Development of a genetically programed vanillin-sensing bacterium for high-throughput screening of lignin-degrading enzyme libraries.
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 ofEscherichia colicells 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 engineeredE. colicell 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 engineeredE. colicell could potentially be used as a host cell for screening lignin-degrading enzymes that can convert lignin to vanillin.
p53 Laboratory, Agency for Science Technology And Research (ASTAR), 8A Biomedical Grove, #06-04/05 Neuros/Immunos, Singapore, 138648 Singapore.
Barindra
B
Sana
Genome Institute of Singapore, 60 Biopolis Street, Genome, #02-01, Singapore, 138672 Singapore.
Kuan Hui Burton
KHB
Chia
p53 Laboratory, Agency for Science Technology And Research (ASTAR), 8A Biomedical Grove, #06-04/05 Neuros/Immunos, Singapore, 138648 Singapore.
Sarada S
SS
Raghavan
Institute of Chemical and Engineering Sciences, 8 Biomedical Grove, Neuros, #07-01, Singapore, 138665 Singapore.
Balamurugan
B
Ramalingam
Genome Institute of Singapore, 60 Biopolis Street, Genome, #02-01, Singapore, 138672 Singapore.
Niranjan
N
Nagarajan
Institute of Chemical and Engineering Sciences, 8 Biomedical Grove, Neuros, #07-01, Singapore, 138665 Singapore.
Jayasree
J
Seayad
p53 Laboratory, Agency for Science Technology And Research (ASTAR), 8A Biomedical Grove, #06-04/05 Neuros/Immunos, Singapore, 138648 Singapore.
Farid J
FJ
Ghadessy
Biorefinery; Directed evolution; Enzyme engineering; Fluorescence-activated cell sorting; High-throughput screening; Lignin; Microbial biosensor; RNA sequencing; Vanillin; Vanillin-inducible promoter