Production of polyketides in bacteria and yeast

Details Production of polyketides in bacteria and yeast Production of polyketides in bacteria and yeast

1999-10-21

2001-07-10

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C435S252300, C435S254110, C435S419000, C435S325000, C435S252330, C435S254200, C435S252600, C435S320100, C435S183000

Reexamination Certificate

active

06258566

ABSTRACT:

TECHNICAL FIELD
The invention relates to production of polyketides in microbial hosts such as yeast and
E. coli
and to preparation of libraries containing a variety of functional polyketide synthases (PKSs) and the resulting variety of polyketides. More specifically, it concerns supplying portions of the polyketide synthase systems on separate vectors for simplicity in mixing and matching these portions to create a variety of PKS. This permits production of libraries of polyketide syntheses and polyketides through a combinatorial approach rather than manipulation focused on a single production system.
BACKGROUND ART
Polyketides represent a singularly useful group of natural products which are related by their general pathway of biosynthesis. Representative members include the macrolide antibiotics, for example, erythromycin, spiramycin and tylosin, immunosuppressants such as rapamycin and FK506, antiparasitics such as the avermectins, antifungal agents such as amphotericin B and nystatin, anticancer agents such as daunorubicin and doxorubicin and anticholesterolemics such as mevinolin. Polyketides generally are secondary metabolites of the actinomycetes including the genera Streptomyces, Actinomyces, Actinomadura, Micromonospora, Saccharopolyspora, and Nocardia. It was estimated that in 1986 about 6,000 antibiotics of microbial origin had been characterized of which 70 were in clinical use; an additional 1100 metabolites were reported between 1988 and 1992, approximately 40% of which were polyketides.
Despite the multiplicity of polyketide structures available from nature, there remains a need to expand the repertoire of available polyketides and to synthesize a multiplicity of polyketides in the form of libraries so that there is a convenient substrate for screening to identify polyketides that are relevant to a specific target of interest. The present invention provides solutions to these needs.
Polyketides generally are synthesized by condensation of two-carbon units in a manner analogous to fatty acid synthesis. In general, the synthesis involves a starter unit and extender units; these “two-carbon” units are derived from acylthioesters, typically acetyl, propionyl, malonyl or methylmalonyl coenzyme-A thioesters. There are two major classes of polyketide synthases (PKSs) which differ in the “manner” in which the catalytic sites are used—the so-called “aromatic” PKS and the modular PKS. The present invention employs coding sequences from both these classes as will further be explained below.
Recombinant production of heterologous functional PKS—i.e., a PKS which is capable of producing a polyketide—has been achieved in Streptomyces and hybrid forms of aromatic PKSs have been produced in these hosts as well. See, for example, Khosla, C. et al.
J Bacteriol
(1993) 175:2194-2204; Hopwood, D. A. et al.
Nature
(1985) 314:642-644; Sherman, D. H. et al.
J Bacteriol
(1992) 174:6184-6190. In addition, recombinant production of modular PKS enzymes has been achieved in Streptomyces as described in PCT application WO 95/08548. In all of these cases, the PKS enzymes have been expressed from a single vector. A single vector which carried genes encoding PKS catalytic sites was transformed into
E.
coil by Roberts, G. A., et al.,
Eur J Biochem
(1993) 214:305-311, but the PKS was not functional, presumably due to lack of pantetheinylation of the acyl carrier proteins.
The present invention provides double or multivector systems for production of PKS and the resultant polyketides in a variety of hosts. The use of multiple vectors provides a means more efficiently to enhance the number of combinatorial forms of PKS and polyketides that can be prepared. Addition of the machinery for pantetheinylation of the acyl carrier proteins (i.e., a holo ACP synthase) permits production of polyketides in a wide spectrum of hosts.
DISCLOSURE OF THE INVENTION
The invention relates to recombinant materials for the production of polyketides in a wide variety of hosts and of libraries of PKS enzymes and the resultant polyketides based on a multiple vector system. The use of a multivector system facilitates the construction of combinatorial libraries and permits more flexibility in designing various members thereof. The invention also relates to such libraries which are essentially self-screening due to an autocrine system involving polyketide-responsive receptors.
Thus, in one aspect, the invention relates to a recombinant host cell and libraries thereof when the host cell is modified to contain at least two vectors, a first vector containing a first selection marker and a first expression system and the second vector containing a second selection marker and a second expression system and optionally additional vectors containing additional selectable markers and expression systems, wherein the expression systems contained on the vectors encode and are capable of producing at least a minimal PKS system. If the minimal PKS system is an aromatic system, the minimal system will comprise a ketosynthase/acyl transferase (KS/AT) catalytic region, a chain length factor (CLF) catalytic region and an acyl carrier protein (ACP) activity. If the minimal PKS system is a modular system, the system will contain at least a KS catalytic region, an AT catalytic region, and an ACP activity. For modular systems, these activities are sufficient provided intermediates in the synthesis are provided as substrates; if de novo synthesis is to be required, a loading acyl transferase should be included, which will include another AT and ACP region.
In one specific embodiment of this aspect of the invention, the recombinant host cell will be modified to contain: (a) a first vector comprising a first selectable marker and an expression system comprising a nucleotide sequence encoding a ketosynthase/acyl transferase (KS/AT) catalytic region of an aromatic PKS operably linked to a promoter operable in said cell; (b) a second vector comprising a second selectable marker and an expression system comprising a nucleotide sequence encoding a chain length factor (CLF) catalytic domain operably linked to a promoter operable in said cell; and (c) a third vector containing a third selectable marker and an expression system which comprises a nucleotide sequence encoding an acyl carrier protein (ACP) activity operably linked to a promoter operable in said cell, and to libraries comprised of colonies of such cells. Alternatively, two of the vectors can be combined so that the host cell contains only two vectors; the vector containing two expression systems may maintain these as separate expression systems or two open reading frames may be placed under the control of a single promoter.
In another specific embodiment, the invention relates to a cell modified to contain a first vector containing a first selectable marker and an expression system for at least one minimal module of a modular polyketide synthase (PKS) operably linked to a promoter operable in said cell; and a second vector containing a second selectable marker and a nucleotide sequence encoding at least a second minimal module of a modular polyketide synthase operably linked to a promoter operable in said cell, and to libraries comprising colonies of such cells.
In another variation, one or more expression systems for a defined portion of a PKS system is integrated into the host chromosome and at least one additional expression system resides on a replicable vector. Thus, in the case of aromatic PKS, an expression system for one of the open reading frames may first be integrated into the chromosome and expression systems for other open reading frames may reside on vectors. In the case of a modular PKS, an expression system for one or more modules may reside on the chromosome and additional expression systems for one or more modules reside on vectors. The integration of such expression systems into the chromosome can occur either through known phage-mediated integration or by homologous recombination.
The invention also is directed to novel polyketides produced by the methods of the

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