Heterologous Expression And Functional Characterization Of Fostriecin Biosynthetic Gene FosH

Polyketides are bioactive natural products synthesized principally by soil microorganisms like Streptomyces and related mycelial bacteria, but also by fungi and plants. There are about 10,000 known polyketides, from which numerous pharmaceutical products in many therapeutic areas have been derived, including:antibiotics, antifungals, antiparasitic, antitumor compounds, cholesterol-lowering agents and immunosuppressants.One valuable class of polyketides includes Fostriecin and its analogs, which are synthesized by modular PKS and modification enzymes. Fostriecin (CI-920), originally isolated from the fermentation beer of Streptomyces pulveraceus (subspecies fostreus), is a structurally novel phosphate ester natural product which exhibits efficacious in vivo antitumor activity against a wide spectrum of tumor cells and has excellent activity against L1210 and P388 leukemias in vivo.This antitumor activity is believed to resulted from selective inhibition of protein phosphatase 2A(PP2A) and protein phosphatase 4(PP4). Due to these properties, Fostriecin has attracted much interest as a lead compound and the biosynthesis of it has become a hot research topic.In order to achieve biology transformation of Fostriecin, a total biosynthesis gene cluster of Fostriecin should be studied to reveal all of its functions.The molecular biological background of Fostriecin's biosynthesis is almost clear now. In 2005,researchers of Kosan Biosciences first reported Fostriecin's biosynthesis gene cluster,although a few of modification gene cluster have not been characterized yet. Therefore now what we have to do is to design PKS gene clusters in heterologous hosts such as Streptomyces and E.coli to establish high population genetic engineering strains using genetic recombination technology and express new products to provide a target for post modification,thereby set a technological base for production and preparation of an independent intellectual property rights of the stable new Fostriecin derivatives. Secondly, the modification gene clusters which have not been fully located should be characterized.Therefore, gene function location of fosH is our main work in this study, which will provide new insights into the studies of Fostriecin and its analogs. Development of heterologous expression systems along with the ability to express and purify these products from Escherichia coli is an effective way. In this paper, the modification enzymes gene has been heterologous expressed in Escherichia coli cells. The protein has been obtained in a soluble and catalytically active form, and its properties have been determined.The fosH gene, encoding a putative phosphokinase, was identified in the fostriecin biosynthetic gene cluster of Streptomyces pulveraceus subspecies fostreus ATCC 31906. The fosH gene in the fostriecin biosynthetic gene cluster is located downstream of fosG, it is one of the fostriecin's post PKS modification gene clusters. Homology analysis showed that it has 43% identity with PlmT5, which is from the phoslactomycin biosynthetic cluster. Amino acid sequence analysis of the deduced fosH product revealed a high degree of similarity of this polypeptide to bacterial kinases, including those involved in antibiotic biosynthesis. Although the putative function of FosH is polyketide kinase, the experimental evidence for this assumption was lacking. Repeated attempts to inactivate fosH by inserting the neomycin resistance gene had not succeeded yet. So we expressed the target gene and used alkaline phosphatase to prepare the substrate for the target proteins. There are two atg codons in the same open reading frame in the fosH gene, which are 120bp's away from each other. PCR produced two DNA fragments, fosHl and fosH2, which were 1084-bp and 964-bp respectively. The fosH gene was cloned to the expression vector pET-28b by traditional method. The protein FosHl and FosH2 were expressed heterologously in Escherichia coli strain BL21(DE3) as His-tagged proteins and used in an enzyme assay with DP-Fosytiecin (dephosphofostriecin) as a substrate, which was prepared by chromatographing on a C18-revers column.Spectrometer analysis together with nuclear resonance magnetic spectrometry (1H NMR) verified the correctness of the structures of fostriecin and DP-Fostriecin.Purification and an in vitro activity assay for fosH encoded proteins FosH1 and FosH2 demonstrated their phosphokinase activity. These studies provides foundations for other genes' function location,it perfects the mechanism of Fostriecin biosynthesis and provides new opportunities for engineered biosynthesis of novel Fostriecin analogues.