理性代谢工程改造促进谷氨酸棒杆菌高效合成L-谷氨酰胺

Efficient Synthesis of L-Glutamine by Corynebacterium glutamicum Promoted via Rational Metabolic Engineering

摘要:

L-谷氨酰胺（L-glutamine，L-Gln）是人体液中含量最丰富的一种半必需氨基酸，具有多种营养和药理功能，被广泛应用于医药、食品添加剂、营养保健品、饲料等领域。目前，微生物发酵法是生产L-Gln的主要方法，发酵产酸效率低、副产物多、菌种性能欠缺等问题严重限制了其工业化应用。为解决这一问题，作者以实验室前期构建的L-Gln生产菌株CGQ03为出发菌株，通过强化谷氨酸脱氢酶表达及辅因子NADPH供应促进前体L-谷氨酸合成、增强辅因子ATP胞内含量、过表达溶氧相关蛋白VHb提高细胞携氧能力、增强关键酶谷氨酰胺合成酶催化活性及发酵工艺优化等策略提高了L-Gln产量。最终的基因工程菌CGQ08/pDXW10-glnA<>supSc-gdh^Cg在5 L发酵罐上补料分批发酵66 h，L-Gln的产量最高达（94.5±1.8） g/L，糖酸转化率为34.8%，生产强度为1.43 g/（L·h）。本研究为L-Gln及其相关化合物的工业化生产提供了借鉴。

Abstract:

L-glutamine is the most abundant semi-essential amino acid in human fluids. It has a variety of nutritional and pharmacological functions and is widely used in medicine, food additives, nutritional health products, feed and other fields. At present, microbial fermentation is the main method to produce L-Gln, but its industrial application is seriously limited by the problems of low fermentation acid production efficiency, many by-products, and lack of bacterial performance. In order to solve those problems, the L-Gln production strain CGQ03 constructed early in our laboratory was used as the starting strain in this study. The synthesis of precursor L-glutamate was promoted by enhancing the expression of glutamate dehydrogenase and the supply of cofactor NADPH. The yield of L-Gln production was increased by enhancing the intracellular content of cofactor ATP, overexpressing the dissolved oxygen-related protein VHb to improve the oxygen carrying capacity of cells, enhancing the catalytic activity of key enzyme glutamine synthetase, and optimizing the fermentation process. The final genetically engineered strain CGQ08/pDXW10-glnA^Sc-gdh^Cg was fed-batch fermented in a 5 L fermenter for 66 h, and the yield of L-Gln was up to (94.5±1.8) g/L, with the sugar-acid conversion rate of 34.8% and the production intensity of 1.43 g/(L·h). This study could provide an important reference for the industrial production ofL-Gln and its related compounds.