高产吡咯喹啉醌扭脱甲基杆菌的高通量选育

High-throughput screening of Methylobacterium extorquens for high production of pyrroloquinoline quinone

摘要:

吡咯喹啉醌 (PQQ) 是一种细菌脱氢酶的辅酶，具有促进机体生长、调节机体自由基水平等功能，应用于食品、医药等领域。由于化学合成法成本较高，微生物发酵法生产PQQ受到关注。目前，发酵法生产PQQ产量较低，限制了其工业应用。然而，由于对PQQ菌株的合成与调控机制尚缺乏深入理解，以及对野生型菌株缺乏必要的基因工程改造手段，目前采用代谢工程强化PQQ合成菌株还缺乏相关基础。因此，本研究以扭脱甲基杆菌Methylobacterium extorquens I-F2为研究对象，整合常压室温等离子体诱变、流式细胞术分选和高通量筛选策略，对样品制备和流式分选过程进行优化，最终筛选出一株PQQ高产突变菌株1-C6，PQQ产量比出发菌株I-F2提高98.02%。本文所述的流式细胞术结合高通量筛选方法能简单、快速地获得高产突变菌株，相比于基因工程改造和传统筛选方法，具有提升效果明显且易于实施等优势。

Abstract:

Pyrroloquinoline quinone (PQQ) is a bacterial dehydrogenase coenzyme. PQQ can promote body growth and regulate the function of free radical level of the body. It could be applied in food, medicine and other fields. Due to the extremely high cost of chemical synthesis, the production of PQQ by microbial fermentation attracted more and more attention. At present, the production titer of PQQ by fermentation method is too low to achieve industrial application. Due to the lack of a thorough understanding of the PQQ biosynthesis and its regulation mechanisms, and the lack of necessary genetic engineering modification methods for wild type strains, metabolic engineering of microorganisms to enhance PQQ production still lacks essential requirements. In this study, a PQQ-producing bacterium, Methylobacterium extorquens I-F2, was employed as a model strain. By integration of Atmospheric and room temperature plasma (ARTP) mutagenesis, flow cytometry sorting and high-throughput screening strategies, optimization of sample preparation and flow sorting process, a high-titer PQQ mutant strain was obtained. The titer of PQQ was increased by 98.02% compared with that of M. extorqunens I-F2. The process described here showed that the combination of the flow cytometry with high-throughput screening method can be used to obtain high-titer mutants more simply and rapidly, compared with genetic engineering and traditional screening methods.