Engineering the “Missing Link” in Biosynthetic (−)-Menthol Production: Bacterial Isopulegone Isomerase

The realization of a synthetic biology approach to microbial (1R,2S,5R)-(−)-menthol (1) production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the Mentha piperita biosynthetic pathway as yet unidentified. We demonstrate that Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida can act as an IPGI, producing (R)-(+)-pulegone ((R)-2) from (+)-cis-isopulegone (3). Using a robotics-driven semirational design strategy, we identified a key KSI variant encoding four active site mutations, which confer a 4.3-fold increase in activity over the wild-type enzyme. This was assisted by the generation of crystal structures of four KSI variants, combined with molecular modeling of 3 binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions with downstream Mentha enzymes pulegone reductase and (−)-menthone:(−)-menthol reductase to generate 1 from 3. This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of 1 in microorganisms.

. Pulegone (2) standard curve. S4 Construction of the (+)-cis-Isopulegone (3) to (-)-Menthol (1) Enzyme Cascade Construct Table S3. Design of the ribosomal binding site (RBS) sequences. S5 KSI Crystallisation methods Table S4. KSI data collection and refinement statistics. S6 KSI Molecular Simulation methods Figure S2. Overlay of energy minimised structures. S7 Chiral Analysis of KSI Biotransformations Table S5. Product enantiomeric identity of wild-type and variant KSI with 3 by chiral GC. S8 Robotic Directed Evolution Strategy Figure S3. The robotics-driven directed evolution workflow. S9 Crystal Structures and Molecular Simulation Discussion Figure S4. Superimposition of the crystal structures of wild type and four variant KSI enzymes. Figure S5. Distances restrained during MD simulations of wild type KSI. S10 Cascade Biocatalysis Products Identification Figure S6. Isopulegol isomer identification by GC-MS. Figure S7. Separation of 1 and precursors by GC-MS.

S2.5 SpeedyGenes Protocol
Oligonucleotides one and eight were utilised as forward and reverse primers, respectively (600 nM each), while the remaining oligonucleotides were mixed together to form the template (30 nM). The reaction (50 µL) also contained 0.2mM dNTPs, Q5 reaction buffer and 0.02 U/µL Q5 hot-start polymerase (New England Biolabs). The reaction constituted an initial denaturation at 98 °C for 90 s, followed by 35 cycles of 98 °C for 20 s, 60 °C for 20 s and 72 °C for 30 s. Full-length genes were then purified by electrophoresis and gel extraction kit (Macherey-Nagel).

S3.1 Extraction and purification of enzymes
Cultures in deep well plates were pelleted by centrifugation (3000 x g), resuspended in lysis buffer (50mM Tris pH 8.0 containing 50% Bugbuster, 0.1 mg/mL lysozyme, protease inhibitors cocktail and 0.1 mg/mL DNase) and agitated at 30 °C 20 min at 1000 rpm. Insoluble material was pelleted by centrifugation and the soluble protein was bound to Ni-NTA resin suspension (Qiagen) (50 µL per variant). The resin was washed, and KSI was eluted with buffer (50 mM Tris pH 7.5 + 250 mM NaCl) containing 10 mM and 250 mM imidazole, respectively. Purified enzyme eluates were buffer exchanged into 50 mM Tris-HCl pH 7.0 and concentrated using Vivaspin 500 (Sartorius) prior to enzyme assays.

S3.2 GC/GC-MS analysis of Mentha monoterpenoids
GC-MS achiral quantitative analysis was conducted on a 7890B GC coupled to a 5975 series MSD quadrupole mass spectrometer and equipped with a 7693 autosampler (Agilent, Technologies, UK). Samples (1 µL) were injected onto a DB-WAX column (30 m x 0.320 mm x 0.25 µm; Agilent Technologies) with an inlet temperature of 240 °C and a split ratio of 20:1. Helium was used as the carrier gas with a flow rate of 1.5 mL/min and a pressure of 1.5603 psi. The chromatography was programmed to begin at 40 °C with a hold time of 1 minute, followed by an increase to 150 °C at a rate of 10 °C/min, then a subsequent increase to 210 °C at a rate of 80°C/min and a final hold time of 1 min. The total run time for the analysis was 13.75 min. The MS was equipped with an electron impact ion source using 70eV ionisation and a fixed emission of 35 µA. The mass spectrum was collected for the range 35-550 mz with a scan speed of 3,125 (N=1).
Chiral product analysis was performed by analysing reactions by GC using an Agilent Technologies 7890A GC system with an FID detector and a Chirasil-DEX-CB column (Agilent; 25 m, 0.32 mm, 0.25 µm). In this method the injector temperature was at 180 °C with a split-less injection. The carrier gas was helium with a flow rate of 1 mL/min and a pressure of 5.8 psi. The program began at 70 °C with an increase of temperature to 150 °C at a rate of 20 °C/minute and a hold for 3 min. This was followed by an increase of temperature to 190 °C at a rate of 2 °C/minute and a hold for 3 min.
For all GC-MS analyses, sec-butylbenzene was used as an internal standard to allow for accurate quantification. sec-Butylbenzene (0.01%) was added to all samples and the quantification of members of the menthol pathway was calculated relative to the peak area of this internal standard. Calibration curves were constructed for accurate quantification and calibration standards were analysed in a random sequence in the same analytical run as the standards. Vendor binary files were converted to open mzXML data format 1 using ProteoWizard msConvert. 2 Automated peak profiling and quantification was conducted using in-house scripts written in R.
Analysis of the isopulegol isomers was conducted on a 7890B GC coupled to a 5975 series MSD quadrupole mass spectrometer and equipped with a 7693 autosampler (Agilent, Technologies, UK). The sample (2 µL) was injected onto a DB-5 column (20 m x 0.1 mm x 0.1 µm; Agilent Technologies) with an inlet temperature of 250 °C and a split ratio of 2:1. Helium was used as the carrier gas with a flow rate of 0.1 mL/min and a pressure of 22.303 psi. The chromatography was programmed to begin at 60 °C with a hold time of 2 minutes, followed by an increase to 130 °C at a rate of 40 °C/min, a subsequent increase to 150 °C at a rate of 2 °C/min followed by an increase to 200 °C at a rate of 40 °C/min and a final hold time of 2 min. The total run time for the analysis was 17 min. The MS was equipped with an electron impact ion source using 70 eV ionisation and a fixed emission of 34.6 µA. The mass spectrum was collected for the range 50-550 mz with a scan speed of 3,125 (N=1).

S3.3 Steady state reactions
Product concentrations from steady state reactions were determined by comparison to a pulegone standard curve. Reactions were monitored over a 1 hour time period, and corrected for losses over time. The standard curve shows the absorbance at the start of the incubation.

S6. KSI Molecular Simulation Methods
Molecular dynamics simulations were carried out using Gromacs 4.6.1 with the Gromos 53A6 force field and periodic boundary conditions. [3][4] The enzyme was placed in a solvation box of minimum 10 Å around the protein. The simulation protocol was as follows: after energy minimization the system was thermalized 300 K for 100 ps using NVT dynamics, and the pressure was then equilibrated for 100 ps using NPT dynamics; the protein and substrates were constrained during these steps. All constraints and pressure couplings were then switched off and harmonic constraints of 100 kJ/mol/Å 2 were applied to keep the (+)-cis-isopulegone (3) near a reactive conformation, as defined by the distances in the energy minimised DFT model for the variant: 2.0 Å for the distance between the carbonyl oxygen of 3 and the carboxylic/hydroxyl H of D/S103 and 3.5 Å for the distance between substrate C and carboxylate O of D40 ( Figure S5). The system was then relaxed at 250 K, 280 K, 290 K and 300 K for 1 ns each, before the 50 ns production MD runs at 300 K. Representative structures for illustrative purposes were selected as those with the lowest RMSD for the substrate and residues 40 and 103 relative to the average, following structural alignment to the protein backbone.   Figure S3. The robotics-driven directed evolution workflow. The process included manual (green) and automated (blue) procedures. Following library synthesis, vector ligation and transformation into E. coli (1), colonies expressing single KSI variants were picked, grown and the expression induced (2). His-tagged KSI proteins were purified by IMAC (3), followed by quantification and desalting (4). Reactions were processed for GC-MS analysis by solvent extraction (5 & 6), followed by automated data processing (7) and analysis for the design of the next library (8). This data, together with structural information, was utilized to design subsequent variants for screening. Figure S4. Superimposition of the crystal structures of wild type and four variant KSI enzymes. The structures are shown as grey, green, red, orange and blue cartoons for wild-type (PDB 1OPY) 6 and variants D103S, V88I/L99V, V88I/L99V/D103S and V88I/L99V/V101A/D103S, respectively. Variant residues are shown as sticks in the same respective colours.