表达元件优化促进重组胶原蛋白在谷氨酸棒杆菌中的表达  

Efficient production of recombinant collagen in Corynebacterium glutamicum by expression elements optimization

作　　者：程逸凡 张萌[1] 许菲 CHENG Yifan;ZHANG Meng;XU Fei(Key Lab of Ministry of Education of Industrial Biotechnology,School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《食品与发酵工业》2024年第14期1-9,共9页Food and Fermentation Industries

基　　金：国家自然科学基金(22078129)。

摘　　要：重组胶原蛋白是一种具有广泛应用潜力的生物材料,近年来引起了生物医学、组织工程等众多领域的关注。胶原蛋白的3股螺旋结构赋予其独特的生物学功能和生物相容性,但也增加了其在微生物系统中表达的复杂性。该研究以细菌胶原蛋白V-B作为模式蛋白,通过对表达元件的优化,促进重组胶原蛋白在谷氨酸棒杆菌中的表达。首先通过启动子筛选和发酵时长优化,获得介导V-B表达的最优启动子tac-R0。接着利用RBS calculator设计核糖体结合位点(ribosomal binding site,RBS)和间隔序列(aligned spacing,AS)的突变文库,得到与tac-R0启动子搭配组合的最优RBS和AS,使V-B的产量提高至514 mg/L。此外,通过多基因表达盒的串联组合策略,将多个目的基因串联,最终V-B的产量较初始水平提高了8.4倍,达697 mg/L,该研究为重组胶原蛋白的工业化生产提供了基础。Recombinant collagen is a biopolymer with broad potential applications in various fields,such as biomedicine and tissue engineering.The triple-helix structure of collagen imparts unique biological functions and biocompatibilit but also increases the complexity of its expression in microbial systems.In this study,bacterial collagen V-B was used as a model protein to promote the expression of recombinant collagen in Corynebacterium glutamicum through optimization of expression elements.Firstly,the most efficient promoter tac-R0 was obtained through promoter screening and fermentation duration optimization.Subsequently,the RBS calculator was used to design a mutation library for ribosome binding sites(RBS)and aligned spacing(AS).The RBS and AS with the highest strength were identified,which increased the yield of V-B to 514 mg/L.Furthermore,through the tandem combination of multi-gene expression cassettes,the final yield of V-B increased by 8.4-fold compared to the initial level,reaching 697 mg/L.This study lays a foundation for the industrial production of recombinant collagen.

关 键 词：重组胶原蛋白 谷氨酸棒杆菌 启动子 核糖体结合位点 串联表达 

分 类 号：TQ920.1[轻工技术与工程—发酵工程] Q78[生物学—分子生物学]