P. Kiatpapan et Y. Murooka, Construction of an expression vector for propionibacteria and its use in production of 5-aminolevulinic acid by Propionibacterium freudenreichii, APPL MICR B, 56(1-2), 2001, pp. 144-149
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.