Heterologous Expression Of Fostriecin Biosynthesis Gene Cluster

Fostriecin (CI-920) is a unique naturally occurring phosphate monoester compound produced by Streptomyces pulveraceus ATCC31906. Fostriecin inhibits DNA topoisomerase II and is also a potent and selective inhibitor of protein phosphatases1(PP1), PP2A, and PP4. It shows in vivo antitumor activity against a wide spectrum of tumor cells; also it has excellent activity against P388and L1210leukemias cell lines. Therefore, fostriecin has attracted extensive interest as a lead compound and it was evaluated as an antitumor drug in clinical trials. Although phase I clinical trials of fostriecin were halted due to its unpredictable chemical purity and storage instability, comprehensive chemical synthesis of fostriecin and its analogs is under development to address these limitations and to further develop this specific antitumor agent. Due to the benefits of biosynthesis, there is a need for using improved biosynthetic engineering technologies to produce fostriecin and its analogs. Here, to elucidate the biosynthetic machinery associated with fostriecin production, we have characterized the fostriecin biosynthetic gene cluster, and heterologously express the genes in E. coli and Streptomyces. The results of the study were as follows:(1) A putative fostriecin gene cluster was isolated from the cosmid library of S. pulveraceus ATCC31906, and the sequence was analyzed. The entire73kb gene cluster contains coding sequences for six modular type I polyketide synthases genes (fosABCDEF); seven tailoring enzymes genes (fosGHIJKLM), and eight other ORF genes (orfl-8) were deduced.(2) One-step Red/ET recombination method was used to construct vector pRC with both positive and negative selectable markers, which can be used for subsequent cloning and screening positive clones in the multiple gene replacement. Besides, the vectors pMT and pCT containing different resistance genes were constructed for more flexible screening in different hosts. Finally, expression vector pACYC184was modified by inserting a promoter and a terminator sequence, respectively; which can be used for co-expression with other replicon expression vector in the same host. All these vectors can be widely used in gene cloning operations, and provide tools for subsequent cloning and expression of heterologous expression of Fostriecin polyketide synthase gene cluster.(3) The56kb PKS genes (fosABCDEF) of fostriecin biosynthesis were engineered into co-expression vectors pMT and p184using Red/ET recombination for heterologous expression in E. coli BG11-01and BAP1to gain BG11-01/p8-fosAB-pM-fosCDEF and BAP1/p8-fosAB-pM-fosCDEF. Although there was no structurally related products of fostriecin in the fermentataion broth by HPLC analysis, the results provide baisis for further design and construction of possible model compounds in the heterologous host, and also benefit further analysis of polyketide synthase function and mechanism of fostriecin biosynthesis.(4) The56kb PKS gene cluster (fosABCDEF) of fostriecin biosynthesis were engineered into integrating expression vector p126and autonomously replicating expression p124using Red/ET recombination for heterologous expression in S. lividans TK24and S. coelicolor M512to gain TK24/p6-fosAB-p4-fosCDEF and M512/p6-fosAB-p4-fosCDEF. Fermentation of resultant two Streptomyces mutants revealed activation of pigment production by the introduced genes, and fermentation products were further analyzed by LC-MS. One proposed fostriecin precursor compound was detected in the fermentation broth of M512/p6-fosAB-p4-fosCDEF, but the signal was weak. RT-PCR analysis of M512/p6-fosAB-p4-fosCDEF cells showed that the fostriecin polyketide synthase gene cluster is indeed expressed.(5) The in vitro enzymatic functional study of a putative phosphokinase FosH showed that it is responsible for the phosphorylation of fostriecin precursor or its analogs on C-9, which indicated that FosH acts as a novel kinase with relaxed substrate specificity and is involved in the process of fostriecin biosynthesis.Study of the heterologous expression of fostriecin gene cluster in this work provide basis for further design and construction of possible genetic engineering strain for natural product biosynthesis in the heterologous host. The results in this study facilitate further analysis of the functions of fostriecin polyketide synthases and fostriecin generation mechanism, and offer new opportunities for the biosynthesis of novel fostriecin analogues.