• Microbiology and Biotechnology Letters
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• v.41 no.2
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• pp.153-159
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• 2013

A mannitol dehydrogenase (MDH; EC 1.1.1.67) gene was cloned from the Sinorhizobium meliloti 1021 (KCTC 2353) genome and expressed in Escherichia coli. It was seen to have an open reading frame consisting of 1,485 bp encoding 494 amino acids (about 54 kDa), which shares approximately 35-55% of amino acid sequence identity with some known long-chain dehydrogenase/ reductase family enzymes. The recombinant S. meliloti MDH (SmMDH) showed the highest activity at $40^{\circ}C$, and pH 7.0 (D-fructose reduction) and pH 9.0 (D-mannitol oxidation), respectively. SmMDH could catalyze the oxidative/reductive reactions between D-mannitol and D-fructose in the presence of $NAD^+/NADH$ as a coenzyme, but not with NADP+/NADPH. These results indicate that SmMDH is a typical $NAD^+/NADH$-dependent mannitol dehydrogenase.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.156-162
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• 2012

A mannitol dehydrogenase (StMDH) gene was cloned from Salmonella typhimurium LT2 (KCTC 2421) and overexpressed in Escherichia coli. It has a 1,467 bp open reading frame encoding 488 amino acids with deduced molecular mass of 54 kDa, which shares approximately 36% of amino acid identity with known long-chain dehydrogenase/reductatse (LDR) family enzymes. The recombinant StMDH showed the highest activity at $30^{\circ}C$, and pH 5.0 and 10.0 for D-fructose reduction and D-mannitol oxidation, respectively. On the contrary, it has no activity on glucose, galactose, xylose, and arabinose. StMDH can catalyze the oxidative/reductive reactions between D-fructose and D-mannitol only in the presence of $NAD^+$/NADH as coenzymes. These results indicate that StMDH is a typical $NAD^+$/NADH-dependent mannitol dehydrogenase (E.C. 1.1.1.67).

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2009-2018
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• 2018

Leuconostoc mesenteroides can be used to produce mannitol by fermentation, but the mannitol productivity is not high. Therefore, in this study we modified the chromosome of Leuconostoc mesenteroides by genetic methods to obtain high-yield strains for mannitol production. In this study, gene knock-out strains and gene knock-in strains were constructed by a two-step homologous recombination method. The mannitol productivity of the pat gene (which encodes phosphate acetyltransferase) deletion strain (${\Delta}pat::amy$), the fk gene (which encodes fructokinase) deletion strain (${\Delta}fk::amy$) and the stpk gene (which encodes serine-threonine protein kinase) deletion strain (${\Delta}stpk::amy$) were all increased compared to the wild type, and the productivity of mannitol for each strain was 84.8%, 83.5% and 84.1%, respectively. The mannitol productivity of the mdh gene (which encodes mannitol dehydrogenase) knock-in strains (${\Delta}pat::mdh$, ${\Delta}fk::mdh$ and ${\Delta}stpk::mdh$) was increased to a higher level than that of the single-gene deletion strains, and the productivity of mannitol for each was 96.5%, 88% and 93.2%, respectively. The multi-mutant strain ${\Delta}dts{\Delta}ldh{\Delta}pat::mdh{\Delta}stpk::mdh{\Delta}fk::mdh$ had mannitol productivity of 97.3%. This work shows that multi-gene knock-out and gene knock-in strains have the greatest impact on mannitol production, with mannitol productivity of 97.3% and an increase of 24.7% over wild type. This study used the methods of gene knock-out and gene knock-in to genetically modify the chromosome of Leuconostoc mesenteroides. It is of great significance that we increased the ability of Leuconostoc mesenteroides to produce mannitol and revealed its broad development prospects.