出芽短梗霉发酵生产聚苹果酸的代谢通量及关键酶活性分析

Metabolic Flux Analysis and Key Enzymes Activities Determination for the Production of Poly Malic Acid using Aureobasidium pullulans

野生型出芽短梗霉菌株TKPM00006及其诱变菌株CGMCC30007在相同条件下,使用5 L罐进行聚苹果酸发酵,分析这2株菌在相同发酵状态下发酵中后期代谢网络的代谢流分布和关键酶活变化,对出芽短梗霉合成聚苹果酸的机理进行探究。结果表明,菌株TKPM00006和菌株CGMCC30007的菌体生长情况相似,但产酸量分别为20.54 g/L和30.2 g/L。.通过代谢通量分析及关键酶活性的测定可知,丙酮酸羧化途径及乙醛酸途径是PMLA合成的主要途径,TCA循环途径在发酵后期比较弱,该结论通过添加代谢抑制剂及中间代谢物实验加以证明。酶活分析同时还证明了高产菌株比出发菌株的PMLA合成能力强主要是因为丙酮酸羧化途径的加强。根据实验分析可在丙酮酸节点进行靶点改造或通过发酵调控改变丙酮酸节点处碳架的分配,通过加强丙酮酸羧化途径来减少因副产物的生产而造成的碳架流失,达到增加聚苹果酸生物合成的目的。

In this paper, metabolic flux and enzyme activity analysis for wild type Aureobasidium pullulans TKPM00006 and its mutant strain CGMCC3337 was conducted in 5 L bioreactor in the same conditions. The mechanism of biosynthesis of PMLA using Aureobasidium pullulans was investigated. The results showed that the biomasses in the two strains during the fermentation process were almost the same, but the accumulated yield was 20.54 g/L and 30.2 g/L, respectively. Through the metabolic flux analysis and key enzyme activity determination, it was found that pyruvate carboxylation pathway and glyoxylate pathway were the main pathways in PMLA synthesis, while TCA pathway was weak in the two strains at the post fermentation. This was confirmed by adding metabolic inhibitors and intermediate metabolites into medium. Enzyme activity analysis also confirmed that pyruvate carboxylation pathway was strengthened in the high yield strain whose PMLA synthesis ability stronger than the wild strain. The carbon flux distribution on pyruvate node could be manipulated by genetic manipulation and fermentation control. The enhanced flux in pyruvate carboxylation pathway decreased other pathways flux, thereby reducing the byproduct generation and improving PMLA yield.