Terminal Alkenes from Acrylic Acid Derivatives via Non-Oxidative Enzymatic Decarboxylation by Ferulic Acid Decarboxylases

Abstract

Fungal ferulic acid decarboxylases (FDCs) belong to the UbiD-family of enzymes and catalyse the reversible (de)carboxylation of cinnamic acid derivatives through the use of a prenylated flavin cofactor. The latter is synthesised by the flavin prenyltransferase UbiX. Herein, we demonstrate the applicability of FDC/UbiX expressing cells for both isolated enzyme and whole-cell biocatalysis. FDCs exhibit high activity with total turnover numbers (TTN) of up to 55000 and turnover frequency (TOF) of up to 370 min^-1. Co-solvent compatibility studies revealed FDC's tolerance to some organic solvents up 20 % v/v. Using the in-vitro (de)carboxylase activity of holo-FDC as well as whole-cell biocatalysts, we performed a substrate profiling study of three FDCs, providing insights into structural determinants of activity. FDCs display broad substrate tolerance towards a wide range of acrylic acid derivatives bearing (hetero)cyclic or olefinic substituents at C3 affording conversions of up to >99 %. The synthetic utility of FDCs was demonstrated by a preparative-scale decarboxylation.

Keywords: Biocatalysis; Decarboxylation; Ferulic acid decarboxylase; Prenylated flavin; Terminal alkenes.

Scheme 1

Enzymatic decarboxylation of α,β‐unsaturated carboxylic acids.

Scheme 2

Substrates (1 a–37 a) decarboxylated by FDCs and their corresponding products (1 b–37 b).

Figure 1

Substrates rejected by FDC (conversion <1 %), for standard conditions see Table 1.

Figure 2

Mechanism and substrate scope of ferulic acid decarboxylases (FDCs). a) Active site of Aspergillus niger FDC (AnFDC) in complex with α‐fluorocinnamic acid (PDB code 4ZAB). A transparent surface reveals the solvent accessible surface on the re side of the prFMN that is complementary in shape to the substrate. In addition, a water filled cavity is present near the cofactor ribityl moiety (indicated by circle), providing ample space for m‐ and p‐substitutions of the aromatic ring. Potential steric constraint occurs with cinnamic acid derivatives bearing bulky substituents at the α‐carbon (R¹) to the carboxylate or o‐substitutions of the aromatic ring (R⁴). b) A general mechanism proposed for reversible decarboxylation of acrylic acid derivatives by prFMN in FDC enzymes via 1,3 dipolar cycloaddition.

Figure 3

Decarboxylation of sorbic acid (36 a) by ScFDC in the presence of organic solvents. Reaction conditions: NaP_i (100 mM, pH 6.0), whole lyophilised cells of E. coli containing ScFDC (30 mg mL⁻¹), substrate (10 mM), organic co‐solvents (5–20 % v/v), 30 °C, 120 rpm, 18 h. Conversions were determined by calibrated RP‐HPLC.

Scheme 3

Substrates tested in the carboxylation direction with ScFDC (conversions <1 %). The arrow indicates the expected carboxylation site. Reaction conditions using KHCO₃: NaP_i (100 mM, pH 5.5), whole lyophilised cells of E. coli containing ScFDC (30–50 mg mL⁻¹), 10 mM substrate (17 b, 36 b, 61, 62), KHCO₃ (0.5–3 M), 30 °C, 120 rpm, 18–20 h, 5–20 % v/v DMSO or DME. Reaction conditions using CO₂ (gas): NaP_i (250 mM, pH 7.5), whole lyophilised cells of E. coli containing ScFDC (100 mg mL⁻¹), 10 mM substrate (17 b), 30 bar CO₂, 30 °C, 50 rpm, 18 h, 5 % v/v DMSO. Conversions were determined by calibrated RP‐HPLC.