Engineering Lactococcus lactis for Production of Mannitol: High Yields from Food-Grade Strains Deficient in Lactate Dehydrogenase and the Mannitol Transport System

Abstract

Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTS Mtl ). Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L. lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (⌬ldh⌬mtlA and ⌬ldh⌬mtlF) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo 13 C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTS Mtl . Disruption of this system completely blocked mannitol transport in L. lactis, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.Mannitol, a six-carbon sugar polyol, is synthesized by many eukaryotes and a few bacteria, but apparently not by archaea. Mannitol is also a well-known compatible solute that accumulates mainly in yeast, fungi, and plants, but rarely in bacteria, in response to osmotic and/or heat stresses (5,16,21,23). Like several other compatible solutes, mannitol protects proteins and cells against different stressing conditions. Recently, its ability to enhance survival of starter cultures of Lactococcus lactis subjected to drying has been demonstrated (6).Mannitol is assumed to have several health-promoting properties; thus the enrichment of foods with mannitol by in situ production during fermentation could be a positive, clean strategy to obtain healthier fermented food products. In the human gut, mannitol can be converted to short-chain fatty acids (such as butyrate), which have been claimed to confer protection against the development of colon cancer (42). Moreover, mannitol is a scavenger of hydroxyl radicals and a low-calorie sweetener that is partially and slowly absorbed in the small intestine (8,40).Lactic acid bacteria (LAB) are a group of microorganisms widely used as starter cultures in dairy fermentations. Hence, these food-associated organisms could be ideal agents for the in situ production of mannitol in foods. The ability of heterofermenta...

Sugar alcohols—their role in the modern world of sweeteners: a review

“…Moreover, it acts as a scavenger of hydroxyl radicals and is claimed to protect against the development of colon cancer [68,80]. Mannitol is also presumed to be health promoting, and thus, its addition to foods can result in extra nutritional value [72].…”

Availability of public goods shapes the evolution of competing metabolic strategies

“…S8 and S9). An increase in transcription levels upon transposition of IS905 into other promoter regions of MG1363 has been described earlier (19,20). The transpositions of IS905 can therefore explain the enhanced acidification rate phenotype of the revertant strains HB61 and HB63.…”

Overproduction of Heterologous Mannitol 1-Phosphatase: a Key Factor for Engineering Mannitol Production byLactococcus lactis

“…Recently, inactivation of the mannitol transport system in an LDH-deficient L. lactis strain resulted in high extracellular mannitol production. About onethird of glucose was converted into mannitol by nongrowing cells, and no undesired mannitol utilization after glucose depletion was observed (12). In these strains, mannitol was produced to fulfill the redox balance during sugar metabolism, since NAD is regenerated in the conversion of fructose 6-phosphate into mannitol 1-phosphate by M1PDH.…”