Metabolic engineering of mannitol production in Lactococcus lactis

Mannitol is a sugar alcohol that is produced by a wide variety of (micro) organisms. It is assumed to have several beneficial effects as a food additive. It can serve as an antioxidant and as a low-calorie sweetener which can replace high-calorie sugars such as sucrose, lactose, glucose and fructose. In addition, it has been shown that mannitol has a protective effect for lactic acid bacteria, such as Lactococcus lactis , when they are subjected to drying and/or freezing. The viability of starter cultures containing these lactic acid bacteria may thus be enhanced by inducing mannitol production in these strains. In addition the use of a mannitol producing L. lactis may result in fermented products with extra nutritional value.The metabolic engineering of mannitol production in L. lactis, using a combined approach of predicting the control points for mannitol production in L. lactis by a kinetic glycolysis model, and experimental metabolic engineering steps, are described in this thesis. Based on the model predictions, we combined engineering steps such as knocking out lactate dehydrogenase, reduction of phosphofructokinase and fructokinase activity, and overexpression of genes involved in mannitol biosynthesis, such as mannitol 1-phosphate dehydrogenase, mannitol 1-phosphatase, and mannitol dehydrogenase. This resulted in different levels of mannitol production in several L. lactis strains. Especially the combination of overexpression of genes encoding mannitol 1-phosphate dehydrogenase and a mannitol-1-phosphatase resulted in high mannitol production by L. lactis . Moreover, it was found that mannitol 1-phosphatase has a high control on the mannitol production, and a clear correlation between the activity of this enzym and the mannitol production was shown.The research described in his thesis is a text book example of metabolic engineering. It contributes to the understanding of mannitol biosynthesis in L. lactis , and how the mannitol production can be improved by metabolic engineering. Moreover, it provides suggestions for food-grade applications, especially in the field of the production of low-calorie sweeteners.