Dataset Information

Generation of 4-vinylguaiacol through a novel high-affinity ferulic acid decarboxylase to obtain smoke flavours without carcinogenic contaminants.

ABSTRACT: Traditional smoke flavours bear the risk of containing a multitude of contaminating carcinogenic side-products. Enzymatic decarboxylation of ferulic acid released from agro-industrial side-streams by ferulic acid esterases (FAE) enables the sustainable generation of pure, food grade 4-vinylguaiacol (4-VG), the impact compound of smoke flavour. The first basidiomycetous ferulic acid decarboxylase (FAD) was isolated from Schizophyllum commune (ScoFAD) and heterologously produced by Komagataella phaffii. It showed a molecular mass of 21 kDa, catalytic optima at pH 5.5 and 35°C, and a sequence identity of 63.6% to its next relative, a FAD from the ascomycete Cordyceps farinosa. The ScoFAD exhibited a high affinity to its only known substrate ferulic acid (FA) of 0.16 mmol L-1 and a turnover number of 750 s-1. The resulting catalytic efficiency kcat KM-1 of 4,779 L s-1 mmol-1 exceeded the next best known enzyme by more than a factor of 50. Immobilised on AminoLink Plus Agarose, ScoFAD maintained its activity for several days. The combination with FAEs and agro-industrial side-streams paves the way for a new generation of sustainable, clean, and safe smoke flavours.

SUBMITTER: Detering T

PROVIDER: S-EPMC7751879 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Generation of 4-vinylguaiacol through a novel high-affinity ferulic acid decarboxylase to obtain smoke flavours without carcinogenic contaminants.

Detering Thorben T Mundry Katharina K Berger Ralf G RG

PloS one 20201221 12

Traditional smoke flavours bear the risk of containing a multitude of contaminating carcinogenic side-products. Enzymatic decarboxylation of ferulic acid released from agro-industrial side-streams by ferulic acid esterases (FAE) enables the sustainable generation of pure, food grade 4-vinylguaiacol (4-VG), the impact compound of smoke flavour. The first basidiomycetous ferulic acid decarboxylase (FAD) was isolated from Schizophyllum commune (ScoFAD) and heterologously produced by Komagataella ph ...[more]

PMID: 33347481

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Project description:The interest towards ferulic acid decarboxylase (FDC), piqued by the enzyme's unique 1,3-dipolar cycloaddition mechanism and its atypic prFMN cofactor, provided several applications of the FDC mediated decarboxylations, such as the synthesis of styrenes, or its diverse derivatives, including 1,3-butadiene and the enzymatic activation of C-H bonds through the reverse carboligation reactions. While rational design-based protein engineering was successfully employed for tailoring FDC towards diverse substrates of interest, the lack of high-throughput FDC-activity assay hinders its directed evolution-based protein engineering. Herein we report a toolbox, useful for the directed evolution based and/or structure-guided protein engineering of FDC, which was validated representatively on the well described FDC, originary from Saccharomyces cerevisiae (ScFDC). Accordingly, the developed fluorescent plate-assay allows in premiere the FDC-activity screens of a mutant library in a high-throughput manner. Moreover, using the plate-assay for the activity screens of a rationally designed 23-membered ScFDC variant library against a substrate panel comprising of 16, diversely substituted cinnamic acids, revealed several variants of improved activity. The superior catalytic properties of the hits revealed by the plate-assay, were also supported by the conversion values from their analytical scale biotransformations. The computational results further endorsed the experimental findings, showing inactive binding poses of several non-transformed substrate analogues within the active site of the wild-type ScFDC, but favorable ones within the catalytic site of the variants of improved activity. The results highlight several 'hot-spot' residues involved in substrate specificity modulation of FDC, such as I189, I330, F397, I398 or Q192, of which mutations to sterically less demanding residues increased the volume of the active site, thus facilitated proper binding and increased conversions of diverse non-natural substrates. Upon revealing which mutations improve the FDC activity towards specific substrate analogues, we also provide key for the rational substrate-tailoring of FDC.

Project description:Effects of waterpipe smoking on lung pathobiology and carcinogenesis remain sparse despite the worldwide emergence of this tobacco vector. To address this gap, we investigated the effects of chronic waterpipe smoke (WPS) exposure on lung pathobiology, host immunity, and tumorigenesis using an experimental animal model that is prone to tobacco carcinogens and an exploratory observational analysis of human waterpipe smokers and nonsmokers. Mice exhibited elevated incidence of lung tumors following heavy WPS exposure (5 days/week for 20 weeks) compared to littermates with light WPS (once/week for 20 weeks) or control air. Lungs of mice exposed to heavy WPS showed augmented CD8+ and CD4+ T cell counts along with elevated protumor immune phenotypes including increased IL17A in T/B cells, PD-L1 on tumor and immune cells, and the proinflammatory cytokine IL1β in myeloid cells. RNA-sequencing (RNA-seq) analysis showed reduced antitumor immune gene signatures in animals exposed to heavy WPS relative to control air. We also performed RNA-seq analysis of airway epithelia from bronchial brushings of cancer-free waterpipe smokers and nonsmokers undergoing diagnostic bronchoscopy. Transcriptomes of normal airway cells in waterpipe smokers, relative to waterpipe nonsmokers, harbored gene programs that were associated with poor clinical outcomes in patients with lung adenocarcinoma, alluding to a WPS-associated molecular injury, like that established in response to cigarette smoking. Our findings support the notion that WPS exhibits carcinogenic effects and constitutes a possible risk factor for lung cancer as well as warrant future studies that can guide evidence-based policies for mitigating waterpipe smoking.Prevention relevancePotential carcinogenic effects of waterpipe smoking are very poorly understood despite its emergence as a socially acceptable form of smoking. Our work highlights carcinogenic effects of waterpipe smoking in the lung and, thus, accentuate the need for inclusion of individuals with exclusive waterpipe smoking in prevention and smoking cessation studies.

Dataset Information

Generation of 4-vinylguaiacol through a novel high-affinity ferulic acid decarboxylase to obtain smoke flavours without carcinogenic contaminants.

Publications

Generation of 4-vinylguaiacol through a novel high-affinity ferulic acid decarboxylase to obtain smoke flavours without carcinogenic contaminants.

Similar Datasets

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