以Acetobacter pasteurianus CICIM B7003及其高产突变株A. pasteurianus CICIM B7003-02为研究对象,通过2株巴氏醋酸杆菌发酵过程中胞内微环境分析来揭示其耐酸机制。在7.5 L发酵罐中比较分批和半连续发酵过程中其发酵动力学以及和胞内微环境变化。结果显示,分批发酵中其平均产酸速率达0.836 g/(L·h),相对亲本菌株提高29.6%。半连续发酵中酸启动时间缩短了31 h,微环境分析表明,突变株中与产酸直接相关的乙醇脱氢酶(alcohol dehydrogenase, ADH)和乙醛脱氢酶(acetaldehyde dehydrogenase, ALDH)的最高酶活分别提高27.0%和15.2%,辅酶Q9含量提高69.5%。突变株拥有更高比例的十八碳烯酸,比亲本株高71.4%。突变株最高胞内ATP含量是亲本株2.33倍,胞内ATP与比生长速率呈正相关,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%。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
This study was conducted to investigate changes in intracellular microenvironment of Acetobacter pasteurianus CICIM B7003 and its high-yield mutant A.pasteurianus CICIM B7003-02 to unravel the acid-tolerant mechanisms of A. pasteurianus. The fermentation kinetics of parent and mutant strain during batch and semi-continuous fermentation in a 7.5 L fermenter under acidic stress, as well as changes in their intracellular microenvironment were compared. It was found that during batch fermentation, the average fermentation efficiency of the mutant strain reached 0.836 g/(L·h), which increased 29.6% compared against that of the parent strain. Moreover, the acid start-up time decreased 31 h during semi-continuous fermentation. Furthermore, enzymes that are directly related to acid production in the mutant strain, such as alcohol dehydrogenase and acetaldehyde dehydrogenase, increased their highest activities by 27.0% and 15.2%, respectively. Besides, the content of coenzyme Q9 increased by 69.5%. In addition, the mutant strain had a higher proportion of octadecenoic acid, which was 71.4% higher than that of the parent strain. The maximum intracellular ATP content of the mutant strain was 2.33-fold higher, and intracellular ATP was positively correlated with the specific growth rate. Also, intracellular glutamate and aspartic acid increased by 10.7% and 18.3%, respectively. In conclusion, the mutant strain was mainly relied on the synergistic effects of strengthening ethanol respiratory chain, ATP synthesis, and critical amino acid metabolism to improve its acid resistance.
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