CLONING, NUCLEOTIDE-SEQUENCE, AND OVEREXPRESSION OF SMOS, A COMPONENTOF A NOVEL OPERON ENCODING AN ABC TRANSPORTER AND POLYOL DEHYDROGENASES OF RHODOBACTER-SPHAEROIDES SI4

The gene coding for sorbitol dehydrogenase (SDH) of Rhodobacter sphaer oides Si4 was located 55 nucleotides upstream of the mannitol dehydrog enase gene (mtlK) within a previously unrecognized polyol operon, This operon probably consists of all the proteins necessary for transport and metabolization of various polyols, The gene encoding SDH (smoS) wa s cloned and sequenced, Analysis of the deduced amino acid sequence re vealed homology to enzymes of the short-chain dehydrogenase/reductase protein family, For structure analysis of this unique bacterial enzyme , smoS was subcloned into the overexpression vector pET-24a(+) and the n overproduced in Escherichia coli BL21(DE3), which yielded a specific activity of 24.8 U/mg of protein and a volumetric yield of 38,000 U/l iter. Compared to values derived with the native host, R. sphaeroides, these values reflected a 270-fold increase in expression of SDH and a 971-fold increase in the volumetric yield, SDH was purified to homoge neity,with a recovery of 49%, on the basis of a three-step procedure. Upstream from smoS, another gene (smoK), which encoded a putative ATP- binding protein of an ABC transporter, was identified.