Knockout of the p-coumarate decarboxylase gene from Lactobacillus plantarum reveals the existence of two other inducible enzymatic activities involved in phenolic acid metabolism

Abstract

Lactobacillus plantarum NC8 contains a pdc gene coding for p-coumaric acid decarboxylase activity (PDC). A food grade mutant, designated LPD1, in which the chromosomal pdc gene was replaced with the deleted pdc gene copy, was obtained by a two-step homologous recombination process using an unstable replicative vector. The LPD1 mutant strain remained able to weakly metabolize p-coumaric and ferulic acids into vinyl derivatives or into substituted phenyl propionic acids. We have shown that L. plantarum has a second acid phenol decarboxylase enzyme, better induced with ferulic acid than with p-coumaric acid, which also displays inducible acid phenol reductase activity that is mostly active when glucose is added. Those two enzymatic activities are in competition for p-coumaric and ferulic acid degradation, and the ratio of the corresponding derivatives depends on induction conditions. Moreover, PDC appeared to decarboxylate ferulic acid in vitro with a specific activity of about 10 nmol. min(-1). mg(-1) in the presence of ammonium sulfate. Finally, PDC activity was shown to confer a selective advantage on LPNC8 grown in acidic media supplemented with p-coumaric acid, compared to the LPD1 mutant devoid of PDC activity.

FIG. 1

Physical map of the pdc locus in the wild-type strain LPNC8 (a) and the mutant strain LPD1 (b). Long horizontal arrows represent the two ORFs and their orientations. The start sites are indicated by vertical arrows and the stop codons by T. The positions and orientations of the primers are indicated by short horizontal arrows, and restriction sites that were created are noted between brackets.

FIG. 2

UV spectra of p-coumaric acid and metabolic derivatives: 1, 60 μM p-coumaric acid; 2, 60 μM 4-vinyl phenol; 3, 60 μM phloretic acid; 4, 60 μM 4-ethyl phenol; 5, 30 μM 4-vinyl phenol with 30 μM phloretic acid. Arrows point to the maximum absorbance for each compound.

FIG. 3

Growth of strain LPNC8 (squares) and the LPD1 mutant (circles) supplemented (filled symbols) or not (open symbols) with 1.2 mM p-coumaric acid at pH 6.5. Residual p-coumaric acid concentrations in LPNC8 (filled triangles) and in LPD1 (open triangles) were measured by UV spectrophotometry (see Materials and Methods).

FIG. 4

UV spectra resulting from the conversion of 0.6 mM p-coumaric acid by induced whole cells of LPNC8 (0.2 g/liter) or LPD1 (5 g/liter) incubated 1 h without glucose in 25 mM phosphate buffer. t₀, UV spectra corresponding to the sample taken at the start of kinetic reaction. t₁, UV spectra corresponding to the sample taken after 1 h of kinetic reaction. (a) LPNC8 induced with p-coumaric or ferulic acid (1.2 or 3 mM); (b) LPD1 induced with 1.2 mM p-coumaric acid; (c) LPD1 induced with 3 mM p-coumaric acid.

FIG. 5

Growth of (filled symbols) and degradation of p-coumaric acid by (open symbols) LPNC8 (a) and LPD1 (b) at different p-coumaric acid concentrations (⧫, 0 mM; ■, 0.6 mM; ●, 3 mM; ▴, 6 mM) at pH 6.5. Samples were taken during growth to determine the biomass (OD₆₀₀) and p-coumaric acid degradation using UV spectrophotometry (see Materials and Methods).

FIG. 6

Growth of LPNC8 (a) and LPD1 (b) at different p-coumaric acid concentrations (⧫, 0 mM; ■, 1.2 mM; ●, 3 mM; ▴, 6 mM) at pH 4.5.

FIG. 7

Proposed pathway for the degradation of p-coumaric acid in L. plantarum. The arrow thickness represents the relative intensity of enzymatic activity. PDC, p-coumaric acid decarboxylase; PDC2: phenolic acid decarboxylase; PAR, phenolic acid reductase; VPR, putative 4-vinyl phenol reductase; DEC, putative phloretic acid decarboxylase.