Reassembly of anthramycin biosynthetic gene cluster by using recombinogenic cassettes

The reassembly and heterologous expression of complete gene clusters in shuttle vectors has enabled investigations of several large biosynthetic pathways in recent years. With a gene cluster in a mobile construct, the interrogation of gene functions from both culturable and nonculturable organisms is greatly accelerated and large pathway engineering efforts can be executed to produce new natural products. However, the genetic manipulation of complete natural product biosynthetic gene clusters is often complicated by their sheer size (10-200 kbp), which makes standard restriction/ligation-based methods impracticable. To circumvent these problems, alternative recombinogenic methods, which depend on engineered homology-based recombination have recently arisen as a powerful alternative. Here, we describe a new general technique that can be used to reconstruct large biosynthetic pathways from overlapping cosmids by retrofitting each cosmid with a recombinogenic cassette that contains a shared homologous element and orthogonal antibiotic markers. We employed this technique to reconstruct the anthramycin biosynthetic gene cluster of the thermotolerant actinomycete Streptomyces refuineus, from two > 30 kbp cosmids into a single cosmid and integrate it into the genome of Streptomyces lividans. Anthramycin production in the heterologous Streptomyces host confirmed the integrity of the reconstructed pathway and validated the proposed boundaries of the gene cluster. Notably, anthramycin production by recombinant S. lividans was seen only during growth at high temperature-a property also shown by the natural host. This work provides tools to engineer the anthramycin biosynthetic pathway and to explore the connection between anthramycin production and growth at elevated temperatures.