Research Of The Genes Encoding Key Enzymes And Reactivating Factor In The 3-hydroxy-propionic Acid Biosynthesis Pathway In Escherichia Coli

3-hydroxypropionic acid (3-HP) is an important platform chemical from which several commodity and specialty chemicals can be generated. Biosynthetic pathways for 3-hydroxypropionic production has broad prospect of application because of its low cost, mild conditions and environmental. Along with the development of genetic engineering technology, genetically modified metabolic pathways are thus required to realize biosynthetic pathways for 3-hydroxypropionic production. At present the research about biological method production 3-hydroxyl propionic acid is still in initial stage. The topic of this study is to research the genes encoding key enzymes and reactivating factor in the 3-hydroxy propionic acid biosynthesis pathway use genetic engineering.The structure gene aldH from Saccharomyces cerevisiae W303 encoding acetaldehyde dehydrogenase and gene dhaB from Klebsiella pneumoniae were amplified using PCR method to construct the plasmid pEtac-dhaB-tac-aldH. The recombinant E. coli/pEtac-dhaB -tac-aldH was obtained by transformation of pEtac-dhaB-tac-aldH. The results from SDS-PAGE analysis show that the recombinant products were consistent with the molecular weight predicted from genes sequence.The key enzyme activity, recombinant plasmid stability and yield of 3-HP were contrasted between E.coli JM109/pEtac-dhaB, pUC-tac-aldH and E. coli JM109/pEtac- dhaB-tac-aldH. Comparied with E.coli JM109/pEtac-dhaB, pUC-tac-aldH, the glycerol dehydratase activity and aldehyde dehydrogenase activity in recombinant E. coli JM109/ pEtac-dhaB-tac-aldH were increased 41.5% and 3.5%. The plasmid stability in E. coli JM109 /pEtac-dhaB-tac-aldH obviously improved 29.4%. The novel recombinant E. coli harboring pEtac-dhaB-tac-aldH could transform glycerol to 3-hydroxypropionic acid under aerobic condition. Its yield of 3-HP was 0.58 g/L, which was 52.6% higher than the recombinant E. coli JM109/pEtac-dhaB, pUC-tac-aldH. Research results show that Co-expression of the key enzyme genes in favor of the recombinant strain E. coli synthesize 3-HP.Glycerol dehydratase was the critical enzyme in the metabolic pathway of converting glycerol to 3-hydroxypropionic acid. It was subject to suicide inactivation by the natural substrate glycerol during catalysis. The inactivated glycerol dehydratase will influence the production of 3-hydroxypropionic acid. Therefore, the gdrA, gdrB gene encoding glycerol dehydratase-reactivating factor from Klebsiella pneumoniae were amplified using PCR method to construct the recombinant strain E. coli JM109/pEtac-dhaB-gdrAB-tac-aldH. The effects of gdrA, gdrB expression on 3-hydroxypropionic acid production from glycerol were investigated. When cultivated aerobically on medium, the recombinant E. coli JM109/pEtac-dhaB-gdrAB-tac-aldH produced 3-HP at amaximum of 2.51 g/L which was increased by 3.3-fold compared with E. coli JM109/pEtac-dhaB-tac-aldH. The recombinant strain E. coli JM109/pEtac-dhaB-gdrAB-tac-aldH yield of 3-HP was increasd 60% after optimize the fermentation conditions of the recombinant strain. The results show that the gdrA, gdrB expression could reactive the activation of the inactivated glycerol dehydratase and increase the yield of 3-HP.