In Vivo Manipulation of the Bleomycin Biosynthetic Gene Cluster in Streptomyces verticillus ATCC15003 Revealing New Insights into Its Biosynthetic Pathway

Ute Galm,Liyan Wang,Evelyn Wendt-Pienkowski,Runying Yang,Wen Liu,Meifeng Tao,Jane M Coughlin,Ben Shen

doi:10.1074/jbc.m804971200

Abstract

Bleomycin (BLM), an important clinically used antitumor compound, and its analogs are challenging to prepare by chemical synthesis. Genetic engineering of the biosynthetic pathway in the producer strain would provide an efficient and convenient method of generating new derivatives of this complex molecule in vivo. However, the BLM producing Streptomyces verticillus ATCC15003 has been refractory to all means of introducing plasmid DNA into its cells for nearly two decades. Several years after cloning and identification of the bleomycin biosynthetic gene cluster, this study demonstrates, for the first time, genetic accessibility of this pharmaceutically relevant producer strain by intergeneric Escherichia coli-Streptomyces conjugation. Gene replacement and in-frame deletion mutants were created by lambdaRED-mediated PCR targeting mutagenesis, and the secondary metabolite profile of the resultant mutants confirmed the identity of the BLM biosynthetic gene cluster and established its boundaries. Ultimately, the in-frame blmD deletion mutant strain S. verticillus SB5 resulted in the production of a bleomycin intermediate. The structure of this compound, decarbamoyl-BLM, was elucidated, and its DNA cleavage activity was compared with the parent compounds.

Highlights

The bleomycins (BLMs)3 (1, 2) are important clinically used hybrid peptide-polyketide antitumor compounds
Several years after cloning and identification of the bleomycin biosynthetic gene cluster, this study demonstrates, for the first time, genetic accessibility of this pharmaceutically relevant producer strain by intergeneric Escherichia coli-Streptomyces conjugation
We have been studying the biosynthesis of BLM in Streptomyces verticillus ATCC15003 (9 –16) and the closely related compounds tallysomycin in Streptoalloteichus hindustanus E465–94 ATCC31158 (17) and zorbamycin in Streptomyces flavoviridis ATCC21892 (18)

Summary

Introduction

The bleomycins (BLMs)3 (1, 2) are important clinically used hybrid peptide-polyketide antitumor compounds. S. verticillus occurred with the highest frequency on modified the NRPS encoding blmIV gene by the aac(3)IV gene on ISP-4 agar freshly supplemented with 20 mM MgCl2 when 109– pBS38 generated 18.9-kb upstream and a 12.9-kb down1010 S. verticillus recipient cells and E. coli S17-1 donor cells stream flanking regions accessible for homologous recombiharvested from 10 ml of culture grown to an A600 of 0.4 – 0.6 nation in pBS43 (Fig. 1A).

Results

Conclusion