Combinatorial polyketide libraries produced using a modular PKS gene cluster as scaffold

#The invention relates to the field of combinatorial libraries, to novel polyketides and antibiotics and to methods to prepare them. More particularly, it concerns construction of new polyketides and to libraries of polyketides synthesized by polyketide synthases derived from a naturally occurring PKS, as illustrated by the erythromycin gene cluster.

#Polyketides represent a large family of diverse compounds ultimately synthesized from 2-carbon units through a series of Claisen-type condensations and subsequent modifications. Members of this group include antibiotics such as tetracyclines, anticancer agents such as daunomycin, and immunosuppressants such as FK506 and rapamycin. Polyketides occur in many types of organisms including fungi and mycelial bacteria, in particular, the actinomycetes.

#A number of lactones of keto acids have been synthesized using standard organic chemistry. These include a series of unsaturated ketolactones synthesized by Vedejes et al., J. Am Chem Soc (1987) 109:5437-5446, shown as formulas 201, 202 and 203 in FIG. 11 herein. Additional compounds of formulas 204 and 205, also shown in FIG. 11 were synthesized as reported by Vedejes et al J Am Chem Soc (1989) 111:8430-8438. In addition, compounds 206-208 (FIG. 11) were synthesized by Borowitz______(1975)______; compound 209 has been synthesized by Ireland et al., J Org Chem (1980) 45:1868-1880.

#FIG. 1A is a diagram of the erythromycin PKS complex from S. erythraeus showing the function of each multifunctional protein, and also shows the structure of 6 dEB and of D-desosamine and L-cladinose.

#FIG. 1B shows a diagram of the post-PKS biosynthesis of erythromycins A-D.

#FIG. 2 is a diagram-of DEBS-1 from S. erythraeus showing the functional regions separated by linker regions.

#It may be helpful to review the nature of the erythromycin PKS complex and the gene cluster that encodes it as a model for modular PKS, in general.

#FIG. 1A is a diagrammatic representation of the gene cluster encoding the synthase for the polyketide backbone of the antibiotic erythromycin. The erythromycin PKS protein assembly contains three high-molecular-weight proteins (>200 kD) designated DEBS-1, DEBS-2 and DEBS-3, each encoded by a separate gene (Caffrey et al., FEBS Lett (1992) 304:225). The diagram in FIG. 1A shows that each of the three proteins contains two modules of the synthase—a module being that subset of reactivities required to provide an additional 2-carbon unit to the molecule. As shown in FIG. 1A, modules 1 and 2 reside on DEBS-1; modules 3 and 4 on DEBS-2 and modules 5 and 6 on DEBS-3. The minimal module is typified in module 3 which contains a ketosynthase (KS), an acyltransferase (AT) and an acyl carrier protein (ACP). These three functions are sufficient to activate an extender unit and attach it to the remainder of the growing molecule. Additional activities that may be included in a module relate to reactions other than the Claisen condensation, and include a dehydratase activity (DH), an enoylreductase activity (ER) and a ketoreductase activity (KR). The first module also contains repeats of the AT and ACP activities because it catalyzes the initial condensation, i.e. it begins with a “loading domain” represented by AT and ACP, which determine the nature of the starter unit. Although not shown, module 3 has a KR region which has been inactivated by mutation. The “finishing” of the molecule is regulated by the thioesterase activity (TE) in module 6. This thioesterase appears to catalyze cyclization of the macrolide ring thereby increasing the yield of the polyketide product.

#The product in this case is 6 dEB; the structure and numbering system for this molecule are shown in FIG. 1A. Conversion to the antibiotics erythromycin A, B, C and D would require glycosylation generally by D-desosamine or L-mycarose, which may ultimately be converted to cladinose at appropriate locations. FIG. 1B diagrams the post-PKS biosynthesis of the erythromycins through addition of glycosyl groups.