基于辅因子全局调控改善巴氏醋酸杆菌酸胁迫抗性的生理机制研究

The effect of acid stress has always been a bottleneck of restricting the yield and production intensity during the acetic acid fermentation. In this project, an industrial vinegar brewing strain named Acetobacter pasteurianus Huniang 1.01, was used as a research model. A series of promoter elements with different strength were excavated from A. pasteurianus genome, and then applied to the dynamic regulation of cofactor NADPH and PQQ regeneration pathway for improving the fitness of Acetobacter under acetic acid stress condition. The effect of intracellular NADPH and PQQ concentration on acetic acid production and tolerance was investigated by analyzing cell specific growth rate, acetification rate, survival rate under acetic acid stress condition. The optimal combination of NADPH and PQQ metabolism was determined to improve maximally acetic acid production and tolerance. Differences in transcriptional level and enzyme activity of carbon metabolic pathway genes were analyzed to reveal the physiological mechanism of intracellular carbon metabolic flux rebalanced. Additionally, the physiological mechanism of intracellular cofactor for improving acid tolerance was explored by analysis of cell physiological, and transcriptional level between wild strain and mutants under acetic acid stress condition. This project is expected to increase not only acetic acid production and tolerance of Acetobacter, the mechanism to be illustrated and method might also provide some guidance and reference for improving the acid tolerance of other microorganisms to efficiently produce organic acid.

针对工业液态醋酸酿造过程中由于酸胁迫导致的醋酸产率和生产强度难以提高这一瓶颈问题，本研究以一株食醋工业酿造菌株巴氏醋酸杆菌（Acetobacter pasteurianus）沪酿1.01为研究模型，从定向改善A. pasteurianus酸胁迫抗性能力提高产酸速率入手，应用分子生物学手段挖掘A. pasteurianus中的梯度组成型和酸诱导型启动子；借助代谢工程策略对微生物代谢网络中的关键辅因子NADPH和PQQ合成水平进行动态调控，揭示辅因子水平与细胞比生长速率、产酸速率和酸胁迫抗性之间的作用规律，重点探讨辅因子NADPH和PQQ全局调控的最佳适配关系，探究胞内碳代谢流再平衡的生理机制，最大程度地提高醋酸产量和酸胁迫抗性；并从细胞微环境和基因转录水平对辅因子组合调控改善酸胁迫抗性的生理机制进行解析，为改进其他工业微生物酸胁迫抗性提高生物技术过程经济性提供理论依据。

本文针对工业液态醋酸酿造过程中由于酸胁迫导致的醋酸产率和生产强度难以提高这一瓶颈问题，以一株食醋工业酿造菌株巴氏醋酸杆菌（Acetobacter pasteurianus）沪酿1.01为研究模型，从定向改善A. pasteurianus酸胁迫抗性提高生产效率入手，首先利用RNA-Seq测序分析了菌株A. pasteurianus CICIM B7003（沪酿1.01）发酵过程中全基因组转录水平，搭建了以耐酸红色荧光蛋白mRFP为报告子的A. pasteurianus启动子筛选平台，筛选出了具有高强度启动活性的组成型启动子Ptuf和酸驱动型启动子PgroES；其次，通过乙醇呼吸链关键脱氢酶ADH和ALDH和辅因子PQQ的协同调控，明确了脱氢酶与PQQ组合代谢调控的最佳适配关系；不仅能够提高巴氏醋酸杆菌乙酸产量，还能改善细胞对初始乙酸的适应度。获得了一株具有优良性状的工程菌株A. pasteurianus/pT-adhA-pqqABCDE，半连续发酵启动时间从原始菌株116 h缩短至99 h，发酵周期也缩短至34~35 h，全过程的平均酸化率达0.99 g/L/h，较原始菌株提高了32%；最后，对高产酸耐酸的工程突变菌株的生理、转录水平分析发现高产酸耐酸菌株中乙醇脱氢酶(Alcohol dehydrogenase, ADH)和乙醛脱氢酶(Acetaldehyde dehydrogenase, ALDH)的最高酶活较原始菌株分别提高27.0%和15.2%，辅酶Q9含量提高69.5%。胞内微环境分析表明，突变株中十八碳烯酸含量比亲本株高31.5%，与比生长速率呈正相关的胞内ATP含量是亲本株2.33倍，胞内谷氨酸和天冬氨酸含量分别比亲本株提高10.7%和18.3%。供能途径中呼吸链基因，三羧酸循环基因以及应激蛋白基因不同酸度下转录水平大部分呈上调趋势。突变株主要依靠加强乙醇呼吸链，增强ATP合成和关键氨基酸代谢等机制的协同作用提高酸胁迫抗性。通过本研究的实施，为改进其他工业微生物酸胁迫抗性提高生物技术过程经济性提供理论依据。