Cloning And Functional Analysis Of Gene Cluster Involved In Toyocamycin Biosynthesis In Streptomyces Diastatochromogenes 1628

Streptomyces diastatochromogenes 1628 was shown to produce the nucleoside antibiotic toyocamycin?TM?,which is a promising fungicide utilized in controlling the occurrence of plant fungal diseases in the agricultural industry field.In order to investigate the regulatory mechanism of the TM biosynthesis in Streptomyces diastatochromogenes 1628,we have cloned the TM biosynthetic gene cluster and verified its function.Subsequently,pathway-specific regulatory gene toyA in the TM gene cluster was cloned and studied.The results revealed that toyA gene was involved in the positive regulation of TM biosynthesis.Finally,the transcriptional levels and their applications of the different promoters in strain 1628were compared and analyzed by using structural gene toyF located in the TM gene cluster and gusA as reporter gene.Main research contents are as follows:Firstly,10.4 kb TM biosynthetic gene cluster?toy cluster??GenBank accession No.KT291433.1?was cloned from S.diastatochromogenes 1628 genome DNA by using specific primers and Gibsen Assembly method.The similarity of toy cluster with the TM biosynthetic gene cluster of Streptomyces rimosus and Streptomyces ahygroscopicus were 75%and 99%,respectively.Recombinant strain S.albus J1074-CLU which harbored heterologous expression of toy cluster was obtained by conducting intergeneric conjugation.From the data of TM fermentation and HPLC detection of wild-type strain S.albus J1074 and the recombinant strain J1074-CLU,the TM could be detected in fermentation broth of J1074-CLU.Recombinant strain1628-CLU,in which toy cluster was over-expressed in 1628 strain,was also constructed.The recombinant strain 1628-CLU showed a 105.7%increase in TM production,compared with wild-type strain 1628.Secondly,the TOYA protein which was encoded by toyA gene in the toy cluster was recognized by bioinformatics,and it belongs to the LAL family which is a kind of pathway-specific regulator in Streptomyces.A 2877 bp toyA gene?GenBank accession No.KT291433.1?,which was located in toy cluster,was cloned from S.diastatochromogenes 1628 genome DNA by using specific primers.Then the toyA gene was placed under the control of the constitutive promoter permE^*to construct p IB139-toyA.Recombinant strain 1628-TOYA was obtained by conducting intergeneric conjugation.The TM production of strain 1628-TOYA increased 80.6%compared with that of wild-type strain S.diastatochromogenes 1628.Moreover,the transcriptional levels of toyB,toyM,toyG and toyF in strain 1628-TOYA were significantly higher than those of wild-type strain.The recombinant plasmid pET28a-adpA_sd was constructed to express protein ADPA_sd in E.coli Rosseta,and protein ADPA_sd was purified.The electrophoretic mobility shift assays?EMSA?result showed that protein ADPA_sdd could directly bind the promoter regions of toyA gene.In this study,the transcriptional level of promoters permE^*,kasOp^*,SPL-21 and SPL-57 were compared and analyzed by using the gusA as the reporter gene.Recombinant plasmids pGUS-ermE,pDR4-GUS,pGUS-SPL-21 and p GUS-SPL-57were constructed and integrated into the chromosome of S.diastatochromogenes1628 by using intergeneric conjugation to yield recombinant strains 1628-ES,1628-RS,1628-21S and 1628-57S,respectively.The results indicated that promoter SPL-21 showed the strongest transcriptional and expression level and gave rise to increase 5.2-fold in GUS activity compared with that of permE^*.To elucidate whether toyF gene which encoded adenylosuccinate lyase in the toy cluster was involved in TM biosynthesis,the knock-out plasmid pKC1132-toyF'was constructed and integrated into the chromosome of S.diastatochromogenes 1628 by using intergeneric conjugation to yield toyF-disrupted mutant 1628-?TOYF.The results showed that TOYF activity and TM production of strain 1628-?TOYF decreased66.7%and 87.5%comparing with those of wild-type strain S.diastatochromogenes1628,respectively.Moreover,recombinant strain 1628-?TOYF/toyF in which toyF gene was complementary expressed was constructed.The recombinant strain1628-?TOYF/toyF showed an increase of 2.7-fold and 8.3-fold in specific enzyme activity of TOYF and TM production compared with those of mutant 1628-?TOYF,respectively.These results suggest that toyF is positively involved in TM biosynthesis in S.diastatochromogenes 1628.Finally,the aforementioned promoters permE^*,kasOp^*,SPL-21 and SPL-57 were also applied in overexpression of toyF gene to construct the recombinant strains 1628-EF,1628-RF,1628-21F and 1628-57F.As a result,among all different recombinant strains,the recombinant strain 1628-21F,in which overexpression of toyF gene was driven by SPL-21,exhibited the largest increase in TOYF activity and TM production.In 5-l fermentor,the recombinant strain 1628-21F produced 108.05%in increase of TM comparing with that of the wild-type strain.