利用甘油发酵耦联生产3-羟基丙酸及1,3-丙二醇重组菌的构建及筛选

Construction and screening of novel recombinant Klebsiella pneumoniae capable of co-producing 3-hydroxypropionic acid and 1,3-propanediol from glycerol

摘要:

在肺炎克雷伯杆菌(Klebsiella pneumoniae)代谢甘油生产1,3-丙二醇(1,3-PD)的过程中, 为了减少有毒中间产物3-羟基丙醛(3-HPA)的积累, 可将其转化为3-羟基丙酸(3-HP), 从而实现1,3-丙二醇和3-羟基丙酸的联产。克隆来自于酿酒酵母的NAD+依赖型的乙醛脱氢酶(ALDH)的基因aldh4, 构建了表达载体pKP-aldh, 转化K. pneumoniae, 得到了有效表达乙醛脱氢酶的重组肺炎克雷伯杆菌(K. pneumoniae A+)。在此基础上, 使用紫外诱变联合菌种驯化的方法对K. pneumoniae A+进行筛选, 获得了可耐受较高3-HP浓度(≥35 g/L)的重组肺炎克雷伯杆菌K. pneumoniae A+5-3。发酵实验结果表明, K. pneumoniae A+5-3可将3-HPA转化为3-HP, 能够同时利用甘油耦联生产3-HP和1,3-PD, 产量分别达到5.0 g/L和74.5 g/L。

Abstract:

3-HPA was a toxic intermediate metabolite in the 1,3-PD pathway of Klebsiella pneumoniae and its accumulation could inhibit cell growth and glycerol utilization. In order to reduce the 3-HPA accumulation and produce 3-HP simultaneously, the aldh gene from a Saccharomyces cerevisiae en-coding NAD+-dependent acetaldehyde dehydrogenase (ALDH) was cloned and the expression vector pKP-aldh harboring aldh gene was constructed and transformed into K. pneumoniae. The resulting re-combinant strain K. pneumoniae A+ could express the ALDH effectively. The bacteria was mutated by UV lights and screened in 3-HP tolerance plates to yield K. pneumoniae A+5-3. In the fed-batch fermenta-tion of K. pneumoniae A+5-3, the final 3-HP and 1,3-PD concentration reached 5.0 g/L and 74.5 g/L, re-spectively, which were higher than those of wild-type K. pneumoniae.