Expression in Escherichia coli of native and chimeric phenolic acid decarboxylases with modified enzymatic activities and method for screening recombinant E. coli strains expressing these enzymes

Four bacterial phenolic acid decarboxylases (PAD) from Lactobacillus plantarum, Pediococcus pentosaceus, Bacillus subtilis, and Bacillus pumilus were expressed in Escherichia coli, and their activities on p-coumaric, ferulic, and caffeic acids were compared. Although these four enzymes displayed 61% amino acid sequence identity, they exhibit different activities for ferulic and caffeic acid metabolism. To elucidate the domain(s) that determines these differences, chimeric PAD proteins were constructed and expressed in E. coli by exchanging their individual carboxy-terminal portions. Analysis of the chimeric enzyme activities suggests that the C-terminal region may be involved in determining PAD substrate specificity and catalytic capacity. In order to test phenolic acid toxicity, the levels of growth of recombinant E. coli displaying and not displaying PAD activity were compared on medium supplemented with different concentrations of phenolic acids and with differing pHs. Though these acids already have a slight inhibitory effect on E. coli, vinyl phenol derivatives, created during decarboxylation of phenolic acids, were much more inhibitory to the E. coli control strain. To take advantage of this property, a solid medium with the appropriate pH and phenolic acid concentration was developed; in this medium the recombinant E. coli strains expressing PAD activity form colonies approximately five times smaller than those formed by strains devoid of PAD activity.     

Sensitivity to vinyl phenol derivatives produced by phenolic acid decarboxylase activity in Escherichia coli and several food-borne Gram-negative species

Ferulic, p-coumaric, and caffeic acids are phenolic acids present in soil, food, and gut, which have antimicrobial effects. Some Gram (+) bacteria metabolize these phenolic acids into vinyl derivatives due to phenolic acid decarboxylase activity (PAD) involved in the phenolic acid stress response (PASR). In this study, the antimicrobial activity of phenolic acids and their vinyl derivatives was tested on a panel of desirable and undesirable food-borne bacteria, especially Gram (?) species of Salmonella, Enterobacter, Klebsiella, and Pseudomonas, most of them without PAD activity. Native and engineered Escherichia coli strains either expressing or not PAD activity were included. Gram (?) bacteria of the panel were not significantly inhibited by phenolic acids at 3 mM, but were dramatically inhibited by the corresponding vinyl derivatives. On the contrary, Gram (+) bacteria displaying the PASR face the toxicity of phenolic acids by PAD activity and are not inhibited by vinyl phenols. In E. coli, the genes aaeB and marA, encoding efflux pumps for antimicrobial compounds, are upregulated by the addition of p-coumaric acid, but not by its derivative 4-vinyl phenol (p-hydroxystyrene). These results suggest that phenolic acids and their vinyl phenol derivatives produced by PAD (+) species could have a significant impact on undesirable or pathogenic food-borne Gram (?) bacteria in complex microbial ecosystems.     

Structure-function relationships of the antibacterial activity of phenolic acids and their metabolism by lactic acid bacteria

Aims: To determine structure–function relationships of antibacterial phenolic acids and their metabolites produced by lactic acid bacteria (LAB). Methods and Results: Minimum inhibitory concentrations (MICs) of 6 hydroxybenzoic and 6 hydroxycinnamic acids were determined with Lactobacillus plantarum, Lactobacillus hammesii, Escherichia coli and Bacillus subtilis as indicator strains. The antibacterial activity of phenolic acids increased at lower pH. A decreasing number of hydroxyl groups enhanced the activity of hydroxybenzoic acids, but had minor effects on hydroxycinnamic acids. Substitution of hydroxyl groups with methoxy groups increased the activity of hydroxybenzoic, but not of hydroxycinnamic, acid. Metabolism of chlorogenic, caffeic, p‐coumaric, ferulic, protocatechuic or p‐hydroxybenzoic acids by L. plantarum, L. hammesii, Lactobacillus fermentum and Lactobacillus reuteri was analysed by LC‐DAD‐MS. Furthermore, MICs of substrates and metabolites were compared. Decarboxylated and/or reduced metabolites of phenolic acids had a lower activity than the substrates. Strain‐specific metabolism of phenolic acids generally corresponded to resistance. Conclusions: The influence of lipophilicity on the antibacterial activity of hydroxybenzoic acids is stronger than that of hydroxycinnamic acids. Metabolism of phenolic acids by LAB detoxifies phenolic acids. Significance and Impact of the Study: Results allow the targeted selection of plant extracts for food preservation, and selection of starter cultures for fermented products.     

Engineering E. coli for caffeic acid biosynthesis from renewable sugars

Caffeic acid is a valuable aromatic compound that possesses many important pharmacological activities. In structure, caffeic acid belongs to the hydroxycinnamic acid family and can be biosynthesized from the aromatic amino acid tyrosine. In the present paper, the caffeic acid biosynthesis pathway was reconstituted in engineered Escherichia coli to produce caffeic acid from simple biomass sugar glucose and xylose. Different engineering approaches were utilized to optimize the production. Specifically, two parallel biosynthesis routes leading from tyrosine to caffeic acid were studied. The copy number of the intermediate biosynthesis genes was varied to find appropriate gene doses for caffeic acid biosynthesis. Three different media, including a MOPS medium, a synthetic medium, and a rich medium, were also examined to improve the production. The highest specific caffeic acid production achieved was 38 mg/L/OD. Lastly, cultivation of engineered E. coli in a bioreactor resulted in a production of 106 mg/L caffeic acid after 4 days.     

Gene cloning,expression, and characterization of phenolic acid decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> RM84

Phenolic acid decarboxylase (PAD) catalyzes the synthesis of vinyl phenols from hydroxycinnamic acids. The gene encoding PAD from Lactobacillus brevis was cloned and expressed as a fusion protein in Escherichia coli. The recombinant PAD enzyme is a heat-labile enzyme that functions optimally at 22°C and pH 6.0. The purified enzyme did not show thermostability at temperatures above 22°C. L. brevis PAD is able to decarboxylate exclusively the hydroxycinnamic acids, such as p-coumaric, caffeic, and ferulic acids, with K _m values of 0.98, 0.96, and 0.78 mM, respectively. The substrate specificity exhibited by L. brevis PAD is similar to the PAD isolated from Bacillus subtilis and B. pumilus, but different from that of L. plantarum and Pediococcus pentosaceus. As the C-terminal region may be involved in determining PAD substrate specificity and catalytic capacity, amino acid differences among these proteins could explain the differences observed. The substrate specificity shown by L. brevis PAD shows promise for the synthesis of high-added value products from plant wastes.     

Antibiotic potential of plant growth promoting rhizobacteria (PGPR) against Sclerotium rolfsii

High performance liquid chromatographic (HPLC) analysis of culture filtrates of plant growth promoting rhizobacteria (PGPR) and medium of inhibitory zone of interaction of Sclerotium rolfsii with PGPR, viz. Pseudomonas aeruginosa, Pseudomonas fluorescens 4, Pseudomonas fluorescens 4 (new) and Pseudomonas sp. varied from sample to sample. In all the culture filtrates of PGPRs, P. aeruginosa had nine phenolic acids in which ferulic acid (14.52 μg/ml) was maximum followed by other phenolic acids. However, the culture filtrates of P. fluorescens 4 had six phenolic acids with maximum ferulic acid (20.54 μg/ml) followed by indole acetic acid (IAA), caffeic, salicylic, o-coumeric acid and cinnamic acids. However, P. fluorescens 4 culture filtrate had seven phenolic acids in which salicylic acid was maximum (18.03 μg) followed by IAA, caffeic, vanillic, ferulic, o-coumeric and cinnamic acids. Pseudomonas sp. also showed eight phenolic acids where caffeic acid (2.75 μg) was maximum followed by trace amounts of ferulic, salicylic, IAA, vanillic, cinnamic, o-coumeric and tannic acids. The analysis of antibiosis zone of PGPRs showed fairly rich phenolic acids. A total of nine phenolic acids were detected in which caffeic acid was maximum (29.14 μg/g) followed by gallic (17.64 μg/g) and vanillic (3.52 μg/g) acids but others were in traces. In P. aeruginosa, antibiosis zone had seven phenolic acids where IAA was maximum (3.48 μg/g) followed by o-coumeric acid (2.08 μg/g), others were in traces. The medium of antibiosis zone of P. fluorescens 4 and P. fluorescens 4 new had eight phenolic acids in which IAA was maximum with other phenolic acids in traces.     

GC/MS and LC/MS analysis,and antioxidant and antimicrobial activities of various solvent extracts from Mirabilis jalapa tubers

The antioxidant and antimicrobial activities of various solvent extracts from Mirabilis jalapa tubers (MJT) were investigated using various in vitro assays. The total phenolic and flavonoid contents varied from 21.45 to 364.6 mg gallic acid equivalent (GAE)/g dried extract and 5.2 to 71.6 mg quercetin/g dried extract, respectively. Water extract of MJT was the most potent antioxidant in all assays used, followed by methanol extract. The five solvent extracts were screened for antibacterial and antifungal activities. Water extract was the most effective with minimum inhibitory concentration <200 μg/ml against Staphylococcus aureus, Micrococcus luteus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus cereus and Enterococcus faecalis. Only water extract showed antifungal activity against Aspergillus niger, Fusarium solani, Fusarium oxysporium and Fusarium granularium. GC/MS analysis of MJT dichloromethane and methanol extracts showed that oleic acid and β-sitosterol were, respectively, the major compounds. LC/MS analysis of the aqueous extract showed a high content of flavanol and flavonol compounds. Phenolic acids such as ferulic and caffeic acid were also detected.To our knowledge, this is the first report on the chemical composition, and antioxidant and antimicrobial activities of phenolic extracts from M. jalapa tubers (MJT). The results of the present work indicate that MJT extracts could be used as natural antioxidant and antimicrobial agents in the food preservation and human health.     

Heterologous synthesis of 4-ethylphenol in engineered Escherichia coli

4-Ethylphenol (4-EP) is an industrially versatile commodity chemical widely applied in the pharmaceutical and food industries. In this study, an artificial biosynthetic pathway was constructed in Escherichia coli for production of 4-ethylphenol from simple sources of carbon. The pathway consists of the tal, pad and vpr genes, which encode tyrosine ammonia lyase (TAL), phenolic acid decarboxylase (PAD) and vinylphenol reductase (VPR), respectively. Our results confirmed that the TAL from Saccharothrix espanaensis possessed higher catalytic activity than the TAL from Rhodobacter sphaeroides for biosynthesis of p-hydroxycinnamic acid. The low solubility of Lactobacillus plantarum VPR (LpVPR) in E. coli was a critical factor limiting its availability in the biosynthetic pathway. The solubility of LpVPR was improved by E. coli strain and induction condition optimization. Under the optimized conditions, the engineered E. coli TransB-TPV produced as high as 110 mg/L 4-EP at 37 ℃ in Terrific Broth (TB) medium with glycerol as carbon source after cultivation of 48 h. This study provided a new and feasible strategy for biosynthesis of 4-EP from simple sugars, which may provide a basis for future large-scale industrial application.     

Changes in the composition of phenolic compounds and antioxidant properties of grapevine roots and leaves (Vitis vinifera L.) under continuous of long-term drought stress

Grapevine seedlings Vitis vinifera L. were grown in a greenhouse under optimum conditions (soil moisture ca 70 %) and under drought stress (soil moisture ca 30 %). Drought stress caused reduction in total phenolic compounds in grapevine leaves and roots, where were identified tree phenolic acids: caffeic acid, p-coumaric acid and ferulic acid. All acids found in leaves and roots occurred in the ester-bound form. Only caffeic acid in leaves appeared in the free and ester-bound form. Caffeic acid was present in the highest concentrations. The content of ferulic acid was the lowest in both tissues. The levels of all phenolic acids in leaves and roots decreased significantly under the drought stress. All the extracts from grapevine leaves and roots had antioxidative properties, but the antiradical activity of the extracts obtained from roots subjected to drought stress was lower to the control. The results of the analysis revealed that long-term drought stress caused a decrease in selected elements of secondary metabolism in such a different plant tissues that are the leaves and roots of the grapevine.     

Interaction of Moisture Stress and Three Phenolic Compounds on Lettuce Seed Germination

No interactions between water stress and three phenolic acids(p-coumaric, caffeic and ferulic acids) on lettuce (Lactucasativa L. var. Grand Rapids) seed germination were found. Probitanalysis indicated that mechanisms of action of water stressand the phenolic inhibitors were similar. The relative effectivenessof the compounds was p-coumaric > ferulic > caffeic. Nointeraction was found between p-coumaric and ferulic acid, whereasantagonism was found between caffeic acid and each of the othertwo phenolic acids. Lactuca sativa L., lettuce, germination, phenolic compounds, moisture stress, allelopathy, seed     

Effects of Common Forage Phenolic Acids on Escherichia coli O157:H7 Viability in Bovine Feces

Ruminant animals are carriers of Escherichia coli O157:H7, and the transmission of E. coli O157:H7 from cattle to the environment and to humans is a concern. It is unclear if diet can influence the survivability of E. coli O157:H7 in the gastrointestinal system or in feces in the environment. Feces from cattle fed bromegrass hay or corn silage diets were inoculated with E. coli O157:H7, and the survival of this pathogen was analyzed. When animals consumed bromegrass hay for <1 month, viable E. coli O157:H7 was not recovered after 28 days postinoculation, but when animals consumed the diet for >1 month, E. coli O157:H7 cells were recovered for >120 days. Viable E. coli O157:H7 cells in feces from animals fed corn silage were detected until day 45 and differed little with the time on the diet. To determine if forage phenolic acids affected the viability of E. coli O157:H7, feces from animals fed corn silage or cracked corn were amended with common forage phenolic acids. When 0.5% trans-cinnamic acid or 0.5% para-coumaric acid was added to feces from silage-fed animals, the E. coli O157:H7 death rate was increased significantly (17-fold and 23-fold, respectively) compared to that with no addition. In feces from animals fed cracked corn, E. coli O157:H7 death rates were increased significantly with the addition of 0.1% and 0.5% trans-cinnamic acid (7- and 13-fold), 0.1% and 0.5% p-coumaric acid (3- and 8-fold), and 0.5% ferulic acid (3-fold). These data suggest that phenolic acids common to forage plants can decrease viable counts of E. coli O157:H7 shed in feces.     

Phenolic acid content,antioxidant and cytotoxic activities of four Kalanchoë species

Phenolic acid composition, antioxidant, and cytotoxic activities in leaves of four Kalanchoe (Crassulaceae) species were evaluated. Determination of phenolic acid contents were conducted by an optimized LC–ESI-MS/MS method. The results show that Kalanchoe daigremontiana Raym.-Hamet & H. Perrier (using ASE extraction) and Kalanchoe pinnata (Lam.) Pers. contain the highest amounts of phenolic acids, while Kalanchoe nyikae Engl. the lowest ones. Among phenolic acids ferulic, caffeic and protocatechuic acids were occurring in the highest quantities in the analysed species. The greatest amounts of ferulic and protocatechuic acids were found in K. daigremontiana and K. pinnata. Moreover, the antiradical and cytotoxic activities of Kalanchoe extracts were investigated. All tested extracts possessed antioxidant activity. The obtained IC₅₀ values (μg/mL) ranged from 49.9 μg/mL to 1410 μg/mL, indicating a large variation of the activity of the analysed extracts. Cytotoxicity assays revealed dose-dependent effects in the cells lines tested. Only K. pinnata extract showed a high cytotoxicity against the H-9 human T cell line. Other extracts (K. daigremontiana, Kalanchoe milloti, K. nyikae) showed more pronounced cytotoxicity towards J45.01 cells (human acute lymphoblastic leukaemia T cells).The present study demonstrated that Kalanchoe extracts have significant antioxidant and cytotoxic effects. This suggests that these species can be used as new sources of natural antioxidants and potential anticancer compounds.     

Biotransformation of Ferulic Acid to 4-Vinylguaiacol by Enterobacter soli and E. aerogenes

We investigated the conversion of ferulic acid to 4-vinylguaiacol (4-VG), vanillin, vanillyl alcohol, and vanillic acid by five Enterobacter strains. These high-value chemicals are usually synthesized by chemical methods but biological synthesis adds market value. Ferulic acid, a relatively inexpensive component of agricultural crops, is plentiful in corn hulls, cereal bran, and sugar-beet pulp. Two Enterobacter strains, E. soli, and E. aerogenes, accumulated 550–600?ppm amounts of 4-VG when grown in media containing 1,000?ppm ferulic acid; no accumulations were observed with the other strains. Decreasing the amount of ferulic acid present in the media increased the conversion efficiency. When ferulic acid was supplied in 500, 250, or 125?ppm amounts E. aerogenes converted ~72?% of the ferulic acid present to 4-VG while E. soli converted ~100?% of the ferulic acid to 4-VG when supplied with 250 or 125?ppm amounts of ferulic acid. Also, lowering the pH improved the conversion efficiency. At pH 5.0 E. aerogenes converted ~84?% and E. soli converted ~100?% of 1,000?ppm ferulic acid to 4-VG. Only small, 1–5?ppm, accumulations of vanillin, vanillyl alcohol, and vanillic acid were observed. E. soli has a putative phenolic acid decarboxylase (PAD) that is 168 amino acids long and is similar to PADs in other enterobacteriales; this protein is likely involved in the bioconversion of ferulic acid to 4-VG. E. soli or E. aerogenes might be useful as a means of biotransforming ferulic acid to 4-VG.     

In vitro and in vivo conjugation of dietary hydroxycinnamic acids by UDP-glucuronosyltransferases and sulfotransferases in humans

Hydroxycinnamic acids are a class of phenolic antioxidants found widely in dietary plants. Their biotransformation in the human organism primarily involves Phase II conjugation reactions. In this study, activities of UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) towards major dietary hydroxycinnamic acids (caffeic, dihydrocaffeic, dihydroferulic, ferulic and isoferulic acids) were investigated. Conjugate formation was evaluated using human liver and intestinal S9 homogenates, and in vitro characterization was carried out using recombinant human UGTs and SULTs. Analysis of the kinetics of hydroxycinnamic acid conjugation in human S9 homogenates revealed that intrinsic clearance (V_max/K_m) is much greater for sulfation than for glucuronidation. Assessment of activity using a panel of recombinant human SULTs showed that SULT1A1 is most active in the sulfation of caffeic, dihydrocaffeic and isoferulic acids, while SULT1E1 is most active in the sulfation of ferulic and dihydroferulic acids. Only isoferulic acid was significantly glucuronidated by human liver S9 homogenates, explained by the high activity of liver-specific UGT1A9. Studies on the kinetics of active SULTs and UGTs demonstrated a markedly lower K_m for SULTs. To further corroborate our findings, we carried out an intervention study in healthy humans to determine the hydroxycinnamic acid conjugates in urine after consumption of hydroxycinnamate-rich coffee (200 ml). Analysis showed that sulfates are the main conjugates in urine, with the exception of isoferulic acid, which is mainly glucuronidated. These data suggest that sulfates are the predominant hydroxycinnamic acid conjugates in humans, and that SULT mediated sulfation is a major factor determining the bioavailability of hydroxycinnamic acids in vivo.     

Highly Efficient Biotransformation of Eugenol to Ferulic Acid and Further Conversion to Vanillin in Recombinant Strains of Escherichia coli

The vaoA gene from Penicillium simplicissimum CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in Escherichia coli XL1-Blue under the control of the lac promoter, together with the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp. strain HR199, respectively. Resting cells of the corresponding recombinant strain E. coli XL1-Blue(pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter⁻¹. This biotransformation was scaled up to a 30-liter fermentation volume. The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture. The maximum concentration of ferulic acid obtained was 14.7 g liter⁻¹ after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol. In a two-step biotransformation, E. coli XL1-Blue(pSKvaomPcalAmcalB) was used to produce ferulic acid from eugenol and, subsequently, E. coli(pSKechE/Hfcs) was used to convert ferulic acid to vanillin (J. Overhage, H. Priefert, and A. Steinbüchel, Appl. Environ. Microbiol. 65:4837-4847, 1999). This process led to 0.3 g of vanillin liter⁻¹, besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter⁻¹. The genes ehyAB, encoding eugenol hydroxylase of Pseudomonas sp. strain HR199, and azu, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in E. coli XL1-Blue.     

Methods for the isolation and purification of ethanol-insoluble,phenolic esters in Mentha arvensis

Previous kinetic, isotopic studies have suggested that ‘insoluble’ phenolic esters may be precursors of lignin. Heretofore, the ‘insoluble’ esters have been detected by the chromatographic examinations of gross hydrolysis products of ethanol-insoluble resides and/or acetone powders. We have developed new methods for the isolation and purification of certain of the ethanol-insoluble, phenolic esters of Mentha arvensis. ‘Insoluble’ conjugates of caffeic, ferulic and p-coumaric acids were purified and were shown to be electro-phoretically and chromatographically homogeneous. These compounds were distinguished on the basis of their anionic mobility at pH 1·9. A second pool of caffeic acid was associated with a high MW fraction. Two acylated anthocyanins containing p-coumaric acid and caffeic acid were also obtained from acetone powders.     

Antioxidant activity of extracts from Thalictrum minus suspension culture

Low meadow-rue (Thalictrum minus L.) antioxidant complex was studied in cell extracts and culture medium. Its activity was expressed as total polyphenol content in ferulic acid equivalents. In these model systems (cell extracts and culture medium) the inhibition of lipid oxidation and diphenylpicrylhydrazine reduction (EC₅₀ = 12–15 μg/ml) were observed. At the phenolic compound concentration of 8–15 μg/ml, the reducing capacity of cell extracts was equivalent to 1.5 mM ascorbic acid. At the same time, berberine, a major alkaloid synthesized by the culture, manifested a low antioxidant activity. The analysis of phenolic acid composition in low meadow-rue showed that one of the main components of its antioxidant system were caffeic, gallic, chlorogenic, and ferulic acids.