High-Level Folate Production in Fermented Foods by the B12 Producer Lactobacillus reuteri JCM1112

We observed that Lactobacillus reuteri JCM1112 produces B₁₂and folate. However, the folate/B₁₂ mass ratio found was farbelow that desired for human consumption ( $~$ 170:1). We used metabolicengineering applying genetic and physiological approaches toimprove this ratio and developed a generic and natural processthat significantly increases folate production.

INTRODUCTION

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INTRODUCTION

Humans have an auxotrophic requirement for vitamin B₁₂ and folate,and the recommended intakes of these nutrients for healthy adultsare 2.4 and 400 µg/day, respectively (7). Suboptimal intakeof either of these compounds has been linked to cardiovasculardisease, neuropathy, birth defects, cancer, and different typesof anemia, among other pathologies (4). Remarkably, the onsetof vitamin B₁₂ deficiency symptoms is often delayed by an increasedintake of folate (20). This masking of B₁₂ deficiency has resultedin the restriction of folate intake levels and prevented folatefortification in many countries (7). Strict vegetarian dietaryregimens tend to be poor in vitamin B₁₂ and rich in folic acid,increasing the risk of vitamin B₁₂ deficiency masking. Thishas boosted the popularity of fortifying vegetarian foodstuffswith B₁₂ (3).

Coenzyme B₁₂ is synthesized by a few members of the bacterialand archaeal groups (13). In situ microbial B₁₂ production isa convenient strategy to achieve natural enrichment of fermentedfoods, notably from vegetable sources. Lactobacillus reuteriis a gram-positive, heterofermentative lactic acid bacteriumwith a long history of safe use by the food industry (10). Thismicroorganism ferments several sugars, and this flexibilityleads to its capacity to thrive on several substrates of vegetableorigin (14). Strain CRL1098 has been reported to produce differentforms of B₁₂ (18, 25), and the draft genome sequence of strainJCM1112 (accession no. CP000705) (http://www.jgi.doe.gov/) suggeststhat it is able to produce folate, as well as B₁₂. In this study,we investigated the possibility of using L. reuteri for thecombined production of both vitamins at a ratio desired forhuman consumption, $~$ 170:1 (wt/wt).

In silico analysis of the folate biosynthesis genes of L. reuteri JCM1112.

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In silico analysis of...

Folate is a tripartite molecule assembled from GTP, para-aminobenzoicacid (PABA), and one or more L-glutamate moieties. The biosynthesispathway has been extensively characterized in several lacticacid bacteria, including Lactobacillus plantarum WCFS1 (Fig.1). The predicted product of each folate biosynthesis gene ofthis bacterium was used to search the genome of L. reuteri JCM1112using the BLAST algorithm (2). The sequence identity of thebidirectional best hit was calculated on the nucleotide andamino acid levels based on separate Needleman-Wunsch globalalignments (15) determined using the needle script includedin EMBOSS (European Molecular Biology Open Software Suite) (17)with default settings. Gene order was analyzed using the ERGObioinformatics suite (http://ergo.integratedgenomics.com/ERGO/)(16). The two clusters are very similar, as expected from theclose phylogenetic relationship of their hosts (Table 1). Sequenceidentity is high at both the amino acid and nucleotide levels(on average, 43 and 51%, respectively). Gene order is completelyconserved throughout the entire length of the approximately4.5-kb cluster composed of six genes.

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FIG. 1. Folate biosynthesis pathway in L. plantarum WCFS1.

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TABLE 1. Presence of folate biosynthesis genes in the genome of L. reuteri JCM1112 as determined by homology searches with L. plantarum WCFS1

Characterization of B₁₂ and folate production in CDM by L. reuteri JCM1112 and derivatives of this strain.

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Characterization of...

The human isolate L. reuteri JCM1112 (type strain) was obtainedfrom the Japanese Collection of Microorganisms (Riken, Japan).It was cultured at 37°C in chemically defined medium (CDM)containing 10 mg/liter of PABA and lacking vitamin B₁₂ and folicacid (26). Folate in stationary-phase cultures was quantifiedas described previously (8) with a bioassay using Lactobacilluscasei ATCC 7469 as the indicator strain, which included enzymaticdeconjugation of polyglutamate tails (23). The vitamin B₁₂ contentwas determined as described in the Official Methods of Analysisof AOAC International, using the Lactobacillus delbrueckii subsp.lactis ATCC 7830 vitamin B₁₂ assay (9). Cell extracts of stationary-phasecultures used for B₁₂ analysis were prepared as previously described(18). In CDM L. reuteri JCM1112 produces around 20 µgof folate·liter^–1·unit of optical densityat 600 nm (OD₆₀₀)^–1 at an approximately 1:1 (wt/wt) ratiowith B₁₂ (Fig. 2).

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FIG. 2. Folate (open bars) and B₁₂ (shaded bars) production by L. reuteri wild-type and derivative strains and by L. plantarum WCFS1 in different media. Plasmid pNZ7021 is the empty plasmid, and plasmid pNZ7026 contains the folate biosynthesis gene cluster of L. plantarum. Each bar represents the average of three biological replicates, and the error bars show standard deviations. All experiments were repeated with at least two different batches of media with similar results.

We used a metabolic engineering strategy as proof of principlefor the possibility that the ratio of production of these twovitamins was influenced. We aimed at increasing folate productionthrough the overexpression of the complete folate biosynthesisgene cluster, as described previously for other lactic acidbacteria (29, 30), ideally leaving the native B₁₂ productionunchanged. The constructs used in this study cannot be directlyused by the food industry, but the use of food-grade alternativesis possible. A wide variety of food-grade systems have beendeveloped for lactic acid bacteria (namely, for representativesof the genus Lactobacillus) (5). L. reuteri was transformedby electroporation as described elsewhere (27) with plasmidspNZ7021 (empty vector) and pNZ7026 harboring the folate biosynthesisgene cluster of L. plantarum WCFS1 under control of the pepNpromoter (28). The derivatives of JCM1112 were cultured andanalyzed for folate and B₁₂ content in a fashion similar tothat used for the parent strain. Chloramphenicol was used asa selection marker at a final concentration of 10 µg/ml.The constitutive overexpression of the folate biosynthesis genesof L. plantarum WCFS1 in cultures of L. reuteri JCM1112/pNZ7026resulted in an almost 100-fold increase in folate levels (Fig.2), while the control (L. reuteri JCM1112/pNZ7021) did not showany change in folate and B₁₂ production. The overproductionof folate was found to have a very small effect on B₁₂ production(<10% reduction), resulting in a folate/B₁₂ ratio of approximately100:1 (wt/wt), which was stable over five consecutive transfers(data not shown). The high levels of folate overproduction forthe strain transformed with pNZ7026 were expected, providedthat PABA was supplied in the medium. The same construct hasbeen tested with L. plantarum, resulting in similar folate productionlevels (28), and similar results were obtained when the samestrategy was applied to Lactococcus lactis (30) and Lactobacillusgasseri (29).

Characterization of B₁₂ and folate production in fruit fermentations.

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We assessed the applicability of the principle of improvingfolate/B₁₂ ratios through genetic engineering to media otherthan CDM. Most (sub)tropical fruits are perishable and sensitiveto chill damage, leading to losses of up to 40% in industrializedcountries and far greater than 50% in less economically developednations (6, 12). Fermentation is a secular process of food preservation,which in this case could increase the vitamin content of a rawmaterial. Juice derived from two Cucumis spp. (melon and cucumber)was selected for natural enrichment, since this material islow in folate and deficient in B₁₂ according to the USDA NationalNutrient Database for Standard Reference (http://www.ars.usda.gov/ba/bhnrc/ndl.).Melon juice medium was made from Cucumis melo var. reticulatusafter peeling and removal of seeds. The pulp was liquefied usinga kitchen blender (Masterchef 370; Moulinex, France), and theresulting paste was squeezed through a cotton cloth. The flowthroughwas centrifuged twice at 8,000 x g for 10 min using a Sorvallcentrifuge (Newton, CT). The supernatant was stored at –20°Cuntil it was used. Before inoculation, the melon juice was dilutedat a 4:1 (vol/vol) ratio with potassium phosphate buffer (finalconcentration, 0.1 M; pH 5.8). Further dilution was found toresult in growth impairment (data not shown). The final pH wasadjusted to 6.0, and the melon juice medium was forced througha 0.22-µm filter to ensure sterility. Cucumber juice mediumwas prepared from intact cucumber (Cucumis sativus) and wassterilized using the procedure described above for melon mediumwith the following modifications: (i) an additional filtrationstep using a cellulose filter (0.15 mm) was used before centrifugation,and (ii) the cucumber juice was diluted in 1 volume of potassiumphosphate buffer (final concentration, 0.1 M; pH 5.8). Whenmentioned below, both media were supplemented with 10 mg/literPABA. This concentration of PABA does not conflict with exitingfood legislation as PABA is listed as a generally regarded assafe compound with an upper intake limit of 30 mg/day (http://www.cfsan.fda.gov/ $~$ dms/opa-appa.html).If appropriate, 10 µg/ml chloramphenicol was also added.Biomass formation in the different growth media is indicatedin Table 2.

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TABLE 2. Biomass formation in the different growth media

Folate and B₁₂ contents were determined for cultures of L. reuteritransformed with pNZ7026 and pNZ7021. The background folatelevels in melon and cucumber media were found to be 22.5 ±0.9 and 10.0 ± 0.4 µg/liter, respectively. As expected,B₁₂ could not be detected in these media. The overexpressionof the folate biosynthesis cluster of WCFS1 in L. reuteri JCM1112/pNZ7026led to production of a high level of folate (2,518.2 ±182.1 µg·liter^–1·OD₆₀₀ unit^–1)and a folate/B₁₂ ratio of $~$ 250:1 (wt/wt), but only when PABAwas added (Fig. 2). PABA availability has been shown to limitfolate biosynthesis in several lactic acid bacteria (24, 30).The control experiment using L. reuteri JCM1112 with the emptyvector pNZ7021 resulted in the production, in melon medium,of 131.7 ± 5.5 µg·liter^–1·OD₆₀₀unit^–1 of folate, which is more than five times greaterthan the production in CDM (P < 0.001, pairwise t test).In cucumber medium, folate production by JCM1112/pNZ7021 wasnegatively affected compared to the production in CDM, regardlessof the addition of PABA (Fig. 2). The overexpression of thefolate biosynthesis genes had an effect similar to that describedfor CDM, but the final folate/B₁₂ ratios were 1 order of magnitudelower than desired. The twofold reduction in B₁₂ productionobserved for the melon juice fermentation can be attributedto the amount of sugars present, $~$ 1.5% glucose and $~$ 2% fructoseas determined by high-performance liquid chromatography analysesperformed as described elsewhere (21). Such concentrations havebeen shown in previous studies to repress B₁₂ biosynthesis atthe transcriptional level (1, 19).

The remarkable feature of melon fermentation in comparison toCDM and cucumber fermentation is the 5- to 10-fold-grater productionof folate by the strain carrying the empty plasmid (pNZ7021).To establish the unique ability of melon juice to induce productionof a high level of folate, we tested the parent strain, L. reuteriJCM1112, and another lactic acid bacterium, L. plantarum WCFS1(11). Both L. reuteri and L. plantarum showed a 5- to 10-foldincrease in folate production in melon juice medium comparedto CDM (Fig. 2). Folate biosynthesis relies on three buildingblocks (Fig. 1) whose availability does not seem to explainthis unsuspected observation. We have experimentally ruled outPABA and L-glutamate since both of these compounds are presentin excess in CDM. Regarding the other building block, it hasbeen shown that GTP is not the rate-limiting substrate in folatebiosynthesis (24), which can be explained by the small fluxfrom GTP to folate in comparison to the total GTP pool. Thisimplies that an increase in GTP availability for folate synthesiscannot reasonably explain the increase in folate productionobserved in melon juice. Folate production is tightly regulatedon both the transcriptional and translational levels (22, 23,28). We suspect that there might be an interaction between acompound present in melon juice and one of these regulatoryfactors. However, the nature of the postulated interaction isunclear and remains to be elucidated.

In this study, we demonstrated that it is possible to combinethe production of folate and the production of B₁₂ in L. reuteri.We used, as proof of principle, a metabolic engineering strategyto optimize the ratio of production of these two vitamins andassessed its applicability to fruit fermentations. This resultedin the development of a natural fermentation process to increasefolate production by lactobacilli to levels substantially higherthan those previously described (24). The findings reportedhere may lead to the development of (fermented) foods basedon perishable fruits, such as melons, with extended durabilityand higher nutritional value. A good-tasting fermented melonjuice or melon squash containing high folate and vitamin B₁₂levels could be the start of a product line with a longer shelf-lifethat especially targets vitamin-deficient populations.

FOOTNOTES

* Corresponding author. Mailing address: P.O. Box 20, 6710 BA Ede, The Netherlands. Phone: 31 318 65 95 40. Fax: 31 318 65 04 00. E-mail: jeroen.hugenholtz{at}nizo.nl

Published ahead of print on 14 March 2008.

F.S. and A.W. contributed equally to this work.

REFERENCES

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