谷氨酸棒杆菌一步法发酵糖质原料生产γ-聚谷氨酸

γ-Polyglutamic acid production in Corynebacterium glutamicum using sugar by one-step fermentation

摘要:

γ-聚谷氨酸在食品、化妆品、生物医药等领域具有广泛的应用，目前主要的生产菌株是谷氨酸依赖型菌株，在生产过程中需要添加谷氨酸作为前体，因而生产γ-聚谷氨酸的成本较高。文中主要研究从糖质原料一步法发酵合成γ-聚谷氨酸的生产工艺。首先，从产γ-聚谷氨酸的菌株枯草芽孢杆菌中克隆γ-聚谷氨酸合成酶的基因簇pgsBCA，在谷氨酸棒杆菌模式菌株ATCC13032中进行诱导型和组成型表达，结果显示，仅诱导型表达菌株可以积累γ-聚谷氨酸，产量为1.43 g/L。进一步对诱导条件进行优化，确定诱导时间为2 h，IPTG浓度为0.8 mmol/L，γ-聚谷氨酸产量为1.98 g/L。在此基础上，在一株高产谷氨酸的谷氨酸棒杆菌F343中外源表达pgsBCA，对重组菌进行发酵，结果表明，在摇瓶发酵中γ-聚谷氨酸产量达到10.23 g/L，在5 L发酵罐中产量达到20.08 g/L；继而对γ-聚谷氨酸进行分子量测定，结果显示，产自F343重组菌的γ-聚谷氨酸的重均分子量比产自枯草芽孢杆菌的提高34.77%。文中构建了一步法发酵糖质原料生产γ-聚谷氨酸的新途径，同时为开发其潜在应用奠定了基础。

Abstract:

γ-polyglutamic acid (γ-PGA) is widely used in food processing, cosmetic production, medicinal industry, etc. Currently, the production strains used in fermentation process are commonly glutamic acid-dependent, which results in extra cost. In this study, a de novo way of producing γ-PGA from sugars was reported. To this end, the γ-polyglutamate synthase gene cluster pgsBCA was cloned from the natural γ-PGA-producing strain Bacillus subtilis (ATCC 6051-U), and was constitutively and inducibly expressed in Corynebacterium glutamicum ATCC 13032. Only inducible expression of pgsBCA can lead to the generation of γ-PGA with a titer of 1.43 g/L from glucose, without any supplementation of glutamic acid. The production was further elevated to 1.98 g/L upon optimization of the induction conditions with the induction time at 2 h post-inoculation and the IPTG concentration of 0.8 mmol/L. Moreover, to achieve a higher titer of γ-PGA, pgsBCA was inducibly expressed in C. glutamicum F343, which shows a paramount glutamate production capacity. The final γ-PGA production reached 10.23 g/L in shake flasks and 20.08 g/L in a 5-L fermentor using glucose as the substrate. The weight-average molecular weight (Mw) of γ-PGA from recombinant strain F343 showed 34.77% higher than that produced by B. subtilis. This study provides a novel way of producing γ-PGA from sugars directly and potentiates new applications of γ-PGA in the future.