Technical Abstract: The commercial production of mannitol involves high-pressure hydrogenation of fructose using a nickel catalyst, a costly process. Mannitol can be produced through fermentation by microorganisms. Currently, a few Lactobacillus strains are used to develop an efficient process for mannitol bio-production; most of the strains produce mannitol from fructose with other products. An approach toward improving this process would be to genetically engineer Lactobacillus strains to increase fructose to mannitol conversion with decreased production of other products. In this study, the gene mtlK encoding mannitol-2-dehydrogenase (EC 1.1.1.67) that catalyzes the conversion of fructose to mannitol was cloned from L. brevis using genomic PCR. The mtlK clone contains 1328 bp DNA sequence including 1002 bp ORF that consisted of 333 amino acids with a predicted molecular mass of about 36 kDa. The functional mannitol 2 dehydrogenase was produced by overexpressing mtlK via pRSETa vector in E. coli BL21pLysS upon IPTG induction. The fusion protein containing about 3 kDa additional N-terminal sequences from the vector, is able to catalyze the reduction of fructose to mannitol at pH 5.35, can use both NADH or NADPH as cofactor and similar rates of catalytic reduction were observed using either one of the cofactors under in vitro assay conditions. Genetic engineered L. plantarum TF103 carrying mtlK gene of L. brevis indicated increased mannitol production from glucose. The evaluation of mixed sugar fermentation and mannitol production by this strain are in progress.