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.
作者
程逸凡
张萌
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)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
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)。
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
recombinant collagen
Corynebacterium glutamicum
promoter
ribosome binding sites
multi-gene expression cassettes
作者简介
第一作者:程逸凡,硕士研究生;共同通信作者:许菲,教授,E-mail:feixu@jiangnan.edu.cn;共同通信作者:张萌,助理研究员,E-mail:mzhang@jiangnan.edu.cn。
