醋酸胁迫对Acetobacter pasteurianus产酸呼吸链活力的影响

Effects of acetic acid stress on the activity of acetic acid respiratory chains in Acetobacter pasteurianus

摘要:

【目的】以巴氏醋酸杆菌工业菌株沪酿1.01和模式菌株Acetobacter pasteurianus ATCC 33445为研究对象，研究工业菌株与模式菌株在高浓度醋酸胁迫下的产酸发酵时呼吸链酶活、相关基因转录水平的变化规律。【方法】检测两种菌株在0、1%、2%、3%不同初始醋酸浓度下的生物量、产酸以及酶活，并通过实时荧光定量PCR检测呼吸链相关酶合成基因的相对转录水平变化。【结果】两种菌株在1%初始醋酸浓度下产酸能力最强，发酵48 h平均产酸速度达到0.667 g/(L·h)；两种菌株的ADH (乙醇脱氢酶)和ALDH (乙醛脱氢酶)酶活也达到最高，平均为12.01和9.77 U/mg；相关酶合成基因的相对转录水平较无底酸均提高。当初始醋酸浓度上升至2%和3%时，菌体酶活、产酸能力逐渐下降，呼吸链上的adh、cyt bd、cyt o和fapA基因转录水平上升，其余基因都降低。【结论】确定了巴氏醋酸杆菌在高初始浓度醋酸条件下，菌体会自发提高adh基因的转录水平，启动底物磷酸化，并将醋酸泵出到胞外；同时aldh基因的表达受到抑制，降低产酸，从而维持体内较低醋酸浓度。此外，呼吸链上其他外排相关酶转录水平也会提升，如cyt bd和cyt o，辅助底物磷酸化进程，加快释放能量。

Abstract:

[Objective] To analyze the regularity of the activity of respiratory chains and the transcriptional levels of related genes in Acetobacter pasteurianus under the stress of high acetic acid concentration, A. pasteurianus CICC 20001 and A. pasteurianus ATCC 33445 were studied. [Methods] The biomass, acid production and enzyme activity of the two strains have been analyzed in different initial acetic acid concentrations of 0, 1%, 2% and 3%. Real-time quantitative PCR was used to analyzed changes of relative transcriptional levels of respiratory chains related enzyme synthesis genes in different concentrations. [Results] It was found that acid production capacity of two strains was the strongest when initial acetic acid concentration was 1%; and their average acid production rate reached 0.667 g/(L·h) during 48 hours fermentation. At the same time, enzyme activity levels of ADH (alcohol dehydrogenase) and ALDH (acetaldehyde dehydrogenase) also rise to the highest of 12.01 and 9.77 U/mg on average. And the relative transcriptional level of the respiratory chain related enzyme synthesis genes were also increased compared with initial acid of 0. The enzyme activity and acid production capacity decreased gradually when initial acetic acid concentration increased to 2% and 3%. The adh, cyt bd, cyt o and fapA gene on respiratory chain maintained a relatively high transcriptional level while the transcriptional level of rest genes decreased. [Conclusion] It is identified that under high-intensity acetic acid conditions, the transcriptional level of adh gene is increased spontaneously, which activates the substrate phosphorylation and pump acetic acid out. At the same time, the aldh gene’s expression is inhibited, which causing the reducing of acid production and maintaining rather low concentration of acetic acid. In addition, the level of other efflux enzymes in the respiratory chain will also increase, such as cyt bd and cyt o, which enhance the substrate phosphorylation in metabolic process and accelerate the release of energy.