Tailoring enzyme-rich environmental DNA clones: a source of enzymes for generating libraries of unnatural natural products

Abstract

A detailed bioinformatics analysis of six glycopeptide biosynthetic gene clusters isolated from soil environmental DNA (eDNA) megalibraries indicates that a subset of these gene clusters contains collections of tailoring enzymes that are predicted to result in the production of new glycopeptide congeners. In particular, sulfotransferases appear in eDNA-derived gene clusters at a much higher frequency than would be predicted from the characterization of glycopeptides from cultured Actinomycetes . Enzymes found on tailoring-enzyme-rich eDNA clones associated with these six gene clusters were used to produce a series of new sulfated glycopeptide derivatives in both in vitro and in vivo derivatization studies. The derivatization of known natural products with eDNA-derived tailoring enzymes is likely to be a broadly applicable strategy for generating libraries of new natural product variants.

Figure 1

A.) Environmental DNA libraries are predicted to contain thousands of unique biosynthetic gene clusters. Many of these previously inaccessible clusters likely encode the biosynthesis of new members of previously characterized natural product families. Tailoring enzyme-rich eDNA clones from these clusters will encode collections of enzymes that have evolved to specifically interact with a conserved natural product core structure. These enzymes should therefore be useful for generating new collections of unnatural natural products using either in vitro or in vivo biosynthetic strategies. B.) Glycopeptides are derived from a small number of oxidatively cross-linked heptapeptides. In this manuscript we describe the use of eDNA derived glycopeptide tailoring enzymes to generate a family of new glycopeptide congeners.

Figure 2

A. Many sequenced glycopeptide gene clusters show a similar overall gene organization. The balhimycin, chloroeremomycin and teicoplanin gene clusters were cloned from cultured soil bacteria while the VEG and TEG gene clusters were cloned directly from soil. Open reading frames have been color coded according their predicted functions. B. Glycopeptide gene clusters recovered from the CA eDNA mega-library are shown. C. AZ205, a tailoring enzyme-rich eDNA cosmid clone, recovered from the AZ library is shown. Dashed lines are used to indicate the region from each gene cluster that corresponds to the tailoring enzyme-rich clone used in this study.

Figure 3

New glycopeptide congeners produced using eDNA derived tailoring enzymes. Compounds 2-5 were produced in vivo by S. toyocaensis transformed with tailoring enzyme-rich eDNA clones. Compounds 13-16 were generated in vitro using recombinantly expressed, eDNA-derived sulfotransferases.

Figure 4

New sulfated glycopeptide derivatives produced by either in vivo or in vitro methods using eDNA derived tailoring enzymes.

Figure 5

Structures A and B correspond to the cross-linked core peptides that are predicted to arise from eDNA derived NRPS systems. An inventory of the number and type of tailoring enzymes (or functional group transferases) found in sequenced gene clusters from cultured soil bacteria as well as gene clusters cloned from eDNA is shown. For gene clusters associated with known metabolites, the number of tailoring enzyme derived functional groups found on the encoded metabolite is also shown.