外源添加VB_(12)对肺炎克雷伯氏菌代谢3-羟基丙酸的影响  

Effect on Metabolism of 3-hydroxypropionic Acid by Klebsiella pneumoniae:Exogenous Addition of VB_(12)

作　　者：敖国旭 田甜 葛菁萍[1,2] 平文祥 AO Guoxu;TIAN Tian;GE Jingping;PING Wenxiang(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 Molecular Biology,College of Heilongjiang Province/School of Life Sciences,Heilongjiang University,Harbin 150080;Hebei University of Environmental Engineering,Hebei Key Laboratory of Agroecological Safety,Qinhuangdao,Hebei 066102)

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,黑龙江省普通高校分子生物学重点实验室,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,河北秦皇岛066102

出　　处：《中国农学通报》2024年第12期85-93,共9页Chinese Agricultural Science Bulletin

基　　金：黑龙江省自然科学基金重点项目“乙酸代谢与副干酪乳杆菌群体感应互作探究及对细菌素产生的影响”(ZD2020C008);黑龙江省农业废弃物综合利用体系岗位专家项目“黑龙江省现代农业产业技术协同创新推广体系作物秸秆低温降解菌株的研制”;黑龙江省生态环境厅2022年度生态环境保护科研项目“人工重组细菌菌群对黑土磷障碍的解控机制”(HST2022TR004);龙江省高校基本科研业务费“群体感应介导下重组根圈细菌菌群对土壤磷养分循环的调控机制”(2023-KYYWF-1448)。

摘　　要：为了探究外源添加维生素B12(Vitamin B12,VB_(12))对Klebsiella pneumoniae HD79和K.pneumoniae HD79-T利用甘油还原途径生产3-羟基丙酸(3-hydroxypropionic acid,3-HP)的影响以及摸索VB_(12)对K.pneumoniae HD79和K.pneumoniae HD79-T的阈值上限,将不同浓度(0.01、0.02、0.03、0.04、0.05 g/L)的VB_(12)添加到K.pneumoniae HD79及K.pneumoniae HD79-T的发酵培养基中,利用HPLC检测其底物消耗及产物产生情况、qRT-PCR检测还原途径相关基因的mRNA表达情况以及酶联免疫试剂盒检测代谢相关酶活性。结果表明,VB_(12)对K.pneumoniae HD79和K.pneumoniae HD79-T的阈值为0.01 g/L和0.03 g/L。与未添加VB_(12)相比,菌株K.pneumoniae HD79和K.pneumoniae HD79-T的3-HP产量分别提高了24.39%,8.86%;醛脱氢酶基因puuC表达量分别提高了2.49倍和1.68倍;ALDH、GDHt和PDOR的酶活力分别提高了50.24%、40.36%和18.29%,及30.49%、37.84%和13.56%。说明通过外源添加辅酶因子VB_(12)对肺炎克雷伯氏菌高产3-HP是可行策略。To investigate the effect of exogenous addition of Vitamin B12(VB_(12))on the production of 3-hydroxypropionic acid(3-HP)through glycerol reduction pathway in Klebsiella pneumoniae HD79 and K.pneumoniae HD79-T,and to explore the threshold upper limit of VB_(12) on the above strains,different concentrations(0.01,0.02,0.03,0.04,and 0.05 g/L)of VB_(12) were added to the fermentation medium of K.pneumoniae HD79 and K.pneumoniae HD79-T.The consumption of substrates and production of products were detected by HPLC,mRNA expression of genes related to the reduction pathway was detected by qRT-PCR,and enzyme activities related to metabolism were detected by enzyme-linked immunosorbent assay(ELISA).The results showed that the thresholds of VB_(12) for K.pneumoniae HD79 and K.pneumoniae HD79-T was 0.01 g/L and 0.03 g/L,respectively.Compared with the absence of VB_(12),the productions of 3-HP of K.pneumoniae HD79 and K.pneumoniae HD79-T were increased by 24.39%and 8.86%,respectively;the expression levels of aldehyde dehydrogenase gene puuC were increased by 2.49-fold and 1.68-fold,respectively;the enzyme activities of ALDH,GDHt,and PDOR increased by 50.24%,40.36%,and 18.29%in K.pneumoniaeHD79,and by 30.49%,37.84%,and 13.56%in K.pneumoniae HD79-T,respectively.These results indicated that exogenous addition of coenzyme factor VB_(12) was a feasible strategy for high production of 3-HP in Klebsiella pneumoniae.

关 键 词：维生素B_(12) 肺炎克雷伯氏菌 3-羟基丙酸 醛脱氢酶基因puuC 甘油脱水酶 

分 类 号：S182[农业科学—农业基础科学]