Abstract
Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block. As an alternative to fossil fuel derived isobutene, we here develop a modified mevalonate pathway for the production of isobutene from glucose in vivo. The final step in the pathway consists of the decarboxylation of 3-methylcrotonic acid, catalysed by an evolved ferulic acid decarboxylase (Fdc) enzyme. Fdc belongs to the prFMN-dependent UbiD enzyme family that catalyses reversible decarboxylation of (hetero)aromatic acids or acrylic acids with extended conjugation. Following a screen of an Fdc library for inherent 3-methylcrotonic acid decarboxylase activity, directed evolution yields variants with up to an 80-fold increase in activity. Crystal structures of the evolved variants reveal that changes in the substrate binding pocket are responsible for increased selectivity. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group.
Highlights
Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block
A recent communication describes a pathway for the production of butadiene in E. coli where the decarboxylation of cis,cis-muconic acid is catalysed by ferulic acid decarboxylase (Fdc) from Saccharomyces cerevisiae enhanced by rational design[20]
Initial in vivo screening tested 15 UbiD homologues co-expressed with UbiX (E. coli K12) in E. coli for conversion of 3-methylcrotonic acid into isobutene as detected by gas chromatography
Summary
Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block. We report on discovery and optimization through directed evolution of Fdc decarboxylation activity with 3-methylcrotonic acid to produce isobutene. F404Y T405M V445P Q448W No crystal structure (Supplementary Table 1) and a directed evolution approach was taken to generate a variant of TaFdc with superior isobutene production activity and selectivity for 3-methylcrotonic acid over cinnamic acid (schematically presented in Supplementary Fig. 1).
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