The Study On The Biosynthesis Of Phoslactomycin In Streptomyces Auratus AGR0001

Neophoslactomycin A(PLM A),discovered from Streptomyces auratus AGR0001 in our laboratory,is a novel natural product with high efficiency,low toxicity,non-irritation,and low sensitization.PLM A possesses potent activities against phytopathogenic fungi,virus,and tumor,which has potential applications in pesticide and medicine.However,the yield of PLM A is extremely low,which hampered its further development.The study on the biosynthetic pathway of PLMs and its regulatory mechanism would lay the foundation for further increasing the production of PLM A or acquiring novel structural analogs through genetic engineering.In the present work,a preliminary study on the biosynthesis of phoslactomycins in S.auratus AGR0001 was conducted.The whole genome of the phoslactomycin producing strain S.auratus AGR0001 was sequenced and analyzed.The location of the phoslactomycin biosynthetic gene cluster in the genome was determined by sequence alignment and analysis.The sequence of the whole phoslactomycin biosynthetic gene cluster was obtained by assembling and re-sequencing the gap fragments.Key genes involved in the biosynthetic process of phoslactomycins were analyzed.The genetic organization of the PLM biosynthetic gene cluster was illustrated,and the synthetic route for phoslactomycin was proposed.The methods for genetic manipulation in S.auratus AGR0001 were explored,and the best way to introduce foreign DNA into S.auratus AGR0001 was established.Twenty one genes involved in the biosynthesis of phoslactomycins were cloned,and 15 of them were knocked out in Escherichia coli.The knockouts were introduced into the wild-type S.auratus AGR0001 through conjugation,and four knockout mutants of S.auratus AGR0001 were constructed.Neither phoslactomycins nor antifungal activity was detected in the ferments of the four mutants,which indicated that these four genes were involved in the biosynthesis of phoslactomycins.The regulatory roles of regulators orf5 and nsdA in the expression of other genes in the phoslactomycin biosynthetic gene cluster were analyzed by using RT-PCR.From the first to the ninth day,all genes in the cluster were expressed in the wild-type strain,while none of the post modification genes was expressed in the mutant strains through the third to the ninth day,indicating that the expression of these genes were positively regulated by Orf5 and NsdA.Regulatory gene orf27 was not expressed in the orf5 mutant,which indicated that orf27 might also be regulated by Orf5.Regulatory genes orfl and orf3 were both expressed on the third and the fifth day in the two mutants,which suggested that their expression was not under the control of Orf5 and NsdA.The fact that orf5 gene was expressed at high levels except for the first day in the nsdA mutant implied that NsdA did not regulate the expressionof orf5.In addition,all of the genes involved in the biosynthesis of phoslactomycins were expressed at very low levels,which was consistent with the low production of phoslactomycins in the wild-type strain.Five regulatory genes were heterologously expressed in E.coli,all in the form of inclusion bodies.Three of the expressed proteins were purified(Orfl,Orf4 and Orf5).The binding of these three regulatory proteins to their postulated target sequences was analyzed by using EMSA.It was found that they were not able to bind to the postulated targets in vitro under the conditions used.In conclusion,a preliminary study on the biosynthesis of phoslactomycins in S.auratus AGR0001 was conducted in this work.The genetic manipulation system was set up for the phoslactomycin producing strain S.auratus AGR0001.The complete phoslactomycin biosynthetic gene cluster was obtained through whole genome sequencing and analysis.The function of several key genes involved in the biosynthesis of PLMs was studied.The work would provide a starting point for further studying the biosynthetic pathway of PLMs and their regulatory mechanism,and would facilitate future work to increase the production of PLM A and generate novel analogs through genetic engineering.