OVERPRODUCTION OF MANNITOL DEHYDROGENASE IN RHODOBACTER-SPHAEROIDES

Mannitol dehydrogenase (MDH) from Rhodobacter sphaeroides Si4 was over produced by constructing a strain that overexpresses the MDH gene and by producing high cell concentrations via fed-batch cultivation in a b ioreactor. With the gene of mannitol dehydrogenase (mtlK) cloned into the expression vector pKK223-3 expression of MDH in Escherichia cell w as obtained, but the specific enzyme activity was lower than in R. sph aeroides Si4. In order to overexpress mtlK in R. sphaeroides, plasmid pAK82 was constructed by cloning a DNA fragment carrying mtlK into the broad-host-range expression vector pRK415. When pAK82 was introduced into R. sphaeroides Si4 the specific mannitol dehydrogenase activity i n the strain obtained was 0.48 unit (U)mg(-1), 3.4-fold higher than in the wild type. In this way the enzyme yield from cultivation in a bio reactor could be improved from 110 Ul(-1) to 350 Ul(-1). A further inc rease in productivity was obtained by fed-batch cultivation of R. spha eroides Si4 [pAK82]. Using this cultivation method an optical density of 27.6 was reached in the bioreactor, corresponding to a dry mass of 16.6 g l(-1). Since MDH formation correlated with biomass production, the MDH yield could be raised to 918 Ul(-1), an 8.3-fold increase in c omparison to batch cultivation of the wild-type strain.