Controlled modulation of folate polyglutamyl tail length by metabolic engineering of Lactococcus lactis

Abstract

The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates >90% of the produced folate intracellularly, predominantly in the polyglutamyl form. Approximately 10% of the produced folate is released into the environment. Overexpression of folC in L. lactis led to an increase in the length of the polyglutamyl tail from the predominant 4, 5, and 6 glutamate residues in wild-type cells to a maximum of 12 glutamate residues in the folate synthetase overproducer and resulted in a complete retention of folate in the cells. Overexpression of folKE, encoding the bifunctional protein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP-cyclohydrolase I, resulted in reduction of the average polyglutamyl tail length, leading to enhanced excretion of folate. By simultaneous overexpression of folKE and folC, encoding the enzyme folate synthetase or polyglutamyl folate synthetase, the average polyglutamyl tail length was increased, again resulting in normal wild-type distribution of folate. The production of bioavailable monoglutamyl folate and almost complete release of folate from the bacterium was achieved by expressing the gene for gamma-glutamyl hydrolase from human or rat origin. These engineering studies clearly establish the role of the polyglutamyl tail length in intracellular retention of the folate produced. Also, the potential application of engineered food microbes producing folates with different tail lengths is discussed.

FIG. 1.

Folate chromatograms of 5-formyl tetrahydrofolate standards (A) and cell extracts of L. lactis NZ9000 harboring pNZ8048 (control strain with empty vector) (B), pNZ7010 (overexpressing folKE) (C), or pNZ7011 (overexpressing folKE and folC) (D) monitored by fluorescent detection. Cells were induced with 2 ng of nisin/ml as described in Materials and Methods. The numbers correspond to the polyglutamyl tail lengths of 5-formyl tetrahydrofolate derivatives. For 5-formyl tetrahydromonoglutamyl folate, both the S and Rdiastereoisomeres can be distinguished.

FIG. 2.

Folate chromatograms of cell extracts of L. lactis NZ9000, harboring pNZ7016 with increased overexpression of folC, induced with 0 (A), 0.01 (B), 0.1 (C), and 1 (D) ng of nisin/ml and monitored at 360 nm. The numbers correspond to thepolyglutamyl tail lengths of 5,10-methenyl tetrahydrofolate derivatives. mAU, milliabsorbance units.

FIG. 3.

Functional expression of the gene coding for MG-HGH in a cell extract of L. lactis pNZ9000 harboring pNZ7001 (▪) or pNZ8048 (negative control) (▴) monitored in vitro using yeast extract as a source of polyglutamyl folate at pH 7.0. Folate concentrations were measured by a microbiological assay and corrected for the production of folate by the L. lactis strain. The error bars indicate the standard deviation of the microbiological assay (measurements were done in duplicate).

FIG. 4.

(A) Growth curves (dashed lines) and functional expression of the MG-HGH gene by monitoring the extracellular concentrations of folate (solid lines) in growing cells of L. lactis NZ9000 harboring pNZ7001 (▪) or pNZ8048 (negative control) (•). (B) Growth curves (dashed lines) and functional expression of the MG-HGH gene by monitoring the intracellular concentrations of folate (solid lines) in growing cells of L. lactis NZ9000 harboring pNZ7001 (▪) or pNZ8048 (♦). Higher intracellular folate levels were detected in the control strain after the deconjugation of polyglutamyl folate (•). The cells were induced with nisin at an OD₆₀₀ of 0.5. Folate concentrations were measured by a microbiological assay. The error bars indicate the standard deviation of the microbiological assay (measurements were done in duplicate). Open symbols indicate cell mass.

FIG. 5.

Chromatography of 5-formyl tetrahydrofolate standards (A), folates extracted at the end of the exponential phase from fermentation broth of L. lactis harboring pNZ7001 induced with nisin (C) or not induced with nisin (B), and intracellular folates present in cell extracts of L. lactis harboring pNZ7001 induced with nisin (D) or not induced with nisin (E). Fluorescence detection: λ_ex, 310 nm; λ_em, 352 nm. The numbers correspond to the polyglutamyl tail lengths of 5-formyl tetrahydrofolate derivatives. For 5-formyl tetrahydromonoglutamyl folate, the S and R diastereoisomeres can be distinguished.