AUTHOR=Ma Zheng , Hu Yefeng , Liao Zhijun , Xu Jie , Xu Xianhao , Bechthold Andreas , Yu Xiaoping 

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

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.02074

DOI=10.3389/fmicb.2020.02074

ISSN=1664-302X

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