【摘要】 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.更多还原

【Abstract】 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 clones1were 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 SDSPAGE,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 JM109/pEtac-dhaB-gdrA-gdrB had the best ability in glycerol dehydratase expression and 3-HPA production.Compared with Klebsiella pneumoniae,the byproducts 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.更多还原