puuC基因过表达对肺炎克雷伯氏菌发酵甘油生产3-HP的影响  

Effect of overexpressed puuC on 3-HP production by fermented glycerol of Klebsiella pneumoniae

作　　者：涂绣军 田甜 葛菁萍[1,2] TU Xiujun;TIAN Tian;GE Jingping(Engineering Research Center of Agricultural Microbiology Technology,Ministry of Education/Heilongjiang Provincial Key Laboratory of Plant Genetic Engineering and Biological Fermentation Engineering for Cold Region/Key Laboratory of Microbiology,College of Heilongjiang Province,School of Life Sciences,Heilongjiang University,Harbin 150080,China;Hebei Key Laboratory of Agroecological Safety,Hebei University of Environmental Engineering,Qinhuangdao 066102,China)

机构地区：[1]黑龙江大学生命科学学院农业微生物技术教育部工程研究中心/黑龙江省寒区植物基因与生物发酵重点实验室/黑龙江省普通高校微生物重点实验室,哈尔滨150080 [2]河北环境工程学院河北省农业生态安全重点实验室,秦皇岛066102

出　　处：《黑龙江大学自然科学学报》2024年第4期443-452,共10页Journal of Natural Science of Heilongjiang University

基　　金：国家自然科学基金面上项目(32071519);黑龙江省自然科学基金重点项目(ZD2020C008);黑龙江省生态环境厅生态环境保护科研项目(HST2022TR004)。

摘　　要：为获得高产3-羟基丙烯酸(3-hydroxypropionic acid,3-HP)的基因重组菌,从肺炎克雷伯氏菌(Klebsiella pneumoniae,K.pneumoniae)HD79中克隆乙醛脱氢酶基因puuC,进行同源过表达,探究其对K.pneumoniae HD79甘油还原途径中乙醛脱氢酶(Aldehyde dehydrogenase,ALDH)活性与3-HP产量的影响。结果表明,成功构建了同源过表达puuC的重组菌株K.pneumoniae HD79-T,过表达puuC基因后提高了K.pneumoniae HD79的3-HP生产能力。K.pneumoniae HD79-T的3-HP最高产量为(2.05±0.03)g·L^(-1),比K.pneumoniae HD79((1.47±0.02)g·L^(-1))提高了39.46%。K.pneumoniae HD79-T中puuC基因、甘油脱水酶基因(dhaB)和1,3-丙二醇氧化还原酶基因(dhaT)的相对表达量较K.pneumoniae HD79分别提高94.35、1.72和1.35倍。K.pneumoniae HD79-T中ALDH、甘油脱水酶(Glycerol dehydratase,GDHt)和1,3-丙二醇氧化还原酶(1,3-propanediol oxidoreductase,1,3-PDOR)的酶活性相比,K.pneumoniae HD79分别提高了27.40%、18.85%和5.10%。本研究构建的K.pneumoniae HD79-T提高了甘油还原途径关键酶ALDH的酶活性,是高效利用甘油发酵生产3-HP的前景菌株。To augment the 3-hydroxypropionic acid(3-HP)production capability in Klebsiella pneumoniae HD79,the aldehyde dehydrogenase gene puuC was cloned and overexpressed.This overexpression was investigated for its influence on aldehyde dehydrogenase(ALDH)activity and 3-HP production in the glycerol reduction pathway.The experiment confirms successful enhancement of a recombinant strain,K.pneumoniae HD79-T,which shows improved 3-HP production due to the overexpression of puuC.The maximum 3-HP yield from the HD79-T strain is(2.05±0.03)g·L^(-1),displaying a striking increase of 39.46%compared to the typical K.pneumoniae HD79 yield((1.47±0.02)g·L^(-1)).The relative gene expression levels in the HD79-T strain,including puuC,the glycerol dehydrase gene dhaB,and the 1,3-propanediol oxidoreductase gene dhaT,amplifiy by multiples of 94.35,1.72,and 1.35 respectively.Correspondingly,enzyme activities of ALDH,glycerol dehydrase(GDHt),and 1,3-propanediol oxidoreductase(1,3-PDOR)rise by 27.40%,18.85%,and 5.10%respectively.Thus,the HD79-T strain develops here substantially enhanced ALDH enzyme activity,rendering it a potential strain for effective glycerol fermentation to yield 3-HP.

关 键 词：肺炎克雷伯氏菌 3-羟基丙酸 乙醛脱氢酶 过表达 

分 类 号：Q78[生物学—分子生物学]