Anisyl alcohol

De Novo Biosynthesis of Anisyl Alcohol and Anisyl Acetate in Engineered Escherichia coli.[Pubmed:36779799]

J Agric Food Chem. 2023 Feb 13.

Anisyl alcohol and its ester anisyl acetate are both important fragrance compounds and have a wide range of applications in the cosmetics, perfumery, and food industries. The currently commercially available Anisyl alcohol and anisyl acetate are based on chemical synthesis. However, consumers increasingly prefer natural fragrance compounds. Therefore, it is of great significance to construct microbial cell factories to produce Anisyl alcohol and anisyl acetate. In this study, we first established a biosynthetic pathway in engineered Escherichia coli MG1655 for the production of Anisyl alcohol from simple carbon sources. We further increased the Anisyl alcohol production to 355 mg/L by the increasing availability of erythrose-4-phosphate and phosphoenolpyruvate. Finally, we further demonstrated the production of anisyl acetate by overexpressing alcohol acetyltransferase ATF1 for the subsequent acetylation of Anisyl alcohol to produce anisyl acetate. To our knowledge, this is the first report on the biosynthesis of Anisyl alcohol and anisyl acetate directly from a renewable carbon source.

Interfacial Composition of Surfactant Aggregates in the Presence of Fragrance: A Chemical Trapping Study.[Pubmed:35889205]

Molecules. 2022 Jul 6;27(14):4333.

In recent years, there has been increasing interest in daily-use chemical products providing a pleasant scent. The added fragrance molecules may induce microstructural transitions of surfactant aggregates, which further affect the physical and chemical properties of the products. Here, the effects of four types of aromatic alcohols (cinnamyl alcohol, phenyl ethanol, phenyl methanol and Anisyl alcohol) on cetyltrimethylammonium bromide (CTAB)/KBr aggregates were studied. The combined results from rheology, dynamic light scattering, and transmission electron microscopy measurements showed that cinnamyl alcohol induced significant micellar growth, while increases in micellar growth were less obvious for the other aromatic alcohols. The changes in the interfacial molarities of water, aromatic alcohol, and bromide ions during such transitions were studied using the chemical trapping method. Transitions resulting from added cinnamyl alcohol were accompanied by significant declines in interfacial water and bromide ion molarities, and a rise in interfacial alcohol molarity. The marked decrease in interfacial water molarity was not observed in previous studies of the octanol induced formation of wormlike micelles and vesicles, indicating that a different mechanism was presented in the current system. Nuclear magnetic resonance investigation showed that pi-pi stacking between cinnamyl alcohols, but not cation-pi interactions between alcohols and CTAB headgroups, facilitated the tight packing of alcohol molecules in CTAB aggregates and the repulsion of water from the interfacial region. The current study may provide a theoretical basis for the morphological regulation of surfactant aggregates in the presence of additives.

Agar plate assay for rapid screening of aryl-alcohol oxidase mutant libraries in Pichia pastoris.[Pubmed:35122934]

J Biotechnol. 2022 Feb 20;346:47-51.

Directed evolution is a powerful tool for developing biocatalysts with tailor-made properties for biocatalytic applications. High-throughput activity screening of large mutant libraries generated by e.g. means of directed evolution is usually performed in 96-well microtiter plates requiring, however, time-consuming and laborious enzyme expression, cell harvesting and activity measurements. In addition, automated liquid handling systems are needed to cope with the high number of colonies to be screened. Herein, we developed an agar plate-based assay for simple and fast screening of H(2)O(2)-producing aryl-alcohol oxidase (AAO) mutant libraries in Pichia pastoris. Buffered minimal methanol agar plates were supplemented with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), horseradish peroxidase (HRP) and the target substrate. AAO activity is visualized by formation of green zones around AAO-secreting P. pastoris colonies due to ABTS oxidation by HRP which is fueled with H(2)O(2) by AAO in course of substrate oxidation. Colonies were screened within 24 h for AAO activity using different AAO substrates like veratryl alcohol, p-Anisyl alcohol or trans,trans-2,4-hexadien-1-ol and even low AAO activity towards 5-hydroxymethylfurfural could be detected within 48 h. The developed agar plate-based assay can be extended to other substrates and might also be applied for fast and substrate-specific screening of other H(2)O(2)-producing oxidases in P. pastoris.

Comparison of the Phytochemical Composition and Antibacterial Activities of the Various Extracts from Leaves and Twigs of Illicium verum.[Pubmed:34206777]

Molecules. 2021 Jun 26;26(13):3909.

Previous studies have revealed the numerous biological activities of the fruits of Illicium verum; however, the activities of its leaves and twigs have remained undiscovered. The study aimed to investigate the phytochemical components and antibacterial activity of the various extracts from the leaves and twigs of Illicium verum. The herbal extracts were prepared by supercritical CO(2) extraction (SFE) and 95% ethanol extraction, followed by partition extraction based on solvent polarity. Analysis of antimicrobial activity was conducted through the usage of nine clinical antibiotic- resistant isolates, including Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii. Among the tested samples, the SFE extracts exhibited broader and stronger antibacterial activities against the test strains, with a range of MIC between 0.1-4.0 mg/mL and MBC between 0.2-4.5 mg/mL. Observations made through scanning electron microscopy revealed potential mechanism of the antimicrobial activities involved disruption of membrane integrity of the test pathogens. Evaluation of the chemical composition by gas chromatography-mass spectrometry indicated the presence of anethole, anisyl aldehyde, anisyl acetone and Anisyl alcohol within the SFE extracts, demonstrating significant correlations with the antibacterial activities observed. Therefore, the leaves and twigs of Illicium verum hold great potential in being developed as new natural antibacterial agents.

Production of Protocatechuic Acid from p-Hydroxyphenyl (H) Units and Related Aromatic Compounds Using an Aspergillus niger Cell Factory.[Pubmed:34154420]

mBio. 2021 Jun 29;12(3):e0039121.

Protocatechuic acid (3,4-dihydroxybenzoic acid) is a chemical building block for polymers and plastics. In addition, protocatechuic acid has many properties of great pharmaceutical interest. Much research has been performed in creating bacterial protocatechuic acid production strains, but no protocatechuic acid-producing fungal cell factories have been described. The filamentous fungus Aspergillus niger can produce protocatechuic acid as an intermediate of the benzoic acid metabolic pathway. Recently, the p-hydroxybenzoate-m-hydroxylase (phhA) and protocatechuate 3,4-dioxygenase (prcA) of A. niger have been identified. It has been shown that the prcA deletion mutant is still able to grow on protocatechuic acid. This led to the identification of an alternative pathway that converts protocatechuic acid to hydroxyquinol (1,3,4-trihydroxybenzene). However, the gene involved in the hydroxylation of protocatechuic acid to hydroxyquinol remained unidentified. Here, we describe the identification of protocatechuate hydroxylase (decarboxylating) (PhyA) by using whole-genome transcriptome data. The identification of phyA enabled the creation of a fungal cell factory that is able to accumulate protocatechuic acid from benzyl alcohol, benzaldehyde, benzoic acid, caffeic acid, cinnamic acid, cinnamyl alcohol, m-hydroxybenzoic acid, p-hydroxybenzyl alcohol, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, p-Anisyl alcohol, p-anisaldehyde, p-anisic acid, p-coumaric acid, and protocatechuic aldehyde. IMPORTANCE Aromatic compounds have broad applications and are used in many industries, such as the cosmetic, food, fragrance, paint, plastic, pharmaceutical, and polymer industries. The majority of aromatic compounds are synthesized from fossil sources, which are becoming limited. Plant biomass is the most abundant renewable resource on Earth and can be utilized to produce chemical building blocks, fuels, and bioplastics through fermentations with genetically modified microorganisms. Therefore, knowledge about the metabolic pathways and the genes and enzymes involved is essential to create efficient strategies for producing valuable aromatic compounds such as protocatechuic acid. Protocatechuic acid has many pharmaceutical properties but also can be used as a chemical building block to produce polymers and plastics. Here, we show that the fungus Aspergillus niger can be engineered to produce protocatechuic acid from plant-derived aromatic compounds and contributes to creating alternative methods for the production of platform chemicals. .

High-level expression of aryl-alcohol oxidase 2 from Pleurotus eryngii in Pichia pastoris for production of fragrances and bioactive precursors.[Pubmed:32949280]

Appl Microbiol Biotechnol. 2020 Nov;104(21):9205-9218.

The fungal secretome comprises various oxidative enzymes participating in the degradation of lignocellulosic biomass as a central step in carbon recycling. Among the secreted enzymes, aryl-alcohol oxidases (AAOs) are of interest for biotechnological applications including production of bio-based precursors for plastics, bioactive compounds, and flavors and fragrances. Aryl-alcohol oxidase 2 (PeAAO2) from the fungus Pleurotus eryngii was heterologously expressed and secreted at one of the highest yields reported so far of 315 mg/l using the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). The glycosylated PeAAO2 exhibited a high stability in a broad pH range between pH 3.0 and 9.0 and high thermal stability up to 55 degrees C. Substrate screening with 41 compounds revealed that PeAAO2 oxidized typical AAO substrates like p-Anisyl alcohol, veratryl alcohol, and trans,trans-2,4-hexadienol with up to 8-fold higher activity than benzyl alcohol. Several compounds not yet reported as substrates for AAOs were oxidized by PeAAO2 as well. Among them, cumic alcohol and piperonyl alcohol were oxidized to cuminaldehyde and piperonal with high catalytic efficiencies of 84.1 and 600.2 mM(-1) s(-1), respectively. While the fragrance and flavor compound piperonal also serves as starting material for agrochemical and pharmaceutical building blocks, various positive health effects have been attributed to cuminaldehyde including anticancer, antidiabetic, and neuroprotective effects. PeAAO2 is thus a promising biocatalyst for biotechnological applications. KEY POINTS: * Aryl-alcohol oxidase PeAAO2 from P. eryngii was produced in P. pastoris at 315 mg/l. * Purified enzyme exhibited stability over a broad pH and temperature range. * Oxidation products cuminaldehyde and piperonal are of biotechnological interest. Graphical abstract.

Aqueous solution photocatalytic synthesis of p-anisaldehyde by using graphite-like carbon nitride photocatalysts obtained via the hard-templating route.[Pubmed:35515447]

RSC Adv. 2020 May 21;10(33):19431-19442.

Graphite-like carbon nitride (GCN)-based materials were developed via the hard-templating route, using dicyandiamide as the GCN precursor and silica templates. That resulted in urchin-like GCN (GCN-UL), 3D ordered macroporous GCN (GCN-OM) and mesoporous GCN (GCN-MP). The introduction of silica templates during GCN synthesis produced physical defects on its surface, as confirmed by SEM analysis, increasing their specific surface area. A high amount of nitrogen vacancies is present in modified catalysts (revealed by XPS measurements), which can be related to an increase in the reactive sites available to catalyse redox reactions. The textural and morphological modifications induced in GCN an enhanced light absorption capacity and reduced electron/hole recombination rate, contributing to its improved photocatalytic performance. In the photocatalytic conversion of p-Anisyl alcohol to p-anisaldehyde in deoxygenated aqueous solutions under UV-LED irradiation, the GCN-UL was the best photocatalyst reaching 60% yield at 64% conversion for p-anisaldehyde production after 240 min of reaction. Under oxygenated conditions (air), the process efficiency was increased to 79% yield at 92% conversion only after 90 min reaction. The GCN-based photocatalyst kept its performance when using visible-LED radiation under air atmosphere. Trapping of photogenerated holes and radicals by selective scavengers showed that under deoxygenated conditions, holes played the primary role in the p-anisaldehyde synthesis. Under oxygenated conditions, the process is governed by the effect of reactive oxygen species, namely superoxide radicals, with a significant contribution from holes.

High turnover in electro-oxidation of alcohols and ethers with a glassy carbon-supported phenanthroimidazole mediator.[Pubmed:28989674]

Chem Sci. 2017 Sep 1;8(9):6493-6498.

Glassy carbon electrodes covalently modified with a phenanthroimidazole mediator promote electrochemical alcohol and ether oxidation: three orders of magnitude increase in TON, to approximately 15 000 in each case, was observed compared with homogeneous mediated reactions. We propose the deactivation pathways in homogeneous solution are prevented by the immobilization: modified electrode reversibility is increased for a one-electron oxidation reaction. The modified electrodes were used to catalytically oxidize p-Anisyl alcohol and 1-((benzyloxy)methyl)-4-methoxybenzene, selectively, to the corresponding benzaldehyde and benzyl ester, respectively.

Emission characteristics of allergenic terpenols in PM(2.5) released from incense burning and the effect of light on the emissions.[Pubmed:28131447]

Sci Total Environ. 2017 Apr 15;584-585:495-504.

This study investigated allergenic terpenol compounds in incense powder and smoke. The powder of two Thai brands contained higher concentrations of terpenols up to 6.15 times higher than those of two Taiwanese brands. Consequently, Thai incense makers face a higher potential risk of contact dermatitis than Taiwanese incense makers do. d-Limonene was the primary terpenol compound in the powder of Thai B (64.0%) and Thai Y (31.5%), sold in Thailand. By contrast, Anisyl alcohol was the primary terpenol compound in the powder of LST (40.3%) and SC (37.7%), sold in Taiwan. After the four brands of incense were ignited, their mean PM(2.5) emission factor was 18.02+/-6.20mgg(-1) incense. The PM(2.5) mass emission factors of the Taiwanese brands were far higher than those of the Thai brands, and so were the PM(2.5) terpenol emission factors, showing that the smokes of the Taiwanese incense were potentially more allergenic than those of the Thai incense. Geraniol, the most allergenic terpenol compound, was 2.8%-10.7% of total terpenol compounds in the powder of the four brands, yet was the main contributor to PM(2.5), constituting 66.3%-83.5% of terpenol compounds in the smokes of the four brands. Furthermore, geraniol exhibited an IP ratio, defined as the incense/powder (IP) ratio of terpenol-related species, >1 in all four brands, and >5 in the Taiwanese brands, suggesting a greater health risk with the smoke from the Taiwanese incense. The IP ratios of other terpenol species were all <1, indicating decomposition through combustion. Additionally, the light/darkroom ratios of the terpenol species were >1, meaning that the generation of PM(2.5) terpenol compounds was potentially enhanced by indoor lighting.

Characterization of a new aryl-alcohol oxidase secreted by the phytopathogenic fungus Ustilago maydis.[Pubmed:26452496]

Appl Microbiol Biotechnol. 2016 Jan;100(2):697-706.

The discovery of novel fungal lignocellulolytic enzymes is essential to improve the breakdown of plant biomass for the production of second-generation biofuels or biobased materials in green biorefineries. We previously reported that Ustilago maydis grown on maize secreted a diverse set of lignocellulose-acting enzymes including hemicellulases and putative oxidoreductases. One of the most abundant proteins of the secretome was a putative glucose-methanol-choline (GMC) oxidoreductase. The phylogenetic prediction of its function was hampered by the few characterized members within its clade. Therefore, we cloned the gene and produced the recombinant protein to high yield in Pichia pastoris. Functional screening using a library of substrates revealed that this enzyme was able to oxidize several aromatic alcohols. Of the tested aryl-alcohols, the highest oxidation rate was obtained with 4-Anisyl alcohol. Oxygen, 1,4-benzoquinone, and 2,6-dichloroindophenol can serve as electron acceptors. This GMC oxidoreductase displays the characteristics of an aryl-alcohol oxidase (E.C.1.1.3.7), which is suggested to act on the lignin fraction in biomass.