A gene cloning system for ‘Streptomyces toyocaensis’

Abstract

Summary: We explored different methods of introducing DNA into ‘Streptomyces toyocaensis’ and Streptomyces virginiae to construct stable recombinant strains. Plasmid pIJ702 isolated from Streptomyces lividans transformed protoplasts of ‘S. toyocaensis’ at a frequency of 7×103 transformants (μgDNA)-1. pIJ702 prepared from ‘S. toyocaensis’ transformed ‘S. toyocaensis’ protoplasts at a frequency of 1.5×105 (μgDNA)-1. suggesting that ‘S. toyocaensis’ expresses restriction and modification. Plasmid pRHB126 was transduced by bacteriophage FP43 into ‘S. toyocaensis’ at a frequency of 1.2×10−6 (p.f.u.)−1. Plasmids pOJ436 and pRHB304 were introduced into ‘S. toyocaensis’ by conjugation from Escherichia coli S17-1 at frequencies of about 2×10−4 and 1×10−4 per recipient, respectively. Analysis of several exconjugants indicated that pOJ436 and pRHB304 inserted into a unique øC31 attB site and that some of the insertions had minimal deleterious effects on glycopeptide A47934 production. The results indicate that ‘S. toyocaensis’ is a suitable host for gene cloning, whereas S. virginiae does not appear to be.