Members of the genus Propionibacterium are widely used in the production of vitamin B,,, tetrapyrrole compounds, and propionic acid as well as in probiotic and cheese industries. Shuttle vectors were developed in propionibacteria using replicons from endogenous plasmids in Propi-onibacterium and Escherichia coli and an appropriate selection marker. The efficient transformation was achieved using the shuttle vector prepared from Propionibactertium freudenreichii to overcome the high restriction modification system in propionibacteria. Expression vectors with native promoters for use in propionibacteria were also developed. Using this system, cholesterol oxidase, which is used as a diagnostic enzyme, was produced in P freudenreichii. Genes involved in 5-aminolevulinic acid (ALA) and vitamin 13, biosynthesis in propionibacteria were isolated. ALA in propionibacteria could be synthesized via both the C4 pathway (condensation of glycine and succinyl CoA) and the C5 pathway (from glutamate). The hemA gene encoding ALA synthase from Rhodobacter spheroides, was overexpressed and ALA accumulated in P.freudenreichii. Thus, the genetic manipulation systems in propionibacteria will facilitate genetic studies of probioties and the vitamin B-12 biosynthetic pathway.