ToyA, a positive pathway-specific regulator for toyocamycin biosynthesis in Streptomyces diastatochromogenes 1628

The nucleoside antibiotic toyocamycin (TM), which was produced by Streptomyces diastatochromogenes 1628, was found to be highly efficient against a broad range of plant pathogenic fungi. Despite its importance, little is known about the regulation TM biosynthesis. In this study, toyA , located in the TM biosynthetic gene cluster, was identified as a regulatory gene encoding a large ATP-binding regulator of the LuxR family (LAL-family). The role of toyA in TM biosynthesis in S. diastatochromogenes 1628 was investigated by gene deletion, complementation, and over-expression. Gene disruption of toyA resulted in almost loss of TM production. TM production in complemented strain was restored to the level comparable to that in the wild-type strain S. diastatochromogenes 1628. Over-expression of toyA separately controlled by promoter SPL57, SPL21, and p erm E * in wild-type strain S. diastatochromogenes 1628 led to a 2-fold, 1-fold, and 80% increase in TM production compared with wild-type strain S. diastatochromogenes 1628, respectively. Quantitative RT-PCR analysis revealed that the transcriptional level of toy structural genes was downregulated in the ΔtoyA mutant but restored in complemented strain and further upregulated in the toyA over-expression strain. The detection results from GFP reporter system in Escherichia coli and GUS reporter system and GUS activities in S. albus J1074 and S. diastatochromogenes 1628 showed that ToyA activated the expression of toyB and toyE operon directly and activated the expression of other toy structural genes indirectly. These results indicate that ToyA is essential for TM biosynthesis controlling the expression of structural genes.