Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/8/2004
Publication Date: 3/1/2005
Citation: Liu, S., Saha, B.C., Cotta, M.A. 2005. Cloning, expression, purification, and analysis of mannitol dehydrogenase gene mtlK from Lactobacillus brevis. Applied Biochemistry and Biotechnology. 121-124:391-402. Interpretive Summary: D-mannitol (a six carbon polyalcohol) has a sweet taste with lower calories compared to six carbon sugars, like fructose or glucose, and is an ideal sugar substitute in food, medicine, and pharmaceutical products. The industrial production of mannitol is expensive and inefficient: a process that involves high-pressure hydrogenation of fructose using a nickel catalyst. Greater efficiency and more mannitol can be produced via fermentation by microorganisms, which offers an alternative economic mannitol production process. The existing mannitol producing microbial strains produce mannitol from fructose with other products. An approach toward improving this process would be to genetically engineer these and similar Lactobacillus strains to increase mannitol conversion from other sugars; in addition to fructose. In this study, the gene encoding mannitol-2-dehydrogenase, an enzyme that catalyzes the conversion of fructose to mannitol, was cloned from L. brevis using genomic PCR. Genetic engineered L. plantarum TF103 carrying mtlK gene of L. brevis indicated increased mannitol production from glucose. This information will be used to direct further research on genetically engineering lactic acid bacteria for increased mannitol production.
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 22.214.171.124) 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.