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

Abstract

To achieve high mannitol production by Lactococcus lactis, the mannitol 1-phosphatase gene of Eimeria tenella and the mannitol 1-phosphate dehydrogenase gene mtlD of Lactobacillus plantarum were cloned in the nisindependent L. lactis NICE overexpression system. As predicted by a kinetic L. lactis glycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by the L. lactis parental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenasedeficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficient L. lactis strain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.Mannitol is a reduced form of fructose and is produced by a variety of microorganisms including bacteria, yeasts, and fungi. Besides the ability of several organisms to maintain their redox balance by the production of mannitol (9, 21, 22), mannitol has a physiological function in microorganisms as an osmolyte (16) and can serve as a protecting agent. It has been reported that mannitol enhances survival of Lactococcus lactis cells during drying processes (10). The viability of starter cultures of L. lactis, a lactic acid bacterium (LAB) extensively used in dairy industry, may thus be enhanced by mannitol production. In addition, the use of a mannitol-producing L. lactis may result in fermented products with extra value, since mannitol is assumed to have several beneficial effects as a food additive. It can serve as an antioxidant (4,5,25,26) and as a low-calorie sweetener that can replace sucrose (6,8).In heterofermentative LABs such as Leuconostoc mesenteroides, mannitol is formed from fructose in a single conversion by mannitol dehydrogenase, and fructose-to-mannitol conversion rates of Ͼ90% are common (13,24,27). In contrast, most homofermentative LABs, such as Lactococcus lactis, do not normally produce mannitol. Mannitol formation in homofermentative LABs is limited to strains whose ability to regenerate NAD to fulfill the redox balance is severely hampered. In these strains, mannitol 1-phosphate dehydrogenase (M1PDH) and mannitol 1-phosphatase (M1Pase) are the enzymes involved in the mannitol biosynthesis route (Fig. 1). Transient formation of high concentrations of intracellular mannitol (90 mM) and mannitol 1-phosphate (76 mM) were detected in high-density nongrowing cell suspensions of a lactate dehydrogenase (LDH)-deficient L. lactis strain (22). During growth, only small amounts of mannitol (Ͻ0.4 mM) were transiently produced extracellularly (23). Recently, inactivation of the mannitol transport system in an LDH-def...

High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD + Cofactor Recycling

“…In fact, about 42% of the glucose was channeled to mannitol, a value close to the theoretical maximum of 50%, calculated on the assumption that glucose is transported via phosphoenolpyruvate phosphotransferase systems only (9). Higher mannitol yields have been reported in L. lactis NZ9010 by Wisselink et al (61), using a slightly different engineering strategy. Most likely, the reason for the enhanced mannitol production lies in the distinct fermentation conditions used in the two studies (rich medium versus CDM, higher glucose content, different host strains, etc.).…”

“…The resultant constraint at the level of NAD ϩ regeneration can be exploited to increase the production of reduced compounds under anaerobic conditions. This approach has been successfully applied in the production of value-added compounds, such as alanine (25) or mannitol (19,61).…”

Lactobacillus reuteri CRL 1101 highly produces mannitol from sugarcane molasses as carbon source

“…Heterofermentative LAB belonging to the genera Leuconostoc, Lactobacillus and Oenococcus produce mannitol from fructose in a single enzymatic conversion by mannitol 2-dehydrogenase (MDH), thereby producing less ethanol (no further need for NAD + regeneration) and more acetic acid (enabling more ATP production) (Korakli and Vogel 2003;von Weymarn 2002). In contrast, most homofermentative LAB normally do not produce mannitol, and its formation is limited to strains whose ability to regenerate NAD + is hampered (Wisselink et al 2005). A number of heterofermentative LAB strains have been investigated for their production of mannitol (Saha and Racine 2011).…”

Natural sweetening of food products by engineering Lactococcus lactis for glucose production

“…Additionally, L. lactis is a suitable model organism for metabolic pathway engineering (Kleerebezem and Hugenholtz, 2003), since it has a relatively simple carbon metabolism and many molecular cloning tools are available (Kuipers et al, 1998;Leenhouts et al, 1996Leenhouts et al, , 1998. The metabolism of L. lactis has already been successfully engineered, e.g., for the production of the sweet amino acid L-alanine (Hols et al, 1999), the production of the buttery flavor diacetyl (Hugenholtz et al, 2000), the production of mannitol (Gaspar et al, 2004, Wisselink et al, 2005, and for the simultaneous overproduction of the vitamins folate and riboflavin (Sybesma et al, 2004). The aim of the present study was to disrupt glucose uptake and metabolism in L. lactis in such a way that, when growing on lactose it excretes glucose, which can be used as a natural sweetener in dairy products.…”