Recombinant host cells for the production of polyketides

Details Recombinant host cells for the production of polyketides Recombinant host cells for the production of polyketides

1998-10-28

2001-01-23

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S252350

Reexamination Certificate

active

06177262

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides recombinant host cells and methods for producing polyketides. Polyketides are a diverse class of compounds with a wide variety of activities, including activities useful for medical, veterinary, and agricultural purposes. The present invention therefore relates to the fields of molecular biology, chemistry, recombinant DNA technology, medicine, animal health, and agriculture.
BACKGROUND OF THE INVENTION
Polyketides have been produced in a variety of host cells, including Streptomyces, Saccharopolyspora, and Aspergillus for commercial purposes for many years. In particular, these compounds are often found in mycelial bacteria, the actinomycetes, in which the compounds are synthesized by enzymes known as polyketide synthases (PKSs) and produced as secondary metabolites. Typically, a polyketide was first identified as an active but uncharacterized ingredient in a soil or other environmental sample. Once an active ingredient was identified, then the organism that produced the ingredient was isolated and characterized. After the organism was characterized, it was often the subject of an intensive effort to increase the yield of the active ingredient. This effort typically involved successive rounds of subjecting the organism to mutagenic conditions, culturing the mutagenized organisms, and selecting those mutant organisms that produced the active ingredient in higher yields.
With the advent of molecular biology, the genes for the enzymes, called polyketide synthases or PKS(s), that perform the synthesis of certain polyketides became known. In some instances, such as, for example, the PKS enzymes that catalyze the synthesis of modular polyketides, the PKS enzymes are very large, multi-subunit proteins encoded by large gene clusters ranging from 10 kilobases (kb) to more than 100 kb in size. See, e.g., PCT patent publication No. 93/13663 (erythromycin); U.S. Pat. No. 5,098,837 (tylosin); U.S. Pat. No. 5,272,474 (avermectin); U.S. Pat. No. 5,744,350 (triol polyketide); and European patent publication No. 791,656 (platenolide), each of which is incorporated herein by reference. The cloning of these and other genes led to speculation that the yields of polyketide produced by these organisms could be increased by molecular biology techniques. See, e.g., U.S. Pat. No. 5,672,497. Despite these advances, however, there have been few, if any, reports of polyketide-producing strains that have been improved using such techniques.
Others working in the field developed novel methods and host cells not only for producing polyketides in host cells in which naturally occurring polyketide synthase genes had been eliminated or which otherwise did not produce polyketides but also for producing polyketides not otherwise found in nature in recombinant host cells. Thus, 6-deoxyerythronolide B has been produced in a
Streptomyces coelicolor
strain from which the endogenous actinorhodin gene cluster has been eliminated. See U.S. Pat. Nos. 5,672,491 and 5,712,146 and McDaniel et al., 1993, Engineered biosynthesis of novel polyketides,
Science
262:1546-1550, each of which is incorporated herein by reference. In addition, the successful synthesis of a fungal polyketide, 6-methylsalicylic acid (6-MSA), has been reported in
E. coli
and in yeast. See Kealey et al., 1998, Production of a polyketide natural product in nonpolyketide producing prokaryotic and eukaryotic hosts,
Proc. Natl. Acad. Sci. USA
95:505-509, and PCT patent publication No. 98/27203, incorporated herein by reference. Also, methods, reagents, and host cells were developed for producing novel polyketides from starting units not used by polyketide producing organisms in nature. See PCT patent publication No. 97/02358 and PCT patent application No. US98/14911. While the novel polyketides produced by such methods and cells were useful, yields were sometimes low, and rapid application of the technology in new host cells was sometimes hindered by endogenous recombination pathways and restriction—modification systems.
As one example, Kieser et al., December 1989, A mutation of
Streptomyces lividans
which prevents intraplasmid recombination has no effect on chromosomal recombination,
Mol. Gen. Genet
. 220(1): 60-64, reported on a recombination-deficient strain of
S. lividans
, JT46, originally characterized by Tsai and Chen, 1987, Isolation and characterization of
Streptomyces lividans
mutants deficient in intraplasmid recombination,
Mol. Gen. Genet
. 208: 211-218. This strain, however, produced the pigmented antibiotic actinorhodin produced by the unmodified parent strain
S. lividans
and so is not especially preferred for the production of other polyketides.
Other researchers have noted that actinorhodin as well as undecylprodigiosin and A-factor levels can be increased in
Streptomyces lividans
by transforming the strain with a vector having a copy number of 3-4 (but not 1-2) and encoding a phosphotyrosine protein phosphatase gene (ptpA) with its endogenous promoter isolated from
S. coelicolor
. See Umeyama et al., 1996, Expression of the
Streptomyces coelicolor
A3(2) ptpA gene encoding a phosphotyrosine protein phosphatase leads to overproduction of secondary metabolites in
S. lividans, FEMS Microbiology Letters
144: 177-184; and Li and Strohl, January 1996, Cloning, purification, and properties of a phosphotyrosine protein phosphatase from
Streptomyces coelicolor
A3(2),
J. Bacteriology
178(1): 136-142. Unfortunately, actinorhodin has little if any therapeutic value.
There remains a need for host cells that can produce useful polyketides at higher levels than can be achieved with currently available cells, as well as a need for cells that can be transformed at high efficiency and can stably maintain extrachromosomal plasmids containing polyketide synthase genes over many generations. The present invention meets these and other needs.
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a
Streptomyces lividans
host cell that can be transformed at high efficiency with methylated DNA and that does not produce actinorhodin. In a preferred mode, this host cell is
S. lividans
K4-114 or K4-155.
In another embodiment, the present invention provides a
Streptomyces lividans
host cell that lacks an ability to recombine plasmid DNA with other plasmid DNA. In a preferred mode, this host cell is
S. lividans
K27-39.
In another embodiment, the invention provides a method for increasing production of a polyketide other than actinorhodin or undecylprodigiosin in a host cell that produces said polyketide, said method comprising steps of: (a) transforming said host cell with a recombinant vector that comprises a ptpA gene capable of expression in said host cell; and (b) culturing said host cell transformed in step (a) under conditions such that said ptpA gene is expressed and said polyketide is produced. The ptpA gene may be under the control of its endogenous promotor or may be controlled by a heterologous promoter. In a preferred mode, the ptpA gene is under the control of a heterologous promoter from a polyketide biosynthetic gene. In an especially preferred mode the promoter is the actI promoter. The recombinant vector can be an integrating or extrachromosomally maintained vector.
In a preferred mode, the method is practiced with a Streptomyces host cell, such as
S. coelicolor
or
S. lividans
, including, but not limited to
S. coelicolor
CH999
, S. lividans
K4-114
, S. lividans
K4-155, and
S. lividans
K27-39. In another preferred mode, the polyketide is produced by a recombinant polyketide synthase. In another preferred mode, the recombinant polyketide synthase is encoded on a plasmid in said host cell. In a very preferred mode, the polyketide is erythromycin or an erythromycin precursor (i.e., 6-deoxyerythronolide B) that is produced from a recombinant polyketide synthase that differs from an erythromycin PKS by substitution, insertion, or deletion of one or more amino acid residues. As one example, the erythromycin precursor can be 10,11-anhydro,

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