell of  claim 35 , wherein the amount and/or activity of at least one of the following nucleotide sequences selected from the group consisting of
 nucleotide sequence coding for homoserine kinase thrB,   nucleotide sequence coding for threonine dehydratase ilvA,   nucleotide sequence coding for threonine synthase thrC,   nucleotide sequence coding for meso-diaminopimelate-D-dehydrogenase ddh,   nucleotide sequence coding for phosphoenolpyruvate carboxy kinase pck,   nucleotide sequence coding for glucose-6-phosphate-6-isomerase pgi,   nucleotide sequence coding for pyruvate-oxidase poxB,   nucleotide sequence coding for dihydrodipicolinate synthase dapA,   nucleotide sequence coding fro dihydrodipicolinate reductase dapB,   nucleotide sequence coding for diaminopicolinate-decarboxylase lysA,   nucleotide sequence coding for glycosyl transferase and   nucleotide sequence coding for lactate hydrogenase is reduced in comparison to the corresponding parent strain.   
     
     
         40 . A method of producing methionine which comprises
 a) cultivating the host cell of  claim 35 , and   b) isolating the methionine.   
     
     
         41 . A method of producing methionine which comprising:
 a) transfecting the vector of  claim 35  into a host cell,   b) culturing the host cell, and   c) optionally recovering the methionine