Modulation of folate production in lactic acid bacteria

Food fortification has proven to be very useful in reducing health problems associated with mal-intake of essential nutrients, such as the B-vitamin folate. Folate is used as one-carbon donor/acceptor in several biochemical processes like synthesis of DNA, RNA and some amino acids. Sufficient intake of folate is essential for neural tube development in early life but it has also been described to aid to brain power in the elderly. The daily recommended intake level for folate, however, are still not met by the whole human population. Fermentation fortification is a new concept which can help to increase the intake levels of nutrients and vitamins such as folate. By this method, the level of the nutrient of interest in the food product is increased as a result of microbial activity in the fermentation process. In this study we have focused on modulation of folate levels in food products using lactic acid bacteria. To be able to modulate folate levels it is essential to gain insight in the genes, which are involved in the biosynthesis of folate. Moreover, it is important to gain insight in pathways, which are involved in the production of folate. The missing gene in the folate biosynthesis pathway of Lactococcus lactis and Arabidopsis thaliana has been identified. Moreover, the role of pABA biosynthesis in the production of folate in Lactococcus lactis was addressed. It was observed that disruption of pABA biosynthesis abolished the production of folate. In addition we have shown that a folate-consuming Lactobacillus gasseri can be converted into a folate-producing strain by heterologous overexpression of the folate gene cluster of Lactococcus lactis. Moreover, a folate-overproducing Lactobacillus plantarum strain has been constructed and the impact of folate overproduction on the gene expression and metabolite formation was monitored. The observed discrepancy between the limited metabolic response and the reduced growth rate was further investigated. Propagation of the folate-overproducing strain of L. plantarum results in an instable phenotype. We have shown a clear correlation between growth rate, plasmid copy numbers and folate production levels. A minimal medium was developed for L. plantarum, which was used to study the impact of extremely low folate pools on growth. In addition, we found that folate overproduction results in resistance towards the folate antagonist methotrexate (MTX). Remarkably, mutants that showed resistance towards MTX were isolated and one of these was found to produce 70% more folate in comparison to the wild-type. Finally we demonstrated that two lactobacilli (L. reuteri and L. plantarum) can be used to increase the folate content of melon juice by fermentation. This example illustrates the significance of fermentation fortification for increasing the nutritional value of a fermented beverage In this study we have shown that folate production can be modulated from very low levels (1 ng/L per OD600 unit) to very high levels (3 mg/L per OD600 unit). Essential for the modulation of folate levels is the presence and absence of pABA in the growth medium. Another critical factor that influences the folate production pools is the expression of the folate gene cluster.