Isolation Of Coenzyme B₁₂-Producing Microorganisms And Cloning, Expression And Application Of Coenzyme B₁₂ Biosynthetic Genes

In the glycerol reduction process, glycerol dehydratase (GDHt) required coenzyme B12 (adenosylcobalamin, AdoCbl). GDHt catalyzed the free radical mediated conversion in a rate-limiting reation. In the case of glycerol and some other substrates, GDHt is subject to suicide inactivation, which involved irreversible cleavage of the Co-C bond of coenzyme B12. The requirement of high-cost vitamine B12 (cyanocobalamine, CNCbl) supplementation was one of the main limitations in the development of economically feasible 1,3-propanediol (1,3-PDO) production. Attempts to overcome the problem with coenzyme B12 requirement of GDHt had been undertaken. In this study I enhanced the biosynthesis and conversion of coenzyme B12 to research the activity of GDHt. All the work led to a good result.In this paper, 67 strains were isolated from 12 soil samples. I screened of one strain named A24 with the ability of biosynthesis coenzyme B12 via culturing on selective medium using acelamide as the sole source of nitrogen. The strain A24 was classified into Bacillus magaterium according to the morphology, physiological and biochemical characteristics and the 16S rRNA sequence analysis.The key gene cobA of uroporphyrinogenⅢmethyltransferase involved in coenzyme B12 biosynthesis was cloned from B. megaterium which was isolated in this study. Then I cloned the gene hemA of glutamyl-tRNA reductase from Klebsiella pneumonia. After sequencing, I constructed the tandem expression vector pUC18-tac-cobA-tac-hemA.Transforming pUC18-tac-cobA-tac-hemA into Escherichia coli JM109, I got recombinant E. coli JM109(pUC18-tac-cobA-tac-hemA). A lot of porphyrin compounds were tested from the fermentation broth after induced of IPTG, shown that the key genes cobA and hemA were right expressed.Expression vector pUC18-tac-cobA-tac-hemA was transformed into E. coli JM109 with pEtac-dhaB-tac-yqhD including gene dhaB of glycerol dehydratase and gene yqhD of 1,3-propanediol oxidoreductase, got recombinant strain E. coli JM109(pUC18-tac-cobA-tac- hemA/pEtac-dhaB-tac-yqhD). After the optimization of fermentation medium in shake flask, the most suitable vitamine B12 concentration of E. coli JM109(pEtac-dhaB-tac-yqhD) was 0.008 g/L, compared with that of E. coli JM109(pUC18-tac-cobA-tac-hemA/pEtac-dhaB- tac-yqhD) being 0.004 g/L considered the yield of 1,3-propanediol. The result indicated that expression the key genes cobA and hemA decreased the dependence of adding vitamine B12.For further studying on the course of converting inactive vitamine B12 to active coenzyme B12, I cloned ATP:cob(I)alamin adenosyltransferase (ACA) gene btuR and dehydrogenase gene yciK from K. pneumonia, and constructed a dual-promoter expression plasmid pUC18-tac-btuR-tac-yciK including gene btuR and yciK, finally, transferred the plasmid into E. coli JM109 getting recombinant E. coli JM109(pUC18-tac-btuR-tac-yciK). The result of RT-PCR experiment and SDS-PACE analysis of whole proteins of the recombinant showing that the genes btuR and yciK were right expressed.Using an improved assay procedure, the overexpressed proteins of recombinant E. coli JM109(pUC18-tac-btuR-tac-yciK) had the power to convert hydroxocobalamin to deoxyadenosylcobalamin, shown that the proteins had bioactivity. The OD600 value of recombinant E. coli JM109(pUC18-tac-btuR-tac-yciK) was 1.4 times higher than that of E. coli JM109(pUC18) after cultered on the medium using acelamide as the sole source of nitrogen. This was because the expression of btuR and yciK in the recombinant promoted the course of converting inactive vitamine B12 to active coenzyme B12. This study discussed a new way for the research of glycerol dehydratase reactivation in the 1,3-propanediol production.