利用甘油脱水酶基因构建产3-羟基丙醛工程菌及其表达比较

Construction of Recombinant Strains for 3-hydroxypropionaldehyde Biosynthesis and Comparison of Glycerol Dehydratase Gene Expression in Hosts

3-羟基丙醛(3-HPA)是一种重要的化工产品,可由甘油经甘油脱水酶作用后生成.为获得产3-HPA基因工程菌,在已构建含甘油脱水酶基因及其激活因子大亚基质粒pEtac-dhaB-gdrA的基础上,构建了包含小亚基gdrB激活因子的重组质粒pEtac-dhaB-gdrA-gdrB.利用大肠杆菌通用tac启动子将该质粒在不同Escherichia coli BL21、DH5a及JM109中进行表达.阳性转化子经IPTG诱导后,提取总RNA,以cDNA为模板进行RT-PCR发现,目标基因在不同宿主都能较好转录.SDS-PAGE、酶活测定和3-HPA浓度测定结果表明,目标蛋白表达存在差异;酶活分别为4.7(±0.44)、3.5(±0.95)、8.1(±0.66)U/mg;发酵液中3-HPA的含量分别为0.012(±0.0044)、0.014(±0.003)、0.375(±0.018)g L-1,重组E.coli JM109/pEtac-dhaB-gdrA-gdrB具有较好的甘油脱水酶基因表达和产3-HPA性能.该基因工程菌与克雷伯氏菌(Klebsiella pneumoniae)相比,发酵副产物明显较少,有利于后期提取,为生产3-HPA提供了一条新思路.

3-hydroxypropionaldehyde （3-HPA） is an important chemical product, which could be transformed from glycerol by glycerol dehydratase. In order to acquire an engineering strain to produce 3-HPA, gdrB encoding glycerol dehydratase reactivating factor small-subunit was amplified and employed to construct the plasmid pEtac-dhaB-gdrA-gdrB on the basis of the plasmid pEtac-dhaB-gdrA, which contained glycerol dehydratase reactivating factor large-subunit. The common Escherichia coli tac promoter was used for expression of pEtac-dhaB-gdrA-gdrB in different hosts E. coli BL21, DH5a, JM109. The positive clones were induced with IPTG, and the complete cDNA was obtained by RT-PCR using the total RNA as template. It was found that the gene of glycerol dehydratase could be transcribed well in all 3 hosts. The results of SDS- PAGE, enzyme analysis and the fermentation of 3-HPA indicated that the expression of glycerol dehydratase in different hosts were not the same. The specific enzyme activities of recombinant strains with different hosts （E. coli BL21, DH5a, JM109） were 4.7（＋0.44）, 3.5（~0.95） and 8.1（~0.66） U/mg, respectively, and the 3-HPA productions by shake flask fermentation were 0.012（~0.0044）, 0.014（~0.003）and 0.375（~0.018） g L-1, respectively. The recombinant E. coli JMlO9/pEtac-dhaB-gdrA-gdrB had the best ability in glycerol dehydratase expression and 3-HPA production. Compared with Klebsiella pneumoniae, the by- products of recombinant E. coli were much less, which was good for separation and purification. This study provides a new route for the biosynthesis of 3-HPA. Fig 4, Tab 3, Ref 16