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Lactic acid bacteria produce γ-aminobutyric acid (GABA) as an acid stress response. GABA is a neurotransmitter that may improve sleep and resilience to mental stress. This study focused on the selection, identification and optimization of a bacterial strain with high GABA production, for development as a probiotic supplement. The scientific literature and an industry database weresearched for probiotics and potential GABA producers. In silico screening was conducted to identify genes involved in GABA production. Subsequently, 17 candidates were screened for in vitro GABA production using thin layer chromatography, which identified three candidate probiotic strains Levilactobacillus brevis DSM 20054, Lactococcus lactis DS75843and Bifidobacterium adolescentis DSM 24849 as producing GABA. Two biosensors capable of detecting GABA were developed: 1. a transcription factor-based biosensor characterized by the interaction with the transcriptional regulator GabR was developed in Corynebacterium glutamicum; and 2. a growth factor-based biosensor was built in Escherichia coli, which used auxotrophic complementation by expressing 4-aminobutyrate transaminase (GABA-T) that transfers the GABA amino group to pyruvate, hereby forming alanine. Consequently, the feasibility of developing a workflow based on co-culture with producer strains and a biosensor was tested. The three GABA producers identified and the biosensors were encapsulated in nanoliter reactors (NLRs) as alginate beads in defined gut-like conditions. The E. coli growth factor-based biosensor was able to detect changes in GABA concentrations in liquid culture and under gut-like conditions. L. brevis and L. lactis were successfully encapsulated in the NLRs and showed growth under miniaturized intestinal conditions.

Summary Background Urinary tract infections (UTIs) are among the most prevalent microbial diseases and their financial burden on society is substantial. In the context of increasing antibiotic resistance, finding alternative treatments for UTIs is a top priority. We aimed to determine whether intravesical bacteriophage therapy with a commercial bacteriophage cocktail is effective in treating UTI. Methods We did a randomised, placebo-controlled, clinical trial, at the Alexander Tsulukidze National Centre of Urology, Tbilisi, Georgia. Men older than 18 years of age, who were scheduled for transurethral resection of the prostate (TURP), with complicated UTI or recurrent uncomplicated UTI but no signs of systemic infection, were allocated by block randomisation in a 1:1:1 ratio to receive intravesical Pyo bacteriophage (Pyophage; 20 mL) or intravesical placebo solution (20 mL) in a double-blind manner twice daily for 7 days, or systemically applied antibiotics (according to sensitivities) as an open-label standard-of-care comparator. Urine culture was taken via urinary catheter at the end of treatment (ie, day 7) or at withdrawal from the trial. The primary outcome was microbiological treatment response after 7 days of treatment, measured by urine culture; secondary outcomes included clinical and safety parameters during the treatment period. Analyses were done in a modified intention-to-treat population of patients having received at least one dose of the allocated treatment regimen. This trial is registered with ClinicalTrials.gov , NCT03140085 . Findings Between June 2, 2017, and Dec 14, 2018, 474 patients were screened for eligibility and 113 (24%) patients were randomly assigned to treatment (37 to Pyophage, 38 to placebo, and 38 to antibiotic treatment). 97 patients (28 Pyophage, 32 placebo, 37 antibiotics) received at least one dose of their allocated treatment and were included in the primary analysis. Treatment success rates did not differ between groups. Normalisation of urine culture was achieved in five (18%) of 28 patients in the Pyophage group compared with nine (28%) of 32 patients in the placebo group (odds ratio [OR] 1·60 [95% CI 0·45–5·71]; p=0·47) and 13 (35%) of 37 patients in the antibiotic group (2·66 [0·79–8·82]; p=0·11). Adverse events occurred in six (21%) of 28 patients in the Pyophage group compared with 13 (41%) of 32 patients in the placebo group (OR 0·36 [95% CI 0·11–1·17]; p=0·089) and 11 (30%) of 37 patients in the antibiotic group (0·66 [0·21–2·07]; p=0·47). Interpretation Intravesical bacteriophage therapy was non-inferior to standard-of-care antibiotic treatment, but was not superior to placebo bladder irrigation, in terms of efficacy or safety in treating UTIs in patients undergoing TURP. Moreover, the bacteriophage safety profile seems to be favourable. Although bacteriophages are not yet a recognised or approved treatment option for UTIs, this trial provides new insight to optimise the design of further large-scale clinical studies to define the role of bacteriophages in UTI treatment. Funding Swiss Continence Foundation, the Swiss National Science Foundation, and the Swiss Agency for Development and Cooperation. Translations For the Georgian and German translations of the abstract see Supplementary Materials section.

The cognitive skills critical for success have largely been studied in Western populations, despite the fact that children in low- and middle-income countries are at risk to not reach their full developmental potential. Moreover, scientists should leverage recent discovery to explore means of boosting cognition in at-risk populations. This semi-randomized controlled trial examined normative cognitive development and whether it could be enhanced by consumption of a probiotic food in a sample of 251 4- to 7-year-old children in urban schools in Côte d’Ivoire. Participants completed executive functioning measures at baseline (T1) and 5 months later (T2). After T1, children in one school received a probiotic (N = 74) or placebo (N = 79) fermented dairy food every day they were in school for one semester; children in the other school (N = 98) continued their diet as usual. Children improved on all tests across time (Cohen’s d = 0.08–0.30). The effects of probiotic ingestion were inconclusive and are interpreted with caution due to socio-political factors affecting daily administration. Given the general feasibility of the study, we hope that it will serve as an inspiration for future research into child development and sustainable (health-promoting) interventions for school children in developing nations.

IntroductionThe metabolic activity of the gut microbiota plays a pivotal role in the gut-brain axis through the effects of bacterial metabolites on brain function and development. In this study we investigated the association of gut microbiota composition with language development of three-year-old rural Ugandan children.MethodsWe studied the language ability in 139 children of 36 months in our controlled maternal education intervention trial to stimulate children’s growth and development. The dataset includes 1170 potential predictors, including anthropometric and cognitive parameters at 24 months, 542 composition parameters of the children’s gut microbiota at 24 months and 621 of these parameters at 36 months. We applied a novel computationally efficient version of the all-subsets regression methodology and identified predictors of language ability of 36-months-old children scored according to the Bayley Scales of Infant and Toddler Development (BSID-III).ResultsThe best three-term model, selected from more than 266 million models, includes the predictors Coprococcus eutactus at 24 months of age, Bifidobacterium at 36 months of age, and language development at 24 months. The top 20 four-term models, selected from more than 77 billion models, consistently include Coprococcus eutactus abundance at 24 months, while 14 of these models include the other two predictors as well. Mann-Whitney U tests further suggest that the abundance of gut bacteria in language non-impaired children (n = 78) differs from that in language impaired children (n = 61) at 24 months. While obligate anaerobic butyrate-producers, including Coprococcus eutactus, Faecalibacterium prausnitzii, Holdemanella biformis, Roseburia hominis are less abundant, facultative anaerobic bacteria, including Granulicatella elegans, Escherichia/Shigella and Campylobacter coli, are more abundant in language impaired children. The overall predominance of oxygen tolerant species in the gut microbiota of Ugandan children at the age 24 months, expressed as the Metagenomic Aerotolerant Predominance Index (MAPI), was slightly higher in the language impaired group than in the non-impaired group (P = 0.09).ConclusionsApplication of the all-subsets regression methodology to microbiota data established a correlation between the relative abundance of the anaerobic butyrate-producing gut bacterium Coprococcus eutactus and language development in Ugandan children. We propose that the gut redox potential and the overall bacterial butyrate-producing capacity could be factors of importance as gut microbiota members with a positive correlation to language development are mostly strictly anaerobic butyrate-producers, while microbiota members that correlate negatively, are predominantly oxygen tolerant with a variety of known adverse effects.

An increasing body of evidence has shown that gut microbiota imbalances are linked to diseases. Currently, the possibility of regulating gut microbiota to reverse these perturbations by developing novel therapeutic and preventive strategies is being extensively investigated. The modulatory effect of vitamins on the gut microbiome and related host health benefits remain largely unclear. We investigated the effects of colon-delivered vitamins A, B2, C, D, and E on the gut microbiota using a human clinical study and batch fermentation experiments, in combination with cell models for the assessment of barrier and immune functions. Vitamins C, B2, and D may modulate the human gut microbiome in terms of metabolic activity and bacterial composition. The most distinct effect was that of vitamin C, which significantly increased microbial alpha diversity and fecal short-chain fatty acids compared to the placebo. The remaining vitamins tested showed similar effects on microbial diversity, composition, and/or metabolic activity in vitro, but in varying degrees. Here, we showed that vitamins may modulate the human gut microbiome. Follow-up studies investigating targeted delivery of vitamins to the colon may help clarify the clinical significance of this novel concept for treating and preventing dysbiotic microbiota-related human diseases. Trial registration: ClinicalTrials.gov, NCT03668964. Registered 13 September 2018 – Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03668964.

Background: Urinary tract infections (UTIs) are among the most prevalent microbial diseases and their financial burden on the society is substantial. Moreover, the continuing increase of antibiotic resistance worldwide is alarming. We aimed to determine whether intravesical bacteriophage therapy is effective to treat UTI. Methods: In this randomised, placebo-controlled, double-blind clinical trial 97 patients presenting with UTI were enrolled to receive intravesical Pyo bacteriophage (n=28), intravesical placebo solution (n=32), or oral antibiotic therapy (n=37). The primary outcome included clinical and microbiological treatment response. Secondary outcomes included safety parameters and quantitative microbiological urine assessments. Findings: At day seven, the treatment success rates were statistically similar between the three groups: Compared to the Pyo bacteriophage (5/28 (18%)) group, the placebo group had 9/32 (28%) (Odds Ratio (OR) 1.8, 95% confidence interval (CI) 0.52-6.2; p=0.352) and the antibiotic group had 13/37 (35%) (OR 2.49, 95% CI 0.77-8.1; p=0.129) successes. Also, adverse events were statistically similar between the three groups. They were seen in 6/28 patients (21%) in the Pyo bacteriophage group, in 13/32 (41%) in the placebo group (OR 0.4, 95% CI 0.13-1.25; p=0.116) and in 11/37 (30%) in the antibiotic group (OR 0.65, 95% CI 0.21-2.03; p=0.452). Interpretation: Beside antibiotics, bacteriophages and bladder irrigation appear to have specific but limited beneficial effects on UTI control. Moreover, the bacteriophage safety profile seems to be favourable. Although bacteriophages are not yet a valuable treatment option for UTIs, this randomized controlled trial provides new insights to optimize the setting for further large-scale clinical studies to define the role of bacteriophages for treating UTIs. Trial Registration: Trial registration number: ClinicalTrials.gov, number NCT03140085. Funding Statement: Swiss Continence Foundation (www.swisscontinencefoundation.ch) The Swiss National Science Foundation (www.snsf.ch) and the Swiss Agency for Development and Cooperation in the framework of the program SCOPES (Scientific cooperation between Eastern Europe and Switzerland, grant number 152304). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study protocol was approved by the institutional review board and ethical committee at TNCU (TNCU-02/283; Tbilisi, Georgia), complied with International Conference on Harmonization Guideline for Good Clinical Practice and the Declaration of Helsinki.

Chronic exposure of children in sub-Saharan Africa to aflatoxins has been associated with low birth weight, stunted growth, immune suppression, and liver function damage. Lactobacillus species have been shown to reduce aflatoxin contamination during the process of food fermentation. Twenty-three Lactobacillus strains were isolated from fecal samples obtained from a cohort of rural Ugandan children at the age of 54 to 60 months, typed by 16S rRNA gene sequencing, and characterized in terms of their ability to bind aflatoxin B1 in vitro. Evidence for chronic exposure of these children to aflatoxin B1 in the study area was obtained by analysis of local foods (maize flour and peanuts), followed by the identification of the breakdown product aflatoxin M1 in their urine samples. Surprisingly, Lactobacillus in the gut microbiota of 140 children from the same cohort at 24 and 36 months showed the highest positive correlation coefficient with stunting among all bacterial genera identified in the stool samples. This correlation was interpreted to be associated with dietary changes from breastfeeding to plant-based solid foods that pose an additional risk for aflatoxin contamination, on one hand, and lead to increased intake of Lactobacillus species on the other.

A main target in microbiome research is the understanding and ability to safely and effectively modulate the microbiome to improve health. Hereto, we discuss the role of vitamins in relation to the gut microbiome and present a rationale for the modulation of gut microbial communities via selected systemic and colon-targeted vitamin administration.

INTRODUCTION AND OBJECTIVES:Urinary tract infections (UTIs) are among the most widespread microbial diseases and their economic impact on the society is substantial. The continuing increase of anti...

This multidisciplinary expert panel opinion on bacteriophage therapy has been written in the context of a society that is confronted with an ever increasing number of antibiotic resistant bacteria. To avoid the return to a pre-antibiotic era, alternative treatments are urgently needed. The authors aim to contribute to the opinion formation of relevant stakeholders on how to potentially develop an infrastructure and legislation that paves the way for the acceptance and re-implementation of bacteriophage therapy.;

Here, we elaborate on the natural origin and use of enzymes and cultures in sustainable food processing. We also illustrate how enzymatically treated or fermented food can contribute to solving challenges involving nutrition and health, such as aging, malnutrition, obesity, and allergy.

© 2016 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter can be used for the production of fermented milk and other fermented foods either at home or at small-scale in rural settings. For the fresh starter, 1 L of milk is pasteurized in a pan that fits into a larger pan containing water, placed on a source of heat. In this water bath, the milk is heated and incubated at 85 °C for 30 min. Thereafter, the milk is cooled down to 45 °C, transferred to a vacuum flask, inoculated with the dried bacteria and left for at least 16 hr between 30 °C and 45 °C. For the purpose of frequent home production, the fresh starter is frozen into ice cubes, which can be used for the production of small volumes of up to 2 L of fermented milk. For the purpose of small-scale production in resource-poor countries, pasteurization of up to 100 L of milk is conducted in milk cans that are placed in a large sauce pan filled with water and heated on a fire at 85 °C for 30 min, and subsequently cooled to 45 °C. Next, the 100 L batch is inoculated with the 1 L freshly prepared starter mentioned before. To assure an effective fermentation at a temperature between 30 and 45 °C, the milk can is covered with a blanket for 12 hr. For the production of non-dairy fermented foods, the fresh starter is left in a cheese cloth for 12 hr, and the drained-off whey can be subsequently used for the inoculation of a wide range of food raw materials, including vegetables and cereal-based foods.

The postharvest treatment and processing of fresh coffee cherries can impact the quality of the unroasted green coffee beans. In the present case study, freshly harvested Arabica coffee cherries were processed through two different wet and dry methods to monitor differences in the microbial community structure and in substrate and metabolite profiles. The changes were followed throughout the postharvest processing chain, from harvest to drying, by implementing up-to-date techniques, encompassing multiple-step metagenomic DNA extraction, high-throughput sequencing, and multiphasic metabolite target analysis. During wet processing, a cohort of lactic acid bacteria (i.e., Leuconostoc , Lactococcus , and Lactobacillus ) was the most commonly identified microbial group, along with enterobacteria and yeasts ( Pichia and Starmerella ). Several of the metabolites associated with lactic acid bacterial metabolism (e.g., lactic acid, acetic acid, and mannitol) produced in the mucilage were also found in the endosperm. During dry processing, acetic acid bacteria (i.e., Acetobacter and Gluconobacter ) were most abundant, along with Pichia and non- Pichia ( Candida , Starmerella , and Saccharomycopsis ) yeasts. Accumulation of associated metabolites (e.g., gluconic acid and sugar alcohols) took place in the drying outer layers of the coffee cherries. Consequently, both wet and dry processing methods significantly influenced the microbial community structures and hence the composition of the final green coffee beans. This systematic approach to dissecting the coffee ecosystem contributes to a deeper understanding of coffee processing and might constitute a state-of-the-art framework for the further analysis and subsequent control of this complex biotechnological process. IMPORTANCE Coffee production is a long process, starting with the harvest of coffee cherries and the on-farm drying of their beans. In a later stage, the dried green coffee beans are roasted and ground in order to brew a cup of coffee. The on-farm, postharvest processing method applied can impact the quality of the green coffee beans. In the present case study, freshly harvested Arabica coffee cherries were processed through wet and dry processing in four distinct variations. The microorganisms present and the chemical profiles of the coffee beans were analyzed throughout the postharvest processing chain. The up-to-date techniques implemented facilitated the investigation of differences related to the method applied. For instance, different microbial groups were associated with wet and dry processing methods. Additionally, metabolites associated with the respective microorganisms accumulated on the final green coffee beans.

This perspective paper follows up on earlier communications on bacteriophage therapy that we wrote as a multidisciplinary and intercontinental expert-panel when we first met at a bacteriophage conference hosted by the Eliava Institute in Tbilisi, Georgia in 2015. In the context of a society that is confronted with an ever-increasing number of antibiotic-resistant bacteria, we build on the previously made recommendations and specifically address how the Nagoya Protocol might impact the further development of bacteriophage therapy. By reviewing a number of recently conducted case studies with bacteriophages involving patients with bacterial infections that could no longer be successfully treated by regular antibiotic therapy, we again stress the urgency and significance of the development of international guidelines and frameworks that might facilitate the legal and effective application of bacteriophage therapy by physicians and the receiving patients. Additionally, we list and comment on several recently started and ongoing clinical studies, including highly desired double-blind placebo-controlled randomized clinical trials. We conclude with an outlook on how recently developed DNA editing technologies are expected to further control and enhance the efficient application of bacteriophages.

This study describes the genetic analysis of the riboflavin (vitamin B 2 ) biosynthetic ( rib ) operon in the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NZ9000. Functional analysis of the genes of the L. lactis rib operon was performed by using complementation studies, as well as by deletion analysis. In addition, gene-specific genetic engineering was used to examine which genes of the rib operon need to be overexpressed in order to effect riboflavin overproduction. Transcriptional regulation of the L. lactis riboflavin biosynthetic process was investigated by using Northern hybridization and primer extension, as well as the analysis of roseoflavin-induced riboflavin-overproducing L. lactis isolates. The latter analysis revealed the presence of both nucleotide replacements and deletions in the regulatory region of the rib operon. The results presented here are an important step toward the development of fermented foods containing increased levels of riboflavin, produced in situ, thus negating the need for vitamin fortification.

Lactic acid bacteria display a relatively simple and well-described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies on the level of sugar metabolism, that lead to either the efficient re-routing of the lactococcal sugar metabolism to nutritional end-products other than lactic acid such as L-alanine, several low-calorie sugars and oligosaccharides or to enhancement of sugar metabolism for complete removal of (undesirable) sugars from food materials. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to the production of the B-vitamins folate and riboflavin. An overview of these metabolic engineering activities can be found on the website of the Nutra Cells 5th Framework EU-project (www.nutracells.com). Finally, the impact of the developments in the area of genomics and corresponding high-throughput technologies on nutraceutical production will be discussed.

Within the European Union (EU), the use of genetically modified organisms (GMOs) in food production is not widely applied and accepted. In contrast to the United States of America, the current EU legislation limits the introduction of functional foods derived from GMOs that may bring a clear benefit to the consumer. Genetically modified lactic acid bacteria (GM-LAB) can be considered as a different class of GMOs, and the European Union is preparing regulations for the risk assessment of genetically modified microorganisms. Since these procedures are not yet implemented, the current risk assessment procedure is shared for GMOs derived from micro organisms, plants, or animals. At present, the use of organisms in food production that have uncontrolled genetic alterations made through random mutagenesis, is permitted, while similar applications with organisms that have controlled genetic alterations are not allowed. The current paper reviews the opportunities that genetically modified lactic acid bacteria may offer the food industry and the consumer. An objective risk profile is described for the use of GM-LAB in food production. To enhance the introduction of functional foods with proven health claims it is proposed to adapt the current safety assessment procedures for (GM)-LAB and suggestions are made for the related cost accountability. A qualified presumption of safety as proposed by SANCO (EU SANCO 2003), based on taxonomy and on the history of safe use of LAB applied in food, could in the near future be applied to any kind of LAB or GM-LAB provided that a series of modern profiling methods are used to verify the absence of unintended effects of altered LAB that may cause harm to the health of the consumer.

Removal of pyrophosphate from dihydroneopterin triphosphate (DHNTP) is the second step in the pterin branch of the folate synthesis pathway. There has been controversy over whether this reaction requires a specific pyrophosphohydrolase or is a metal ion-dependent chemical process. The genome of Lactococcus lactis has a multicistronic folate synthesis operon that includes an open reading frame (ylgG) specifying a putative Nudix hydrolase. Because many Nudix enzymes are pyrophosphohydrolases, YlgG was expressed in Escherichia coli and characterized. The recombinant protein showed high DHNTP pyrophosphohydrolase activity with a K(m) value of 2 microM, had no detectable activity against deoxynucleoside triphosphates or other typical Nudix hydrolase substrates, required a physiological level (approximately 1 mM) of Mg(2+), and was active as a monomer. Essentially no reaction occurred without enzyme at 1 mM Mg(2+). Inactivation of ylgG in L. lactis resulted in DHNTP accumulation and folate depletion, confirming that YlgG functions in folate biosynthesis. We therefore propose that ylgG be redesignated as folQ. The closest Arabidopsis homolog of YlgG (encoded by Nudix gene At1g68760) was expressed in E. coli and shown to have Mg(2+)-dependent DHNTP pyrophosphohydrolase activity. This protein (AtNUDT1) was reported previously to have NADH pyrophosphatase activity in the presence of 5 mM Mn(2+) (Dobrzanska, M., Szurmak, B., Wyslouch-Cieszynska, A., and Kraszewska, E. (2002) J. Biol. Chem. 277, 50482-50486). However, we found that this activity is negligible at physiological levels of Mn(2+) and that, with 1 mM Mg(2+), AtNUDT1 prefers DHNTP and (deoxy) nucleoside triphosphates.

A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly. Lactococcus lactis , Streptococcus thermophilus , and Leuconostoc spp. all produced folate, while most Lactobacillus spp., with the exception of Lactobacillus plantarum , were not able to produce folate. Folate production was further investigated in L . lactis as a model organism for metabolic engineering and in S . thermophilus for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production. S . thermophilus excreted more folate than L . lactis , presumably as a result of differences in the number of glutamyl residues of the folate produced. In S . thermophilus 5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in L . lactis 5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in S . thermophilus , but pH had no effect on folate excretion by L . lactis . Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of p -aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis.

The dairy starter bacterium Lactococcus lactis has the potential to synthesize both folate (vitamin B11) and riboflavin (vitamin B2). By directed mutagenesis followed by selection and metabolic engineering we have modified two complicated biosynthetic pathways in L. lactis resulting in simultaneous overproduction of both folate and riboflavin: Following exposure to the riboflavin analogue roseoflavin we have isolated a spontaneous mutant of L. lactis strain NZ9000 that was changed from a riboflavin consumer into a riboflavin producer. This mutant contained a single base change in the regulatory region upstream of the riboflavin biosynthetic genes. By the constitutive overproduction of GTP cyclohydrolase I in this riboflavin-producing strain, the production of folate was increased as well. Novel foods, enriched through fermentation using these multivitamin-producing starters, could compensate the B-vitamin-deficiencies that are common even in highly developed countries and could specifically be used in dietary foods for the large fraction of the Caucasian people (10-15%) with mutations in the methylene tetrahydrofolate reductase (MTHFR).

Lactic acid bacteria display a relatively simple and well described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies that led to the efficient re-routing of the lactococcal pyruvate metabolism to end-products other than lactic acid, including diacetyl and alanine. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to exopolysaccharides and folic acid. Finally, the (future) impact of the developments in the area of genomics and corresponding high-throughput technologies will be discussed.

Folates are essential components in the human diet. They are involved as cofactor in many metabolic reactions, including the biosynthesis of nucleotides, the building blocks of DNA and RNA. Folates are produced in different (green) plants (folium (Latin) = leaf) and by some micro-organisms. Therefore, vegetables and dairy products are the main source of folates for humans. The daily recommended intake for an adult is 200 µg. For pregnant women a double dose is recommended, since folates are known to prevent neural-tube defect in newborns (1). Moreover, folates are reported to protect against some forms of cancer (2). A low folate level in the diet is associated with high homocysteine levels in the blood and, consequently with coronary diseases (3, 4).