Development and substrate specificity screening of an in vivo biosensor for the detection of biomass derived aromatic chemical building blocks

To facilitate the screening of chemical, enzymatic, and cellular processes to degrade and valorize plant biomass a whole cell biosensor was developed to detect lignin-derived substrates.


Material and Methods
. Signal range (max/min) and fitted dose response curve data for the three biosensor systems in BL 21 and Top10F' strains Table S2. Signal range (max/min) and fitted dose response curve data for all responsive compounds tested.

Material and Methods
All cells were grown in LB medium (0.5% yeast extract, 0.5% NaCl, 1.0% Bactotryptone). A BMG CLARIOstar Microplate Reader was used to measure the GFP fluorescence and OD 600 for intact cells.

Vector Engineering
The sequences containing the P LC, P PC and P ferB promoters, a RBS, and a Hexa-Histidine tag, flanked by SphI/NdeI were synthetized (GeneART, ThermoFisher). The sequences were enzyme restricted with SphI/NdeI and cloned in pET44eGFP, 2 upstream to the eGFP gene, replacing the T7 promoter region generating respectively, the pET44P LC eGFP, pET44P PC eGFP and pET44P ferB eGFP vectors.
The FerCA DNA sequence containing the ferA and ferC genes, individually flanked by a PLacI promoter and an rrn_B1 terminator, was synthetized by GeneART TM (ThermoFisher).
The construct was cloned in a p15 plasmid flanked by NaeI/KasI restriction sites generating the p15FerCA vector. The ferA gene was removed by restriction digestion of two XbaI flanking sites. The remaining backbone with ferC was re-circularized to originate the p15FerC vector, and the obtained plasmid was sequenced to confirm identity.

Biosensors Performance and Screening Methods
Reporter controls and the biosensor systems P LC, P PC and P ferB were respectively generated by Compounds for screening were selected using cinnamic acid as a reference structure. The 58 selected compounds were tested with the P LC biosensor in E. coli BL21 cells. The substrate screening assays were performed using concentrations ranging from 0.32 μM to 1000 μM, as described for Ferulic acid. All experimental data are the mean of at least two biological replicates.

Biomass enzymatic degradation and screening
The three biomass sources kraft lignin, wheat flour and micronized oat husk were individually mixed with each feruloyl esterase (CE1) enzyme in a 200:1 weight ratio (10 mg : 0.1 mg).
One control, without enzyme, was made for each source. Phosphate buffer (0.1 M, pH 6.5) was added to 500 µL final volume and the tubes were incubated at 60 ºC with shaking at 1000 RPM (ThermoMixer Epperdorf) for 12 hours. Enzymatic reactions were centrifuged at 13,800 G for 15 minutes and the supernatants were collected.
Screening to detect released phenolic compounds was performed with the pLC biosensor in E. coli BL21. 50 μL of supernatant, phosphate buffer or FA (100mM) were mixed with 200 μL of culture at OD 0.6 in triplicates and the screening was followed as described for the FA induction assay.

Data Processing and Curve Fitting
Biosensor signal output (eGFP expression) was measured as Relative Fluorescence Units (RFU) and normalised to cell density (OD 600 ). The background auto-fluorescence of E. coli was subtracted from RFU/OD and was normalised (%) to the pLC biosensor response curve to ferulic acid for each experiment. The normalised data was plotted and fitted with a doseresponse curve using the Levenberg Marquardt logistic growth/sigmoidal algorithm, using the