School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huaian, China.
Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China.
Appl Microbiol Biotechnol. 2019 Jun;103(11):4467-4481. doi: 10.1007/s00253-019-09784-1. Epub 2019 Apr 15.
Locillomycins are cyclic lipononapeptides assembled by a nonlinear hexamodular NRPS and have strong antibacterial activity. In this study, we genetically engineered Bacillus velezensis FZB42 as a surrogate host for the heterologous expression of the loc gene cluster for locillomycins. The fosmid N13 containing whole loc gene cluster was screened from the B. velezensis 916 genomic library. Subsequently, a spectinomycin resistance cassette, and the cassette fused with an IPTG inducible promoter Pspac, was introduced in the fosmid N13 using λ Red recombination system, respectively. The resulting fosmids, designated N13+Spec and N13+PSSpec, were used for the transformation of B. velezensis FZB42 to obtain derivative strains FZBNPLOC and FZBPSLOC. RT-PCR and qRT-PCR results revealed the efficient heterologous expression of the loc gene cluster in both derivative strains. Particularly, there was positive correlation between the derivative FZBPSLOC strain and the enhanced production of locillomycins upon addition of the inducer IPTG with the highest production of locillomycins at 15-fold more than that of B. velezensis 916. This overproduction of locillomycins was also related to the enhancement of antibacterial activity against methicillin-resistant Staphylococcus aureus, and exhibited moderate changes in its hemolytic activity. Together our findings demonstrate that the nonlinear hexamodular NRPS, encoded by the loc gene cluster from B. velezensis 916, is sufficient for the biosynthesis of cyclic lipononapeptide locillomycins in the surrogate host B. velezensis FZB42. Moreover, the FZBPSLOC strain will also be useful for further development of novel locillomycins derivatives with improved antibacterial activity.
洛西霉素是由非线性六模块 NRPS 组装而成的环状脂环肽，具有很强的抗菌活性。在本研究中，我们通过基因工程将解淀粉芽孢杆菌 FZB42 作为异源表达洛西霉素基因簇的替代宿主。从解淀粉芽孢杆菌 916 基因组文库中筛选出含有完整 loc 基因簇的 fosmid N13。随后，利用 λ Red 重组系统，分别在 fosmid N13 中引入壮观霉素抗性盒和与 IPTG 诱导启动子 Pspac 融合的盒。所得 fosmid，分别命名为 N13+Spec 和 N13+PSSpec，用于转化解淀粉芽孢杆菌 FZB42 以获得衍生菌株 FZBNPLOC 和 FZBPSLOC。RT-PCR 和 qRT-PCR 结果表明，该基因簇在这两个衍生菌株中均得到有效表达。特别是，在添加诱导剂 IPTG 后，衍生菌株 FZBPSLOC 的产量与洛西霉素产量呈正相关，洛西霉素的产量比解淀粉芽孢杆菌 916 高 15 倍。这种洛西霉素的过度产生也与对耐甲氧西林金黄色葡萄球菌的抗菌活性增强有关，并表现出溶血活性的适度变化。总之，我们的研究结果表明，来源于解淀粉芽孢杆菌 916 的 loc 基因簇编码的非线性六模块 NRPS 足以在替代宿主解淀粉芽孢杆菌 FZB42 中合成环状脂环肽洛西霉素。此外，FZBPSLOC 菌株也将有助于进一步开发具有增强抗菌活性的新型洛西霉素衍生物。
