Modulation of Folate Production in Lactic Acid Bacteria Arno Wegkamp Promotor: Prof. dr. Willem M. de Vos Hoogleraar Microbiologie Wageningen Universiteit Copromotor: Dr. Eddy J. Smid Projectleider NIZO food research, Ede Leden van de promotiecommisie: Prof. dr. R.J. Bino Wageningen Universiteit Prof. dr. J. Kok Rijksuniversiteit Groningen Dr. D. van Sinderen University College Cork, Ireland Prof. dr. P. Hols Catholic University of Louvain-la-Neuve, Belgium Dit onderzoek is uitgevoerd binnen de onderzoekschool VLAG 2 Modulation of Folate Production in Lactic Acid Bacteria Arno Wegkamp Proefschrift Ter verkrijging van de graad van doctor Op gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. M.J. Kropff, in het openbaar te verdedigen op vrijdag 8 februari 2008 des namiddags te half twee in de Aula 3 Modulation of Folate Production in Lactic Acid Bacteria Arno Wegkamp Ph.D. thesis Wageningen University, Wageningen, The Netherlands (2008) 176 pages, with summary in Dutch ISBN 978 90 8504 859 6. 4 Abstract Wegkamp, A. (2008). Modulation of Folate Production in Lactic Acid Bacteria. Ph.D. thesis. Laboratory of Microbiology, Wageningen University, The Netherlands. 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. 5 Contents Chapter 1 Introduction, Aim and Outline of the Thesis 9 Chapter 2 The Role of Tetrahydrofolates and Tetrahydromethanopterins in Bacteria and Archaea 19 Chapter 3 A Nudix Enzyme Removes Pyrophosphate from Dihydroneopterin Triphosphate in the Folate Synthesis Pathway of Bacteria and Plants 35 Chapter 4 Characterization of the Role of para-Aminobenzoic Acid Biosynthesis in Folate Production by Lactococcus lactis 53 Chapter 5 Transformation of Folate-Consuming Lactobacillus gasseri into a Folate Producer 73 Chapter 6 Plasmid Maintenance in Lactobacillus plantarum WCFS1 Engineered for Folate Overproduction 79 Chapter 7 Physiological Responses to Folate Overproduction in Lactobacillus plantarum WCFS1 95 Chapter 8 Development of a Minimal Growth Medium for Lactobacillus plantarum WCFS1 115 Chapter 9 Folate Overproduction in Lactobacillus plantarum WCFS1 Causes Methotrexate Resistance 129 Chapter 10 Combined Production of B12 and Folate by Lactobacillus reuteri JCM1112 for the Natural Enrichment of Fermented Foods 141 6 Chapter 11 Summary and Concluding Remarks 149 Appendix : 160 Samenvatting en Conclusies 161 Dankwoord 170 Curriculum Vitae 172 Publications and Patents 173 Activities in the Frame of VLAG Research School 175 175 7 8 Chapter 1 Introduction, Aim and Outline of the Thesis Arno Wegkamp Chapter 1 Introduction Tetrahydrofolate is a one-carbon carrier that plays a key role in the synthesis of purines, pyrimidines, amino acids and formylation of tRNAMet. The property of folate biosynthesis can be found in two of the three domains of the phylogenetic tree. The exception is the domain of the Archaea. Species in this domain use tetrahydromethanopterin as one- carbon carrier (25, 27). However, exceptions of the ability to synthesize folate can also be found within the group of the Bacteria and Eukarya. In particular, the position of Animalia is remarkable since members of this Kingdom require folate but are not capable of synthesis (26). Species that can not synthesize folate are therefore dependent on the consumption of folate rich food sources like: meat, plants, fruits and fermented food products. Dietary intake of folate is not the only source of biological available folate. Studies with folate-depleted rats have shown that the folate supply for the animals could be increased through folate coming from cecal-bacteria (20, 23). Folate deficiency in humans is associated with health problems, such as cancer, cardiovascular diseases as well as neural tube defects in newborns (13, 18, 31, 36). The daily recommended intake (DRI) in European Union (EU) is set at 200 and 400 µg/day for adults and women in the periconceptional period, respectively (5). This increase in folate intake for women in the periconceptional period is important since, only 10% of women at childbearing age have a folate level in red blood cells that is sufficiently high to decrease the risk of neural tube defects (11). There are several ways to increase the folate levels of food products: i) fortification of food products, ii) selection of special plant cultivars, or fruits with increased folate pools, iii) fermentation fortification. Methods such as fortification have proven to be very useful in reducing health problems associated with folate mal-intake. The addition of folate to food products in the USA and Canada has reduced the prevalence of neutral tube defects with 25% and 46%, respectively (3, 10). However, it was recently shown that high-level intake of chemically synthesized folate might have some adverse health effects (29). It was proposed that these problems could be circumvented by consumption of a mixed diet containing folate-rich foods (29, 38). Bacterial strain selection can be an important contributor to increased folate intake levels, e.g. in fermented food products. The ability to produce folate can differ remarkably between different lactic acid bacteria. The levels may vary from 2 to 214 μg/L folate (34). Another way to increase folate content in food products is by fermentation fortification. The natural folate content of rye sourdoughs is 6.5 μg/100g and after fermentation by yeasts the folate content was shown to have increased approximately three- fold (19). This increased folate content is a direct result of folate that is produced by the yeast present in the rye sourdough. Fermentation fortification is a new concept which can be improved in several ways: i) bacterial strain selection, ii) delivery engineering and iii) metabolic engineering. There is a growing interest in using lactic acid bacteria for improvement of the nutritional value of fermented food products and/or adding health benefits to these products. Several known lactic acid bacteria are marketed as probiotics, defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host (WHO (2)). Also these probiotic lactic acid bacteria can be used for fermentation fortification to increase the nutritional value of the food products. 10 Introduction Lactic acid bacteria Lactic acid bacteria are a remarkable group of bacteria. Mankind has used lactic acid bacteria