低pH胁迫小白链霉菌高效积累ε-聚赖氨酸生理机制研究

ε-Poly-L-lysine (ε-PL), a newly approved biological food preservative by our country, is a key product for promoting biological food preservative replace of chemical ones in food industry. Low pH value is an important prerequisite and common feature of producing strain for the ε-PL accumulation in fermentation. However, the relationship between the low pH value and the ε-PL accumulation is still unclear in-depth until now. Recently, we have successfully achieved the ε-PL production to 54.7 g/L by Streptomyces albulus using a low pH stress strategy in a short time during fed-batch fermentation process, which is the highest ε-PL production reported in the worldwide. In this project, the effect of low pH on intracellular micro-environment, the profiles of gene transcription and protein expression in S. albulus were investigated by the combination of physiological & biochemical analysis and –omics technology, to reveal the physiological mechanism of S. albulus response to the low pH environmental stress. Meanwhile, some key trans-acting factors involved in the regulation of ε-PL biosynthesis would be elucidated, and their interaction modes with ε-PL synthase gene (pls) also need exploited through qRT-PCR experiment, a series mutants construction with corresponding genes deletion-recovering-overexpression, and in vitro electrophoretic mobility shift assay (EMSA). Those results will demonstrated the mechanism of trans-acting factor(s) regulation of ε-PL biosynthesis, and finally to reveal the relationship between the physiological mechanism of S. albulus response to low pH stress and efficient ε-PL accumulation. The result of this project will help people to understand the physiology and regulation mechanisms of efficient ε-PL accumulation dependence on low pH in the view point of environmental stress. It is also provide guidance to continually improve the production of ε-PL by metabolic engineering and biochemical engineering approaches. Therefore, this project has important theoretical and practical significances.

ε-聚赖氨酸（ε-PL）作为我国最新批准的生物食品防腐剂，是推动生物型取代化学型食品防腐剂的关键品种。低pH值发酵环境是产生菌积累ε-PL的前提和共有特征。然而，二者之间的关系，目前还缺乏全局性和机理性认识。最近，项目组利用低pH值胁迫策略，实现小白链霉菌ε-PL发酵水平达到54.7 g/L，为国际报道最高水平。基于此，本项目借助生理生化分析和组学技术，从胞内微环境、基因转录和蛋白质表达水平，解析小白链霉菌响应低pH值胁迫的生理机制；并利用qRT-PCR、基因敲除-回补-过表达和凝胶阻滞等实验方法，明确参与ε-PL合成调控的关键反式作用因子及其作用ε-PL合成酶基因的方式，阐明小白链霉菌响应低pH值胁迫的生理机制与ε-PL高效积累的关系。研究结果有助于从环境胁迫角度深入理解产生菌依赖低pH高效积累ε-PL的生理和调控机制，为后续利用代谢工程和生化工程手段继续提高ε-PL发酵水平提供指导。

ε-聚赖氨酸（ε-PL）作为我国最新批准的生物食品防腐剂，是推动生物型取代化学型食品防腐剂的关键品种。低pH值发酵环境是产生菌积累ε-PL的前提和共有特征。然而，二者之间的关系缺乏全局性和机理性认识。本项目在生理和基因转录水平研究了小白链霉菌对低pH值胁迫的响应机制，发现小白链霉菌主要依赖改变细胞膜脂肪酸不饱和度和脂肪酸链长来增强细胞膜流动性，通过积累精氨酸、赖氨酸和组氨酸等碱性氨基酸，增强H+-ATPase活力和胞内ATP浓度以及尿素酶系统等来维持胞内pH稳定性。同时，在低pH环境中，小白链霉菌除了改变众多代谢途径关键酶基因表达，也上调了大量分子伴侣和ATP依赖性蛋白酶编码基因的表达量以修复和降解错误折叠的蛋白质；并上调了负责核苷酸切除、同源重组和非同源末端连接等参与DNA修复的关键酶基因表达量以应对DNA的损伤，为细胞在酸性环境中的存活提供了保障。另外，首次在小白链霉菌中发现调控ε-PL合成酶基因（pls）的信号调控途径MprAB。在分子水平研究了关键反式作用因子MprAB调控pls的方式，发现mprA、mprB和pepD的失活导致ε-PL产量显著下降，分别为52.11%、60.56%和55.63%。通过基因敲除、回补、过表达和发酵等实验，证实了mprA、mprB和pepD对pls具有正调控作用。进一步荧光定量PCR分析发现，MprB能够正调控MprA的表达，MprA与PepD之间也存在正调控关系，且三个基因均能够正调控pls的表达，从而明确了MprAB参与ε-PL合成调控及其初步作用pls的方式。研究结果有助于从环境胁迫角度深入理解产生菌依赖低pH高效积累ε-PL的生理和调控机制，为后续利用代谢工程和生化工程手段继续提高ε-PL发酵水平提供指导。