Efficient synthesis of tyrosol from L-tyrosine via heterologous Ehrlich pathway in Escherichia coli

For the efficient conversion of L-tyrosine (L-Tyr) to tyrosol, which is an aromatic compound widely used in the pharmaceutical and chemical industries, a novel four-enzyme cascade pathway based on the Ehrlich pathway of Saccharomyces cerevisiae was designed and reconstructed in Escherichia coli. Then, the expression levels of the relevant enzymes were coordinated using a modular approach and gene duplication after the identification of the pyruvate decarboxylase from Candida tropicalis (CtPDC) as the rate-limiting enzymatic step. In situ product removal (ISPR) strategy with XAD4 resins was explored to avoid product inhibition and further improve tyrosol yield. As a result, the titer and conversion rate of tyrosol obtained were 35.7 g·L^-1 and 93.6%, respectively, in a 3-L bioreactor. Results presented here provide a potential enzymatic process for industrial production of tyrosol from cheap amino acids.     

深共熔溶剂-水混合物同步提取红景天苷和酪醇的响应面优化及其分离回收

以深共熔溶剂-水混合物（LAEG40）作为提取溶剂，红景天苷提取率为主要指标，酪醇提取率为辅助指标，在前期单因素实验的基础上，利用响应面法考察了液料比、提取温度、提取时间三个主要因素对红景天中红景天苷和酪醇同步提取的影响。得到的最佳提取条件为: 液料比12.05:1(mL/g)，提取温度60 ℃，提取时间35 min。在此条件下LAEG40对红景天苷的提取率可达到19.3552?0.6604 mg/g，酪醇提取率可达到1.7211?0.0585 mg/g，远高于传统溶剂水、乙醇对红景天苷和酪醇的提取率。为实现LAEG40提取红景天苷和酪醇的回收，进行了大孔树脂吸附分离的研究。经过一系列优化，获得的最佳吸附分离条件为：选用SP-825树脂装柱，每10g树脂上样30 mL LAEG40提取液，洗脱剂选用80 %乙醇，洗脱体积40 mL，洗脱流速1 mL/min，在此条件下红景天苷和酪醇的回收率分别可达60.47 %和85.07 %。     

Removal of phenols from aqueous solutions by emulsion liquid membranes

The present study deals with the extraction of phenols from aqueous solutions by using the emulsion liquid membranes technique. Besides phenol, two derivatives of phenol, i.e., tyrosol (2-(4-hydroxyphenyl)ethanol) and p-coumaric acid (4-hydroxycinnamic acid), which are typical components of the effluents produced in olive oil plants, were selected as the target solutes. The effect of the composition of the organic phase on the removal of solutes was examined. The influence of pH of feed phase on the extraction of tyrosol and p-coumaric was tested for the membrane with Cyanex 923 as an extractant. The use of 2% Cyanex 923 allowed obtaining a very high extraction of phenols (97-99%) in 5-6 min of contact time for either single solute solutions or for their mixtures. The removal efficiency of phenol and p-coumaric acid attained equivalent values by using the system with 2% isodecanol, but the removal rate of tyrosol was found greatly reduced. The extraction of tyrosol and p-coumaric acid from their binary mixture was also analysed for different operating conditions like the volume ratio of feed phase to stripping phase (sodium hydroxide), the temperature and the initial concentration of solute in the feed phase.     

Reconstruction of tyrosol synthetic pathways in Escherichia coli

Tyrosol is a pharmacologically active phenolic compound widely used in the medicine and chemical industries. Traditional methods of plant extraction are complicated and chemical synthesis of tyrosol is not commercially viable. In this study, a recombinant Escherichia coli strain was constructed by overexpressing the phenylpyruvate decarboxylase ARO10 from Saccharomyces cerevisiae, which could produce tyrosol from glucose. Furthermore, genes encoding key enzymes from the competing phenylalanine and tyrosine synthesis pathways and the repression protein TyrR were eliminated, and the resulting engineered strain generated 3.57 mmol·L^-1 tyrosol from glucose. More significantly, codon optimization of ARO10 increased expression and tyrosol titer. Using the novel engineered strain expressing codon-optimized AR10 in shake-flask culture, 8.72 mmol·L^-1 tyrosol was obtained after 48 h. Optimization of the induction conditions improved tyrosol production to 9.53 mmol·L^-1 (1316.3 mg·L^-1). A higher titer of tyrosol was achieved by reconstruction of tyrosol synthetic pathway in E. coli.     

Modulation of the superoxide anion production and MMP-9 expression in PMA stimulated THP-1 cells by olive oil minor components: Tyrosol and hydroxytyrosol

Extra virgin olive oil has been associated with a reduced incidence of risk factors for coronary heart disease also owing to the presence of antioxidant biophenols. Reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) have been implicated in numerous somatic illnesses, including cardiovascular disorders and cancer. The aim of this work was to study the capacity of virgin olive oil tyrosol (T) and hydroxytyrosol (HT) at impairing superoxide production and MMP-9 expressions in monocyte cells (THP-1) conveniently differentiated into adherent macrophages, taken as a model of human macrophages implicated in atheroma.O₂⁻ production was evaluated in THP-1 cells by using lucigenin as a specific chemiluminescent probe. Cells, after differentiation for 72 h, were preincubated in the presence of HT and T at increasing concentrations for 4, 15 and 24 h, and then, monocyte-like cells were stimulated by phorbol myristate acetate (PMA) and the O₂⁻-dependent luminescence was immediately recorded at 37 °C by means of a Luminometer. Enzymatic activity of MMP-9 derived from a medium of cells preincubated, or not, with T or HT was tested by zymography.As compared to the cells without treatment, cells preincubated with HT, showed a decrease of O₂⁻ production (50%) at 1 ??M for 15 h of preincubation time. Tyrosol fully prevented ROS overproduction at 15 h and, like HT displayed a high degree of protection but at higher concentrations and later time points (24 h). Gelatin zymograms revealed a reduction of the expression of MMP-9 in conditioned medium derived from T and HT-treated cells. These findings give further evidence in favour of olive oil consumption to counteract cardiovascular diseases.     

Enzymatic Synthesis of Tyrosol-Based Phenolipids: Characterization and Effect of Alkyl Chain Unsaturation on the Antioxidant Activities in Bulk Oil and Oil-in-Water Emulsion

Oxidative stability of lipids is one of the most important parameters affecting their quality. Lipase‐catalyzed lipophilic tyrosyl esters with an equivalent carbon alkyl chain but different degrees of unsaturation (C18:0 to C18:4n3) were prepared, characterized, and used as antioxidants. Free fatty acids and fatty acid ethyl esters (substrate molar ratio tyrosol: acyl donor, 1:10) were used as acyl donors and immobilized lipase from Candida antarctica was the biocatalyst (10 %). The phenolipids were isolated and characterized using ESI–MS, ¹H‐NMR, and ¹³C‐NMR. Peroxide value (PV) and para‐anisidine value (p‐AV) were measured to evaluate their antioxidant activities in bulk oil structured lipid (SL) and in an oil‐in‐water emulsion (SL‐based infant formula). No significant difference was found in yield and reaction time between the two types of acyl donors. However, as the unsaturation of the fatty acids increased the reaction time also increased. In SL, tyrosyl esters exhibited lower antioxidant activity than tyrosol whereas the addition of an alkyl chain enhanced the antioxidant efficiency of tyrosol in infant formula. Tyrosyl oleate was the most efficient antioxidant in the emulsion system followed by tyrosyl stearate and tyrosyl linoleate. These results suggest that the synthesized phenolipids may be used as potential antioxidants in lipid‐based products.     

Tyrosol exerts a protective effect against dopaminergic neuronal cell death in in vitro model of Parkinson’s disease

Experimental evidence suggests that tyrosol [2-(4-hydroxyphenyl)ethanol] exhibits potent protective activities against several pathogeneses. In this study, we evaluated the protective effect of tyrosol against 1-methyl-4-phenylpyridinium (MPP⁺)-induced CATH.a neuron cell death. Tyrosol dose-dependently protected CATH.a cells from MPP⁺-induced cell death and the protection was more apparent after prolong incubation (48 h). The data showed that tyrosol treatment suppressed the reduction of phospho-tyrosine hydroxylase level in CATH.a cells. Further, the compound repressed MPP⁺-induced depletion of mitochondrial membrane potential (Δψ_m) and thereby maintained intracellular ATP production in the cell. The cellular signalling pathway studies revealed that tyrosol protected CATH.a cells from MPP⁺-induced apoptotic signalling, most likely via activation of PI3K/Akt signalling pathway along with up-regulation of anti-oxidative enzymes (SOD-1 and SOD-2) and DJ-1 protein in the cell. Collectively, present study demonstrates that tyrosol significantly protects dopaminergic neurons from MPP⁺-induced degradation, and reveals potential neuroprotective mechanism of tyrosol.     

Photocatalytic promoted oxidation of phenolic mixtures: an insight into the operating and mechanistic aspects

The photocatalytic oxidation of a phenolic mixture (gallic acid, tyrosol and syringic acid) has been carried out in the presence of titanium dioxide and a selected inorganic peroxide, namely persulphate, monopersulphate or hydrogen peroxide. The results obtained in UVA irradiated solutions reveal that hydrogen peroxide is the most effective option. The influence of some relevant operating parameters (i.e. initial concentration of phenols, hydrogen peroxide or titanium dioxide) has been investigated. In all cases a saturation-like behaviour has been observed. Thus, an increase in oxidation efficiency is observed when raising, up to a certain value, the initial amounts of reagents/catalyst added to the reactor. Nevertheless, a further increase of any of the previous parameters does not lead to the expected enhancement of the process. From the concave shape of the parent compounds’ depletion profiles, it is suggested that the system proceeds through an autocatalytic route, likely involving the action of organic radicals. A pseudoempirical model has been used to acceptably model the experimental data obtained.     

Kinetics Of Competitive Ozonation Of Some Phenolic Compounds Present In Wastewater From Food Processing Industries

Ozonation of four phenolic compounds found in wastewater effluents from food manufacturing processes: Gallic (G-Ac) and p-hydroxybenzoic (pHB-Ac) acids, (+)-catechin ((+)-Cat) and tyrosol (Ty), has been carried out in ultrapure water. The results showed that the direct reaction between ozone and the organic compound seems to be the exclusive way of phenolic compounds elimination. A kinetic study of these reactions was completed by using a high concentration of phenolic substances (up to 3 g L^?1 total phenolic content) to simulate typical amounts of these compounds found in real wastewater. By means of a competitive method, rate constants of the direct reaction with ozone were determined at different pH. The following reactivity was found depending on pH: pHB-Ac < Ty < G-Ac < (+)-Cat in acidic conditions, pHB-Ac < Ty < G-Ac < (+)-Cat in neutral conditions and pHB-Ac < Ty for basic conditions. Finally, validation of the calculated rate constants was completed by checking the kinetic regime in which competitive reactions were developed.