质粒复制原点在地衣芽孢杆菌中的功能鉴定及理性设计

Development and application of strong origin of replication of Bacillus licheniformis

食品安全级工业微生物地衣芽胞杆菌(Bacillus licheniformis)与模式微生物相比,严重缺乏质粒载体等合成生物学标准化遗传工具,导致其研究与应用受到限制。该研究首先建立了质粒特性在地衣芽孢杆菌中的评价方法,由此鉴定获得了高效复制原点。在此基础上进一步解析了复制原点中茎环结构影响质粒拷贝数和稳定性的分子机制,并理性设计了系列人工复制原点。结果表明,枯草芽孢杆菌来源的复制原点ORIT5拷贝数是pHY300内源复制原点的2.15倍(达到40.2),同时具有更高的稳定性。3′端增加1个motif为TCCGCC茎环的人工复制原点pT1,拷贝数进一步提升至62.2,稳定性提高36%。以麦芽寡糖基海藻糖合成酶为报告蛋白,携带pT1复制原点的重组菌发酵最高酶活力达到8726.7 U/mL,比出发菌株提高了43.9%。人工复制原点的开发与应用为地衣芽孢杆菌表达系统的改进和完善奠定了基础。

Compared to model microorganisms,the food-safety grade industrial bacteria Bacillus licheniformis has a serious lack of standardized genetic tools for synthetic biology,such as plasmid vectors,which limits its use in research and applications.Using a technique we developed for assessing plasmid characteristics in Bacillus licheniformis,we were able to identify and obtain extremely effective replication primordia in this work.A variety of artificial replication origins were logically created based on the additional analysis of the molecular process by which the stem-loop structure in the replication origin influences plasmid copy number and stability.According to the findings,the replication origin ORIT5 from B.subtilis had a copy number that was 2.15 times larger and more stable than the endogenous replication origin pHY300(up to 40.2).With the addition of a TCCGCC stem-loop at the 3′end,pT1,an artificial replication origin,enhanced stability by 36%and raised copy number to 62.2.The maximal enzyme activity of the recombinant bacterial fermentation bearing the pT1 replication origin was 8726.7 U/mL,which was 43.9%greater than that of the starting strain,using malt oligosaccharide-based alginate synthase as the reporter protein.The creation and use of the artificial replication origin set the stage for Bacillus licheniformis′s expression system to be enhanced and perfected.