Cloning and Overexpression of the Toy Cluster for Titer Improvement of Toyocamycin in Streptomyces diastatochromogenes .

The nucleoside antibiotic toyocamycin (TM) is a potential fungicide that can control plant diseases, and it has become an attractive target for research.Streptomyces diastatochromogenes1628, a TM-producing strain, was isolated by our laboratory and was considered to be a potent industrial producer of TM. Recently, the putative TM biosynthetic gene cluster (toycluster) inS. diastatochromogenes1628 was found by genome sequencing. In this study, the role oftoycluster for TM biosynthesis inS. diastatochromogenes1628 was investigated by heterologous expression, deletion, and complementation. The extract of the recombinant strainS. albusJ1074-TC harboring a copy oftoycluster produced TM as shown by HPLC analysis. The Delta cluster mutant completely lost its ability to produce TM. TM production in the complemented strain was restored to a level comparable to that of the wild-type strain. These results confirmed that thetoycluster is responsible for TM biosynthesis. Moreover, the introduction of an extra copy of thetoycluster intoS. diastatochromogenes1628 led to onefold increase in TM production (312.9 mg/l vs. 152.1 mg/l) as well as the transcription of alltoygenes. Thetoygene cluster was engineered in which the native promoter oftoyAgene,toyMgene,toyBDoperon, andtoyEIoperon was, respectively, replaced bypermE*or SPL57. To further improve TM production, the engineeredtoygene cluster was, respectively, introduced and overexpressed inS. diastatochromogenes1628 to generate recombinant strainsS. diastatochromogenes1628-EC and 1628-SC. After 84 h,S. diastatochromogenes1628-EC and 1628-SC produced 456.5 mg/l and 638.9 mg/l TM, respectively, which is an increase of 2- and 3.2-fold compared with the wild-type strain.