Heterologous expression and antitumor activity analysis of syringolin from Pseudomonas syringae pv. syringae B728a

Syringolin, synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase in Pseudomonas syringae pv. syringae (Pss) B728a, is a novel eukaryotic proteasome inhibitor. Meanwhile, directly modifying large fragments in the PKS/NRPS gene cluster through traditional DNA engineering techniques is very difficult. In this study, we directly cloned the syl gene cluster from Pss B301D-R via Red/ET recombineering to effectively express syringolin in heterologous hosts.

A 22 kb genomic fragment containing the sylA-sylE gene cluster was cloned into the pASK vector, and the obtained recombinant plasmid was transferred into Streptomyces coelicolor and Streptomyces lividans for the heterologous expression of syringolin. Transcriptional levels of recombinant syl gene in S. coelicolor M145 and S. lividans TK24 were evaluated via RT-PCR and the production of syringolin compounds was detected via LC-MS analysis. The extracts of the engineered bacteria showed cytotoxic activity to B16, 4T1, Meth-A, and HeLa tumor cells. It is noteworthy that the syringolin displayed anticancer activity against C57BL/6 mice with B16 murine melanoma tumor cells. Together, our results herein demonstrate the potential of syrinolin as effective antitumor agent that can treat various cancers without apparent adverse effects.

This present study is the first to report the heterologous expression of the entire syl gene cluster in Streptomyces strains and the successful expression of syringolin in both S. coelicolor M145 and S. lividans TK24. Syringolin derivatives demonstrated high cytotoxicity in vitro and in vivo. Hence, this paper provided an important foundation for the discovery and production of new antitumor compounds.