Functional Characterization Of Several Genes For Fostriecin Biosynthesis

Fostriecin (FST) is a phosphate ester polyketide produced by Streptomyces pulveraceus with promising antitumor activity. FST is synthesized by a modular FST polyketide synthase (PKS) and a series of post-PKS modifying reactions, including hydroxylation, phosphorylation and so on. Recently, the studies on FST mainly exist in biological activity and chemical synthesis. It has not been reported on the FST biosynthetic mechanism. In this study, we established and optimized genomic transfer system for FST producer S. pulveraceus, delineated the functions of post-PKS modification genes by gene knockout inactivation, and elucidated the pathway for post-PKS modification in FST biosynthesis. The main results and conclusions are listed as follows:(1) We established and optimized gemonic transfer system for FST producer S. pulveraceus by conjugal transfer method. The optimal conditions were spore suspension as conjugation recipients, Ms agar medium with5%glycin,50℃heat shock10min and flooding apramycin (20μg/mL) and nalidixic acid (25μg/mL) after18h.(2) To enhance the electrotransformation efficiency, we optimized electrotransformation system of Red/ET recombination system. The optimal electrotansformation parameters as follows:500ng foreign DNA, OD600for E. coli reaches0.4-0.6, competent cell concentration is0.7×107/μL, and final concentration of L-alabinose is20mmol/L, inducing3h.(3) The BLAST analysis of the FST biosynthetic gene cluster revealed that it includes five genes putatively involved in post-PKS modifications, named fosG, fosH, fosJ, fosK and fosM. To elucidate the functions of these genes, we inactivated their functions by gene knockout experiments. Compared with wild-type strain, fosG disrupted strain failed to produce PD113271and accumulated FST as the major product; fosJ disrupted strain accumulated five new FST analogues,1-5; fosH and fosK disrupted strains accumulated two new FST analogues,6,7and8, PD113270, respectively; fosM disrupted strains only accumulated one new FST analogue,9. Structures of these new derivatives were identified through mass and’H NMR spectroscopic analysis. On the basis of the structures of these compounds, we verified the functions of these post-PKS modification genes. FosG-, fosJ-and fosK-encoded cytochrome P450monooxygenases belonged to hydroxylases, are responsible for C-4C-8and C-18hydroxylation, respectively. FosH encoded phosphokinase are involved in C-9phosphorylation. FosM might be the crucial enzyme involved in formation of double bond at C2-C3in FST biosynthesis. (4) Genetic complementations to the mutant strains were subsequently carried out to confirm the metabolites were not influenced by other factors. All target genes were amplified and inserted into pSET153-tsr to yield gene complemented vectors. Then these complemented plasmids were introduced into the corresponding mutant strains by conjugation. When cultured these complemented strains, HPLC analysis suggested that all target genes could be expressed in the corresponding mutant strains. We observed that FST was accumulated by fosJ～fosM complementations and PD113271was restored with fosG complementation.(5) Many poliketides contain one or more double bonds in their structures that are generated by ketoreductase-dehydratase (KR-DH) domains within the PKS modules. It is unable to form the unsaturated double bond of the lactone ring due to the lack of a cognate DH domain in the FST PKS terminal module. Through the determination of function offosM gene, we found that the formation of an unsaturated double bond is dependent upon FosM, not a DH domain in the module PKS. All the post-PKS modification steps in FST biosynthesis can occur with the polyketide chain bearing a malonyl ester at the C-3position. FosM is responsible for the formal elimination of malonate to generate the unsaturated lactone in FST biosynthesis only when the C-18hydroxylation catalyzed by FosK is completed.(6) According to the chemical structures of a series of new analogues achieved from the five post-PKS modification gene disruption experiments, we elucidated the pathway for post-PKS modifications in FST biosynthesis. The malonylated compound4, liberated from PKS elongation by TE, was catalyzed by FosJ to form compound6. Then, FosH and FosK could catalyze the phosphorylation at C-9and hydroxylation at C-18to create the malonylated compound8and9, respectively. Finally, FosM performs its function on elimination of malonic acid and formation of double bond between C-2and C-3. When the biosynthesis of FST is completed, FosG starts to play its role in C4-hydroxylation of FST to afford PD113271.