Ethylphenol Formation by Lactobacillus plantarum: Identification of the Enzyme Involved in the Reduction of Vinylphenols

Abstract

Ethylphenols are strong odorants produced by microbial activity that are described as off flavors in several foods. Lactobacillus plantarum is a lactic acid bacterial species able to produce ethylphenols by the reduction of vinylphenols during the metabolism of hydroxycinnamic acids. However, the reductase involved has not been yet uncovered. In this study, the involvement in vinylphenol reduction of a gene encoding a putative reductase (lp_3125) was confirmed by the absence of reduction activity in the Δlp_3125 knockout mutant. The protein encoded by lp_3125, VprA, was recombinantly produced in Escherichia coli VprA was assayed against vinylphenols (4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol), and all were reduced to their corresponding ethylphenols (4-ethylphenol, 4-ethylcatechol, and 4-ethylguaiacol). PCR and high-performance liquid chromatography (HPLC) detection methods revealed that the VprA reductase is not widely distributed among the lactic acid bacteria studied and that only the bacteria possessing the vprA gene were able to produce ethylphenol from vinylphenol. However, all the species belonging to the L. plantarum group were ethylphenol producers. The identification of the L. plantarum VprA protein involved in hydroxycinnamate degradation completes the route of degradation of these compounds in lactic acid bacteria.IMPORTANCE The presence of volatile phenols is considered a major organoleptic defect of several fermented alcoholic beverages. The biosynthesis of these compounds has been mainly associated with Brettanomyces/Dekkera yeasts. However, the potential importance of lactic acid bacteria in volatile phenol spoilage is emphasized by reports describing a faster ethylphenol production by these bacteria than by yeasts. The genetic identification of the bacterial vinylphenol reductase involved in volatile phenol production provides new insights into the role of lactic acid bacteria in the production of these off flavors. The development of a molecular method for the detection of ethylphenol-producing bacteria could be helpful to design strategies to reduce the bacterial production of vinylphenols in fermented foods.

Keywords: aroma; cider; ethylguaiacol; ethylphenol; lactic acid bacteria; off flavors; phenolic compounds; spoilage; volatile phenols; wine.

FIG 1

Effect of disruption of hcrA (lp_1424), hcrB (lp_1425), and lp_3125 in L. plantarum WCFS1 on the reduction of an hydroxycinnamic acid (m-coumaric acid) and 4-vinylphenol. HPLC chromatograms of L. plantarum cultures incubated in 1.5 mM m-coumaric acid or 4-vinylphenol are shown for L. plantarum WCFS1 (wild type [wt]), L. plantarum WCFS1(pUCE191-hcrA) (ΔhcrA mutant), L. plantarum WCFS1(pUCE191-hcrB) (ΔhcrB mutant), and L. plantarum WCFS1(pUCE191-lp_3125) (Δlp_3125 mutant). Results for uninoculated medium are also shown (control). The m-coumaric acid (mCA), 4-vinylphenol (VP), 3-(3-hydroxyphenyl) propionic acid (3-HPPA), and 4-ethylphenol (EP) detected are indicated. Chromatograms were recorded at 280 nm. mAU, milli-absorbance units.

FIG 2

Genetic organization of the L. plantarum WCFS1 chromosomal region containing the vinylphenol reductase encoding genes. (A) (NCBI accession number NC_004567, positions 2788661 to 2798829). Arrows indicate genes. The shaded genes encode genes putatively involved in vinylphenol reductase (vpr) activity. The location of putative promoters and transcription terminators are also indicated. The size and direction of the transcripts revealed by reverse transcription are also shown. (B) Transcriptional analysis by RT-PCR of the L. plantarum WCFS1 genome in the vinylphenol reductase locus. RT-PCR amplification was performed with primers designed to amplify internal gene regions or intergenic regions, as follows: lp_3123 (primers 1810 and 1811, 617 bp) (1), lp_3123-vprR (1684 and 1685, 613 bp) (2), vprR (1686 and 1687, 596 bp) (3), vprR-vprA (1688 and 1689, 800 bp) (4), vprA (891 and 892, 472 bp) (5), hcrB-hcrC (1385 and 1052, 778 bp) (6), and hcrC (1051 and 1952, 384 bp) (7). Left lane, 100-bp molecular size ladder. Numbers indicate some of the molecular sizes.

FIG 3

Purification and enzymatic activity of E. coli extracts expressing L. plantarum vprA and recombinant VprA protein. (A) SDS-PAGE analysis of the expression and purification of the His₆-tagged VprA. Data represent results of analysis of soluble cell extracts of IPTG-induced E. coli BL21(DE3)(pURI3-Cter) (lane 1) or E. coli BL21(DE3)(pURI3-Cter-VprA) (lane 2), flowthrough from the affinity resin (lane 3), or fractions eluted after His affinity resin (lanes 4 to 8). The 10% gel was stained with Coomassie blue. Molecular mass markers are located on the left (SDS-PAGE standards; Bio-Rad). (B) HPLC chromatograms showing vinylphenol reductase activity of soluble cell extracts of IPTG-induced E. coli BL21(DE3)(pURI3-Cter) (control) or E. coli BL21(DE3)(pURI3-Cter-VprA) (VprA) incubated in 1.5 mM 4-vinylphenol and 15 mM NADH. HPLC chromatograms also showed the reductase activity of purified His₆-VprA protein (500 μg) (VprA) or the reaction mix without VprA protein (control). The 4-vinylphenol (VP) and 4-ethylphenol (EP) detected are indicated. Chromatograms were recorded at 280 nm.

FIG 4

Reductase activity of VprA on several vinylphenols. HPLC chromatograms represent E. coli BL21(DE3)(pURI3-Cter) (control) or E. coli BL21(DE3)(pURI3-Cter-vprA) (VprA) cell extracts incubated at 37°C during 16 h. The vinylphenols assayed were vinyl catechol (VC), 4-vinylphenol (VP), and vinylguaiacol (VG). The corresponding ethyl derivatives such as ethyl catechol (EC), 4-ethylphenol (EP), and ethylguaiacol (EG) detected are indicated. Chromatograms were recorded at 280 nm.

FIG 5

Vinylphenol reductase activity in lactic acid bacteria. (A) PCR amplification of the VprA gene. Chromosomal DNA from several lactic acid bacteria was used for PCR amplification with oligonucleotides 1657 and 1658 to amplify 1.3 kb of the vprA gene. PCR products were subjected to gel electrophoresis and stained with GelRed. Left lane, λ/EcoT14I (TaKaRa) molecular size marker. Numbers indicate some of the molecular sizes. (B) HPLC chromatograms of supernatants from lactic acid bacteria grown during 10 days at 30°C in MRS media supplemented with 1.5 mM 4-vinylphenol. The 4-vinylphenol (VP) and 4-ethylphenol (EP) detected are indicated. Chromatograms were recorded at 280 nm. The strains assayed were Enterococcus casseliflavus DSM 20680 (1), E. durans DSM 20633 (2), E. faecalis DSM 20478 (3), E. faecium CECT 4102 (4), E. gallinarum DSM 24841 (5), E. hirae DSM 20160 (6), Lactobacillus brevis CECT 5354 (7), L. fermentum CECT 4007 (8), L. fructivorans CECT 4785 (9), L. paraplantarum DSM 10641 (10), L. plantarum subsp. argentoratensis DSM 16365 (11), L. plantarum subsp. plantarum ATCC 14917 (CECT 748) (12), L. plantarum DSM 10492 (13), L. pentosus DSM 16366 (14), L. sakei subsp. carnosus DSM 15831 (15), Leuconostoc citreum CECT 4025 (16), and Streptococcus gallolyticus UCN34 (17).

FIG 6

Schematic representation of hydroxycinnamic acid metabolism in L. plantarum WCFS1. When R₁ is –OH and R₂ is –H, the represented compounds are p-coumaric acid (B), vinylphenol (C), ethylphenol (D), and phloretic acid (E). The esters (A) are methyl p-coumarate or ethyl coumarate when R₃ is –OCH₃ or –OCH₂CH₃, respectively. When R₁ is –OH and R₂ is –OH, the represented compounds are caffeic acid (B), vinylcatechol (C), ethylcatechol (D), and hydrocaffeic acid (E). The esters (A) are methyl caffeate or ethyl caffeate when R₃ is –OCH₃ or –OCH₂CH₃, respectively. When R₁ is –OH and R₂ is –OCH₃, the compounds are ferulic acid (B), vinylguaiacol (C), ethylguaiacol (D), and hydroferulic acid (E). The esters (A) are methyl ferulate or ethyl ferulate when R₃ is –OCH₃ or –OCH₂CH₃, respectively. When R₁ is –H and R₂ is –OH, only the reduction reaction is carried out, and the represented compounds are m-coumaric acid (B) and 3-(3-hydroxyphenyl) propionic acid (E).