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