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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Directed evolution of a bacterial WS/DGAT acyltransferase: improving tDGAT from Thermomonospora curvata
Omar Santín1, Serena Galié1, Gabriel Moncalián1
1Departamento de Biología Molecular, Universidad de Cantabria and Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), CSIC-Universidad de Cantabria, Santander, Spain.
Abstract:
Some bacteria belonging to the actinobacteria and proteobacteria groups can accumulate neutral lipids expressing enzymes of the wax ester synthase/acyl coenzyme A: diacylglycerol acyltransferase (WS/DGAT) family. tDGAT is a WS/DGAT-like enzyme from Thermomonospora curvata able to produce TAGs and WEs when heterologously expressed in Escherichia coli. In this study, a protocol for the directed evolution of bacterial lipid-producing enzymes based on fluorimetry is developed and tested. tDGAT has been successfully evolved towards the improvement of TAG production with an up to 2.5 times increase in TAG accumulation. Mutants with no ability to produce TAGs but able to accumulate waxes were also selected during the screening. The localization of the mutations that enhance TAG production in the outer surface of tDGAT points out possible new mechanisms that contribute to the activity of this family of enzymes. This Nile red-based high throughput screening provides an evolution platform for other WS/DGAT-like enzymes.
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