Production of polyketides and other natural products

Abstract

The present invention relates to production of polyketides and other natural products and to libraries of compounds and individual novel compounds. One important area is the isolation and potential use of novel FKBP-ligand analogues and host cells that produce these compounds. The invention is particularly concerned with methods for the efficient transformation of strains that produce FKBP analogues and recombinant cells in which cloned genes or gene cassettes are expressed to generate novel compounds such as polyketide (especially rapamycin) FKBP-ligand analogues, and to processes for their preparation, and to means employed therein (e.g. nucleic acids, vectors, gene cassettes and genetically modified strains).

Description

FIELD OF THE INVENTION [0001] The present invention relates to production of polyketides and other natural products and to libraries of compounds and individual novel compounds. One important area is the isolation and potential use of novel FKBP-ligand analogues and host cells that produce these compounds. The invention is particularly concerned with methods for the efficient transformation of strains that produce FKBP analogues and recombinant cells in which cloned genes or gene cassettes are expressed to generate novel compounds such as polyketide (especially rapamycin) FKBP-ligand analogues, and to processes for their preparation, and to means employed therein (e.g. nucleic acids, vectors, gene cassettes and genetically modified strains). BACKGROUND OF THE INVENTION [0002] Rapamycin (sirolimus) (FIG. 1) is a lipophilic macrolide produced by Streptomyces hygroscopicus NRRL 5491 (Sehgal et al., 1975; Vézina et al., 1975; U.S. Pat. No. 3,929,992; U.S. Pat. No. 3,993,749) with a 1,2,...

AI Technical Summary

Benefits of technology

"The present invention provides methods for efficiently transforming strains that contain biosynthetic clusters that encode FKBP-ligands, such as rapamycin, using recombinant DNA techniques. These methods involve constructing a conjugative deletion plasmid in an E. coli strain, growing spores from the strain, and culturing the strain under conditions suitable for polyketide production. The invention also provides recombinant strains containing biosynthetic clusters that encode FKBP-ligands where one or more auxiliary genes have been deleted or inactivated. Additionally, the invention provides methods for expressing combinations of polyketide modification enzymes to produce novel polyketide analogues."

Problems solved by technology

Modification using chemically available positions on the molecule has been addressed, however, this approach has limited utility as the sites available for chemical modification are limited and there is less ability to selectively modify a particular position.

Biological approaches to producing novel rapamycin analogues have been less successful due to the difficulties encountered in working with the organism (Lomovskaya et al., 1997; Kieser et al., 2000) despite the availability of the sequence of the biosynthetic gene cluster of rapamycin from S. hygroscopicus (Schwecke et al., 1995).

However, chemical modification requires significant quantities of rapamycin template and, as a base and acid labile compound, it is difficult to work with.

Where chemical derivatisation can be group selective, it is often difficult to be site selective.

These strains are often low yielding and produce mixtures of rapamycin analogues.

The use of such inhibitors, however, only allows the targeting of a particular enzyme function and is not site selective.

Rational production of a single selected analogue is not possible via this method.

The resulting production of mixtures of rapamycin analogues rather than a single desired product also impacts yield.

These novel rapamycin analogues were produced in competition with the natural starter, 4,5-dihydroxycyclohex-1-enecarboxylic acid, resulting in reduced yields and mixed products.

Furthermore, the site-specific functionality of rapM and rapQ remains unclear, therefore, rational design of rapamycin analogues requiring methylation at C16-OH or C27-OH has not been enabled.

It offers a difficult and protracted process of obtaining engineered strains and has a reduced versatility in comparison to the methodology disclosed within this current patent.

However, the isolation of novel rapamycin analogues using such biological methods has been limited due to the difficulties in transforming the rapamycin-producing organism S. hygroscopicus.

It has been reported that the commonly used methods of transformation with plasmid DNA or conjugal transfer were unsuccessful with the rapamycin producing strain (Lomovskya et al., 1997, Schweke et al., 1995, Kieser et al., 2000).

(1997), a work intensive phage based method that is severely limited by the size of the cloned DNA fragments transferred into S. hygroscopicus (Kieser et al., 2000).

This technology is limited to the transfer of a maximum of 6.4 kb of cloned DNA.

Thus, when complementing a deletion mutant using this technology the artisan is limited to the inclusion of ˜2 functional genes in addition to desired promoter, regions of homology and resistance marker.

The genetic information for the rapamycin biosynthetic gene cluster has been available since 1995 (Schwecke et al., 1995), however, limited progress in this area has been made (Khaw et al., 1998; Chung et al., 2001; WO01 / 34816).

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