一株新的产吡咯喹啉醌甲基营养菌全基因组测序和比较基因组学分析

【背景】由于甲基营养菌被发现的时间较短，而且可以生产吡咯喹啉醌(pyrroloquinoline quinone，PQQ)的甲基杆菌属细菌只有少数菌株的全基因组序列被公布，增加了该类细菌基因组学和生物代谢途径研究的难度。【目的】将本实验室筛选的PQQ生产菌经多种诱变方式处理，用于提高PQQ的发酵产量。对高产突变菌株进行全基因组解析，以探究甲基杆菌PQQ合成的分子机制，为后续分子育种提供序列背景信息。【方法】将野生型PQQ生产菌株进行紫外诱变、亚硝基胍诱变、甲基磺酸乙酯诱变、硫酸二乙酯诱变和紫外-氯化锂复合诱变。将突变菌株利用PromethION三代测序平台和MGISEQ-2000二代测序平台测序，然后进行组装和功能注释。组装得到的全基因组序列与模式菌株扭脱甲基杆菌AM1 (Methylobacterium extorquens AM1)进行比较基因组学分析。【结果】经11轮诱变获得一株突变菌株NI91，其PQQ产量为19.49 mg/L，相较原始菌株提高44.91%。突变菌株NI91的基因组由一个5 409 262 bp的染色体组成，共编码4 957个蛋白，与模式菌株M. extorquens AM1比较发现其PQQ合成过程中剪切加工相关的基因pqqF和pqqG缺失，但首次在甲基营养菌中发现与基因pqqF具有相似功能的基因pqqL，且基因pqqC/D的序列存在较大差异。【结论】为甲基营养类细菌甲基杆菌的功能基因组学研究及PQQ合成机理研究提供了基础数据支持，NI91与模式菌株M. extorquens AM1的比较基因组学分析为揭示PQQ合成的不同机理提供了分子基础。

Whole genome sequencing and comparative genomics analysis of a new pyrroloquinoline quinone-producing methylotroph

Background] Due to the short time of discovery of methylotrophs, only a few genomes of methylobacterium strains that can produce PQQ were sequenced. It increases the difficulty of study the genomics and biological metabolic pathways of methylobacterium. Objective] The PQQ-producing bacteria was screened and treated with various mutagenesis methods to improve the yield of PQQ. Whole genome analysis of high-yield mutant strains was performed to provide sequence background information for studying the molecular mechanism of PQQ synthesis and subsequent molecular breeding of methylobacterium. Methods] The wild-type PQQ production strains were subjected to ultraviolet rays mutagenesis, nitroso-guanidin mutagenesis, ethylmethylsulfone mutagenesis, diethyl sulfate mutagenesis, and ultraviolet rays-lithium chloride compound mutagenesis. The whole genome sequence of the mutant strain obtained by mutagenesis was sequenced using the PromethION sequencing platform and the MGISEQ-2000 sequencing platform. The assembled whole genome sequence was compared with the model strain Methylobacterium extorquens AM1. Results] A mutant strain NI91 was obtained after 11 rounds of mutagenesis with PQQ yield 19.49 mg/L, which was 44.91% higher than the original strain. The genome of the mutant strain NI91 consists of a chromosome of 5 409 262 bp, encoding 4 957 proteins. Compared with the model strain M. extorquens AM1, it was found that the pqqF and pqqG genes that relate to shear processing during PQQ biosynthesis were deleted. Meanwhile, the pqqL was first discovered in methylotrophic bacteria which has a similar function to the pqqF, and the sequences of pqqC/D between the two strains were quite different. Conclusion] This study provides basic data for functional genomics research of the methylotrophic bacterium and the study of PQQ synthesis mechanism. Comparative genomics between NI91 and the model strain M. extorquens AM1 provides a molecular basis for revealing different mechanisms of PQQ synthesis.