Reaearch On Construction And Expression Of Glycerol Dehydratase Recombinant Strains For3-Hydroxypropionaldehyde Biosynthesis

Glycerol dehydratase is the key enzyme in the metabolic pathway of glycerol, and3-hydroxypropionaldehyde （3-HPA） is transformed from glycerol by glycerol dehydratase.3-HPA is an important chemical product, and it is the precursor of acrolein, acrylic acid and1,3-propanediol.3-HPA is also an effective antimicrobial, which can be used for foodpreservative and medicate treatment. Therefore, the construction of glycerol dehydrataseengineering bacteria is very important, which is good for the biosynthetic of3-HPA and thedevelopment of other high value product from glycerol. However, the report on constructionof glycerol dehydratase gene engineering bacteria for3-HPA biosynthesis is rare, and there isno research on the transcription level of glycerol dehydratase in different recombinants. Inorder to find out the expression property of glycerol dehydratase, the expression, transcriptionand fermentation property in recombinants of different hosts and promoters were studied,which provide theoretical foundation for high expression of glycerol dehydratase.In order to acquire glycerol dehydratase engineering bacteria of different hosts andpromoters, gdrB encoding glycerol dehydratase reactivating factor small-subunit wasamplified and employed to construct the plasmid pEtac-dhaB-gdrA-gdrB on the basis of theplasmid pEtac-dhaB-gdrA, which contained glycerol dehydratase reactivating factorlarge-subunit. The pET28a-dhaB-gdrA-gdrB and pUC-tac-dhaB-gdrA-gdrB wereconstructed at the same time. The plasmid pEtac-dhaB-gdrA-gdrB was transformed toEscherichia coli BL21（DE3）、DH5a、JM109respectively and constructed different hostsrecombinant Escherichia coli strains. The pET28a-dhaB-gdrA-gdrB and pUC-tac-dhaB-gdrA-gdrB were transformed to E. coli BL21and acquire different promoters recombinant E.coli BL21were obtained.Different recombinants were induced. The SDS-PAGE result and specific enzymeactivities of different recombinants were analyzed indicated that: the expression of targetprotein were not the same in different E. coli hosts. The target protein was expressed best in E.coli JM109, and the enzyme activities of glycerol dehydratase was also highest in therecombinant E. coli JM109（8.1（±0.66） U/mg）. The expression of glycerol dehydratase wasworest in E. coli DH5a. It showed that the best host of tac promoter was E. coli JM109. Theexpression of target protein were different in E. coli BL21（DE3） under different promoters, E.coli BL21/pET28a-dhaB-gdrA-gdrB was the best one, and the specific enzyme activities ofrecombinant E. coli BL21/pEtac-dhaB-gdrA-gdrB was also highest （7.3（±0.86） U/mg）), Ingeneral, results of enzyme activities in different recombinants were consistent with the resultsof SDS-PAGE. The transcription level of different recombinants were compared at the same time. It wasshowed by the ratio between the IOD of dhaB and the IOD of the internal gene16S:C_dhaB/C_16S. The C_dhaB/C_16Sof different hosts （E. coli BL21, DH5a, JM109） were1.105（±0.031）,1.456（±0.119）,1.063（±0.032） respectively, and the transcription of target genein the E. coli BL21was best. The C_dhaB/C_16S of recombinant E. coli BL21/pEtac-dhaB-gdrA-gdrB, E. coli BL21/pET28a-dhaB-gdrA-gdrB and E. coli BL21/pUC-tac-dhaB-gdrA-gdrB were1.456（±0.119）,1.770（±0.218）,1.048（±0.033） respectively. Thetranscription level of T7promoter is better than tac promoter, and the transcription level oftac promoter in different plasmid was also different.The fermentation of different recombinants was researched. the productions of3-HPA byshake flask fermentation were tested. The result indicated that the recombinant E. coliJM109/pEtac-dhaB-gdrA-gdrB has the best ability in3-HPA production. Although E. coliBL21has better abilities of glycerol dehydratase transcription and expression, it was not goodin3-HPA production. The fermentation medium and culture condition were optimized. Underthe best conditions, the production of3-HPA by recombinant E. coli JM109/pEtac-dhaB-gdrA-gdrB was0.531g/L, the production was increased2.6fold. The recombinant E.coli JM109/pEtac-dhaB-gdrA-gdrB has the ability of3-HPA production. Compared with theK. pneumoniae, the incubation period was shorten by30h, and the by-products were muchless, which is good for separation and purification, and provide a new way for thebiosynthesis of3-HPA.