大肠杆菌苹果酸有氧发酵溢出机制的解析及其应用

Mechanism of overflow metabolism and its application for L-malate synthesis

L苹果酸是一种重要的四碳二羧酸,以可再生生物质为原料,通过微生物发酵高效制备L苹果酸具有重要意义。本研究首先考察了氮限制条件、温度和pH对有氧条件下L苹果酸合成的影响,结果表明氮限制条件是苹果酸大量积累的重要条件,最适的温度为35℃,最优pH为6.8。进一步分析辅因子水平及呼吸链相关基因的转录水平变化后发现,苹果酸的溢出可能是由于电子呼吸链效率下降,导致NADH的氧化不足,NAD+供给不足最终无法催化苹果酸进一步转化为草酰乙酸。通过敲除大肠杆菌中呼吸链NADH脱氢酶Ⅱ编码基因ndh,可降低电子呼吸链效率。最终重组菌E.coli AFP111(Δndh)在氮源充足条件下,丁二酸也能被大量代谢并以苹果酸为主要产物,收率达到0.65 g/g。本研究成果将为通过呼吸链改造提高中间代谢产物的积累提供理论支持和借鉴。

The utilization of renewable biomass to produce L-malic acid,an important C4 dicarboxylic acid,has attracted significant interests in past years.Herein,we investigated the aerobic synthesis of L-malic acid regarding the influence from several key factors including nitrogen limited conditions,temperature,and pH.As a result,nitrogen limited conditions played a pivotal role in the accumulation of L-malic acid.Additionally,the optimal temperature and pH for the synthesis were determined to be 35℃and 6.8,respectively.Based on the analysis of cofactor concentrations and transcription levels for genes associated with the respiratory chain,the excessive accumulation of L-malic acid might be attributed to the declining efficiency of the electron respiratory chain.In other words,inadequate oxidation of NADH and insufficient availability of NAD+impeded the catalytic conversion of L-malic acid to oxaloacetic acid,leading to the excretion of L-malic acid into the extracellular environment.As the removal of gene ndh in NADH dehydrogenaseⅡcould decrease the electron respiratory chain efficiency,the recombinant E.coli AFP111(Δndh)exhibited the ability to metabolize a significant quantity of succinic acid,affording L-malic acid as the primary product with a yield of 0.65 g/g under nitrogen sufficient conditions.Our research demonstrated that the respiratory chain modification could enhance the overflow metabolism of intermediate metabolites,offering valuable theoretical support and guidance in this field.