Enzymatic decarboxylation and carboxylation are emerging as prospective processes to produce high-value compounds under mild conditions. Ferulic acid decarboxylase Fdc1 catalyzes broad substrate tolerance against α, β-unsaturated carboxylic acids, and provides green routes for carbon dioxide fixation with the reversible carboxylation, while the activity of the enzyme is limited by the indispensable cofactor prenylated flavin (prFMN), which is unstable and is rarely detected in nature. In this study, a prFMN efficient synthesis route was built using six exogenous enzymes introduced intoE. colicells, leading to the construction of a powerful cell catalyst named SC-6. Based on the metabolic analysis, the results indicated that the reduction of FMN to FMNH2was the bottleneck in prFMN synthesis pathway, and introducing FMN reductase increased the production of prFMN 3.8-fold compared with the common flavin prenyltransferase UbiX overexpression strain. Using SC-6 cell catalyst, the decarboxylation activity of Fdc1 increased more than 20 times with cinnamic acid and 4-acetoxycinnamic acid as substrates. Furthermore, the reversible carboxylation reaction was carried out, and the cell catalyst presented 20 times carbon dioxide fixation activity using styrene to produce cinnamic acid. Finally, the maximum yield of cinnamic acid catalyzed by SC-6 achieved 833.68 ± 34.51 mM·mg−1in two hours. The constructed prFMN pathway in vivo provides fundamentals for efficient decarboxylation and carbon fixation reactions catalyzed by prFMN-dependent enzymes.Keywords:cell catalyst;prFMN;decarboxylase;carboxylation;cinnamic acid
Sichuan Paocai is a kind of fermented vegetable with rich microorganism and enzyme resources. Based on our previous metatranscriptomics and flavor-oriented metabolomics studies, three potential phenolic acid decarboxylases Lv PAD, Dh FDC1 and Lv UbiD were mined from environmental RNA of industrial Sichuan Paocai, and their catalytic function were verified by heterologous expression . Lv PAD, which was derived from Lactobacillus vermolensis and had the highest catalytic activity among the three enzymes, was selected for further purification and characterization. It was optimally active at pH 6.0 and 35 °C, had good thermal stability and pH tolerance, and did not require specific cofactors . Kinetic parameters showed that Lv PAD could catalyze the decarboxylation of p -coumaric acid, ferulic acid and caffeic acid with the k cat / K m values of 460.6 ± 0.1, 23.7 ± 0.09 and 118.2 ± 0.1 mM −1 s −1 , respectively, and the products of the first two substrates were 4-vinylphenol and 4-vinylguaiacol with aromatic characteristics. Moreover, Lv PAD was successfully used to preliminarily strengthen the vinylphenol aroma of industrial Paocai, which could shorten the fermentation time to a certain extent in practical production. Therefore, this work not only validated the important role of Lv PAD in the production of aromatic phenols in Paocai, but also facilitated the mining of more enzymes from Paocai to improve the flavor and production process in the future.
危险品化学品经营许可证（带存储）