适应性驯化选育高产吡咯喹啉醌的生丝微菌突变株

Breeding of Hyphomicrobium denitrificans for high production of pyrroloquinoline quinone by adaptive directed domestication

摘要:

吡咯喹啉醌 (PQQ) 广泛存在于生物体内，具有促进机体生长、维护线粒体功能、促进神经生长因子合成和调节机体自由基水平等生理功能，在医药、食品和化妆品领域具有广阔的应用前景。为提高脱氮生丝微菌Hyphomicrobium denitrificans FJNU-6的PQQ生产性能，文中以高浓度甲醇为拮抗因子进行实验室适应性定向驯化，通过光谱法快速筛选体系，选育PQQ高产正突变株。6轮适应性驯化后，每轮驯化的正向突变率达到90%以上，产量提高1倍的突变株达到10%左右。最后，利用5 L发酵罐对突变株FJNU-R8进行分批补料培养，相较于出发菌株，突变株在不同甲醇浓度下pqq和moxF基因簇的表达量较高且差异较小，甲醇消耗和生长速度较慢，PQQ产量达到1 087 mg/L (143 h)，单位细胞产量提高了1.42倍，展现出良好的工业应用潜力。文中所述的适应性定向驯化结合快速筛选体系能简单、快速地获得高产PQQ的生丝微菌突变菌株，对其他甲基营养菌高产PQQ突变株的高通量筛选具有借鉴作用。

Abstract:

Pyrroloquinoline quinone (PQQ) is widely distributed in organisms and has physiological functions such as boosting body growth, maintaining mitochondrial function, promoting synthesis of nerve growth factor and regulating free radical levels in the body. It has broad application prospects in the fields of medicine, food and cosmetics. In order to improve the PQQ production of Hyphamicrobium denitrificans FJNU-6, the high-concentration methanol was used as the antagonistic factor for laboratory adaptive domestication. The PQQ positive mutants were selected using rapid screening system by spectroscopy. After 6 rounds of adaptive domestication, about 10% mutants were acquired with a doubled yield, and over 90% positive mutation rate of each round of domestication was reached. Subsequently, the mutant strain FJNU-R8 was fermented by 5 L fermenter. Compared with the original strain, the expression of pqq and moxF gene clusters were higher at different methanol concentrations and similar to each other. Meanwhile, the methanol consumption rate and growth rate were slower than the original strain. Finally, the PQQ yield was increased by 1.42 times to 1 087.81 mg/L (143 h), indicating good industrial application potential. The adaptive domestication combined with rapid screening system described in this study can easily and rapidly obtain mutants with high yield of PQQ, which can be used as reference for high-throughput screening of other high-yield PQQ mutants of methylotrophic bacteria.