Method of improving methyltransferase activity

Abstract

Methods for evolving cells or strains towards improved methyltransferase activity, particularly SAM-dependent methyltransferase activity, as well as to cells and strains useful in such methods and methods of using the evolved cells in the production of methylated products.

1 . (canceled) 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . A genetically modified yeast cell comprising an s-adenosyl methionine- (SAM-) dependent methyltransferase which is heterologous to the yeast cell and a downregulation or deletion of an endogenous gene encoding acetylhomoserine sulfhydrylase, a downregulation or deletion of an endogenous gene encoding homoserine O-acetyltransferase, or a downregulation or deletion of an endogenous gene encoding an L-threonine/L-serine ammonia-lyase, wherein the downregulation of a gene reduces the level of enzyme encoded by the gene by at least 95%. 
     
     
         21 . The genetically modified yeast cell of  claim 20 , which is a  Saccharomyces, Candida, Hansenula, Kluyveromyces, Pichia, Schizosaccharomyces  or  Yarrowia  cell. 
     
     
         22 . The genetically modified yeast cell of  claim 21 , which is a  Saccharomyces  cell. 
     
     
         23 . The genetically modified yeast cell of  claim 22 , wherein the endogenous gene encoding acetylhomoserine sulfhydrylase is MET17, the homoserine O-acetyltransferase is MET2, and the L-threonine/L-serine ammonia-lyase is CHA1 or ILV1. 
     
     
         24 . The genetically modified yeast cell of  claim 20 , wherein the SAM-dependent methyltransferase is selected from the group consisting of an O-methyltransferase, a C-methyltransferase, an N-methyltransferase and an S-methyltransferase. 
     
     
         25 . The genetically modified yeast cell of  claim 24 , wherein the SAM-dependent methyltransferase is selected from the group consisting of an acetylserotonin O-methyltransferase (ASMT) and a caffeine synthase. 
     
     
         26 . The genetically modified yeast cell of  claim 25 , wherein the SAM-dependent methyltransferase is Promo  sapiens  ASMT,  Ocimum basilicum  ASMT,  Bos taurus  ASMT,  Takifugu rubripes  ASMT,  Macaca mulatta  ASMT,  Elephantulus edwardii  ASMT,  Oryza sativa  ASMT,  Rattus norvegicus  ASMT,  Gallus gallus  ASMT,  Chromobacterium violaceum  ASMT,  Desulfotomaculum kuznetsovii  DSM 6115 ASMT,  Xenopus  ( Silurana )  tropicalis  ASMT,  Pseudomonas fluorescens  ASMT,  Candidatus Solibacter usitatus  ASMT,  Fenneropenaeus chinensis  ASMT,  Arabidopsis thaiiana  ASMT, or  Coffea arabica  caffeine synthase. 
     
     
         27 . A genetically modified  Saccharomyces  cell comprising a SAM-dependent methyltransferase which is heterologous to the native  Saccharomyces  cell, and
 (a) a downregulation or deletion of MET17;   (b) a downregulation or deletion of MET2;   (c) a downregulation or deletion of MET2 and MET17   (d) a downregulation or deletion of MET17, CHA1 and ILV1;   (e) a downregulation or deletion of MET2, CHA1 and ILV1;   (f) a downregulation or deletion of MET17, MET2, CHA1 and ILV1; or   (g) any one of (a) to (f), further comprising a downregulation or deletion of METE, or   (h) any one of (a) to (g), further comprising a downregulation or deletion of one or more native SAM-dependent methyltransferases, optionally selected from one or more of ERG6, CHO2, OPI3, SET2, SET1 and DOT1,
 wherein the downregulation of a gene reduces the level of enzyme encoded by the gene by at least 95%. 
   
     
     
         28 . The genetically modified  Saccharomyces  cell of  claim 27 , which comprises a downregulation or deletion of MET17 and/or MET2. 
     
     
         29 . The genetically modified  Saccharomyces  cell of  claim 28 , which comprises a downregulation or deletion of MET17, MET2, CHO2, OPI3 and SET2. 
     
     
         30 . The genetically modified  Saccharomyces  cell of  claim 29 , which is a  Saccharomyces cerevisiae  cell. 
     
     
         31 . The genetically modified  Saccharomyces  cell of  claim 30 , wherein the SAM-dependent methyltransferase is selected from the group consisting of an O-methyltransferase, a C-methyltransferase, an N-methyltransferase and an S-methyltransferase. 
     
     
         32 . The genetically modified  Saccharomyces  cell of  claim 31 , wherein the SAM-dependent methyltransferase is selected from the group consisting of an acetylserotonin O-methyltransferase (ASMT) and a caffeine synthase. 
     
     
         33 . The genetically modified  Saccharomyces  cell of  claim 32 , wherein the SAM-dependent methyltransferase is  Homo sapiens  ASMT,  Ocimum basilicum  ASMT,  Bos taurus  ASMT,  Takifugu rubripes  ASMT,  Macaca mulatta  ASMT,  Elephantulus edwardii  ASMT,  Oryza sativa  ASMT,  Rattus norvegicus  ASMT,  Gallus gallus  ASMT,  Chromobacterium violaceum  ASMT,  Desulfotomaculum kuznetsovii  DSM 6115 ASMT,  Xenopus  ( Silurana )  tropicalis  ASMT,  Pseudomonas fluorescens  ASMT,  Candidatus Solibacter usitatus  ASMT,  Fenneropenaeus chinensis  ASMT,  Arabidopsis thaliana  ASMT, or  Coffea arabica  caffeine synthase. 
     
     
         34 . A composition comprising a plurality of the genetically modified  Saccharomyces  cell of  claim 27 . 
     
     
         35 . The composition of  claim 34 , comprising a culture medium comprising methionine, at least one substrate or substrate precursor of the SAM-dependent methyltransferase, and a carbon source. 
     
     
         36 . A method of preparing a genetically modified yeast cell which is growth-dependent on a metabolite selected from cysteine, 2-oxobutanoate and isoleucine, or a combination of any thereof, comprising the steps of
 (a) transforming a yeast cell with a nucleic acid encoding a SAM-dependent methyltransferase;   (b) optionally, transforming the yeast cell with nucleic acids encoding cystathionine-beta-synthase and a cystathionine-gamma-lyase;   (c) reducing or disrupting the expression or activity of at least one enzyme in any endogenous pathway for H 2 S-dependent and/or L-cysteine-dependent biosynthesis of homocysteine in the yeast cell, and   (d) reducing or disrupting the expression or activity of at least one enzyme in any endogenous pathway for cysteine, 2-oxobutanoate or isoleucine, or a combination of any thereof,   
       wherein steps (a) to (d) are performed in any order. 
     
     
         37 . A method of evolving the SAM-dependent methyltransferase activity of a  Saccharomyces  cell, comprising cultivating the genetically modified  Saccharomyces  cell of  claim 27  in a medium comprising an abundance of methionine, at least one substrate or substrate precursor of the SAM-dependent methyltransferase, and a carbon source. 
     
     
         38 . A method of preparing a  Saccharomyces  cell having an improved SAM-dependent methyltransferase activity, comprising the steps of:
 (a) culturing a plurality of the genetically modified  Saccharomyces  cell of  claim 27  in a medium comprising an abundance of methionine, at least one substrate or substrate precursor of the SAM-dependent methyltransferase, and a carbon source; and   (b) selecting any  Saccharomyces  cell having an increased growth rate as compared to the genetically modified  Saccharomyces  cell prior to step (a) as a  Saccharomyces  cell having an improved SAM-dependent methyltransferase activity.   
     
     
         39 . A method of producing a methylated product from a substrate of a SAM-dependent methyltransferase, the method comprising the steps of:
 (a) culturing a plurality of the genetically modified  Saccharomyces  cell of  claim 27  in a medium comprising an abundance of methionine, at least one substrate or substrate precursor of the SAM-dependent methyltransferase, and a carbon source;   (b) selecting any  Saccharomyces  cell having an increased growth rate as compared to the genetically modified cell or composition prior to step (a) as a  Saccharomyces  cell having an improved SAM-dependent methyltransferase activity;   (c) producing the methylated product by fermenting a  Saccharomyces  cell selected in step (b) in a medium comprising methionine, the substrate or substrate precursor of the SAM-dependent methyltransferase, and a carbon source; and   (d) optionally, retrieving the methylated product from the cell or fermentation medium.