ISOLATION OF THE GENE CLUSTER WHICH PRODUCES BORRELIDIN IN STREPTOMYCES ROCHEI

Abstract

Streptomyces rochei is a Gram positive bacterium which produces borrelidin, an antibiotic active against the causative organism in Lyme disease and which has shown some anti-viral and anti-tumor properties. In our laboratory, production of borrelidin was too low to justify large-scale fermentations. Increased output of borrelidin would make it possible to harvest the antibiotic for further research. One means of increasing borrelidin production is to isolate the responsible gene or gene cluster in a high-copynumber vector. Increased copies of these genes might produce increased amounts of antibiotic. With this aim in mind, the following investigation was carried out. Escherichia coli, Streptomyces lividans, and Streptomyces rochei and their associated plasmid/phage vectors were considered for host/vector systems. S. lividans 66 and pIJ702 were chosen for cloning with size-fractionated gDNA of S. rochei. Two screening organisms, an Alternaria sp. and a Cylindrocarpum sp., sensitive to borrelidin but not to any host metabolites, were chosen and used to identify any borr+ clones. As a positive control for the library construction, the cloned plasmids were isolated and retransformed into a second host cell, demonstrating the transfer of morphological characteristics coded for by the plasmid inserts. The screen for borr+ clones turned up no positive colonies out of 1500 transformants. Attempts to demonstrate a varied genomic library by looking for restriction bands of the cloned plasmids were less clear. A borr- S. rochei mutant was isolated for possible use in future cloning by complementation studies.