P-coumaric Acid Decarboxylase Research Articles

Expression of genes involved in metabolism of phenolic compounds by Lactobacillus pentosus and its relevance for table-olive fermentations

Genes with the potential to code for enzymes involved in phenolic compound metabolism were detected in the genome of Lactobacillus pentosus IG1, isolated from a green olive fermentation. Based on homology, these genes could code for a 6-P-β Glucosidase, two different Tannases, a Gallate decarboxylase and a p-Coumaric decarboxylase. Expression of up to seven of these genes was studied in L. pentosus IG1 (olive fermentation) and CECT4023T (corn silage), including responses upon exposure to relevant phenolic compounds and different olive extracts. Genes potentially coding Tannase, Gallate decarboxylase and p-Coumaric acid decarboxylase significatively increased their expression upon exposure to such compounds and extracts, although it was strain dependent. In general, both the genetic organization and the characteristics of gene expression resembled very much those described for Lactobacillus plantarum. In accordance to the observed induced gene expression, metabolism of specific phenolic compounds was achieved by L. pentosus. Thus, methyl gallate, gallic acid and the hydroxycinamic acids p-coumaric, caffeic and ferulic were metabolized. In addition, the amount of phenolics such as tyrosol, oleuropein, rutin and verbascoside included in a minimal culture medium was noticeably reduced, again dependent on the strain considered.

Characterization of the p-Coumaric Acid Decarboxylase from Lactobacillus plantarum CECT 748T

It was previously reported that cell cultures from Lactobacillus plantarum CECT 748 (T) were able to decarboxylate phenolic acids, such as p-coumaric, m-coumaric, caffeic, ferulic, gallic, and protocatechuic acid. The p-coumaric acid decarboxylase (PDC) from this strain has been overexpressed and purified. This PDC differs at its C-terminal end when compared to the previously reported PDC from L. plantarum LPCHL2. Because the C-terminal region of PDC is involved in enzymatic activity, especially in substrate activity, it was decided to biochemically characterize the PDC from L. plantarum CECT 748 (T). Contrarily to L. plantarum LPCHL2 PDC, the recombinant PDC from L. plantarum CECT 748 (T) is a heat-labile enzyme, showing optimal activity at 22 degrees C. This PDC is able to decarboxylate exclusively the hydroxycinnamic acids p-coumaric, caffeic, and ferulic acids. Kinetic analysis showed that the enzyme has a 14-fold higher K(M) value for p-coumaric and caffeic acids than for ferulic acid. PDC catalyzes the formation of the corresponding 4-vinyl derivatives (vinylphenol and vinylguaiacol) from p-coumaric and ferulic acids, respectively, which are valuable food additives that have been approved as flavoring agents. The biochemical characteristics showed by L. plantarum PDC should be taken into account for its potential use in the food-processing industry.

Overexpression, purification, crystallization and preliminary structural studies of p-coumaric acid decarboxylase from Lactobacillus plantarum.

The substrate-inducible p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum has been overexpressed in Escherichia coli, purified and confirmed to possess decarboxylase activity. The recombinant His(6)-tagged enzyme was crystallized using the hanging-drop vapour-diffusion method from a solution containing 20%(w/v) PEG 4000, 12%(w/v) 2-propanol, 0.2 M sodium acetate, 0.1 M Tris-HCl pH 8.0 with 0.1 M barium chloride as an additive. Diffraction data were collected in-house to 2.04 A resolution. Crystals belonged to the tetragonal space group P4(3), with unit-cell parameters a = b = 43.15, c = 231.86 A. The estimated Matthews coefficient was 2.36 A(3) Da(-1), corresponding to 48% solvent content, which is consistent with the presence of two protein molecules in the asymmetric unit. The structure of PDC has been determined by the molecular-replacement method. Currently, the structure of PDC complexed with substrate analogues is in progress, with the aim of elucidating the structural basis of the catalytic mechanism.

Purification and characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum

Abstract Lactobacillus plantarum cells displayed substrate-inducible decarboxylase activities on p-coumaric and ferulic acids of 0.6 and 0.01 μmol min−1 mg−1, respectively. Activity in uninduced cells or corresponding cell extracts was undetectable (

Enhancing volatile phenol concentrations in wine by expressing various phenolic acid decarboxylase genes in Saccharomyces cerevisiae.

Phenolic acids, which are generally esterified with tartaric acid, are natural constituents of grape must and wine and can be released as free acids (principally p-coumaric, caffeic, and ferulic acids) by certain cinnamoyl esterase activities during the wine-making process. Some of the microorganisms present in grape can metabolize the free phenolic acids into 4-vinyl and 4-ethyl derivatives. These volatile phenols contribute to the aroma of wine. The Saccharomyces cerevisiae phenyl acrylic acid decarboxylase gene (PAD1) is steadily transcribed, but its encoded product, Pad1p, shows low activity. In contrast, the phenolic acid decarboxylase (PADC) from Bacillus subtilis and the p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum display substrate-inducible decarboxylating activity in the presence of phenolic acids. In an attempt to develop wine yeasts with optimized decarboxylation activity on phenolic acids, the padc, pdc, and PAD1 genes were cloned under the control of S. cerevisiae's constitutive phosphoglyceratekinase I gene promoter (PGK1(P)()) and terminator (PGK1(T)()) sequences. These gene constructs were integrated into the URA3 locus of a laboratory strain of S. cerevisiae, Sigma1278b. The overexpression of the two bacterial genes, padc and pdc, in S. cerevisiae showed high enzyme activity. However, this was not the case for PAD1. The padc and pdc genes were also integrated into an industrial wine yeast strain, S. cerevisiae VIN13. As an additional control, both alleles of PAD1 were disrupted in the VIN13 strain. In microvinification trials, all of the laboratory and industrial yeast transformants carrying the padc and pdc gene constructs showed an increase in volatile phenol formation as compared to the untransformed host strains (Sigma1278b and VIN13). This study offers prospects for the development of wine yeast starter strains with optimized decarboxylation activity on phenolic acids and the improvement of wine aroma in the future.

Molecular characterization of the phenolic acid metabolism in the lactic acid bacteria Lactobacillus plantarum

The lactic acid bacteria Lactobacillus plantarumdisplays substrate-inducible decar- boxylase activities on p-coumaric, caffeic and ferulic acids. Purification of the p-coumaric acid decarboxylase (PDC) was performed. Sequence of the N-terminal part of the PDC led to the cloning of the corresponding pdc gene. Expression of this gene in Escherichia colirevealed that PDC displayed a weak activity on ferulic acid, detectable in vitro in the presence of ammonium sulfate. Transcrip- tional studies of this gene in L. plantarum demonstrated that the pdc transcription is phenolic acid- dependent. A mutant deficient in the PDC activity, designated LPD1, was constructed to study phe- nolic acid alternate pathways in L. plantarum. LPD1 mutant strain remained able to metabolize weakly p-coumaric and ferulic acids into vinyl derivatives or into substituted phenyl propionic acids. These results indicate that L. plantarum has a second acid phenol decarboxylase enzyme and also displays inducible acid phenol reductase activity. Finally, PDC activity was shown to confer a selective advantage for LPNC8 grown in acidic media supplemented with p-coumaric acid, compared to the LPD1 mutant devoid of PDC activity.

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

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.

Molecular characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum: gene cloning, transcriptional analysis, overexpression in Escherichia coli, purification, and characterization.

By using degenerate primers designed from the first 19 N-terminal amino acids of Lactobacillus plantarum p-coumaric acid decarboxylase (PDC), a 56-bp fragment was amplified from L. plantarum in PCRs and used as a probe for screening an L. plantarum genomic bank. Of the 2,880 clones in the genomic bank, one was isolated by colony hybridization and contained a 519-bp open reading frame (pdc gene) followed by a putative terminator structure. The pdc gene is expressed on a monocistronic transcriptional unit, which is transcribed from promoter sequences homologous to Lactococcus promoter sequences. No mRNA from pdc and no PDC activity were detected in uninduced cell extracts, indicating that the expression is transcriptionally regulated by p-coumaric acid, which corresponds to an activation factor up to 6,000. The pdc gene was overexpressed constitutively in Escherichia coli, and the recombinant enzyme was purified and characterized.

Purification and characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum

Lactobacillus plantarum cells displayed substrate-inducible decarboxylase activities on p-coumaric and ferulic acids of 0.6 and 0.01 μmol min −1 mg −1, respectively. Activity in uninduced cells or corresponding cell extracts was undetectable (<10 −4 μmol min −1 mg −1). Specificity of induction indicates that at least two phenolic acid decarboxylases are produced in this bacterium. SDS-PAGE of partially purified protein extract from p-coumaric acid-induced cells showed one band of 23.5 kDa that was absent in the extract from uninduced cells. The native molecular mass of 93 kDa indicates that the enzyme is a homotetramer. The 1276-fold purified enzyme had a K m of 1.4 mM, a V m of about 766 μmol min −1 mg −1, and a K cat of 10 3 s −1 for p-coumaric and caffeic acids, but did not display any detectable activity on ferulic acid. Maximum activity was at 30°C, at pH 5.5–6. Cofactors or metal ions were not required for activity.