Barindra Sana, Balamurugan Ramalingam, Sarada Raghavan, Chia Kuan Hui Burton, Niranjan Nagarajan, Jayasree Seayad and Farid J Ghadessy
Institute of Chemical and Engineering Sciences, Singapore
Genome Institute of Singapore, Singapore
Posters & Accepted Abstracts: J Biosens Bioelectron
Lignin is a potential renewable raw material for synthesis of various value-added chemicals that can substitute fossil-derived consumer products. Huge amounts of lignin is produced as a by-product of paper industry while cellulosic components of plant biomass are utilized for the production of paper pulp. Inspite of vast potential, lignin remains the least exploited component of plant biomass due to its extremely complex cross-linked three dimensional structures. Nature has provided a few enzymes known to degrade lignin biomass. However, till date there are no efficient processes available for enzymatic degradation of these extremely complex molecules. Development of effective lignin degrading enzymes may be possible by amending activity of some currently available enzymes, using protein engineering techniques. Directed evolution is one such protein engineering tool that could be used for this purpose but application of this technique for improving efficiency of potential lignin degrading enzymes is limited due to lack of an effective high throughput screening method. With an objective of detecting the lignin degradation products (LDPs), we identified E. coli promoters that are up-regulated by vanillin and a few other potential lignin degradation products. Seven potential promoters were identified by RNA-Seq analysis of E. coli BL21 cells pre-exposed to a sub-lethal dose of vanillin for different exposure times. A very green fluorescence protein (vGFP) gene was recombinantly placed under control of these promoters within a customized plasmid and transformed in E. coli BL21 cells to generate the whole cell biosensors. Fluorescence of two biosensors enhanced significantly while grown in the presence of the lignin degradation products (e.g. vanillin, acetovanillone and guaiacol) which was detected by fluorescence-activated cell sorting (FACS) analysis. The sensors did not show any increase of fluorescence by the presence of lignin, lignin model compounds or non-specific chemicals. The fluorescence change by the presence of LDPs was dose-dependent; one sensor can detect vanillin at the concentration as low as 0.5mM.
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