Strains, plasmids, and media
E. coli DH10B (New England BioLabs, Ipswich, MA, USA) was used for all routine cloning and directed evolution. All biosensor systems were characterized in E. coli DH10B. E. coli BL21 DE3 (New England BioLabs, Ipswich, MA, USA) was used for protein expression. LB-Miller (LB) media (BD, Franklin Lakes, NJ, USA) was used for routine cloning, fluorescence assays, directed evolution, and orthogonality assays unless specifically noted. Terrific broth (TB) (Thermo Fisher Scientific, CAT#: 22711022) was used for protein purification. LB + 1.5% agar (BD, Franklin Lakes, NJ, USA) plates were used for routine cloning and directed evolution. The plasmids described in this work were constructed using Gibson assembly and standard molecular biology techniques. Synthetic genes, obtained as gBlocks, and primers were purchased from IDT. Relevant plasmid sequences are provided in Supplementary Table 4.
Benzylisoquinoline alkaloids
Cells were induced with the following chemicals: norlaudanosoline (NOR) (HDH Pharma Inc. CAT#: 29030); tetrahydropapaverine (THP) (Tokyo Chemical Company, product#: N0918); papaverine (PAP) (MP Biomedicals LLC. CAT#: 190261); glaucine (GLAU) (Carbosynth Ltd. product#: FG137572); rotundine (ROTU) (Alfa Aesar, product#: J63328); noscapine (NOS) (Aldrich, SKU: 363960-5G); norreticuline (NRT) (Selena Chem Ltd. product#: CSC000735172).
Chemical transformation
For routine transformations, strains were made competent for chemical transformation. 5 mL of an overnight culture of DH10B cells were subcultured into 500 mL of LB media and grows at 37°C, 250 r.p.m. for 3 h. Cultures were centrifuged (3,500 g, 4 °C, 10 min), and pellets were washed in 70 mL of chemical competence buffer (10% glycerol, 100mM CaCl2) and centrifuged again (3,500 g, 4°C, 10 min). The resulting pellets were resuspended in 20 mL of chemical competence buffer. After 30 minutes on ice, cells were divided into 250 μL aliquots and flash frozen in liquid nitrogen. Competent cells were stored at −80 °C until use.
Promoter design and biosensor response assay
Promoters for TtgR and QacR were derived from the literature16,19. For the RamR promoter, a region 60 base pairs upstream the known operator sequence as well as the operator itself was extracted from the Salmonella typhimurium genome (WP_000113609.1). NalD and SmeT are homologs of TtgR, therefore modifications from the Pttgr promoter were made to match the sequence of the NalD operator20 and SmeT operator21. For the Pbm3r1, the known Bm3R1 operator19 was placed immediately after the -10 region of a synthetic medium strength promoter. All promoter sequences are listed in SI Fig. 2. The pReg and pGFP equivalents for each regulator were co-transformed into DH10B cells and plated on an LB agar plate with appropriate antibiotics. Three separate colonies were picked for each transformation and were grown overnight. The following day, 20 μL of each culture was then used to inoculate six separate wells within a 2 mL 96-deep-well plate (Corning, Product #: P-DW-20-C-S) sealed with an AeraSeal film (Excel Scientific, Victorville, CA, USA) containing 900 μL of LB media, one for each test ligand and a solvent control. After two hours of growth at 37°C cultures were induced with 100uL of LB media containing either 10 μL of DMSO or 100 μL of LB media containing one of the five target BIAs dissolved in 10 μL of DMSO. Cultures were grown for an additional 4 hours at 37°C, 250 r.p.m and subsequently centrifuged (3,500 g, 4°C, 10 min). Supernatant was removed and cell pellets were resuspended in 1 mL of PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4. pH 7.4). 100 μL of the cell resuspension for each condition was transferred to a 96 well microtiter plate (Corning, Product #: 3904), from which the fluorescence (Ex: 485 nm, Em: 509 nm) and absorbance (600 nm) was measured using the Tecan Infinite M1000 plate reader.
RamR library design and construction
Five semi-rational libraries were designed, each targeting three inward-facing residues on one of five helices of the RamR ligand binding pocket (Fig. 1d and SI Fig. 4). Libraries were generated using overlap PCR with redundant NNS codons using Accuprime Pfx (Thermo Fisher, CAT#: 12344024) and cloned into pReg. E. coli DH10B bearing pSelis was transformed with the resulting library. Transformation efficiency always exceeded 106 for each round of selection, indicating several fold coverage of the library. Transformed cells were grown in LB media overnight at 37°C in carbenicillin and chloramphenicol.
Directed evolution of RamR biosensors
Twenty μL of cell culture bearing the sensor library was seeded into 5 mL of fresh LB containing appropriate antibiotics, 100 μg/mL zeocin (Thermo Fisher. CAT#: R25001), and 100 μM of non-target BIAs (for rounds three and four) and were grown at 37°C for seven hours. Following incubation, 0.5 μL of culture was diluted into 1 mL of LB media, from which 100 μL was further diluted into 900 μL of LB media. 300 μL of this mixture was then plated across three LB agar plates containing carbenicillin, chloramphenicol and the target BIA dissolved in DMSO. Plates were incubated overnight at 37°C. The following day the brightest colonies were picked and grown overnight in 1 mL of LB media containing appropriate antibiotics within a 96-deep-well plate sealed with an AeraSeal film at 37°C. A glycerol stock of cells containing pSelis and pReg bearing the parental RamR variant was also inoculated in 5mL of LB for overnight growth.
The following day, 20 μL of each culture was used to inoculate two separate wells within a new 96-deep-well plate containing 900 μL of LB media. Additionally, eight separate wells containing 1 mL of LB media were inoculated with 20 μL of the overnight culture expressing the parental RamR variant. A typical arrangement would have 44 unique clones on the top half of the plate, duplicates of those clones on the bottom half of the plate, and the right-most column occupied by cells harboring the parental RamR variant. After 2 hours of growth at 37°C the top half of the 96-well plate was induced with 100 μL of LB media containing 10uL of DMSO whereas the bottom half of the plate was induced with 100 μL of LB media containing the target BIA dissolved in 10 μL of DMSO. The concentration of BIA used for induction is typically the same concentration used in the LB agar plate for screening during that particular round of evolution. Cultures were grown for an additional 4 hours at 37°C, 250 r.p.m and subsequently centrifuged (3,500 g, 4°C, 10 min). Supernatant was removed and cell pellets were resuspended in 1mL of PBS. 100 μL of the cell resuspension for each condition was transferred to a 96 well microtiter plate, from which the fluorescence (Ex: 485 nm, Em: 509 nm) and absorbance (600 nm) was measured using the Tecan Infinite M1000. Clones with the highest signal-to-noise ratio were then sequenced and subcloned into a fresh pReg vector.
For sensor variant validation, the subcloned pReg vectors expressing the sensor variants were transformed into DH10B cells bearing pGFP. These cultures were then assayed, as described “Response function measurements” using eight different concentrations of the target BIA. Sensor variants that displayed a combination of a low background, a reduced EC50 for the target BIA, and a high signal/noise ratio were used as templates for the next round of evolution.
Dose response measurements
Glycerol stocks (20% glycerol) of strains containing the plasmids of interest were inoculated into 1 mL of LB media and grown overnight at 37 °C. 20 μL of overnight culture was seeded into 900 μL of LB media containing ampicillin and chloramphenicol within a 2 mL 96-deep-well plate sealed with an AeraSeal film. Following growth at 37°C, 250 r.p.m. for two hours, cultures were induced with 100 μL of a LB media solution containing appropriate antibiotics and the inducer molecule dissolved in 10 μL of DMSO. Cultures were grown for an additional four hours at 37 °C, 250 r.p.m and subsequently centrifuged (3,500 g, 4 °C, 10 min). Supernatant was removed and cell pellets were resuspended in 1 mL of PBS. 100 μL of the cell resuspension for each condition was transferred to a 96-well microtiter plate, from which the fluorescence (Ex: 485 nm, Em: 509 nm) and absorbance (600 nm) was measured using the Tecan Infinite M1000 plate reader.
Orthogonality assays
For each evolutionary lineage (for example, WT, THP1, THP2, THP3, THP4) all regulators were expressed on the pReg plasmid using the same promoter, which is P114-RBS(riboJ), P114-RBS(riboJ), P103-RBS(elvJ), P114-RBS(riboJ), and P103-RBS(riboJ) for the GLAU, NOS, PAP, ROTU, and THP lineages, respectively. These plasmids were co-transformed with pGFP and the following day three individual colonies were picked into LB and grown overnight. Fluorescence assays were performed as in the “Dose response measurements” section above, but either 100 μM of each BIA in 1% DMSO or 1% DMSO itself was used for induction.
Protein purification
Coding sequences for RamR variants were cloned into an ampicillin resistant pUC plasmid with a T7 RNA polymerase promoter driving the gene of interest with an N-terminal His6-3C tag. Plasmids were transformed into electrocompetent BL21 DE3 cells and single transformants were grown to saturation in LB supplemented with 1,000 μg/mL carbenicillin. Cultures were diluted 1/250 in terrific broth supplemented with antibiotics in baffled flasks and incubated at 37 °C with agitation (250 r.p.m.) until reaching mid-log phase. Protein expression was induced by addition of IPTG to achieve a final concentration of 0.5 mM. For PAP4 only, papaverine was also added during IPTG induction to reach a final concentration of 100 μM. Cells were cultured for 18 hours at 18 °C. Cells were harvested by centrifugation at 8,000 g for 10 min and the cell pellets were resuspended in 25 mL of wash buffer (50 mM K2HPO4, 300 mM NaCl, and 10% glycerol at pH 8.0) with protease inhibitor cocktail (cOmplete, mini EDTA free, Roche) and lysozyme (0.5 mg/mL). Cells were incubated for 20 min at 4 °C with gentle agitation and lysed by sonication (Model 500, Fisher Scientific). Lysate was repeatedly clarified by centrifugation (35,000xg for 30 min), and protein was recovered by immobilized metal ion affinity chromatography (IMAC) using Ni-NTA resin and gravity flow columns. Eluate was concentrated and dialyzed, with 3C protease added to the dialysis cassette, into the appropriate buffer followed by purification to apparent homogeneity by size exclusion fast protein liquid chromatography (FPLC). All RamR variants were dialyzed into 20 mM Tris (pH 8.0), 200 mM NaCl and 3 mM DTT.
X-ray crystallography
To form co-crystals of RamR variants in complex with individual ligands, 1mM substrate was added to 10 mg/ml of purified protein and incubated overnight at 4°C except for PAP4 protein, which already formed complex with papaverine during the protein expression step. Rod-shaped co-crystals grew by using sitting-drop vapor diffusion method at room temperature for PAP4, ROTU, GLAU4, and NOS4 in conditions containing 0.1M MES (pH 6.0 – 7.5), 14 – 23% PEG 3350, 0.2 M Ammonium Sulfate, and 0.1 M Sodium Chloride. Individual crystals were flash-frozen directly in liquid nitrogen after brief incubation with a reservoir solution supplemented with 25% (v/v) glycerol. X-ray diffraction data were collected at BL 5.0.1 beamline in ALS (Berkeley, CA). X-ray diffraction was processed to 1.6Å, 1.8Å, 2.0Å, and 2.2Å resolution for PAP4 with papaverine, ROTU4 with rotundine, GLAU4 with glaucine, and NOS4 with noscapine using HKL2000. In Phenix software, phases were obtained by molecular replacement using a previously solved RamR wildtype structure as the initial search model (PDB code 3VVX). The molecular replacement solutions for each structure were iteratively built using Coot and Phenix refine package. The quality of the final refined structures was evaluated by MolProbity. The final statistics for data collection and structure determination are shown in SI Table 2.
Biosensor-linked O-Methyltransferase activity assay
All OMTs were expressed with the P114-RBS(riboJ) promoter/RBS on the pReg plasmid backbone (no regulator present). Cells were co-transformed with both the OMT plasmid and the THP reporter plasmid and plated on an LB agar plate containing appropriate antibiotics. Three individual colonies from each transformation were picked into LB and grown overnight. Resulting cultures were diluted 50-fold into 1mL of LB media containing norlaudanosoline and 1 mg/mL of ascorbic acid in a 96-deep-well plate and were grown at 30 °C for 18 hours. Subsequently, the fluorescence of cultures was measured in the same manner as previously described in “Dose response measurement” above.
O-Methyltransferase evolution
The OMT coding region of the GfOMT1 expression vector was targeted for random mutagenesis introducing an average of two mutations relative to the template. Libraries were assembled using Gibson assembly and were subsequently transformed into chemically competent cells containing the THP reporter plasmid. In parallel, the template OMT was transformed into the same cells for downstream comparative analysis. Cultures were then plated on an LB agar plate containing norlaudanosoline. Highly fluorescent colonies were picked and grown overnight in appropriate antibiotics. The following day, cultures were grown with norlaudanosoline and 1 mg/mL of ascorbic acid at 30 °C for 18 hours. Fluorescence of the resulting cultures were measured, as described above and variants that were significantly more fluorescent than the template OMT were sequenced. Unique top-performing OMT variants were then subcloned into the pReg vector and transformed into cells bearing the THP reporter plasmid. Three individual colonies from each transformation were subcultured into fresh LB media and the fluorescence of each culture in the presence of norlaudanosoline (refer to SI Fig. 15) was subsequently measured, as described above. The top performing OMT variant was then used as the template for the following round of evolution.
Homology model
The homology model of the GEN5 GfOMT1 variant was constructed using SWISS-MODEL (https://swissmodel.expasy.org/). 5ICE was used as a template for structure generation.
Liquid-chromatography/Mass spectrometry
Cells containing the plasmid expressing each OMT variant with the P114-RBS(RiboJ) promoter were transformed and plated onto an LB agar plate containing appropriate antibiotics. The following day, three colonies from each plate were cultured overnight in LB and subsequently diluted 50-fold into 1mL of LB containing 10 μM of NOR and 1mg/mL ascorbic acid. These cultures were grown for 18 hours at 30 °C, centrifuged at 16,000xg for 1 minute, and the resulting supernatant was filtered using a 0.2 μM filter. The samples were then diluted 1:100 fold into filter sterilized water prior to LC/MS analysis. Samples were analyzed using an Agilent 6546 Q-TOF LC/MS with a dual Agilent Jet Stream electrospray ionization (ESI) source in positive mode. Chromatographic separations were obtained under gradient conditions by injecting 5 μL onto an Agilent RRHD Eclipse Plus C18 column (50 x 2.1 mm, 1.8 micron particle size) with an Agilent Zorbax Eclipse Plus C18 narrow bore guard column (12.5 x 2.1 mm, 5 micron particle size) on an Agilent 1260 Infinity II liquid chromatography system. The mobile phase consisted of eluent A (water + 0.1% formic acid) and eluent B (acetonitrile). The gradient was as follows: 5% B to 95% B from 0 to 6 min (0.3 mL/min), held at 95% B from 6 to 7 min (0.3 mL/min), 95% B to 5% B from 7 to 7.1 min (0.3 mL/min), and held at 5% B from 7.1 to 9 min (0.45 mL/min). The sample tray and column compartment were set to 7 °C and 30 °C, respectively. The fragmentor was set to 180 V. Q-TOF data was processed using the Agilent MassHunter Qualitative Analysis software.
To create chromatograms shown in SI Fig. 18, signal count from the EIC within a window +/- 0.05 minutes relative to the retention time for the corresponding alkaloid, see SI Table 3, was extracted for each scan (m/z ratios of the following: 288.1230, 302.1387, 316.1543, 330.1700, and 344.1856) and added together. Since a significant background peak was detected around the expected retention time for THP, the sigal count in this window of time was removed for all samples that had a count at or below this background level. The resulting chromatograms were then used to create Fig. 5d.
Statistical analysis and reproducibility
All data in the manuscript are displayed as mean ± s.e.m. unless specifically indicated. Bar graphs, fluorescence/growth curves, dose response functions, and orthogonality matrices were all plotted in Python 3.6.9 using matplotlib and seaborn. Dose response curves and EC50 values were estimated by fitting to the hill equation y = d + (a-d)*xb / (cb + xb) (where y = output signal, b = hill coefficient, x = ligand concentration, d = background signal, a = the maximum signal, and c = the EC50), with the scipy.optimize.curve_fit library in Python.