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High-folate-producing Lactococcus lactis strain

High folate-producing Lactococcus lactis

Through the strategy of metabolic engineering, high folate-producing Lactococcus lactis variants were constructed. In the first year, threefold increase in folate production was achieved by overexpression of the first genes in folate biosynthesis, folKE. Also, reduced folate production was achieved by overexpression of another gene, folA. In the third year, much higher (200-fold) folate production was achieved by overexpression of all folate genes, except folA. This high folate production (10 - 15mg/L) only occurred in growth medium containing para-aminobenzoic acid, a precursor for folate.

Another goal of the metabolic engineering strategy was to control bioavailability of the produced folate. By cloning of the human (and rat) folate deconjugase gene in L.lactis, the polyglutamyl-taillength of the produced folate was reduced, leading to more efficient excretion of the folate in the growth medium. By overexpression of the folC gene in L. lactis, coding for polyglutamyl folate synthetase, the produced folate was had much longer polyglutamyl tails and the folate was no longer excreted into the medium.

The gene vectors that were used to achieve high folate production in L. lactis, were also transfered to other lactic acid bacteria. In this way, the non-folate producer Lactobacillus gasseri, was transformed into a folate-producer.

An additional scientific result of the metabolic engineering was the discovery of a new gene involved in folate biosynthesis, folQ.

Finally, a transcriptome analysis was performed on the folate-overproducing Lactococcus lactis. This provided some leads for future metabolic engineering strategies.

These results have been described in thirteen scientific publication, three contributions (chapters) to text books, one PhD Thesis (Wilbert Sybesma) and several popular publications and have been presented at a large number of international scientific meetings.

The high-folate-producing Lactococcus lactis strains are not (yet) commercialised, for the obvious reason that they are GMO's. However, since we are dealing with homologous recombination in almost all results, these strains do not need to labeled as GMO's in some non-European countries such as the USA, and could reach these markets in the near future. For this reason, it was checked, in thefinal year of the project, if these high-folate-producers are an effective source of folate. In animal trials using rats fed on a folate-depleted diet, it was convincingly shown that the bacterial folate is a good, and bioavailable, source of folate.

As to other implementation of the results, the metabolic engineering strategy has clearly indicated how high folate production can be achieved. It also presented some possibilities to increase folate by changes in the medium composition (para-aminobenzoic acid!) or by controlled fermentation (slow growth leads to high folate!)

Reported by

NIZO food research
P.O. Box 20
6710 BA Ede
Netherlands
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