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

作　　者：王凤寰[1] 杨建国[2] 王晓楠[2] 谭天伟[2] 

机构地区：[1]北京工商大学食品学院,北京100048 [2]北京化工大学生命科学与技术学院,北京100029

出　　处：《微生物学通报》2011年第11期1611-1617,共7页Microbiology China

基　　金：国家自然科学基金项目(No.20906003);中国博士后基金项目(No.20080440302)

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。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 recombinant 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,respectively,which were higher than those of wild-type K.pneumoniae.

关 键 词：3-羟基丙酸 1 3-丙二醇 乙醛脱氢酶 重组肺炎克雷伯杆菌 

分 类 号：TQ923[轻工技术与工程—发酵工程]