Engineering the Biosynthesis of prFMN Promotes the Conversion between Styrene/CO2 and Cinnamic Acid Catalyzed by the Ferulic Acid Decarboxylase Fdc1

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 & alpha;, & beta;-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 into E. coli cells, 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 FMNH2 was 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 & PLUSMN; 34.51 mM & BULL;mg(-1) in two hours. The constructed prFMN pathway in vivo provides fundamentals for efficient decarboxylation and carbon fixation reactions catalyzed by prFMN-dependent enzymes.

Automated summary

Enzymatic decarboxylation and carboxylation processes are promising for producing high-value compounds under mild conditions. This study developed an efficient prFMN synthesis pathway by introducing six exogenous enzymes into E. coli cells, resulting in a powerful cell catalyst named SC-6. Using SC-6, the decarboxylation activity of Fdc1 increased by over 20 times and the reversible carboxylation reaction was achieved with high carbon dioxide fixation activity. The maximum yield of cinnamic acid catalyzed by SC-6 reached 833.68±34.51 mM·mg(-1). This constructed prFMN pathway provides the basis for efficient decarboxylation and carbon fixation reactions catalyzed by prFMN-dependent enzymes.