ControlledModulation of Folate Polyglutamyl Tail Length by Metabolic Engineeringof Lactococcuslactis

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 ␥-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.

Tomato γ-Glutamylhydrolases: Expression, Characterization, and Evidence for Heterodimer Formation

“…A similar result has been reported in the bacterium Lactococcus lactis; expressing mammalian GGH led to complete folate deglutamylation, but not to growth reduction (Sybesma et al, 2003). Such findings suggest that the polyglutamyl tail might be less metabolically crucial than generally thought (e.g.…”

Folate Cross-Feeding Supports Symbiotic Homoacetogenic Spirochetes

“…However, it is interesting that the amount secreted by L. lactis ZFX-2 was higher than the amounts observed for 16 strains of L. lactis isolated from a variety of sources (5 to 46 ng/ml), which retained the majority of the folate produced intracellularly (52). It has recently been shown that the degree of folate polyglutamation is the primary factor that affects the extent of folate secretion; folate derivatives with longer polyglutamate tails are more likely to be retained intracellularly (53). It is thus likely that the higher level of folate secretion observed in L. lactis ZFX-2 is the result of production of the triglutamate form of 5-formyl-THF rather than the more extensively glutamated forms (four to six residues) common in other L. lactis strains (52).…”

Characterization of the Role of para- Aminobenzoic Acid Biosynthesis in Folate Production by Lactococcus lactis

“…It is known that monoglutamate THF molecules diffuse more readily out of the cell than the more highly charged polyglutamate THF derivates (19,22,34). In L. lactis, it was found that overexpression of the gene coding for folylpolyglutamate synthetase (folC) resulted in increased polyglutamate tails, which caused higher intracellular retention (42).…”

“…The pABA production levels were determined using a procedure based on the high-performance liquid chromatography (HPLC) method described previously (41,42). Instead of using an HPLC separation gradient of 75% elution liquid B (MilliQ plus 1.5% formic acid) and 25% elution liquid C (80% MilliQ-20% methanol plus 1.5% formic acid), a ratio of 60% elution liquid B to 40% elution liquid C was used for the proper separation of pABA on the column.…”