摘要
为了探究外源添加维生素 B12(Vitamin B12,VB12)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VB12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VB12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,VB12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VB12相比,菌株K.pneumoniaeHD79和K.pneumoniaeHD79-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VB12对肺炎克雷伯氏菌高产3-HP是可行策略.
Abstract
To investigate the effect of exogenous addition of Vitamin B12(VB112)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 VB12 on the above strains,different concentrations(0.01,0.02,0.03,0.04,and 0.05 g/L)of VB12 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 VB12 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 VB12,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 VB12 was a feasible strategy for high production of 3-HP in Klebsiella pneumoniae.
基金项目
黑龙江省自然科学基金重点项目(ZD2020C008)
黑龙江省农业废弃物综合利用体系岗位专家项目()
黑龙江省生态环境厅2022年度生态环境保护科研项目(HST2022TR004)
龙江省高校基本科研业务费(2023-KYYWF-1448)
国家科技期刊平台
NSTL
万方数据