Hunan Provincial Key Laboratory of Microbial Molecular Biology, State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha, 410081, People's Republic of China.
Microb Cell Fact. 2018 Feb 26;17(1):31. doi: 10.1186/s12934-018-0859-1.
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.
丁香菌素是由丁香假单胞菌 pv.丁香（Pss）B728a 中混合非核糖体肽合酶/聚酮合酶合成的一种新型真核蛋白酶体抑制剂。同时，通过传统的 DNA 工程技术直接修饰 PKS/NRPS 基因簇中的大片段非常困难。在本研究中，我们通过 Red/ET 重组直接从 Pss B301D-R 中克隆 syl 基因簇，有效地在异源宿主中表达丁香菌素。
从 sylA-sylE 基因簇中克隆出一个 22kb 的基因组片段，将其克隆到 pASK 载体中，获得的重组质粒转入变铅青链霉菌和变红红链霉菌中进行丁香菌素的异源表达。通过 RT-PCR 评估重组 syl 基因在变铅青链霉菌 M145 和变红红链霉菌 TK24 中的转录水平，并通过 LC-MS 分析检测丁香菌素化合物的产生。工程菌的提取物对 B16、4T1、Meth-A 和 HeLa 肿瘤细胞表现出细胞毒性。值得注意的是，丁香菌素对携带 B16 黑色素瘤肿瘤细胞的 C57BL/6 小鼠具有抗癌活性。总之，我们的研究结果表明，丁香菌素具有作为有效抗癌药物的潜力，可以治疗各种癌症，而没有明显的不良反应。
本研究首次报道了整个 syl 基因簇在链霉菌菌株中的异源表达，以及丁香菌素在变铅青链霉菌 M145 和变红红链霉菌 TK24 中的成功表达。丁香菌素衍生物在体外和体内均显示出高细胞毒性。因此，本研究为发现和生产新型抗肿瘤化合物提供了重要基础。
