PCR-Independent Method of Transformation-Associated Recombination Reveals the Cosmomycin Biosynthetic Gene Cluster in an Ocean Streptomycete

Abstract

The transformation-associated recombination cloning methodology facilitates the genomic capture and heterologous expression of natural product biosynthetic gene clusters (BGCs). We have streamlined this procedure by introduction of synthetic DNA gene blocks for the efficient capture of BGCs. We show the successful capture and expression of the aromatic polyketide antitumor agent cosmomycin from streptomycete bacteria and the discovery of new cosmomycin analogues by mass spectral molecular networking.

Figure 1. Capture Vector Assembly

(A) Cluster specific capture arms, one kilobase in size, are amplified from genomic DNA and subsequently assembled using PCR. (B) Cluster specific capture arms (360 bp) are synthesized commercially as a single 750 bp dsDNA gene block including restriction sites for insertion and linearization.

Figure 2. Gene Map of Biosynthetic Gene Clusters Associated with the Biosynthesis of Cosmomycins C and D

The cosmomycin BGCs from (A) Streptomyces sp. CNS-615, (B) Streptomyces sp. CNT-302, and (C) Streptomyces olindensis are >90% similar on the amino acid sequence level. A gene by gene comparison can be found in Table S2. Homology of the S. sp. CNT-302 and the S. olindensis BGCs is restricted to the first 40 kb of the captured sequence (denoted by dotted lines), suggesting the last 14 kb of genomic material captured in this study is not necessary for cosmomycin production. Genes marked with a ‘*’ have been interrogated by gene knockout experiments in previous studies.

Figure 3. Cosmomycin Cluster Identified by Molecular Networking

Nodes matching the masses for cosmomycins C (1) and D (2) network with nodes matching values of cosmomycin derivatives lacking methyl groups and/or oxygen. These include deoxy-cosmomycin C (3), 13-desmethyl-cosmomycin C (A), and desmethyl, deoxy-cosmomycin C (B).