Directed evolution of an enzyme requires a unique evaluation method to determine specificity and kinetics. Often, the monitoring technique must be adapted for each enzyme engineered. Raman spectroscopy has the capability to provide a high-throughput and universally applicable evaluation method for engineered enzyme activity. A major advantage of this approach is that is can be applied directly to E. coli colonies expressing an enzyme of interest on agar plates flooded with substrate. Among the challenges with using Raman intensity data directly to monitor enzyme activity include (i) the variable nature of procuring Raman spectra and (ii) the lack of relationships between molecular structure and concentration with signal intensities at different wavenumbers. In response, we have implemented a computational pipeline that applies discriminant analysis of principal components (DAPC) of Raman spectra to elucidate critical similarities and differences among spectra to probe enzyme activity. As a proof of concept, DAPC was applied to β-galactosidase expression by E. coli to illustrate the ability to detect concentration dependence of substrate, time dependence in solution, and time dependence of overexpressed enzyme activity. This technique has also been applied to investigate engineered enzyme substrate promiscuity of L-malyl-CoA lyase and malonyl-CoA reductase. The computational pipeline has been encoded in MATLAB and is now distributable as a Raman Data Analysis (RDA) Toolbox.

Monitoring directed evolution of enzymes with Raman spectroscopy

253rd National Meeting (2017)

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