Expression of a Thermomonospora fusca Cellulase Gene in Streptomyces lividans and Bacillus subtilis
AUTOR(ES)
Ghangas, Gurdev S.
RESUMO
A cellulase gene from Thermomonospora fusca coding for endocellulase E5 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 E5. The enzyme produced by the S. lividans transformant was identical to pure T. fusca E5 in its electrophoretic mobility and was completely inhibited by antiserum to E5. Shuttle plasmids containing the E5 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 E5 activity, even in those B. subtilis transformants that retained the E5 gene.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=203894Documentos Relacionados
- Cloning, sequencing, and expression of a Thermomonospora fusca protease gene in Streptomyces lividans.
- Cloning of a Thermomonospora fusca xylanase gene and its expression in Escherichia coli and Streptomyces lividans.
- DNA sequences and expression in Streptomyces lividans of an exoglucanase gene and an endoglucanase gene from Thermomonospora fusca.
- Cloning of the Thermomonospora fusca Endoglucanase E2 Gene in Streptomyces lividans: Affinity Purification and Functional Domains of the Cloned Gene Product
- Studies of Streptomyces reticuli cel-1 (cellulase) gene expression in Streptomyces strains, Escherichia coli, and Bacillus subtilis.