Expression of a Thermomonospora fusca Cellulase Gene in Streptomyces lividans and Bacillus subtilis

Abstract

A cellulase gene from Thermomonospora fusca coding for endocellulase E 5 was introduced into Streptomyces lividans by using shuttle plasmids that can replicate in either S. lividans or Escherichia coli. Plasmid DNA isolated from E. coli was used to transform S. lividans , selecting for thiostrepton resistance. The transformants expressed and excreted the endocellulase, but the ability to produce the endocellulase was unstable. This instability was shown to result from deletion of the endocellulase gene from the plasmid. Plasmid DNA prepared from a culture in which plasmid modification had occurred was used to transform E. coli , selecting for Amp + cells, and all of the transformants were cellulase positive, showing that pBR322 and T. fusca DNA were deleted together. When a plasmid was constructed containing only T. fusca DNA in plasmid pIJ702, the transformants were more stable, and the level of endocellulase activity produced in the culture supernatant after growth on 0.2% glucose was close to the level produced by T. fusca cultures grown on 0.2% cellulose. About 50% of the total protein in the culture supernatant of the S. lividans transformant was endocellulase E 5 . The enzyme produced by the S. lividans transformant was identical to pure T. fusca E 5 in its electrophoretic mobility and was completely inhibited by antiserum to E 5 . Shuttle plasmids containing the E 5 gene that could replicate in Bacillus subtilis and E. coli were also constructed and used to transform B. subtilis. Again there was extensive deletion of the plasmid DNA during transformation and growth in B. subtilis. There was no evidence of E 5 activity, even in those B. subtilis transformants that retained the E 5 gene.