Production of vitamin B₁₂ in genetically engineered Propionibacterium freudenreichii

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.