基于转录组学挖掘兽疫链球菌内源表达元件及高产透明质酸应用  

Screening Endogenous Expression Elements in Streptococcus zooepidemicus via Transcriptomics Analysis and Applications for High Yield of Hyaluronic Acid

作　　者：赵锐 狄靖宜 张广通 刘浩[1] 高伟霞 ZHAO Rui;DI Jing-yi;ZHANG Guang-tong;LIU Hao;GAO Wei-xia(Key Laboratory of Industrial Fermentation Microbiology,College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457)

机构地区：[1]天津科技大学生物工程学院教育部工业发酵微生物重点实验室,天津300457

出　　处：《生物技术通报》2024年第10期296-304,共9页Biotechnology Bulletin

基　　金：重要医用多糖绿色生物制造关键技术及产业化示范(2021YFC2103200)。

摘　　要：【目的】构建兽疫链球菌内源性表达元件文库,并探究其在提高透明质酸产量中的应用。【方法】通过对兽疫链球菌发酵指数期和平台期进行转录组测序分析,初步筛选32种高、中、低3种强度的启动子与RBS组合(表达元件PR);进一步利用绿色荧光蛋白基因转录水平及荧光强度来验证PR元件的强度;最后,利用筛选到的较强元件PR31过表达透明质酸合成关键基因hasA,hasB,hasC,hasD,hasE,并采用2 L发酵罐评价强表达元件在提高透明质酸产量的效果。【结果】上述基因的相对转录水平分别提高至原来的8.17、7.32、3.72、39.48、9倍。其中,过表达hasA及hasD后,透明质酸产量相对于野生型分别提高了43%和31%,达到5.654 g/L和5.283 g/L。【结论】成功构建了兽疫链球菌内源表达元件文库,可用于透明质酸合成途径强化及竞争途径弱化等代谢工程改造。【Objective】This work is aimed at establishing a library of endogenous expression elements of Streptococcus epizooticus,and investigating the application of it in increasing hyaluronic acid yields.【Method】The 32 kinds of high,medium and low strength promoter and RBS combination(expression element,PR)were preliminarily screened,through transcriptomics analysis of S.zooepidemicus in the exponential or stationary stage of fermentation.Then,the strength of the 32 PRs was verified via detecting the transcriptional level of gfp gene and fluorescence intensity of GFP.Eventually,the key hyaluronic acid synthesis gene hasA,hasB,hasC,hasD,and hasE was overexpressed respectively with the selected strong expression element PR31,and the impact of the strong PR on enhancing hyaluronic acid production was assessed through a 2 L fermentation.【Result】The relative transcription of the aforementioned genes were elevated to 8.17,7.32,3.72,39.48 and 9-fold.Among them,the overexpression of hasA and hasD increased the hyaluronic acid yield by 43%and 31%,respectively,compared to the wild type,reaching 5.654 g/L and 5.283 g/L.【Conclusion】A library of endogenous expression elements of S.zooepidemicus had been constructed successfully,which can be employed for metabolic engineering,such as reinforcing the hyaluronic acid synthesis pathway or weakening competitive pathways.

关 键 词：兽疫链球菌 转录组测序 启动子 核糖体结合位点 透明质酸 

分 类 号：Q78[生物学—分子生物学] S852.61[农业科学—基础兽医学]