Li Hongyue, Zeng Weizhu, Zhou Jingwen
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, Jiangsu, China.
School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2018 May 25;34(5):794-802. doi: 10.13345/j.cjb.170440.
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.
吡咯喹啉醌（PQQ）是一种细菌脱氢酶辅酶。PQQ能促进机体生长并调节机体自由基水平的功能。它可应用于食品、医药等领域。由于化学合成成本极高，微生物发酵生产PQQ越来越受到关注。目前，发酵法生产PQQ的产量过低，无法实现工业化应用。由于对PQQ生物合成及其调控机制缺乏深入了解，且缺乏对野生型菌株必要的基因工程改造方法，通过微生物代谢工程提高PQQ产量仍缺乏必要条件。在本研究中，以产PQQ的细菌——扭脱甲基杆菌I-F2作为模式菌株。通过整合常压室温等离子体（ARTP）诱变、流式细胞术分选和高通量筛选策略，优化样品制备和流式分选过程，获得了一株高产PQQ突变菌株。与扭脱甲基杆菌I-F2相比，PQQ产量提高了98.02%。本文所述过程表明，与基因工程和传统筛选方法相比，流式细胞术与高通量筛选方法相结合可更简单、快速地获得高产突变体。
