Biochemistry
Biochemistry
5 years ago

Engineering Erg10 Thiolase from Saccharomyces cerevisiae as a Synthetic Toolkit for the Production of Branched-Chain Alcohols

Engineering Erg10 Thiolase from Saccharomyces cerevisiae as a Synthetic Toolkit for the Production of Branched-Chain Alcohols
Ana García-Sánchez, Fernando López-Gallego, Sonia Huertas, Juande D. Miranda, Federico Gago, Antonio Morreale, Vicente Bernal, Pamela Torres-Salas, Rocío Morales-Jiménez, Victor Barrera, María del Mar González-Barroso, Javier Calvo, Sergio González-Barrera, Aurora Mañas-Fernández, José L. Torres, Ana Alcalde-Bascones, Marta Sagrera Polo, Pedro A. Sánchez-Murcia, Javier Martínez-Crespo, José M. Seoane
Thiolases catalyze the condensation of acyl-CoA thioesters through the Claisen condensation reaction. The best described enzymes usually yield linear condensation products. Using a combined computational/experimental approach, and guided by structural information, we have studied the potential of thiolases to synthesize branched compounds. We have identified a bulky residue located at the active site that blocks proper accommodation of substrates longer than acetyl-CoA. Amino acid replacements at such a position exert effects on the activity and product selectivity of the enzymes that are highly dependent on a protein scaffold. Among the set of five thiolases studied, Erg10 thiolase from Saccharomyces cerevisiae showed no acetyl-CoA/butyryl-CoA branched condensation activity, but variants at position F293 resulted the most active and selective biocatalysts for this reaction. This is the first time that a thiolase has been engineered to synthesize branched compounds. These novel enzymes enrich the toolbox of combinatorial (bio)chemistry, paving the way for manufacturing a variety of α-substituted synthons. As a proof of concept, we have engineered Clostridium’s 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol that is interesting as a branched model compound.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b01186

DOI: 10.1021/acs.biochem.7b01186

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