Cloning, sequencing and analysis of spirotetronate biosynthetic gene clusters

Abstract

Spirotetronate antibiotics are natural products that have been shown to have a broad spectrum of biological activities, including antiviral, antibacterial, antitumor and antimalarial activities. Two representatives of this group are quartromicins and chrolactomycin, which are produced by Amycolatopsis orientalis and Streptomyces sp. 569N-3 respectively. Based on other related natural products with known biosynthetic pathways, the biosynthetic routes for quartromicins and chrolactomycin were proposed and several strategies for locating the putative gene clusters directing the biosynthesis of these natural products were used. An FkbH-like protein was proposed to be specifically involved in the biosynthetic incorporation of a glycerol-derived three-carbon unit into the tetronate moieties of quartromicins and chrolactomycin. It was selected as a target to locate the biosynthetic gene clusters of tetronate natural products. After aligning sequences of known FkbH-like proteins, a set of degenerate oligonucleotide primers for amplification of fkbH-like genes was designed and tested. Fragments of fkbH-like sequences were amplified from several available strains and sequenced. Several genomic libraries were created and screened by PCR using the degenerate fkbH primers. After validating the library, clones containing the putative biosynthetic gene cluster were identified and sequenced. The obtained sequence data was then assembled and analysed. Coding sequences were identified, protein functions assigned and a biosynthetic route was proposed for the biosynthesis of quartromicins and chrolactomycin