Fluostatins produced by the heterologous expression of a TAR reassembled environmental DNA derived type II PKS gene cluster

Abstract

Culture independent approaches for accessing small molecules produced by uncultured bacteria are often hampered by the inability to easily clone environmental DNA (eDNA) fragments large enough to capture intact biosynthetic gene clusters that can be used in heterologous expression studies. Here we show that homology screening of eDNA megalibraries for clones containing natural product biosynthetic genes, coupled with transformation-assisted recombination (TAR) in yeast, can be used to access large, functionally intact, natural product gene clusters from the environment. The eDNA derived gene cluster reported here was functionally reconstructed from two overlapping cosmid clones using TAR. The isolation and structure elucidation of three new fluostatins (F, G, and H) produced by this TAR reconstructed gene cluster is described.

Figure 1

a. Outline of the procedure used to recover and reconstruct the eDNA derived fluostatin gene cluster (red: minimal PKS, green: candidate ring cleavage oxygenases). b. Overview of a general approach for functionally accessing large natural product gene clusters present in soil. c. HPLC traces of organic extracts derived from individual clones as well as the TAR reassembled fluostatin gene cluster.

Figure 2

Clone specific metabolites produced by S. albus transformed with cosmid AB649.

Figure 3

Fluostatins isolated from cultures of S. albus transformed with the TAR assembled BAC AB649/1850.

Figure 4

The fluostatins, like the kinamycins, appear to arise from the excision of C6 from a rabelomycin-like precursor. Ratios of ¹³C integrations calculated from ¹³C NMR spectra of fluostatin G isolated from 1-¹³C acetate fed and unlabeled cultures are shown. Expected positions of ¹³C enrichment are shown as black circles. Observed sites of ¹³C enrichment are shown as green circles.