Identification of plasmids from thermophilic Streptomyces strains and development of a gene cloning system for thermophilic Streptomyces species

Abstract

To develop a host-vector system for use in thermophilic Streptomyces, multi-copy plasmids were screened for thermophilic Streptomyces species using data from public bioresource centers (JCM and NBRC). Of 27 thermophilic Streptomyces strains, 3 harbored plasmids. One plasmid (pSTVU1), derived from S. thermovulgaris NBRC 16615 (= JCM 4520, ATCC 19284, DSM 40444, ISP 5444, NRRL B-12375, and NCIMB 10078), was multi-copy and relatively small in size. Analysis of the sequence of this multi-copy plasmid revealed that it was 7,838 bp and contained at least 10 predicted open reading frames. The plasmid was introduced into 14 thermophilic Streptomyces strains (of 18 strains examined) and several mesophilic Streptomyces strains (S.lividans, S.parvulus, and S.avermitilis). pSTVU1 can be transferred by mixed culture because the plasmid encodes the ORF that regulates the transfer function. Plasmid transfer was observed not only between strains within the same species but also between mesophilic Streptomyces and thermophilic Streptomyces (and vice versa); however, the efficiency of this transfer was extremely low. We also confirmed that a derivative of pSTVU1 can be used as a multi-copy vector in the gene expression system that is expected to exhibit gene-dosage effects, establishing a method for efficient production of thermophilic α-amylase.

One-sentence summary: A multi-copy plasmid was identified in thermophilic Streptomyces and used to develop a gene cloning system for thermophilic Streptomyces species.

Keywords: Broad-host-range plasmid; Development of a gene cloning system; Self-transmissible plasmid; Thermophilic Streptomyces; Transposon mutagenesis.