Method of improving methyltransferase activity
AssigneeDanmarks Tekniske Universitet
InventorsHao LUO, Anne Sofie Laerke HANSEN
Life of this patent
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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.
Claims19
A genetically modified cell which comprises
- (a) a SAM-dependent methyltransferase;
- (b) a biosynthetic pathway converting S-adenosylhomocysteine (SAH) to a metabolite via a homocysteine intermediate, wherein the metabolite is selected from cysteine, 2-oxobutanoate, isoleucine, or a combination of any thereof;
- (c) one or more genetic modifications disrupting any endogenous H2S-dependent and/or L-cysteine-dependent biosynthesis of homocysteine in the cell,
- wherein the growth of the cell is dependent on the production of the metabolite from the biosynthetic pathway in (b).
The genetically modified cell of any one of the preceding claims, comprising one or more genetic modifications disrupting an endogenous pathway for the biosynthesis of the metabolite, other than that in (b).
The genetically modified cell of any one of the preceding claims, which has further been genetically modified to downregulate or delete at least one endogenous gene encoding another SAM-dependent methyltransferase.
The genetically modified cell of any one of the preceding claims, which is a microbial cell.
A genetically modified bacterial cell comprising a SAM-dependent methyltransferase, a heterologous cystathionine-beta-synthase, a heterologous cystathionine-gamma-lyase, and
- (a) a downregulation or deletion of cysE;
- (b) a downregulation or deletion of cysK and cysM;
- (c) a downregulation or deletion of cysE and ilvA;
- (d) a downregulation or deletion of cysE and tdcB;
- (e) a downregulation or deletion of cysE, ilvA and tdcB;
- (f) a downregulation or deletion of cysE, ilvA, tdcB, and metA;
- (g) a downregulation or deletion of cysE, ilvA, tdcB, and metB;
- (h) a downregulation or deletion of metA, ilvA and tdcB;
- (i) a downregulation or deletion of metB, ilvA and tdcB;
- (j) a downregulation or deletion of metC, malY, ilvA and tdcB;
- (k) a downregulation or deletion of metC, malY, ilvA, tdcB and metA;
- (l) a downregulation or deletion of metC, malY, ilvA, tdcB and metB;
- or
- (m) any one of (a) to (l), further comprising a downregulation or deletion of cfa.
The genetically modified cell of any one of the preceding claims, which is an E. coli, B. subtilis, C. glutamicum, S. coelicolor, S. griseus, R. eutropha or C. acetobutylicum cell.
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 MET6, 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.
The genetically modified cell of any one of the preceding claims, 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.
A composition comprising a plurality of the genetically modified cell of any one of the preceding claims.
A method of preparing a genetically modified microbial 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 microbial cell with a nucleic acid encoding a SAM-dependent methyltransferase;
- (b) optionally, transforming the 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 H2S-dependent and/or L-cysteine-dependent biosynthesis of homocysteine in the 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.
Dependencies are inferred from the claim text.