Construction of an expression vector for propionibacteria and its use in production of 5-aminolevulinic acid by Propionibacterium freudenreichii

Several promoters from Propionibacterium freudenreichii subsp. shermanii we re isolated using a promoter probe vector, pCVE1, containing the Streptomyc es cholesterol oxidase gene (choA) as a reporter gene. Three of four promot ers isolated exhibiting a strong, activity in Escherichia coli also express ed a strong activity in P freudenreichii subsp. shermanii IFO12426. Using t wo promoters with a strong activity and a previously constructed shuttle ve ctor, pPK705, shuttling between E. coli and Propionibacterium, we construct ed expression vectors for propionibacteria. To overproduce 5-aminolevulinic acid (ALA), which is the first inter-mediate in the synthesis of porphyrin s, the ALA synthase gene (hemA) from Rhodobacter sphaeroides was recombined with the expression vectors. The activity of ALA synthase in the recombina nt R freudenreichii subsp. shermanii increased about 70-fold that in the st rain without a vector. The recombinant Propionibacterium produced ALA at a maximum concentration of 8.6 mM in the absence of levulinic acid, an inhibi tor of ALA dehydratase, with 1% glucose as a carbon source. The recombinant R freudenreichii accumulated 18.8 mmol/g cells ALA in the presence of I mM levulinic acid and 30 mM glycine. The construction of an efficient express ion vector will facilitate genetic studies of a vitamin B-12 producer, Prop ionibacterium.