紫外突变型盐单胞菌株的基因突变位点与四氢嘧啶高产的分子变异机制  

Mutation sites and high ectoine production mechanism of a Halomonas mutant induced by ultraviolet radiation

作　　者：薛彬娟 韩睿[2] 乔丽娟 李永臻 邢江娃 王嵘 沈国平 朱德锐 XUE Binjuan;HAN Rui;QIAO Lijuan;LI Yongzhen;XING Jiangwa;WANG Rong;SHEN Guoping;ZHU Derui(Department of Basic Medical Sciences,Medical College,Qinghai University,Xining 810016,Qinghai,China;Key Laboratory of Vegetable Genetics and Physiology,Academy of Agriculture and Forestry Sciences,Qinghai University,Xining 810016,Qinghai,China)

机构地区：[1]青海大学医学院,基础医学研究中心,青海西宁810016 [2]青海大学农林科学院,蔬菜遗传与生理重点实验室,青海西宁810016

出　　处：《微生物学报》2024年第12期4902-4917,共16页Acta Microbiologica Sinica

基　　金：国家自然科学基金(32260019);青海中央引导地方科技发展资金项目(2024ZY015)。

摘　　要：利用紫外诱变获得的一株高产四氢嘧啶(ectoine)的突变型坎帕尼亚盐单胞菌(Halomonas campaniensis) G9-72,其突变位点、分子变异和高产四氢嘧啶的机制未知。【目的】探讨野生型菌株XH26与突变菌株G9-72的突变位点与分子遗传变异机制,明确四氢嘧啶积聚量暴发的可能原因。【方法】采用PacBio Sequel II平台进行全基因组测序,分析突变菌株的突变基因位点,结合氨基酸代谢通路分析突变基因与四氢嘧啶合成代谢的关联性,并进行RT-PCR验证。【结果】全基因组测序结果显示野生菌株XH26的基因组4.11 Mb,编码基因3 927个。突变菌株G9-72的基因组存在35个突变位点,包括18个单核苷酸多态性突变、14个插入突变和3个缺失突变。代谢通路的关联分析显示:突变基因argF、coaBC和livH分别编码鸟氨酸氨甲酰基转移酶(NCBI数据库蛋白ArgF相似度100.00%)、磷酸泛酰半胱氨酸脱羧酶(蛋白CoaBC相似度99.28%)和支链氨基酸ABC转运体渗透酶(与蛋白LivH相似度96.27%),分别参与延胡索酸、柠檬酸的合成以及增加支链氨基酸的吸收转运。上游代谢物的流量增加可能是突变菌株四氢嘧啶积聚量暴发的关键原因。RT-PCR验证四氢嘧啶代谢通路相关的20个基因,转录表达水平与预期分析相一致。【结论】突变基因argF、coaBC和livH的过表达增强四氢嘧啶合成的代谢流,与突变菌株四氢嘧啶积聚量的暴发有关,此为后续突变菌株酶分子的反应机制研究和发酵生产提供参考依据。A mutant(G9-72)of Halomonas campaniensis exhibiting high ectoine production was obtained by ultraviolet(UV)mutagenesis.The mutation sites,molecular variations,and high ectoine production mechanism of this mutant remain unknown.[Objective]To investigate the mutation sites and genetic variations of G9-72 compared with the wild type strain XH26 and identify the potential causes of ectoine accumulation outbreak.[Methods]PacBio Sequel II was used for whole-genome sequencing,and the mutation sites in the genome of the mutant were identified based on the sequencing results.The amino acid metabolic pathways were analyzed to reveal the association between mutated genes and ectoine synthesis,and the results were verified by RT-PCR.[Results]The genome of strain XH26 was 4.11 Mb,encoding 3927 genes.Compared with strain XH26,G9-72 showed 35 mutation sites,including 18 single nucleotide polymorphism mutations,14 insertion mutations,and 3 deletion mutations.The mutated genes argF,coaBC,and livH,which encoded ornithine transcarbamylase(100.00%similarity with ArgF proteins in NCBI database),phosphopantothenoylcysteine decarboxylase(99.28%similarity with CoaBC proteins in NCBI database),and branched-chain amino acid ABC transporter permease(96.27%similarity with LivH proteins in NCBI database),were implicated in the synthesis of fumaric acid,citric acid and the absorption and transport of branched-chain amino acids,respectively.The increased flow of upstream metabolites may be the key reason for the sharply increased accumulation of ectoine in the mutant.RT-PCR verified 20 genes related to the ectoine metabolic pathway,and the transcriptional expression levels were consistent with the expected analysis.[Conclusion]The overexpression of genes argF,coaBC,and livH enhanced the anabolic flow of ectoine,which contributed to a significant increase in ectoine accumulation in the mutant.This finding provides a reference point for subsequent studies on the reaction mechanisms of enzymes in the mutant and the fermentation production.

关 键 词：盐单胞菌 紫外诱变 突变基因 比较基因组学 四氢嘧啶 

分 类 号：Q78[生物学—分子生物学] TQ920.6[轻工技术与工程—发酵工程]