Protein Engineering

Biography

Biography: Barindra Sana

Abstract

Lignin is a potential renewable raw material for synthesis of various value-added chemicals that can substitute fossil-derived consumer products. Huge lignin biomass is produced as by-product of paper industry while cellulosic components of plant biomass are utilized in paper pulp. Instead of vast potential, lignin remains the least exploited component of plant biomass due to its extremely complex cross-linked three dimensional structures that cannot be efficiently degraded by currently available enzymes. Effective lignin degrading enzymes could be developed by enzyme engineering. Directed evolution is a strong tool for enhancing activity of currently available enzymes but application of this technique for improving efficiency of lignin degrading enzyme is limited due to unavailability of any high throughput screening method. With an objective of detecting the lignin degradation products, we identified two E. coli promoters that are upregulated by potential lignin degradation products (e.g., vanillin, acetovanillone, guaiacol and veratraldehyde) and recombinantly placed a ‘very green fluorescence protein’ (vGFP) gene under control of these promoters within a customized plasmid. The whole cell sensor was developed by transforming E. coli cell with these constructs. Although the constructs showed some leaky expression, the cells responded by increasing fluorescence while grown in the presence of the lignin degradation products which was detected by FACS. The response was dose-dependent. Both sensors showed best response to vanillin as an individual compound but better response was achieved with the mixture of the lignin degradation products at a certain composition. Once the problem of leaky expression is solved, this technique could be potentially used for directly screening of libraries grown on agar plate.