Selection of an efficient promoter and its application in toyocamycin production improvement in Streptomyces diastatochromogenes 1628.

ABSTRACT

The selection of efficient promoter is usually very crucial for gene expression and metabolic engineering in Streptomycetes. In this study, the synthetic promoters SPL-57and SPL-21, and the engineered promoter kasOp*were selected and their activities were examined by using a reporter gene assay based on GUS. All selected promoters which have been reported to be stronger than promoter permE*, which was used as control promoter. As host we were choosing S. diastatochromogenes 1628, the producer of toyocamycin (TM). Our results indicate that all tested promoters can be used to express genes in S. diastatochromogenes 1628. Interesting, promoter SPL-21 showed the strongest transcriptional and expression level and gave rise to a 5.2-fold increase in GUS activity compared with control. In order to improve TM production, the promoters were used to control expression of toyF. This gene encodes an adenylosuccinate lyase involved in TM biosynthesis. Among all different recombinant strains, the strain 1628-21F, in which over-expression of toyF gene was driven by SPL-21, exhibited the largest increase in TOYF activity and TM production. In a 5-l fermenter this strain produced more than two times more TM compared with the wild-type strain.