Development of a genetically programed vanillin-sensing bacterium for high-throughput screening of lignin-degrading enzyme libraries

Barindra Sana; Kuan Hui Burton Chia; Sarada S Raghavan; Balamurugan Ramalingam; Niranjan Nagarajan; Jayasree Seayad; Farid J Ghadessy

doi:10.1186/s13068-017-0720-5

Development of a genetically programed vanillin-sensing bacterium for high-throughput screening of lignin-degrading enzyme libraries

Biotechnol Biofuels. 2017 Feb 3:10:32. doi: 10.1186/s13068-017-0720-5. eCollection 2017.

Authors

Barindra Sana¹, Kuan Hui Burton Chia², Sarada S Raghavan¹, Balamurugan Ramalingam³, Niranjan Nagarajan², Jayasree Seayad³, Farid J Ghadessy¹

Affiliations

¹ p53 Laboratory, Agency for Science Technology And Research (ASTAR), 8A Biomedical Grove, #06-04/05 Neuros/Immunos, Singapore, 138648 Singapore.
² Genome Institute of Singapore, 60 Biopolis Street, Genome, #02-01, Singapore, 138672 Singapore.
³ Institute of Chemical and Engineering Sciences, 8 Biomedical Grove, Neuros, #07-01, Singapore, 138665 Singapore.

Abstract

Background: Lignin is a potential biorefinery feedstock for the production of value-added chemicals including vanillin. A huge amount of lignin is produced as a by-product of the paper industry, while cellulosic components of plant biomass are utilized for the production of paper pulp. In spite of vast potential, lignin remains the least exploited component of plant biomass due to its extremely complex and heterogenous structure. Several enzymes have been reported to have lignin-degrading properties and could be potentially used in lignin biorefining if their catalytic properties could be improved by enzyme engineering. The much needed improvement of lignin-degrading enzymes by high-throughput selection techniques such as directed evolution is currently limited, as robust methods for detecting the conversion of lignin to desired small molecules are not available.

Results: We identified a vanillin-inducible promoter by RNAseq analysis of Escherichia coli cells treated with a sublethal dose of vanillin and developed a genetically programmed vanillin-sensing cell by placing the 'very green fluorescent protein' gene under the control of this promoter. Fluorescence of the biosensing cell is enhanced significantly when grown in the presence of vanillin and is readily visualized by fluorescence microscopy. The use of fluorescence-activated cell sorting analysis further enhances the sensitivity, enabling dose-dependent detection of as low as 200 µM vanillin. The biosensor is highly specific to vanillin and no major response is elicited by the presence of lignin, lignin model compound, DMSO, vanillin analogues or non-specific toxic chemicals.

Conclusions: We developed an engineered E. coli cell that can detect vanillin at a concentration as low as 200 µM. The vanillin-sensing cell did not show cross-reactivity towards lignin or major lignin degradation products including vanillin analogues. This engineered E. coli cell could potentially be used as a host cell for screening lignin-degrading enzymes that can convert lignin to vanillin.

Keywords: Biorefinery; Directed evolution; Enzyme engineering; Fluorescence-activated cell sorting; High-throughput screening; Lignin; Microbial biosensor; RNA sequencing; Vanillin; Vanillin-inducible promoter.