Since the chemical synthesis of vitamin B12 requires more than 70 steps, the production of vitamin B12 has been achieved by microorganism fermentation with additional brief chemical modifications. In an effort to increase the productivity of vitamin B12, we tried to express 10 genes belonging to the hem, cob and cbi gene families involved in the synthesis of vitamin B12 in Propionibacterium freudenreichii, which is a known producer of vitamin B12. In a recombinant P. freudenreichii clone that harbored the expression vector containing a cobA, cbiLF, or cbiEGH, we obtained an increase in vitamin B12 production of 1.7-, 1.9-, and 1.5-fold higher, respectively, than that in the microorganism without any cloned genes in the expression vector pPK705. The cobU and cobS genes caused a slight increase in the production of vitamin B12. Furthermore, we achieved multigene expression in P. freudenreichii. In a recombinant P. freudenreichii clone that harbored an exogenous gene, hemA, from Rhodobacter sphaeroides and endogenous hemB and cobA genes, we successfully achieved the production of about 1.7 mg/l vitamin B12, 2.2-fold higher than that produced by P. freudenreichii harboring pPK705.
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