Metabolic engineering strategies for caffeic acid production in Escherichia coli

Caffeic acid (CA; 3,4-dihydroxycinnamic acid) is an aromatic compound obtained by the phenylpropanoid pathway. This natural product has antioxidant, antitumor, antiviral, and anti-inflammatory activities. It is also a precursor of CA phenethyl ester (CAPE), a compound with potential as an antidiabetic and liver-protective agent. CA can be found at low concentrations in plant tissues, and hence, its purification is difficult and expensive. Knowledge regarding the pathways, enzymes, and genes involved in CA biosynthesis has paved the way for enabling the design and construction of microbial strains with the capacity of synthesizing this metabolite. In this review, metabolic engineering strategies for the generation of Escherichia coli strains for the biotechnological production of CA are presented and discussed.

Type C feruloyl esterase from Aspergillus ochraceus: a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Background: Aspergillus ochraceus was isolated from coffee pulp and selected as an interesting hydroxycinnamoyl esterase strain producer, using an activity microplate high-throughput screening method. In this work, we purified and characterized a new type C A. ochraceus feruloyl esterase (AocFaeC), which synthesized specifically butyl hydroxycinnamates in a ternary solvent system. Results: AocFaeC was produced by solid state fermentation, reaching its maximal activity (1.1 U/g) after 48 h of culture. After purification, the monomeric protein (34 kDa) showed a specific activity of 57.9 U/mg towards methyl ferulate. AocFaeC biochemical characterization confirmed its identity as a type C feruloyl esterase and suggested the presence of a catalytic serine in the active site. Its maximum hydrolytic activity was achieved at 40°C and pH 6.5 and increased by 109 and 77% with Ca2+ and Mg2+, but decreased by 90 and 45% with Hg2+ and Cu2+, respectively. The initial butyl ferulate synthesis rate increased from 0.8 to 23.7 nmol/min after transesterification condition improvement, using an isooctane:butanol:water ternary solvent system, surprisingly the synthesis activity using other alcohols was negligible. At these conditions, the synthesis specific activities for butyl p-coumarate, sinapinate, ferulate, and caffeate were 87.3, 97.6, 168.2, and 234 U/µmol, respectively. Remarkably, AocFaeC showed 5 folds higher butyl caffeate synthesis rate compared to type B Aspergillus niger feruloyl esterase, a well-known enzyme for its elevated activity towards caffeic acid esters. Conclusions: Type C feruloyl esterase from A. ochraceus is a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Dinámica de polifenoles en ephedra tweediana fisch & C. A. Mey. emend. J. H. Hunz. (Ephedraceae) / Polyphenol dinamics in ephedra tweediana fisch & C. A. Mey. emend. J. H. Hunz. (Ephedraceae)

Ephedra tweediana (Ephedraceae), conocida como “tramontana”, es empleada en la medicina popular como antiasmático. Se analizaron comparativamente los extractos acuosos de los tallos herbáceos, lignificados y partes subterráneas, provenientes de ejemplares femeninos y masculinos. En los tallos lignificados se determinó la presencia de proapigeninidina (cuya identidad fue corroborada por comparación con un testigo de apigeninidina sometido a estudios de FAB-MS, UV, HPLC, 1H-NMR y espectroscopía IR); mientras que en los órganos subterráneos se determinó la presencia de proapigeninidina y propelargonidina (la identidad de la pelargonidina fue establecida por comparación con un testigo de pelargonidina sometido a estudios de TLC/HPTLC y espectroscopía UV-Visible). Estos compuestos no se observaron en los tallos herbáceos. Los tallos herbáceos presentaron las mayores concentraciones de flavonoides y ácidos hidroxicinámicos totales. Los órganos subterráneos presentaron la mayor concentración de taninos y proantocianidinas. En los tallos herbáceos se detectó una reacción fuertemente positiva para flavonoides. No se observó reacción positiva para proantocianidinas. En el tallo aéreo lignificado se observó una reacción positiva para flavonoides y proantocianidinas a nivel de la peridermis. En los órganos subterráneos, los flavonoides y proantocianidinas se localizaron principalmente en los tejidos más externos. Este trabajo constituye el primer aporte a la dinámica de polifenoles de E. tweediana.

Ephedra tweediana (Ephedraceae), known as "tramontana" is used in folk medicine as antiasthmatic. Aqueous extracts obtained from young stems, woody stems and underground parts were analyzed and compared. In lignified stems was detected proapigeninidin (whose identity was confirmed by comparison with a control apigeninidin subjected to FAB-MS, UV, HPLC, 1H-NMR and IR spectroscopy), while underground organs were detected proapigeninidin and propelargonidin (by comparison with a control pelargonidin whose identity was established by studies of TLC/HPTLC, and UV-visible spectroscopy). These compounds were not observed in the herbaceous stems. The herbaceous stems had the highest concentrations of total flavonoids and hydroxycinnamic acids. Uderground organs had the highest concentration of tannins and proanthocyanidins. In herbaceous stems a strong positive reaction for flavonoids was detected. No positive reaction was observed for proanthocyanidins. In the periderm of woody aerial stem a positive reaction for flavonoids and proanthocyanidins were observed. In the underground organs, flavonoids and proanthocyanidins were located mainly in the more external tissues. This work is the first contribution to the dynamic of E. tweediana polyphenols.

Synthesis of hydroxycinnamic acid derivatives as mitochondria-targeted antioxidants and cytotoxic agents

In order to develop agents with superior chemopreventive and chemotherapeutic properties against hepatocellular carcinomas, mitochondria-targeted hydroxycinnamic acids (MitoHCAs) were synthesized by conjugation with a triphenylphosphonium cation. These synthetic compounds were evaluated for their antioxidant activities in hepatic mitochondria, including against OHand ROOinduced lipid peroxidation. HOproduction was decreased significantly by increasing glutathione peroxidase and catalase activities. In addition, cell proliferation data from three cell lines (HepG2, L02 and WI38) indicated that the MitoHCAs were selective for cancer cells. Interestingly, the MitoHCAs both with or without Catriggered mitochondrial dysfunction by inducing mitochondrial swelling, collapsing the mitochondrial membrane potential and causing cytochromerelease. In particular, an inhibitor of the mitochondrial permeability transition pore (mPTP), cyclosporin A, attenuated mitochondrial damage and cell apoptosis, indicating that mPTP may be involved in the antiproliferative activity of MitoHCAs. Further studies focused on structural optimization of these compounds are onging.

Polifenoles y actividad antioxidante en Equisetum giganteum L. (Equisetaceae) / Polyphenols and antioxidant activity in Equisetum giganteum L. (Equisetaceae)

Equisetum giganteum L. (Equisetaceae) is a species native to South America and Central America, which is used in traditional medicine as a diuretic and in treating of various diseases. The aim of this paper was to study the polyphenol profile and antioxidant activity of extracts obtained from lateral branches and main stem of the plants. Quali- and quantitative differences were detected with higher contents of phenols, tannins, condensed tannins, flavonoids and hydroxicinnamic acids in the lateral branches. The proanthocyanidins propelargonidin and procyanidin only were detected in lateral branches. These extracts showed the highest antioxidant activity. Due to their higher concentration and chemical composition, lateral branches could be considered as a sort of source of compounds that would act as antioxidant.

Equisetum giganteum L. (Equisetaceae) es una especie nativa de Sudamérica y América Central, que es empleada en la medicina tradicional como diurético y en el tratamiento de diversas patologías. El objetivo de este trabajo fue estudiar el perfil de polifenoles y la actividad antioxidante de extractos obtenidos de las ramas laterales (tallos finos), tallos entrenudos y tallos basales. Se detectaron diferencias cuali-cuantitativas, con mayor contenido de fenoles, taninos, taninos condensados, flavonoides y ácidos hidroxicinámicos en las ramas laterales. Las proantocianidinas propelargonidina y procianidina sólo se detectaron en las ramas laterales. En estos extractos se detectó la mayor actividad antioxidante. Debido a la alta concentración y composición química, los tallos laterales podrían ser empleados como fuente de compuestos con actividad antioxidante.

Dinámica de polifenoles de “Cedrón” (Aloysia citrodora Palau -Verbenaceae-) en relación al desarrollo foliar / Polyphenol dinamics on “cedrón” (Aloysia citrodora Palau -Verbenaceae-) according to leave development

Leaf infusions from Aloysia citrodora Palau –Verbenaceae-, known as “cedrón” were analyzed in order to establish the possible influence of their development on polyphenols profile. Chromatographic fingerprints of infusions from young and full expanded leaves were performed and total phenols, total tannins, total flavonoids and total hydroxycinnamic acids were quantified. Young leaves had significative higher total phenols, total flavonoids and total hydroxycinnamic acids concentrations than full expanded leaves. This research suggest that the analyzed parameter should be considered when elaborating products from “cedrón” leaves, as the variation in compound concentration may translate in different biological activities.

Se analizaron las infusiones realizadas a partir de las hojas de Aloysia citrodora Palau –Verbenaceae-, conocida vulgarmente como “cedrón”, con el objeto de establecer la influencia del grado de desarrollo foliar en el perfil de polifenoles. Se obtuvieron los perfiles cromatográficos y se cuantificaron los fenoles totales, taninos totales, flavonoides totales y ácidos hidroxicinámicos totales provenientes de los extractos acuosos de las hojas jóvenes y adultas. Se observaron diferencias cuali-cuantitativas en el perfil de polifenoles cuando se comparan las hojas jóvenes con las hojas adultas. En las hojas jóvenes, las concentraciones de fenoles totales, flavonoides totales y ácidos hidroxicinámicos totales resultaron ser significativamente superiores a las determinadas en las hojas adultas. Los estudios realizados sugieren que la variable aquí analizada debe ser considerada al momento de elaborar productos en base a hojas de cedrón, dado que las diferentes concentraciones detectadas podrían traducirse en diferentes actividades biológicas.