Bacterial strains. We used E. coli DH10B, chemically competent NEB Turbo, or electrocompetent One Shot Top10 (Invitrogen) to carry out molecular cloning and to perform preliminary analyses of terpenoid production; we used E. coli BL2-DE31 to express proteins for in vitro studies; and we used E. coli s103061 for our luminescence studies and for all experiments involving terpenoid-mediated growth (i.e., evolution studies).
For all strains, we generated chemically competent cells by carrying out the following steps: (i) We plated each strain on LB agar plates with the required antibiotics. (ii) We used one colony of each strain to inoculate 1 mL of LB media (25 g/L LB with appropriate antibiotics listed in Table S2) in a glass culture tube, and we grew this culture overnight (37℃, 225 RPM). (iii) We used the 1-mL culture to inoculate 100-300 mL of LB media (as above) in a glass shake flask, and we grew this culture for several hours (37℃, 225 RPM). (iv) When the culture reached an OD of 0.3-0.6, we centrifuged the cells (4,000 x g for 10 minutes at 4℃), removed the supernatant, resuspended them in 30 mL of ice cold TFB1 buffer (30 mM potassium acetate, 10 mM CaCl2, 50 mM MnCl2, 100 mM RbCl, 15% v/v glycerol, water to 200 mL, pH=5.8, sterile filtered), and incubated the suspension at 4℃ for 90 min. (v) We repeated step iv, but resuspended in 4 mL of ice cold TFB2 buffer (10 mM MOPS, 75 mM CaCl2, 10 mM RbCl2, 15% glycerol, water to 50 mL, pH=6.5, sterile filtered). (iv) We split the final suspension into 100 μL aliquots and froze them at -80℃ until further use.
We generated electrocompetent cells by following an approach similar to the one above. In step iv, however, we resuspended the cells in 50 mL of ice cold MilliQ water and repeated this step twice—first with 50 mL of 20% sterile glycerol (ice cold) and, then, with 1 mL of 20% sterile glycerol (ice cold). We froze the pellets as before.
Materials. We purchased methyl abietate from Santa Cruz Biotechnology; trans-caryophyllene, tris(2-carboxyethyl)phosphine (TCEP), bovine serum albumin (BSA), M9 minimal salts, phenylmethylsulfonyl fluoride (PMSF), and DMSO (dimethyl sulfoxide) from Millipore Sigma; glycerol, bacterial protein extraction reagent II (B-PERII), and lysozyme from VWR; cloning reagents from New England Biolabs; amorphadiene from Ambeed, Inc.; and all other reagents (e.g., antibiotics and media components) from Thermo Fisher. Taxadiene was a kind gift from Phil Baran of the The Scripps Research Institute. We prepared mevalonate by mixing 1 volume of 2 M DL-mevalanolactone with 1.05 volumes of 2 M KOH and incubating this mixture at 37°C for 30 minutes.
Cloning and molecular biology. We constructed all plasmids by using standard methods (i.e., restriction digest and ligation, Golden Gate and Gibson assembly, Quikchange mutagenesis, and circular polymerase extension cloning). Table S1 describes the source of each gene; Tables S2 and S3 describe the composition of all final plasmids.
We began construction of the B2H system by integrating the gene for HA4-RpoZ from pAB094a into pAB078d and by replacing the ampicillin resistance marker of pAB078d with a kanamycin resistance marker (Gibson Assembly). We modified the resulting “combined” plasmid, in turn, by replacing the HA4 and SH2 domains with kinase substrate and substrate recognition (i.e., SH2) domains, respectively (Gibson assembly), and by integrating genes for Src kinase, CDC37, and PTP1B in various combinations (Gibson assembly). We finalized the functional B2H system by modifying the SH2 domain with several mutations known to enhance its affinity for phosphopeptides (K15L, T8V, and C10A, numbered as in Kaneko et. al.35), by exchanging the GOI for luminescence (LuxAB) with one for spectinomycin resistance (SpecR), and by toggling promoters and ribosome binding sites to enhance the transcriptional response (Gibson assembly and Quickchange Mutagenesis, Agilent Inc.). We note: For the last step, we also converted Pro1 to ProD by using the Quikchange protocol. When necessary, we constructed plasmids with arabinose-inducible components by cloning a single component from the B2H system into pBAD (Golden Gate assembly). Tables S4-S6 list the primers and DNA fragments used to construct each plasmid.
We assembled pathways for terpenoid biosynthesis by purchasing plasmids encoding the first module (pMBIS) and various sesquiterpene synthases (ADS or GHS in pTRC99a) from Addgene, and by building the remaining plasmids. We replaced the tetracycline resistance in pMBIS with a gene for chloramphenicol resistance to create pMBISCmR. We integrated genes for ABS, TXS, ABA, and GGPPS into pTRC99t (i.e., pTRC99a without BsaI sites). Tables S4-S6 list the primers and DNA fragments used to construct each plasmid.
Luminescence assays. We characterized preliminary B2H systems (which contained LuxAB as the GOI) with luminescence assays. In brief, we transformed necessary plasmids into E. coli s1030 (Table S2), plated the transformed cells onto LB agar plates (20 g/L agar, 10 g/L tryptone, 10 g/L sodium chloride, and 5 g/L yeast extract with antibiotics described in Table S2), and incubated all plates overnight at 37°C. We used individual colonies to inoculate 1 ml of terrific both (TB at 2%, or 12 g/L tryptone, 24 g/L yeast extract, 12 mL/L 100% glycerol, 2.28 g/L KH2PO4, 12.53 g/L K2HPO4, pH = 7.3, and antibiotics described in Table S2), and we incubated these cultures overnight (37°C and 225 RPM). The following morning, we diluted each culture by 100-fold into 1 ml of TB media (above), and we incubated these cultures in individual wells of a deep 96-well plate for 5.5 hours (37°C, 225 RPM). (We note: When pBAD was present, we supplemented the TB media with 0-0.02 w/v % arabinose). We transferred 100μL of each culture into a single well of a standard 96-well clear plate and measured both OD600 and luminescence on a Biotek Synergy plate reader (gain: 135, integration time: 1 second, read height: 1 mm). Analogous measurements of cell-free media allowed us to measure background signals, which we subtracted from each measurement prior to calculating OD-normalized luminescence (i.e., Lum / OD600).
Analysis of antibiotic resistance. We evaluated the spectinomycin resistance conferred by various B2H systems in the absence of terpenoid pathways by carrying out the following steps: (i) We transformed E. coli with the necessary plasmids (Table S2) and plated the transformed cells onto LB agar plates (20 g/L agar, 10 g/L tryptone, 10 g/L sodium chloride, 5 g/L yeast extract, 50 μg/ml kanamycin, 10 μg/ml tetracycline). (ii) We used individual colonies to inoculate 1-2 ml of TB media (12 g/L tryptone, 24 g/L yeast extract, 12 mL/L 100% glycerol, 2.28 g/L KH2PO4, 12.53 g/L K2HPO4, 50 μg/ml kanamycin, 10 μg/ml tetracycline, pH = 7.3), and we incubated these cultures overnight (37°C, 225 RPM). In the morning, we diluted each culture by 100-fold into 4 ml of TB media (as above) with 0-500 μg/ml spectinomycin (we used spectinomycin in the liquid culture only for Figure S2), and we incubated these cultures in deep 24-well plates until wells containing 0 μg/ml spectinomycin reached an OD600 of 0.9-1.1. (iv) We diluted each 4-ml culture by 10-fold into TB media with no antibiotics and plated 10-μL drops of the diluent onto agar plates with various concentrations of spectinomycin. (v) We incubated plates overnight (37°C) and photographed them the following day.
To examine terpenoid-mediated resistance, we began with steps i and ii as described above with the addition of 34 μg/ml chloramphenicol and 50 μg/ml carbenicillin in all liquid/solid media. We then proceeded with the following steps: (iii) We diluted samples from 1-ml cultures to an OD600 of 0.05 in 4.5 ml of TB media (supplemented with 12 g/L tryptone, 24 g/L yeast extract, 12 mL/L 100% glycerol, 2.28 g/L KH2PO4, 12.53 g/L K2HPO4, 50 μg/ml kanamycin, 10 μg/ml tetracycline, 34 μg/ml chloramphenicol, and 50 μg/ml carbenicillin), which we incubated in deep 24-well plates (37°C, 225 RPM). (iv) At an OD600 of 0.3-0.6, we transferred 4 ml of each culture to a new well of a deep 24-well plate, added 500 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 20 mM of mevalonate, and incubated for 20 hours (22°C, 225 RPM). (v) We diluted each 4-ml culture to an OD600 of 0.1 with TB media and plated 10 μL of the diluent onto either LB or TB plates supplemented with 500 μM IPTG, 20 mM mevalonate, 50 μg/ml kanamycin, 10 μg/ml tetracycline, 34 μg/ml chloramphenicol, 50 μg/ml carbenicillin, and 0-1200 μg/ml spectinomycin (for both plates, we used 20 g/L agar with media and buffer components described above).
Terpenoid biosynthesis. We prepared E. coli for terpenoid production by transforming cells with plasmids harboring requisite pathway components (Table S2) and plating them onto LB agar plates (20 g/L agar, 10 g/L tryptone, 10 g/L sodium chloride, and 5 g/L yeast extract with antibiotics described in Table S2). We used one colony from each strain to inoculate 2 ml TB (12 g/L tryptone, 24 g/L yeast extract, 12 mL/L 100% glycerol, 2.28 g/L KH2PO4, 12.53 g/L K2HPO4, pH = 7.0, and antibiotics described in Table S2) in a glass culture tube for ~16 hours (37°C and 225 RPM). We diluted these cultures by 75-fold into 10 ml of TB media and incubated the new cultures in 125 mL glass shake flasks (37°C and 225 RPM). At an OD600 of 0.3-0.6, we added 500 μM IPTG and 20 mM mevalonate. After 72-88 hours of growth (22°C and 225 RPM), we extracted terpenoids from each culture as outlined below. Table S9 lists exact sample sizes, culture volumes, and fermentation times.
Protein expression and purification. We expressed and purified PTPs as described previously (Hjortness et al., 2018). Briefly, we transformed E. coli BL21(DE3) cells with pET16b or pET21b vectors (see Table S2 for details), and we induced with 500 μM IPTG at 22°C for 20 hours. We purified PTPs from cell lysate by using desalting, nickel affinity, and anion exchange chromatography (HiPrep 26/10, HisTrap HP, and HiPrep Q HP, respectively; GE Healthcare). We stored the final protein (30–50 μM) in HEPES buffer (50 mM, pH 7.5, 0.5 mM TCEP) in 20% glycerol at −80°C.
Extraction and purification of terpenoids. We used hexane to extract terpenoids generated in liquid culture. For 10-mL cultures, we added 14 mL of hexane to 10 ml of culture broth in 125-mL glass shake flasks, shook the mixture (100 RPM) for 30 minutes, centrifuged it (4000 x g), and withdrew 10 mL of the hexane layer for further analysis. For 4-mL cultures, we added 600 μL hexane to 1 mL of culture broth in a microcentrifuge tube, vortexed the tubes for 3 minutes, centrifuged the tubes for 1 minute (17000 x g), and saved 300-400 μL of the hexane layer for further analysis.
To purify amorphadiene, ⍺-bisabolene, and (+)-1(10),4-cadinadiene, we supplemented 500-1000 mL culture broth with hexane (16.7% v/v), shook the mixture for 30 minutes (100 RPM), isolated the hexane layer with a separatory funnel, centrifuged the isolated organic phase (4000 x g), and withdrew the hexane layer. To concentrate the terpenoid products, we evaporated excess hexane in a rotary evaporator to bring the final volume to 500 μL, and we passed the resulting mixture over a silica gel 1-3 times (Sigma-Aldrich; high purity grade, 60 Å pore size, 230-400 mesh particle size). We analyzed elution fractions (100% hexane) on the GC/MS and pooled fractions with the compound of interest (amorphadiene). Once purified, we dried pooled fractions under a gentle stream of air, resuspended the terpenoid solids in DMSO, and quantified the final samples as outlined below. We repeated the purification process until samples (in DMSO) were >95% pure by GC/MS unless otherwise noted.
GC-MS analysis of terpenoids. We measured terpenoids generated in liquid culture with a gas chromatograph / mass spectrometer (GC-MS; a Trace 1310 GC fitted with a TG5-SilMS column and an ISQ 7000 MS; Thermo Fisher Scientific). We prepared all samples in hexane (directly or through a 1:100 dilution of DMSO) with 20 μg/ml of caryophyllene or methyl abietate as an internal standard. Highly concentrated samples were diluted 10-20x prior to preparation to bring concentrations within the MS detection limit. When the peak area of an internal standard exceeded ± 40% of the average area of all samples containing that standard, we re-analyzed the corresponding samples. For all runs, we used the following GC method: hold at 80°C (3 min), increase to 250°C (15°C/min), hold at 250°C (6 min), increase to 280°C (30°C/min), and hold at 280°C (3 min). To identify various analytes, we scanned m/z ratios from 50 to 550.
We examined sesquiterpenes generated by variants of ADS by using select ion mode (SIM) to scan for the molecular ion (m/z =204). For quantification, we used Eq. 1: where Ai
is the area of the peak produced by analyte i, Astd is the area of the peak produced by Cstd of caryophyllene in the sample, and R is the ratio of response factors for caryophyllene and amorphadiene in a reference sample. Tables S12-14 provide the concentrations of all standards and reference compounds used in this analysis.
We quantified sesquiterpenes generated by variants of GHS by using the aforementioned procedure with several modifications: We used methyl abietate as an internal standard (several mutants of GHS generate caryophyllene as a product); we scanned for both m/z = 204 and m/z = 121, a common ion between sesquiterpenes and methyl abietate; we used a ratio of response factors for amorphadiene and methyl abietate at m/z = 121 for R; and we calculated peak areas at m/z = 121. We focused our analysis on peaks with areas that exceeded 1% of the total area of all peaks at m/z=204.
We quantified diterpenoids by, once again, accompanying our general procedure with several modifications: We scanned for a different molecular ion (m/z = 272) and an ion common to both diterpenoids and caryophyllene (m/z=93); we used a ratio of response factors for pure taxadiene (a kind gift from Phil Baran) and caryophyllene at m/z = 93; and we calculated peak areas m/z = 93. For all analyses, we examined only peaks with areas that exceeded 1% of the total area of all peaks at m/z=272.
We identified molecules by using the NIST MS library and, when necessary, confirmed this identification with analytical standards or mass spectra reported in the literature. We note: The assumption of a constant response factor for different terpenoids (that is, the assumption that all sesquiterpenes and diterpenes ionize like amorphadiene and taxadiene, respectively) can certainly yield error in estimates of their concentrations; our analyses, which are consistent with those of other studies of terpenoid production in microbial systems62,63, supply rough estimates of concentrations for all compounds except amorphadiene and taxadiene (which had analytical standards).
Bioinformatics. We used a bioinformatic analysis to identify a phylogenetically diverse set of terpene synthases. Briefly, we downloaded (i) all constituent genes of PF03936 (the largest terpene synthase family grouped by a C-terminal domain) from the PFAM Database and (ii) all enzymes with Enzyme Commission (EC) number of 4.2.3.# from the Uniprot Database; this string, which defines carbon oxygen lyases that act on phosphates, includes terpene synthases. We cleaned both datasets in Excel (i.e., we ensured that every identifier had only one row), and we used a custom R script to designate each PF03936 member as characterized (i.e., in possession of a Uniprot-based EC number) or uncharacterized. Finally, we used FastTree64 to create a phylogenetic tree of the PF03936 family and the R-package ggtree65 to visualize the resulting tree and function data as a cladogram and heatmap.
After annotating the cladogram by hand, we selected three genes from each of six clades: six with no characterized genes and two with some characterized genes. We avoided clades proximal to known monoterpene synthases or diterpene synthases known to act on GGPP isomers absent in our system (e.g., ent-copalyl diphosphate); these enzymes are unlikely to act on FPP, the primary product of pMBISCmR. When selecting enzymes within clades, we biased our choice towards bacterial/fungal species and selected genes with a minimal number of common ancestors within the clade. The selected genes were synthesized and cloned into the pTrc99a vector by Twist Biosciences and assayed for antibiotic resistance as described above.
Enzyme kinetics. To examine terpenoid-mediated inhibition, we measured PTP1B or TCPTP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) in the presence of various concentrations of terpenoids. Each reaction included PTP (0.05 μM in 50 mM HEPES, 0.5 mM TCEP, 50 µg/ml BSA), pNPP (0.33, 0.67, 2, 5, 10, and 15 mM), inhibitor (with concentrations listed in the figures), buffer (50 mM HEPES pH=7.3, 50 µg/ml BSA), and DMSO at 10% v/v. We monitored the formation of p-nitrophenol by measuring absorbance at 405 nm every 10 seconds for 5 minutes on a SpectraMax M2 plate reader. We report exact sample sizes (i.e., the number of independently prepared reactions) in Supplementary Table 10.
We used a custom MATLAB script to process all raw kinetic data. This script removed all concentration values that fell outside of either (i) the range of our standard curve (absorbance vs. μM; Supplementary Fig. 18) or (ii) the initial rate regime (>10% of the pNPP concentration used in the assay). When this step reduced kinetic dataset to fewer than ten points, we re-measured those datasets to collect at least ten. We fit final datasets, in turn, with a linear regression model (using Matlab’s backslash operator).
We evaluated kinetic models in three steps: (i) We fit initial-rate measurements collected in the absence and presence of inhibitors to Michaelis-Menten and inhibition models, respectively (here, we used the nlinfit and fminsearch functions from MATLAB; Supplementary Table 13). (ii) We used an F-test to compare the mixed model to the single-parameter model with the least sum squared error (here, we used the fcdf function from MATLAB to assign p-values), and we accepted the mixed model when p < 0.05. (iii) We used the Akaike's Information Criterion (AIC) to compare the best-fit single parameter model to each alternative single parameter model, and we accepted the “best-fit” model when the difference in AIC (Δi) exceed 5 for all comparisons.66 We note: For amorphadiene, ⍺-bisabolene, and (+)1-(10),4-cadinadiene this criterion was not met; both noncompetitive and uncompetitive models, however, yielded indistinguishable IC50’s.
We estimated the half maximal inhibitory concentration (IC50) of inhibitors by using the best-fit kinetic models to determine the concentration of inhibitor required to reduce initial rates of PTP-catalyzed hydrolysis of 15 mM of pNPP by 50%. We used the MATLAB function “nlparci” to determine the confidence intervals of kinetic parameters, and we propagated those intervals to estimate corresponding confidence intervals for each IC50.
X-ray crystallography. We prepared crystals of PTP1B by using hanging drop vapor diffusion. In brief, we added 2 μL of PTP1B (~600 μM PTP1B, 50 mM HEPES, pH 7.3) to 6 μL of crystallization solution (100 mM HEPES, 200 mM magnesium acetate, and 14% polyethylene glycol 8000, pH 7.5) and incubated the resulting droplets over crystallization solution for one week at 4°C (EasyXtal CrystalSupport, Qiagen). We soaked crystals with ligand by transferring them to droplets formed with 6 μL of crystallization solution and 1 μL of ligand solution (10 mM in DMSO), which we incubated for 2-5 days at 4°C. We prepared all ligands for freezing by soaking them in cryoprotectant formed from a 70/30 (v/v) mixture of buffer (100 mM HEPES, 200 mM magnesium acetate, and 25% polyethylene glycol 8000, pH 7.5) and glycerol.
We collected X-ray diffraction data through the Collaborative Crystallography Program at Lawrence Berkeley National Lab (ALS ENABLE, beamline 8.2.1, 100 K, 1.00003 Å). We performed integration, scaling, and merging of X-ray diffraction data using the xia2 software package67, and we carried out molecular replacement and structure refinement with the PHENIX graphical interface,68 supplemented with manual model adjustment in COOT69 and one round of PDB-REDO70 (the latter, only for the PTP1B-amorphadiene complex).
Molecular dynamics (MD) simulations. We performed MD simulations using GROMACS 202071. Briefly, we used the CHARMM36m protein force field72, a CHARMM-modified TIP3P water model73, and ligand parameters generated by CGenFF74,75. We solvated each PTP1B-ligand complex (initialized from the corresponding crystal structure) in a dodecahedral box with edges positioned ≥ 10 Å from the surface of the complex, and we added sodium ions (three for amorphadiene and one for ⍺-bisabolol) to neutralize each system. We used the LINCS algorithm76 to constrain all bonds involving hydrogen atoms, the Verlet leapfrog algorithm to numerically integrate equations of motion with a 2-fs time step, and the particle-mesh Ewald summation77 (cubic interpolation with a grid spacing of 0.16 nm) to calculate long-range electrostatic interactions; we used a cutoff of 1.2 nm, in turn, for short-range electrostatic and Lennard-Jones interactions. We independently coupled the protein-ligand complex and solvent molecules to a temperature bath (300K) using a modified Berendsen thermostat78 with a relaxation time of 0.1 ps, and we fixed pressure coupling to 1 bar using the Parrinello–Rahman algorithm79 with a relaxation time of 2 ps and isothermal compressibility of 4.5 × 10−5 bar-1.
For each system, we carried out 30 independent MD simulations to reduce sampling bias. For each MD trajectory, we minimized energy using the steepest decent method followed by 100-ps solvent relaxation in the NVT ensemble and 100-ps solvent relaxation in the NPT ensemble. After an additional 1-ns NPT equilibration, we carried out production runs for 1 ns in the NPT ensemble and registered coordinate data every 10 ps.
Analysis of PTP1B inhibition in HEK293TCells . We prepared HEK293T/17 cells for an enzyme-linked immunosorbent assay (ELISA) by growing them in 75 cm2 culture flasks (Corning) with DMEM media supplemented with 10% FBS, 100 units/ml penicillin, and 100 units/ml streptomycin. We replaced the media every day for 3-5 days until the cells reached 80-100% confluency.
We measured the influence of inhibitors on insulin receptor (IR) phosphorylation by using an IR-specific ELISA (Supplementary Fig. 10a). Briefly, we starved cells for 48 hours in FBS-free media and incubated the with inhibitors (all at 3% DMSO) for 10 minutes. After incubation, we lysed cells with lysis buffer (9803, Cell Signaling Technology) supplemented with 1X halt phosphatase inhibitor cocktail and 1X halt protease inhibitor cocktail (Thermo Fisher Scientific) for 10 min, pelleted the cell debris, and used the lysis buffer to dilute each sample to 60 mg/ml total protein. We measured IR phosphorylation in subsequent dilutions of the 60 mg/ml samples with the PathScan® Phospho-Insulin Receptor β (panTyr) Sandwich ELISA Kit (Cell Signaling Technology; #7082). We note: To identify biologically active concentrations of ⍺-bisabolene and amorphadiene, we screened several concentrations and chose those that gave the highest signal (405 μM for ⍺-bisabolene and 930 μM for amorphadiene); similar concentrations of weak inhibitors did not yield a detectable signal (Supplementary Fig. 10b,c).
Statistical analysis and reproducibility. We determined statistical significance (Figs 3g) with a two-tailed Student’s t-test (details in Supplementary Tables 11 and 15), and we used an F-test to compare one- and two-parameter models of inhibition (Supplementary Table 13).
Reporting Summary. Further information on research design is available in the Nature Research Reporting Summary linked to this data.
Data availability. The plasmids generated in this study are available on Addgene (https://www.addgene.org/) or from the authors. All code generated for data analysis is available upon request. Source data for our figures is available as follows: Supplementary Table 7 (Fig. 1b-d, Supplementary Fig. 1), Supplementary Table 8 (Figs. 1e, 2c, 4b; Supplementary Figs. 2, 3d, 9,11,12), Supplementary Table 9 (Fig. 2g, 4c, 5c; Supplementary Fig. 4a), Supplementary Table 10 (Fig. 2e, 3g, 4e; Supplementary Fig. 5d-o), Supplementary Table 11 (Fig. 4g; Supplementary Fig. 6b-c). The crystal structures determined in this study are available from the RCSB Protein Data Bank (PDB entry 6w30, 6w31). Table 14 provides refinement statistics for both structures.