黄酒酿造乳酸菌ADI途径中瓜氨酸转运方式及其调控机理的研究

Ethyl carbamate is one of the most important factors influencing food safety of Chinese rice wine. Urea and citrulline formed during fermentation process have been demonstrated to be the main precursors of ethyl carbamate. Nowadays methods of controlling or eliminating ethyl carbamate were mainly developed by reducing urea content. In previous work, we found that all lactic acid bacteria strains involved in Chinese rice wine fermentation could accumulate citrulline during arginine degradation. A Lactobacillus brevis 2-34 strain which could re-uptake citrulline was found accidently in previous work. To increase the citrulline uptake and metabolize efficiency of the strain, the current project aims to first identify citrulline transporter and the transport mode, with gene deletion/complement technologies and transport analysis using isotope labelling methodology; and subsequently, to elucidate the physiological motive force of citrulline transport direction transition through physiological index measurement; and finally, to explore the regulatory network of citrulline transport and metabolism by the combination of metabolic flux determination and comparative proteomic analysis based on the Chinese rice wine fermentation technology. The results will feedback clues to promote citrulline uptake of the strain in the genetic level and micro-environmental view, and enable us to thoroughly understand the mechanism of citrulline transport and metabolism in lactic acid bacteria. This project sheds light on developing new strategies to eliminate ethyl carbamate, leading to food safety improvement and technological development of Chinese rice wine industry.

黄酒中氨基甲酸乙酯是影响其食品安全最为重要的因素，尿素和瓜氨酸均为氨基甲酸乙酯的重要前体物质，现有的控制方法大多针对尿素提出。前期工作中，我们发现黄酒酿造乳酸菌在利用精氨酸过程中均能积累瓜氨酸，意外发现其中一株短乳杆菌2-34重吸收瓜氨酸的特点，揭示其应用于黄酒酿造控制氨基甲酸乙酯的潜力。为提高该菌株吸收利用瓜氨酸的效率，本项目结合基因敲除/回补和基于同位素标记的转运分析等技术，明确该菌株瓜氨酸转运蛋白及其转运方式；通过细胞内外在生理指标分析，阐明瓜氨酸转运方向转化的生理动力；参照黄酒酿造工艺，结合代谢流分析和比较蛋白质组学方法，解析瓜氨酸转运代谢的调控网络，为从基因水平和微环境角度调控短乳杆菌2-34吸收瓜氨酸效率反馈策略。研究结果将有助于完善乳酸菌瓜氨酸转运代谢理论，为探索有效控制黄酒中氨基甲酸乙酯的新方法提供理论依据，对提高黄酒饮用的安全性，推动黄酒行业的技术进步具有重要意义。

黄酒中较高含量的致癌物质——氨基甲酸乙酯，成为影响其食品安全的重要因素，其主要由尿素和瓜氨酸等氨甲酰类化合物与乙醇反应形成，目前针对降低黄酒EC的调控策略主要围绕尿素提出，在此基础上，寻找一种有效控制发酵液中瓜氨酸的方法，或将成为进一步降低黄酒中EC含量的最佳途径。本项目针对在黄酒中分离的一株具有瓜氨酸重吸收功能的短乳杆菌2-34，通过在大肠杆菌中构建精氨酸转运代谢相关途径并重组表达推测的瓜氨酸转运蛋白DcuC和AO antiporter，证明了DcuC蛋白具有较强的瓜氨酸吸收功能，而AO antiporter对精氨酸和瓜氨酸均具有一定的转运能力，且其对瓜氨酸的转运活性低于DcuC蛋白；优化了短乳杆菌的转化方法，利用Cre/loxp系统在Lb. brevis 2-34菌株中分别敲除dcuC和ao antiporter基因，证明DcuC和AO antiporter均有吸收精氨酸和瓜氨酸的功能，且DcuC对精氨酸和瓜氨酸的转运能力更强；研究了Lb. brevis 2-34菌株在含有精氨酸的环境中生长时ADI途径相关基因的表达情况，发现精氨酸代谢及瓜氨酸转运相关基因在对数期前期转录水平都较高；至对数中后期，仅dcuC和a/o antiporter基因转录仍维持在较高水平，这与细胞代谢精氨酸和重吸收瓜氨酸的状态相对应；比较分析了DcuC和A/O antiporter的跨膜结构域、核心功能结构域， DcuC和A/O antiporter分别含有13和11个跨膜螺旋，N端位于胞质侧，二者均含有多个与信号传导有关的磷酸化位点和酰化位点，推测其翻译后修饰与转运瓜氨酸的方向转换有关；在黄酒酿造中强化短乳杆菌2-34，可显著降低黄酒中瓜氨酸和氨基甲酸乙酯含量，将该菌株应用于花生粕饲料发酵提升其饲用品质，证明了该菌株的应用前景。此外，在本项目执行过程中，还对短乳杆菌2-34进行了全基因组测序，对其分子遗传和进化进行进一步分析，为完善乳酸菌瓜氨酸转运代谢理论，调控Lb. brevis 2-34菌株提高其吸收瓜氨酸的效率反馈策略，为发展有效控制黄酒中EC的新方法提供理论依据，对提高我国黄酒饮用的安全性，推动黄酒行业的技术进步具有重要意义。