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

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