Exploring prenylated flavin catalysed chemistry in ferulic acid decarboxylases

Abstract

Ferulic acid decarboxylases (Fdc) utilise the prenylated flavin cofactor to catalyse (de)carboxylation of a range of phenylacrylic and sorbic acids. The reaction proceeds via a 1,3-dipolar cycloaddition-cycloelimination mechanism that is driven by enzyme-induced strain. The substrate scope for Fdc has been limited to delta,gamma-unsaturated acrylic acids with no branching at alpha- and beta-carbons. We detect Fdc decarboxylation activity with 3-methylcrotonic acid to yield isobutene and apply directed evolution to an Fdc from Trichoderma atroviride to yield a variant with 80-fold increase in activity. This variant, in part of a modified mevalonate pathway, allows for the production of isobutene from glucose in vivo, as an alternative to fossil fuel derived isobutene. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group with crotonic acid acting as an inhibitor. In addition, we discover that a stable cycloadduct forms between prFMN and prenol, the alcohol equivalent of 3-methycrotonic acid. While the reaction equilibrium of Fdc catalysed (de)carboxylation is strongly towards decarboxylation, we demonstrate the reversibility of the reaction in Aspergillus niger Fdc by converting the carboxylated product to the respective aldehyde by carboxylic acid reductase. This approach leads to efficient functionalization of styrene to corresponding aldehyde through ambient CO2 fixation. We expand Fdc substrate scope by structure-guided laboratory evolution, yielding activity on a range of mono- and bicyclic aromatic compounds through a single mutation. Selected I327X variants demonstrate a 150-fold improvement in the conversion of coumarillic acid to benzofuran and CO2 and unlock reactivity with naphthoic acid.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Munro (Supervisor) & David Leys (Supervisor)