Cloning And Functional Study Of The Phoslactomycin Biosynthetic Gene Cluster

Phoslactomycins(PN, PLMs) , named after the phosphate group and amino-group in the moleculars, are a class of polyketide antifungal and antitumor antibiotics produced by many Streptomyces. As the selective inhibitor of protein phosphorylase2A(PP2A) ,they have the potential for the antineoplastic drugs or antifungal biopesticides, but this prospect of application is limited by their toxicity for mammals. Based on the problem above, we start with the cloning of the biosynthetic gene cluster of PNs to access their biosynthetic machinery in this study, and the findings will set the stage to investigate the genetic and biochemical basis for PNs biosynthesis and make it possible to generate structural diversity for the discovery and development of drugs and pesticides by applying combinatorial biosynthesis to the PNs biosynthetic machinery to find the PLMs analogy with equal or improved bioactivity and less toxicity.We optimized the fermentation conditions reported so that the fermentation yield of PNs is stable at higher level. The genetic system of Streptomyces platensis SAM-0654 was developed by conjugation between Streptomyces platensis SAM-0654 and E. coli ET12567 with high efficiency.The genomic library of Streptomyces platensis SAM-0654 with the efficiency at 8000 cfu/μg DNA was constructed with pCC1FOS as a vector. The degenerate primers were designed according to the homologous gene of the PLMs gene cluster in Streptomyces HK803 reported. The DNA fragments encoding homologous gene were amplified by PCR. Screening the genomic library with the PCR products as probes, we cloned a 100 kb contiguous DNA region from the genomic DNA of Streptomyces platensis SAM-0654.The 100kb contiguous DNA region that contains the entire PLMs biosynthetic gene cluster was sequenced. Bioinformatic analysis of this sequenced region revealed 40 open reading frames, 22 of which from 1 to 22 were proposed to constitute the PNs gene cluster, including 7 genes encoding enzymes for the polyketide skeleton biosynthesis, 2 genes encoding the enzymes for the ethylmalonyl-CoA biosynthesis, 4 genes encoding the cyclohexanoyl-CoA biosynthesis enzymes, 6 genes encoding the tailoring enzymes, as well as 2 regμLatory genes and 1 resistance genes. The PLMs biosynthesis pathway had been proposed based on the bioinformation analysis. The cloned biosynthesis gene cluster was verified to be involved in the PLMs biosynthesis by gene disruption. Comparing the cloned PLMs biosynthesis gene cluster in S. platensis SAM-0654 with that in S. HK803 reported, we find the structual genes are silimar, meanwhile the precursor biosynthesis genes and the regulatory genes differ greatly. So gene replacement via PCR-targeting systems is utilized to investigate the biosynthetic mechanism of two precursor genes- pn P5, pn P6 and two regulatory genes- pn R1, pn R2.In conclusion, the available genetic system of Streptomyces platensis SAM-0654 provides the technical basis of genetic manipulation in vivo. The cloning, sequencing and analysis of the PNs gene cluster provides the genetic basis to access the biosynthetic machinery of PNs, investigation on which will not only shed new insight into PLMs biosynthesis, especially the precursor biosynthesis and the regulation, but also demonstrate the feasibility to evaluate novel biosynthetic pathways of structurally more complex and biologically more active analogs in simple and fermentation-friendly microorganisms.