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The nucleoside antibiotic toyocamycin (TM) is a potential fungicide that can control plant diseases, and it has become an attractive target for research.  Streptomyces diastatochromogenes  1628, 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 ( toy  cluster) in  S. diastatochromogenes  1628 was found by genome sequencing. In this study, the role of  toy  cluster for TM biosynthesis in  S. diastatochromogenes  1628 was investigated by heterologous expression, deletion, and complementation. The extract of the recombinant strain  S. albus J1074-TC harboring a copy of  toy  cluster produced TM as shown by HPLC analysis. The Δcluster 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 the  toy  cluster is responsible for TM biosynthesis. Moreover, the introduction of an extra copy of the  toy  cluster into  S. diastatochromogenes  1628 led to onefold increase in TM production (312.9 mg/l vs. 152.1 mg/l) as well as the transcription of all  toy  genes. The  toy  gene cluster was engineered in which the native promoter of  toyA  gene,  toyM  gene,  toyBD  operon, and  toyEI  operon was, respectively, replaced by  permE  ∗  or SPL57. To further improve TM production, the engineered  toy  gene cluster was, respectively, introduced and overexpressed in  S. diastatochromogenes  1628 to generate recombinant strains  S. diastatochromogenes  1628-EC and 1628-SC. After 84 h,  S. diastatochromogenes  1628-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.

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