Study On Key Genes Involved In Toyocamycin Biosynthesis In Streptomyces Diastatochromogenes1628

Toyocamycin(TM) is a member of the nucleoside antibiotics. It shows antifungal activity and can be used to treat plant diseases. In this paper, because of low TM production of Streptomyces diastatochromogenes 1628, some functional genes involved in TM biosynthesis in S. diastatochromogenes 1628 was cloned.Subsequently, metabolic engineering technology was used to enhance production of TM in S. diastatochromogenes 1628.Firstly, 1443-bp toy F gene(encoding adenylosuccinate lyase)(Gen Bank accession No. JQ267374) and 1284-bp toy G gene(encoding adenylosuccinate synthetase)(Gen Bank accession No. KF517415) involved in toyocamycin biosynthesis were cloned from S. diastatochromogenes 1628 genome DNA by using degenerate primers, respectively. These two genes show the highest similarity to toy F(90%, identical amino acids) and toy G(95%, identical amino acids) of S. venezuelae ATCC 10712, respectively. The toy F and toy G genes was placed under the control of the constitutive promoter Perm E* to constructed p IB139-toy F and p IB139-toy G,respectively. Recombinant strains 1628-TOYF and 1628-TOYG was constructed by conducting intergeneric conjugation, respectively. The wild-type strain and the recombinant strains 1628-TOYF, 1628-TOYG were used for TM fermentation, assay of enzyme activity and cell dry weight(DCW) detection. Both TM production and specific activity of TOYF or TOYG were higher in S. diastatochromogenes1628-TOYF or 1628-TOYG, respectively, comparing to those of original strain. It was worth mentioning that the highest increase rate of TM production was reached26.2% by 1628-TOYG.Secondly, the obtained 558-bp frr gene(Gen Bank accession No. KC416633)encodes a 185 aa long protein, showing the highest similarity to Frr of Streptomyces flavogriseus ATCC 33331(92%, identical amino acids), Frr of Streptomyces venezuelae ATCC 10712(92%, identical amino acids) and Frr of Streptomyces sp.NRRL F-2580(89%, identical amino acids). The recombinant strain 1628-FRR(containing p IB139-frr) was constructed for the over-expression of frr encoding ribosome recycling factor in S. diastatochromogenes 1628. The recombinant strain1628-FRR showed a 21 % increase in cell growth and a 41.1% increase in TM production compared to wild-type strain 1628, and the fermentation time was shortened from 84 to 72 h. Furthermore, by conducting reverse transcriptionpolymerase chain reaction(RT-PCR) analysis, it was shown that the transcriptional levels of toy F and toy G were enhanced in recombinant strain 1628-FRR compared to corresponding values of S. diastatochromogenes 1628, especially toy G. In addition,by using a fluorescent intensity reporter system, which is based on the green fluorescent protein(GFP), it was also suggested that over-expression of frr promoted strongly protein biosynthesis, especially in late growth phase.Finally, the rational combined co-expression of toy G with frr and vgb encoding Vitreoscilla hemoglobin in double or triple was performed to detect its effect on TM production in S. diastatochromogenes 1628. At the end, among all these different recombinant strains, the strain S. diastatochromogenes 1628-VGF, harboring vgb, frr and toy G which was placed under the control of Perm E*, respectively, exhibited the largest increase in TM production. Notably, the TM production of S.diastatochromogenes 1628-VGF reached the highest level at 319.2 mg/L at 72 h,while TM yield of original strain 1628 at 147.1 mg/L at 84 h. The productivity of TM was increased from 1.75 mg?L-1?h-1 to 4.43 mg?L-1?h-1.The high-yield, energy-saving strain S. diastatochromogenes 1628-VGF constructed in this study shall be beneficial for the industrial production of TM.