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

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.ResultsWe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0720-5) contains supplementary material, which is available to authorized users.

A model‐driven approach towards rational microbial bioprocess optimization

“…It has been stated that phenolic compounds such as ferulic acid at higher than 1 g/L exhibited complete arrest of cell growth (Li et al, 2017). Meanwhile, the intermediate feruloyl CoA and the product vanillin formation, as aromatic aldehydes, were also identified to be bacteriostatic which prevent the growth of bacteria and retain them at stationary growth phase rather than bactericidal which could directly kill the bacteria (Parke & Ornston, 2004; Sana et al, 2017). The bacteriostatic effect is reversible whereby the cell growth can recover after the elimination of the bacteriostatic compound.…”

“…CoA and the product vanillin formation, as aromatic aldehydes, were also identified to be bacteriostatic which prevent the growth of bacteria and retain them at stationary growth phase rather than bactericidal which could directly kill the bacteria (Parke & Ornston, 2004;Sana et al, 2017).…”

Bioprospecting Microbial Diversity for Lignin Valorization: Dry and Wet Screening Methods

“…The yeiW promoter was used to build a GFP-based system similar to the one described above and was found to give a concentration-dependent response over a range of 0.2-5 mM vanillin. Furthermore, the output signal was shown to be specific to vanillin, and the sensor did not show a response to polymeric lignin or guaiacol, benzaldehyde, or veratraldehyde, which could also potentially be present in DTL (Sana et al, 2017). Another example of a recently developed vanillin biosensor was engineered with a QacR transcriptional regulator, from the TetR repressor family, which confers resistance to quaternary anionic compounds (Santos et al, 2016).…”

Genetically encoded biosensors for lignocellulose valorization

“…The discovery and characterization of these aromatic-responsive elements have been partly accomplished by different strategies involving gene regulation in response to the target aromatic. For instance, Sana et al [64] utilized an RNA-seq analysis of E. coli cultures treated with vanillin to identify upregulated genes. The identified P yeiW promoter upstream from these genes was thus used to construct a vanillin biosensor by placing it upstream of a reporter gene.…”