Production of polyketides in bacteria and yeast
AssigneeKosan Biosciences, Inc.
InventorsPhilip J. Barr, Daniel V. Santi, Gary W. Ashley, Rainer Ziermann
Life of this patent
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Abstract
Hybrid and novel polyketide synthases and polyketides are produced by use of a multiple vector system. The combinatorial possibilities offered by placing the various catalytic activities of PKS systems on separate vectors permits the construction of improved libraries of PKS and polyketides. In addition, polyketides can be produced in hosts that ordinarily do not produce polyketides by supplying, along with an expression system for the desired PKS, an expression system for holo ACP synthase.
Claims58
A modified recombinant host cell, which, in unmodified form, does not produce polyketides, which cell is modified to contain an expression system for a polyketide synthase (PKS) and an expression system for a holo ACP synthase, wherein
- said PKS comprising at least a ketosynthase/acyl transferase (KS/AT) catalytic region, a chain-length factor (CLF) catalytic region and an acyl carrier protein (ACP) activity of an aromatic PKS;
- or
- said PKS comprising at least a KS catalytic region, an AT catalytic region, and an ACP activity of a modular PKS or a fungal PKS, and
- the nucleotide sequence encoding said holo ACP synthase and the nucleotide sequence encoding said PKS are fused so as to encode a fusion protein.
A recombinant host cell modified to contain either
- a) at least two autonomously replicating vectors, said first vector containing a first selectable marker and a first expression system and said second vector containing a second selectable marker and a second expression system and optionally additional autonomously replicating vectors containing additional selectable markers and expression systems wherein said expression systems contained on said vectors are effective to produce a polyketide synthase (PKS);
- or
- b) at least one autonomously replicating vector and a modified chromosome, said one vector containing a first selectable marker and a first expression system and said modified chromosome containing a second expression system and optionally additional vectors containing additional selectable markers and expression systems wherein said expression systems contained on said vectors in combination with said expression system on said chromosome are effective to produce a PKS;
- said PKS comprising at least a KS catalytic region, an AT catalytic region and an ACP activity from a modular PKS;
- wherein at least one PKS protein produced by said expression system is encoded by less than a complete open reading frame encoding a native modular PKS.
The cell of 13 wherein at least one of said expression systems comprises an actinorhodin promoter.
The cell of 13 wherein at least one of said vectors comprises an SCP2* replicon.
A library of colonies of cells that produce polyketide synthases (PKS) and polyketides which library comprises a panel of individual colonies, wherein in each colony the cells contain either
- a) at least two autonomously replicating vectors;
- said first vector containing a first selectable marker and a first expression system and said second vector containing a second selectable marker and a second expression system and optionally additional autonomously replicating vectors containing additional selectable markers and expression systems wherein said expression systems contained on said vectors are effective to produce a polyketide synthase (PKS), or
- b) at least one autonomously replicating vector and a modified chromosome, said one vector containing a first selectable marker and a first expression system and said modified chromosome containing a second expression system and optionally additional vectors containing additional selectable markers and expression systems wherein said expression systems contained on said vectors in combination with said expression system on said chromosome are effective to produce a PKS;
- said PKS comprising at least a KS catalytic region, an AT catalytic region, and an ACP activity from a modular PKS
- wherein the combination of vectors or of vector(s) and modified chromosome in the cells is different with respect to each colony, and
- wherein at least one PKS protein produced by said expression system is encoded by less than a complete open reading frame encoding a native modular PKS.
A yeast expression vector that contains a selectable marker operable in yeast, and an expression system which comprises the coding region of at least one functional polyketide synthase catalytic activity fused to a coding region for a functional holo ACP synthase so as to encode a fusion protein operably linked to a promoter operable in yeast.
An E. coli expression vector that contains a selectable marker operable in E. coli, and an expression system which comprises the coding region of at least one functional polyketide synthase catalytic activity fused to a coding region for a functional holo ACP synthase so as to encode a fusion protein operably linked to a promoter operable in E. coli.
A method to produce a polyketide in a recombinant E. coli host cell modified to contain an expression system for a polyketide synthase (PKS) and an expression system for a holo ACP synthase, wherein
- said PKS comprising at least a ketosynthase/acyl transferase (KS/AT) catalytic region, a chain-length factor (CLF) catalytic region and an acyl carrier protein (ACP) activity of an aromatic PKS;
- or
- said PKS comprising at least a KS catalytic region, an AT catalytic region, and an ACP activity of a modular PKS or a fungal PKS,
- said method comprising incubating said cells under conditions to promote proper protein folding, or to increase malonyl CoA levels, or both.
A method to produce a polyketide in a recombinant yeast cell which method comprises:
- (i) providing a yeast cell modified to contain an expression system for a polyketide synthase (PKS) and an expression system for a holo ACP synthase, wherein
- said PKS comprises at least a KS catalytic region, an AT catalytic region, and an ACP activity of a modular PKS and
- (ii) incubating said modified cells under conditions such that said polyketide is produced.
A method to produce a polyketide in recombinant Saccharomyces cells, which method comprises:
- (a) providing Saccharomyces cells modified to contain an expression system for a fungal polyketide synthase (PKS) and an expression system for a holo-ACP synthase, wherein said fungal PKS comprises at least a KS catalytic region, an AT catalytic region, and an ACP catalytic region, and
- (b) incubating said modified cells under conditions wherein said polyketide is produced.
Dependencies are inferred from the claim text.