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Articleshttps://doi.org/10.1038/s41929-019-0341-4
Chiral synthesis of LSD1 inhibitor GSK2879552 enabled by directed evolution of an imine reductaseMarkus Schober1,5, Chris MacDermaid2,5, Anne A. Ollis3, Sandy Chang3, Diluar Khan4, Joseph Hosford1, Jonathan Latham 1, Leigh Anne F. Ihnken3, Murray J. B. Brown 1, Douglas Fuerst3, Mahesh J. Sanganee4 and Gheorghe-Doru Roiban 1*
1Synthetic Biochemistry, Medicinal Science & Technology, GlaxoSmithKline Medicines Research Centre, Stevenage, UK. 2Molecular Design, Computational and Modelling Sciences, GlaxoSmithKline, Collegeville, PA, USA. 3Synthetic Biochemistry, Medicinal Science & Technology, GlaxoSmithKline, Collegeville, PA, USA. 4Chemical Development, Medicinal Science & Technology, GlaxoSmithKline, Medicines Research Centre, Stevenage, UK. 5These authors contributed equally: Markus Schober, Chris MacDermaid. *e-mail: [email protected]
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Supplementary Information
Chiral synthesis of LSD1 inhibitor GSK2879552 enabled by directed evolution of an imine reductase Markus Schober1†, Chris MacDermaid2†, Anne A. Ollis3, Sandy Chang3, Diluar Khan4, Joseph Hosford1, Jonathan Latham1, Leigh Anne F. Ihnken3, Murray J.B. Brown1, Douglas Fuerst3, Mahesh J. Sanganee4 and Gheorghe-Doru Roiban1* 1Synthetic Biochemistry, Medicinal Science & Technology, GlaxoSmithKline Medicines
Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom. Email:
2Molecular Design, Computational and Modelling Sciences, GlaxoSmithKline, 1250 South
Collegeville Road, Collegeville, Pennsylvania 19426, United States
3Synthetic Biochemistry, Medicinal Science & Technology, GlaxoSmithKline, 1250 South
Collegeville Road, Collegeville, Pennsylvania 19426, United States
4Chemical Development, Medicinal Science & Technology, GlaxoSmithKline, Medicines
Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom
†These authors contributed equally.
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Table of Contents
Supplementary Methods ......................................................................................................................... 4
Synthesis of starting materials ............................................................................................................ 4
Preparation of (1R,2S)-2-phenylcyclopropan-1-amine (R)-mandelate (1) ...................................... 4 Preparation of aldehyde 3 for Evolution Rd1 ................................................................................... 5 Preparation of aldehyde 3 used for Evolution Rd2 and Rd3 ........................................................... 5
Enzyme identification, backbone selection, and fermentation ............................................................ 5
Origin of reductive amination enzymes ............................................................................................ 5 IR-46 gene acquisition and construction of expression vectors ...................................................... 5 IRED panel screening for the formation of (1R,2S)-4 ...................................................................... 6 Enzyme selection ............................................................................................................................. 7 Desired enzyme operating space .................................................................................................. 10 IR-46 activity in different buffers .................................................................................................... 10 Production of enzyme powders, shake flask procedure ................................................................ 11 Production of enzyme powders, fermentation procedure .............................................................. 11
Directed evolution ............................................................................................................................. 12
General information ....................................................................................................................... 12 Library construction and expression .............................................................................................. 12 Bioinformatics ................................................................................................................................ 12 Library sequencing ......................................................................................................................... 22 HTP Protocols for evolution Rd1 ................................................................................................... 22 HTP Protocols for evolution Rd2 ................................................................................................... 23 HTP Protocols for evolution Rd3 ................................................................................................... 25 Top variants identified after each evolution round ......................................................................... 28
Selected scale-up examples ............................................................................................................. 35
Preparation of (1R,2S)-4 using IR-46 (WT) as catalyst ................................................................. 35 Preparation of (1R,2S)-4 using M1 as catalyst .............................................................................. 36 Preparation of (1R,2S)-4 using M2 as catalyst .............................................................................. 36 Preparation of (1R,2S)-5 using M3 as catalyst .............................................................................. 36 Kilogram make of (1R,2S)-4 using M3 as catalyst ........................................................................ 37
Redox-neutral enzymatic cascade conversion of 2 to (1R,2S)-4...................................................... 38
Selection of the KRED catalyst ...................................................................................................... 38 Screening KREDs for the cascade reaction .................................................................................. 38 Preparation of (1R,2S)-4 using M3/KRED as catalysts ................................................................. 38
Sequence listing ................................................................................................................................ 39
IR-46 .............................................................................................................................................. 39 M1 .................................................................................................................................................. 39 M2 .................................................................................................................................................. 39 M3 .................................................................................................................................................. 40
KRED ................................................................................................................................................ 40
UPLC analysis and selected chromatograms ....................................................................................... 41
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Method 1: High-throughput analytical UPLC method to determine conversion of aldehyde 3 to amine 4 .............................................................................................................................................. 41
Method 2: Achiral analytical HPLC method to determine conversion of aldehyde 3 to amine 4 ...... 41
Method 3: Chiral normal phase HPLC analytical method to determine enantiomeric excess of 4 ... 41
Method 4: Chiral reverse phase UPLC analytical method to determine enantiomeric excess of 4 .. 41
Selected UPLC Chromatograms ....................................................................................................... 42
Example of overlaid chromatograms run using Method 1. ............................................................ 42 Example of chromatogram runs using Method 2. .......................................................................... 42 Example of a chromatogram run of (1R,2S)-5 using Method 3. .................................................... 43 Example of a chromatogram run using Method 4. ......................................................................... 43
NMR of aldehyde (3) and rac-trans-1 mixture in the absence of enzyme ............................................ 46
NMR spectra ......................................................................................................................................... 48 1H NMR (400 MHz, D2O) and 13C NMR (101 MHz, D2O) spectra of rac-trans-1 .............................. 48 1H NMR (400 MHz, DMSO-d6) and 13C NMR (101 MHz, DMSO-d6) spectra of (1R,2S)-1 .............. 49 1H NMR (400 MHz, DMSO-d6) and 13C NMR (101 MHz, DMSO-d6) spectra of 3 ............................ 50 1H NMR (400 MHz, CD3-OD) and 13C NMR (101 MHz, CD3-OD) spectra of (1R,2S)-4 ................... 51
Supplementary References................................................................................................................... 52
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Supplementary Methods Materials: Tranylcypromine sulphate (rac-trans-1), was purchased from Trifarma, Italy and tranylcypromine hydrochloride was acquired from Sigma Aldrich. Aldehyde 3 was prepared in house1 as described below. (1R,2S)-2-Phenylcyclopropan-1-amine (R)-mandelate (1R,2S)-1 was provided by Wuxi, China. D-Glucose dehydrogenase GDH-CDX-901 was acquired from Codexis Inc and NADP+ was purchased from Bontac Engineering, China. All solvents and reagents were purchased from Sigma-Aldrich and Honeywell and used as supplied. Buffers were prepared in house. NMR data were collected using a Bruker Avance 400 Ultrashield instrument using 400 MHz and 101 MHz for 1H, and 13C NMR, respectively. Conversion was determined by achiral UPLC and chiral HPLC on Agilent 1290 systems. For the synthesis of 4, conversion and yield are calculated with respect to the carbonyl compound based on the convention used for reductive amination. This was done as aldehyde 3 is an intermediate and not a starting material like racemic amine 1. Determination of the absolute configuration of 4 [enantiomer (1R,2S) unless stated otherwise] was made after comparison with authentic prepared standard using amine (1R,2S)-1. Fold increase over the parent (FIOP) was calculated by substracting the conversion of the mutant versus backbone in the specific screening conditions. Directed evolution was carried out using the CodeEvolver® protein engineering platform in licenced from Codexis Inc. Bioinformatics: Templates for homology model construction were identified using HHsuite. A homology model was constructed using the Chemical Computing Group’s Molecular Operating Environment (MOE). Scikit-learn was used to spacially cluster the beneficial mutations from round one. Evaluation of the intensive interactions of mutations in the proteins were performed using MOSAIC® in licenced from Codexis Inc. Library construction and expression: Site-saturation mutagenesis libraries were constructed by overlap extension PCR. Combinatorial libraries were constructed using the QuikChange Lightning Multi Site-Directed Mutagenesis kit (Agilent). Expression for high-throughput screening was done in 96-well plates. High throughput screening: High throughput screening reactions were carried out in Costar deep-well 96-well plates, and UPLC analysis performed in shallow well polypropylene plates (Nunc 96). Examples of representative chromatograms are shown below. BioMex FX liquid handling robot (Beckman Coulter, Fullerton, CA) was used for assay setup. Further experimental details are provided herein. Process development and scale-up: Experiments on scale >1g were performed in specialised equipment such as Amigochem or EasyMax. For scale >10 g controlled laboratory reactors (CLRs) with a volume up to 20 L have been used. Detailed experimental procedures of reductive amination reactions are described see below. Cells paste were disrupted using a microfluidiser M110Y from analytikLtd. Lyophilization was performed using a BPS VirTris SP Scientific Advantage Pro lyophilizer.
Synthesis of starting materials Preparation of (1R,2S)-2-phenylcyclopropan-1-amine (R)-mandelate (1)
NH3 resolution
EtOH
(R) COOH
OH
(R) COO
OH
(1R,2S)-1
NH3
(R)
(S)
(R) COO
OHEtOH/H2O
rac-trans-1
NH2
NH2
H2SO4
A solution of rac-trans-1 (1.0 wt) in water was adjusted to pH ≥13 with 2M NaOH solution at 15-25 °C and extracted with dichloromethane (DCM). The separated aqueous layer was adjusted to pH ≥13 with 2M NaOH solution and extracted with further DCM. The combined organic phase was washed with water and concentrated before solvent exchange to ethanol (EtOH) under reduced pressure. (R)-2-Hydroxy-2-phenylacetic acid (~72 g) in EtOH was added at 30-35 °C and then cooled to 15-20 °C. The batch was filtered and the wet cake was washed with EtOH and dried under reduced pressure at 35-45 °C to give the mandelic salt of rac-trans-1. A mixture of the mandelic salt intermediate, EtOH, and water were heated to 60-70 °C and stirred until the solid dissolved completely. The solution was
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cooled to 40-50 °C and, after a hold period, cooled to 15-20 °C and aged. The batch was filtered and the wet cake was washed with EtOH. A mixture of the wet cake, EtOH, and water were heated to 60-70 °C and stirred and then cooled to 40-50 °C. After a hold period, the mixture was cooled to 15-20 °C and aged. The batch was filtered and the wet cake washed with EtOH and dried under reduced pressure at 45-55 °C to give (1R,2S)-1 as a pale white solid (e.e. 99.9%). 1H NMR (DMSO-d6, 400 MHz, 300K): δ 7.42-7.37 (m, 2H), 7.33 (br s, water and exchangeables), 7.29-7.22 (m, 4H), 7.21-7.14 (m, 2H), 7.09-7.05 (m, 2H), 4.67 (s, 1H), 2.63 (ddd, J=7.7, 4.3, 3.6 Hz, 1H), 2.18 (ddd, J=9.7, 6.2, 3.5 Hz, 1H), 1.26 (ddd, J=9.9, 5.7, 4.5 Hz, 1H), 1.06 ppm (dt, J=7.6, 6.0 Hz, 1H). 13C NMR (DMSO-d6, 101 MHz): δ 175.1 (1C), 143.0 (1C), 140.2 (1C), 128.3 (2C), 127.5 (2C), 126.4 (1C), 126.3 (2C), 126.0 (2C), 125.9 (1C), 73.3 (1C), 31.6 (1C), 21.8 (1C), 14.4 (1C) ppm. HRMS (ESI) m/z [M+H]+: molecular ion calculated for C9H21N: 134.0970, found 134.0965 (free base).
Preparation of aldehyde 3 for Evolution Rd1
O
O
N
OH
2
O
O
N
O
3 Alcohol 2 (10.0 g, 1.00 equiv, 1.00 wt), dimethylsulfoxide (DMSO) (50 mL), 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) (0.5 g, 0.10 equiv), CuI (0.6 g, 0.10 equiv), 2,2'-bipyridine (0.5 g, 0.10 equiv) and N-methyl imidazole (0.20 equiv, 0.5 g) were charged to a 100 mL EasyMax vessel fitted with a metal sparger connected to a compressed air line. The reaction mixture was heated to 70 °C and compressed air was sparged into the reaction for 2 h. The crude reaction mixture was then cooled to 25 °C and used directly in the enzymatic reaction. Preparation of aldehyde 3 used for Evolution Rd2 and Rd3 Alcohol 2 (100.0 g, 1.00 equiv), sulfolane (600 mL, 6 vol), 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) (2.5 g, 0.05 equiv), CuI (3.7 g, 0.06 equiv), 2,2'-bipyridine (3.0 g, 0.06 equiv) and N-methyl imidazole (2.4 g, 0.09 equiv) were charged to a 2 L conical CLR fitted with a retreat curve impeller (stirrer speed = 370 rpm) and Mid IR probe. The reaction mixture was heated to 55 °C and compressed air was sparged into the reaction at 350 mL / min (3.5 mL / g*min) using an HPLC cylindrical metal sparger (length = 2 cm, diameter = 1.1 cm). The reaction mixture was cooled to 25 °C and diluted with methyl tert-butyl ether (MTBE) (1.44 L, 14.4 vol). Sodium chloride (31.3 g, 0.31 wt) was added followed by water (720 mL, 7.2 vol). It was stirred for at least 30 min then the phases allowed to separate. The lower aqueous sulfolane phase (pale blue) was separated off. The upper organic phase (pale yellow) was washed three times with water (3 x 600 mL, 3 x 6 vol). The MTBE solution of crude product was distilled to circa 3 vol. DMSO (500 mL, 5 vol) was added to the batch and distillation was continued until no more distillate was collected. The aldehyde 3 – DMSO solution was assayed for yield versus an external standard. Yield 86% and purity by HPLC = 94.6% a/a. For NMR characterization a portion of the MTBE solution of the crude product was vacuumed down and sample submitted to NMR. 1H NMR (400 MHz, DMSO-d6, 300K): δ 9.58 (s, 1H), 7.84 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 3.51 (s, 2H), 2.70-2.64 (m, 2H), 2.31-2.26 (m, 1H), 2.09-2.03 (dt, J=11.1, 2.6 Hz, 2H), 1.82-1.78 (m, 2H), 1.53-1.45 (m, 11H) ppm. 13C NMR (101 MHz, DMSO-d6, 300K): δ 204.7 (1C), 164.8 (1C), 143.8 (1C), 129.9 (1C), 128.9 (2C), 128.7 (2C), 80.4 (1C), 61.7 (1C), 52.0 (2C), 46.8 (1C), 27.8 (2C), 25.0 (2C) ppm. HRMS (ESI) m/z [M+H]+: molecular ion calculated for C18H26NO3: 304.1907, found 304.1907.
Enzyme identification, backbone selection, and fermentation Origin of reductive amination enzymes The enzyme collection has been previously published and most of it’s IRED sequences disclosed2. Sequence of IR-46 is disclosed in this publication.
IR-46 gene acquisition and construction of expression vectors A gene encoding IR-46 codon optimized for expression in E. coli was designed based on the reported amino acid sequence of the Saccharothrix espanaensis DSM 44229 homologue (NCBI Reference
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Sequence: WP_015102218.1). The gene was cloned into the expression vector pCK1109003 under control of a lac promoter. All cloning and expression used an E. coli strain derived from W3110.
IRED panel screening for the formation of (1R,2S)-4 Imine reductase plates were expressed as published previously2. Plates containing frozen cell pellets were allowed to thaw at room temperature for 30 min before re-suspension in lysis buffer (200 µL per well, 0.5 mg/mL polymixin B sulphate, 1.0 mg/mL lysozyme, 10 U/mL benzonase in 100 mM potassium phosphate buffer, pH 7.0). Plates were then sealed using air-permeable nylon seals and incubated with vigorous shaking at room temperature for 2 h. Lysate was then clarified by centrifugation (4000 rpm, 10 min, 4 °C) before use in reactions. General reductive amination panel screening protocol for formation of of (1R,2S)-4 Addition of all stocks to the reaction plate and reaction quenching were done using a BioMek FX liquid handling robot (Beckman Coulter, Fullerton, CA). To a Costar deep 96-well plate was added amine stock (224 µL) followed by cofactor stock solution (156 µL) containing NADP+ (2.77 mg/mL, final concentration 1.03 mg/mL), D-glucose (13.51 mg/mL, final concentration 5.05 mg/mL) and CDX-GDH-901 (6.70 mg/mL, final concentration 2.50 mg/mL) in 100 mM potassium phosphate buffer (pH 7.0). Clarified cell lysate (160 µL) was then transferred to the reaction plate. Reactions were initiated by addition of aldehyde 3 stock to each well (60 µL, 20 mg/mL 3 in DMSO, final concentration 2 mg/mL 3, 10% v/v DMSO). The reaction plate was then sealed using aluminium/polypropylene laminate heat seal tape and incubated at 25 °C with shaking at 800 RPM in an Infors MultiTron shaker. After incubation for 1 h, a 100 µL aliquot was removed from each well of the reaction plate and added to a second Costar deep 96-well plate containing 1M acetic acid (150 µL) and acetonitrile (200 µL). The plate was sealed as described above and shaken at room temperature for 5 min before centrifugation (4000 RPM, 10 min). 200 µL of supernatant was transferred from each well to a flat-bottom Nunc 96-well plate which was sealed before analysis by UPLC. The reaction plate was then re-sealed and incubated as described above. After incubation for a further 16 h, the remaining reaction volume was quenched by addition of 1M acetic acid (200 µL) and acetonitrile (300 µL) to each well of the reaction plate. The reaction plate was then shaken, centrifuged and prepared for analysis by UPLC as described above. Screening of enantiopure (1R,2S)-(1) The IRED panel was screened according to the protocol described above, using a stock of (1R,2S)-1 (7.9 mg/mL in 100 mM potassium phosphate buffer (pH 7.0), 2.1 mg/mL final concentration in reaction, 1.1 equiv (of wanted enantiomer)) as amine stock.
O
O
N
O
IRED panel
N
NH
O
O
2HCl
+
3 (1R,2S)-4
100 mM Phosphate Buffer pH 7.0 DMSO (10%), 30oC
NADP+NADPH
D-GlucoseGluconic acid
NH3
(R)
(S)
(R) COO
OH
(1R,2S)-1
GDH
.
Supplementary Table 1. Top 10 variants resulted of screening the IRED panel plate for conversion of 3 to (1R,2S)-4 using enantiopure (1R,2S)-1 as amine source after 1 h and 17 h. Negative controls showed no conversion.
Enzyme variant Conversion [%] (1 h) Conversion [%] (17 h) IR-5 24.9 65.6 IR-30 11.8 25.5
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IR-32 12.3 39.0 IR-33 7.9 52.8 IR-45 4.6 25.0 IR-46 92.8 94.9 IR-59 6.9 27.9 IR-70 7.6 28.7 IR-82 28.0 63.5 IR-84 66.9 87.4 Screening of racemic 2-phenylcyclopropan-1-amine (rac-trans-1) A reductive amination panel plate was screened according to the protocol described above, using a stock of rac-trans-1 (10 mg/mL in 100 mM potassium phosphate buffer, pH 7.0, 2.6 mg/mL final concentration in reaction, 2.2 equiv (1.1 of wanted enantiomer) as amine stock.
O
O
N
O
IRED panel
N
NH
O
O
rac-trans-1
2HCl
+
3 (1R,2S)-4
NH2+NH2
NH2
H2SO4
100 mM Phosphate Buffer pH 7.0 DMSO (10%), 30oC
NADP+NADPH
D-GlucoseGluconic acid GDH
Supplementary Table 2. Screening results for the IRED panel plate for conversion of 3 and rac-trans-1 to (1R,2S)-4 after 1 h and 17 h. Enantiomeric excess determined after 17 h (desired enantiomer (1R,2S)-4, undesired enantiomer (1S,2R)-4).
Enzyme variant Conversion [%] (1 h) Conversion [%] (17 h) e.e. [%] IR-5 13.6 39.9 36.4 (1R,2S) IR-22 - 34.7 81.2 (1S,2R) IR-30 9.4 29.4 33.4 (1S,2R) IR-33 10.5 46.3 99.9 (1R,2S) IR-37 - 30.7 92.7 (1S,2R) IR-39 - 30.9 61.1 (1S,2R) IR-46 82.1 86.2 99.9 (1R,2S) IR-82 12.4 38.8 99.3 (1R,2S) IR-84 57.1 81.3 43.4 (1R,2S) IR-88 23.7 56.5 96.9 (1S,2R)
Enzyme selection Sequence comparison of IR-33 and IR-46 IR-332 and IR-46 were identified as the best enzymes in the screening process as delivered the desired enantiomer (1R,2S)-4 in high e.e. and conversion. Sequence alignment of IR-33 and IR-46 revealed moderate sequence identity between IR-46 and IR-33 (41.4%). A comparison of these hits to two other recently investigated imine reductases AoIRED4, and AspRedAm5 share 50-68% sequence identity within the active site. Comparison of the residues lining the active site pocket and the adjacent NADPH cofactor binding site gives medium and high identity with 47% and 76% respectively with notable amino acid variations occurring at IR-46 positions L37A/R, V92L/T, A96S/V, F97V/A, I100T/L, Y142G/T, S145A/A, L195F/L, L201M/M, W202Y/W, V236M/V, T255N/T, S256G/D, S257Q/S S240L/F, S258Q/S, L262F/I, N263H/N, G266A/G and L304F/L. Conserved active site positions across IR-33, IR-46 include: G34, L35, G36, M38, G39, N57, K62, T117, I143, M144, V146, D194, L197, L198, Y253 and V257. Notable conservations between these IREDs include IR-46 positions G34, L35, G36, M38 and G39 which comprise the Rossman motif responsible for the recognition of
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NADPH. Positions N57, K62, I143, M144, L197, L198 and Y253 are conserved as well. Comparison of the positions specifically targeted for mutations in the AoIRED and AspRedAm investigations: (IR-46/AoIRED/AspRedAm) S118/S95/N93, W202/Y179/Y177, D194/N171/D169, W232/W209/W210, L262/M240/M239, N263/N241/Q240, shows only low similarities to IR-46.
Preparative scale-up of IR-33 and IR-46 Supplementary Table 3. Preparative-scale reductive aminations catalysed by IR-33 and IR-46. Conversion values for the best enzymes performing the reductive amination 3→ (1R,2S)-4.a
Entry
Enzyme Scale [g]
Enzyme form Enzyme load [% wt/wt]
Timeb [min]
Conversionc [%]
1 IR-33 1.0 Clarified lysate ~30% (1 wt) 1140 43 2 IR-46 1.0 Clarified lysate ~30% (1 wt) 120 73 3 IR-46 0.1 Lyophilised cell free powder 100% (1 wt) 60 76 4 IR-46 0.1 Lyophilised cell free powder 333% (3 wt) 120 100 aReaction parameters: 10 mg/mL 3, 2.1 equiv rac-trans-1, 0.01 equiv NADP+, 2% wt/wt GDH, 1.5 equiv D-glucose at 30 °C. 30% v/v DMSO, potassium phosphate buffer 100 mM, pH 6.0. bTime reaction conversion had plateued. cConversion was quantified through UPLC by integrating the area of amine versus product area. Initial development work using IR-46 Comparison aldehyde 3 directly telescoped from oxidation reaction1 (67.8% purity) and 3 (99.3% purity) with oxidation reagents spiked to the activity of IR-46 to catalyze the reductive amination with rac-trans-1 Reactions were set-up as follows and carried out at 30 °C with shaking at 1000 rpm in a 5 mL tube. 100 mM potassium phosphate buffer, pH 6.5 (K2HPO4/KH2PO4) (3.5 mL) was added, followed by rac-trans-1 (63.1 mg, 2.1 equiv), NADP+ (1.3 mg, 0.1 equiv), D-glucose (44.5 mg, 1.5 equiv), GDH-CDX-901 (1.0 mg, 2% wt/wt) and lyophilised powder of IR-46 (100.0 mg, 200% wt/wt) and dissolved with shaking. Reactions were started by the addition of a solution of DMSO (1.5 mL) containing: A: aldehyde 3 (99.3%) (50.0 mg, 1 equiv), B: aldehyde 3 (99.3%) (50.0 mg, 1 equiv), 2,2'-bipyridine (2.6 mg, 0.1 equiv), C: aldehyde 4 (>97%) (50.0 mg, 1 equiv), 1-methyl-1H-imidazole (2.71 mg, 0.2 equiv), D: aldehyde 3 (>97%) (50 mg, 1 equiv), 2,2,6,6-tetramethylpiperidin-1-yl)oxyl (2.6 mg, 0.1 equiv), E: aldehyde 3 (99.3%) (50.0 mg, 1 equiv), copper iodide (3.1 mg, 0.1 equiv), F: aldehyde 3 (>99.3%) (50.0 mg, 1 equiv), 2,2'-bipyridine (2.6 mg, 0.1 equiv), 1-methyl-1H-imidazole (2.7 mg, 0.2 equiv), 2,2,6,6-tetramethylpiperidin-1-yl)oxyl (2.6 mg, 0.1 equiv), copper iodide (3.1 mg, 0.1 equiv), G: telescoped aldehyde 3 from Stahl oxidation (50.0 mg, 1 equiv). Reactions were sampled (100 µL) at time points (15, 30 and 60 min), samples were quenched by the addition of acetonitrile:1M acetic acid 8:1 (900 µL) and filtered before analysis by HPLC. Reaction conversions were determined by achiral analytical HPLC on an Agilent 1290 LC using Method 2.
Supplementary Table 4. Reaction conversions, calculated by area% product 4 vs. aldehyde 3 at 220 nm.
Time [min] 15 30 60 A - 3 (99.3%) 63 76 85 B - 3 (99.3%) + 2,2'-bipyridine 65 79 86 C - 3 (99.3%) + 1-methyl-1H-imidazole 64 80 89 D - 3 (99.3%) + TEMPO 64 78 86 E - 3 (99.3%) + CuI 64 71 75 F - 3 (99.3%) + All Stahl reagents 60 71 75 G - Telescoped 3 (67.8%) from oxidation of 2 36 43 43
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Supplementary Figure 1. Graph showing conversion to product 4 over time, calculated by area% product 4 vs. aldehyde 3 at 220 nm.
Experiments to establish catalyst stability over time when subject to reaction temperature IR-46 lysate (75 µL) from 1.5 mg wet cell pellet was incubated in reaction buffer (200 mM potassium phosphate buffer, pH 6.5 (K2HPO4 / KH2PO4)) at 30°C for various time points (0 min, 60 min, 120 min, 180 min, 240 min, 1260 min). After which reactions were commenced by the addition of reaction buffer 200 mM potassium phosphate buffer, pH 6.5 (K2HPO4 / KH2PO4) containing the following; rac-trans-1 (1.6 mg), NADP+ (0.26 mg), D-glucose (0.7 mg), GDH-CDX-901 (0.26 mg) and aldehyde 3 (1.0 mg) in DMSO (100 µL). Reactions were sampled (100 µL) at 120 min, samples were quenched by the addition of acetonitrile (500 µL) and filtered before analysis by HPLC. Reaction conversions were determined by achiral analytical HPLC on an Agilent 1290 LC using Method 2.
Supplementary Table 5. Reaction conversions, calculated by area% product 4 vs. aldehyde 3 at 220 nm.
Time catalyst (IR-46) incubated at 30 °C % Conversion to product 4 (area%
product 4 vs. aldehyde 3) 0 96 60 94 120 94 180 93 240 92 1260 10
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70
Con
vers
ion
(%ar
ea a
ldeh
yde
3vs
. pro
duct
4)
)
Time (min)
A - Aldehyde (99.3%)B - Aldehyde (99.3%) + 2,2'-bipyridineC - Aldehyde (99.3%) + 1-methyl-1H-imidazoleD - Aldehyde (99.3%) + TEMPOE - Aldehyde (99.3%) + CuIF - Aldehyde (99.3%) + All Stahl reagentsG - Telescoped aldehyde (67.8%) from Stahl oxidation
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Supplementary Figure 2. Graph showing conversion to product 4 vs. time catalyst (IR-46) incubated at 30 °C. calculated by area% product 4 vs. aldehyde 3 at 220 nm.
Desired enzyme operating space The enzyme had operate under moderate acidic conditions for increased product 4 stability. Acidic pH was also needed to increase the solubility of substrates and product. Due to the very low solubility of aldehyde 3 stepwise addition was favoured. To allow the reaction pH to stay below 6.0 during the stepwise addition a starting pH <5.0 was needed due to the basicity of 3. This was an additional challenge as previous reports of IRED or (reductive aminase) RedAm activity have been conducted in slight basic or neutral pH. Further IR-46 screening showed diminished catalyst activity in potassium phosphate buffer at pH 6.0, with further significant reduction in NaOAc at pH 5.6. Negligible activity was observed in NaOAc buffer at pH 5.0 and 4.6. IR-46 activity in different buffers IR-46 lysate was prepared from 27.5 g wet cell pellet and 20 mM potassium phosphate buffer (112 mL, pH 7.0). Lysate (160 μL) was added to 18 wells of a pre-weighed deep-well 96-well plate. The plate containing lysate was then frozen at -80 oC and lyophilised according to the procedure above. Once lyophilisation was complete, the plate was re-weighed to determine the lyo powder loading in each well. All reaction setup steps were performed using a Beckman Coulter BioMek FX liquid handling robot. Lyophilised lysate was then re-suspended in 400 mM buffer at the required pH (150 μL, see below) and incubated at room temperature with shaking for 10 min. All buffers were added to lyophilised lysate at the same time, by transferring from a separate plate to the lysate plate. Amine stock (187.5 μL) containing racemic 1 (40 mg/mL, 12.5 mg/mL final) in water as added to the plate. Cofactor stock (82.5 μL) containing D-glucose (65 mg/mL, final 8.9 mg/mL), NADP+ (1.9 mg/mL, final 0.26 mg/mL) and CDX-GDH-901 (1.45 mg/mL, final 0.2 g/mL) in water was then added to the plate. DMSO (120 μL) was then added to the plate followed by aldehyde stock (60 μL) containing 3 (100 mg/mL, 10 mg/mL final). The reaction plate was then sealed with aluminium/polypropylene heat seal tape and incubated at 30 oC with shaking at 800 RPM in an Infors MultiTron shaker. After incubation for 16 h, reactions were quenched by addition of 1M acetic acid (300 µL) and acetonitrile (400 µL) to each well of the reaction plate. The reaction plate was then shaken at room temperature before centrifugation (4000 RPM, 10 min, 4 °C). 200 μL of supernatant was then transferred to a fresh plate for analysis by achiral UPLC.
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
0 200 400 600 800 1000 1200 1400
Con
vers
ion
(%ar
ea a
ldeh
yde
3vs
. pro
duct
4)
Time (min)
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Supplementary Table 6. Reaction conversions, calculated by area% product 4 vs. aldehyde 3 at 220 nm.
Reaction Buffer % Conversion to product 4 (area%
product 4 vs. aldehyde 3) 100 mM Potassium phosphate pH 7.0 92 100 mM Potassium phosphate pH 6.5 83 100 mM Potassium phosphate pH 6.0 72
100 mM Sodium acetate pH 5.6 27 100 mM Sodium acetate pH 5.0 2 100 mM Sodium acetate pH 4.6 <1
Conversion of aldehyde 3 to product 4 using IR-46 lyophilised cell free powder in different reaction buffers. Reaction conditions: 10 mg/mL 3, 2.1 equiv rac-trans-1, 0.01 equiv NADP+, 175 w/w% IR-46 lyophilised lysate, 2% wt/wt GDH, 1.5 equiv D-glucose at 30 °C. 30% v/v DMSO. Conversion is an average of 3 reactions. Production of enzyme powders, shake flask procedure A single microbial colony of E. coli containing a plasmid encoding an engineered imine reductase of interest was inoculated into 50 mL Luria Bertoni broth containing 30 µg/mL chloramphenicol and 1% D-glucose. Cells were grown overnight (at least 16 h) in an incubator at 30 °C with shaking at 220 rpm. The culture was diluted into 1000 mL of Terrific broth containing 30 µg/mL chloramphenicol to give an approximate OD600 of 0.1‒0.2 and allowed to grow at 37 °C with shaking at 250 rpm. Expression of the imine reductase gene was induced by addition of isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM when the OD600 of the culture was 0.6 to 0.8 and incubation was then continued overnight (at least 16 h). Cells were harvested by centrifugation (4000 rpm, 20 min, 4 °C), and the supernatant was discarded. Pellets were frozen for 2 h at ‒80 °C. Pellets were then thawed and re-suspended to 3 mL per gram of final pellet mass in 20 mM sodium acetate buffer (e.g., 10 g frozen pellet suspended in 30 mL sodium acetate buffer). The intracellular imine reductase was released from the cells by passing the suspension through a homogenizer fitted with a two-stage homogenizing valve assembly using a pressure of 5000-12000 psi. Cell debris was then removed by centrifugation (9000 rpm, 45 min, 4 °C). The clear lysate supernatant was collected, pooled, and lyophilised to provide a dry cell free powder of crude IRED enzyme.
Production of enzyme powders, fermentation procedure A single microbial colony of E. coli. containing a plasmid with the imine reductase gene of interest was inoculated into 2 mL M9YE broth (1.0 g/L ammonium chloride, 0.5 g/L of sodium chloride, 6.0 g/L of disodium monohydrogen phosphate, 3.0 g/L of potassium dihydrogen phosphate, 2.0 g/L of Tastone-154 yeast extract, 1 L/L de-ionized water) containing 30 μg/mL chloramphenicol and 1% D-glucose. Cells were grown overnight (at least 12 h) in an incubator at 37 °C with shaking at 250 rpm. After overnight growth, 0.5 mL of this culture was diluted into 250 mL M9YE Broth, containing 30 μg/mL chloramphenicol and 1% D-glucose in 1 liter flask and allowed to grow at 37 °C with shaking at 250 rpm. When the OD600 of the culture is 0.5 to 1.0, the cells were removed from the incubator and either used immediately, or stored at 4 °C. Bench-scale fermentations were carried out at 30 °C in an aerated, agitated 10 L fermenter using 6.0 L of growth medium (0.9 g/L ammonium sulphate, 1.0 g/L of sodium citrate; 12.5 g/L of dipotassium hydrogen phosphate trihydrate, 6.2 g/L of potassium dihydrogen phosphate, 3.3 g/L of Tastone-154 yeast extract, 0.083 g/L ferric ammonium citrate, 0.5 mL/L antifoam and 8.3 mL/L of a trace element solution containing 2 g/L of calcium chloride dihydrate, 2.2 g/L of zinc sulphate heptahydrate, 0.5 g/L manganese sulphate monohydrate, 1 g/L cuprous sulphate heptahydrate, 0.1 g/L ammonium molybdate tetrahydrate and 0.02 g/L sodium tetraborate). The vessel was sterilized at 121 °C and 15 PSI for 30 minutes. The fermenter was inoculated with a late exponential culture of E.
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coli W3110 containing a plasmid encoding the imine reductase gene of interest (grown in a shake flask as, described above, to a starting OD600 of 0.5 to 1.0). The fermenter was agitated at 250‒1250 rpm, and air was supplied to the fermentation vessel at 0.6-25 L/min to maintain a dissolved oxygen level of 50% saturation or greater. The pH of the culture was maintained at 7.0 by addition of 20% v/v ammonium hydroxide. Growth of the culture was maintained by addition of a feed solution containing 500 g/L Cerelose dextrose, 12.0 g/L ammonium chloride and 5.1 g/L magnesium sulphate heptahydrate. After the culture reached an OD600 of 80 +/-10, expression of imine reductase was induced by addition of IPTG to a final concentration of 1 mM and fermentation was continued for another 24 h. The culture was then chilled to 8 °C and maintained at that temperature until harvested. Cells were collected by centrifugation at 5000 G for 40 min in a Sorval RC12BP centrifuge at 4 °C. Harvested cell pellets were then frozen at –80 °C and stored until downstream processing and recovery, as described below.
The cell pellets were thawed and re-suspended in 3 volumes of 20 mM sodium acetate buffer, at 4 °C to each volume of wet cell paste. The intracellular imine reductase was released from the cells by passing the suspension through a homogenizer fitted with a two-stage homogenizing valve assembly using a pressure of 5000-12000 psi. The cell homogenate was cooled to 4 °C immediately after disruption. A solution of 11% w/v polyethyleneimine pH 7.2 was added to the lysate to a final concentration of 0.8% w/v. The lysate was then stirred for 30 min. The resulting suspension was clarified by centrifugation at 5000 G in a Sorval RC12BP centrifuge at 4 °C for 30 min. The clear supernatant was decanted and concentrated ten-fold using a cellulose ultrafiltration membrane with a molecular weight cut-off of 30 kD. The final concentrate was dispensed into shallow containers, frozen at –20 °C and lyophilised to cell free powder which was stored at –80 °C.
Directed evolution General information All mutations listed refer to the full-length protein, including the tag. Sampling rate for Rd1 = <88%, and for Rd2 & 3 <1%. The upper limit to the sampling rate is given since not all the variants have been sequenced therefore is impossible to know how many discreet variants we screened and how many duplicates there were. Cv for plate screening was generally <15%. Library construction and expression Site-saturation mutagenesis libraries were constructed by overlap extension PCR6. Gene fragments containing single codon mutations were created using a 1:1.5:3 pool of WKG, VMA, NDT degenerate codons to encode all 20 amino acids. Full-length genes were then generated and amplified using flanking primers, followed by cloning into pCK11009003 using In-Fusion® HD cloning (Clontech). Combinatorial libraries were constructed using the QuikChange Lightning Multi Site-Directed Mutagenesis kit (Agilent). All transformed libraries were plated to single colony density on LB Qtrays containing 34 µg/mL chloramphenicol (cam34) and 1% D-glucose (Teknova).
Expression for high-throughput screening was done in 96-well plates. Single colonies were picked into 180 µL LB containing cam34 and 1% D-glucose in Nunc plates and grown overnight at 30 °C, 200 rpm, in incubators maintaining 85% humidity. A 10 µL aliquot of these saturated overnight cultures was used to inoculate 390 µL TB containing cam34 in deep well plates. Subcultures were grown at 30 °C, 250 rpm, 85% humidity. When OD600 ~0.6–0.8, cultures were induced with 1mM IPTG (final concentration) and incubation continued for 16-18 h. Cells were harvested by centrifugation, and the resulting cell pellets were frozen at –80 °C.
Bioinformatics Template identification and homology model construction To date, there are no experimentally determined structures of IR-46. Therefore, we constructed a homology model to aid in prioritizing and rationalizing positions for mutation. A template search was performed using the HH suite set of tools.7 The sequence profile for IRED was generated using the
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uniprot20 dataset (2016-02), and the search performed against an internally curated redundant PDB dataset (2016-10). For both computations, the default HHblits and HHsearch parameters were used. The search identified 22 potential templates with 100% probability of homology and corresponding e-values less than 10-28. The selected template, 5FWN from Amycolatopsis orientalis, gave a high-quality alignment matching 289 of 296 positions with a single insertion and deletion of one residue, resulting in 42% identity to IR-46 (Supplementary Figure 3). 5FWN was resolved at 2.14 Å resolution, contains the biologically relevant dimeric complex in the asymmetric unit, well resolved density for the NADP+ cofactor and a co-crystallized ligand, (1R)-1-methyl-1,2,3,4-tetrahydroisoquinoline.
Supplementary Figure 3. Sequence alignment of IRED (IR-46) and the selected template 5FWN. HHblits was used to generate the sequence profile and HHsearch to identify the template and associated alignment.
The homology model was constructed using the Chemical Computing Group’s Molecular Operating Environment8 (MOE) using the alignment produced from HHsearch (Supplementary Figure 3). The raw structure was downloaded from the PDB structural database and the dimeric complex prepared using the protein preparation tool. The small molecule ligand and NADP+ cofactors were included in the preparation and model construction. Due to the high homology and contigious alignment, only 25 mainchain models were generated, each with 10 sidechain samples for a total of 250 models. The models were generated stochastically and sidechains were sampled from a distribution defined at 300 K. Intermediate models were refined using the “medium“ setting (used to remove steric strain) with a maximal RMS gradient of 1. The final model was chosen from the best scoring intermediate model and was further refined using the “fine“ setting (full minimization, including electrostatics) with a maximal RMS gradient of 0.1. Refinements used the Amber10-EHT forcefield with the generalized Born implicit solvation model.
Supplementary Figure 4. IR-46 Homology model constructed from 5FWN. Colouring corresponds to the three topological regions, the Rossmann fold (yellow) which recognizes and binds NADPH (green, space filling spheres), the transhelix region (cyan) and the c-terminal bundle (magenta). The co-crystallized ligand from 5FWN, (1R)-1-methyl-1,2,3,4-tetrahydroisoquinoline (magenta, space filling spheres) is present in the active site.
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Rd1 – Identification of positions for investigation by single site-saturation. We took advantage of the well characterized IRED structural family as well as compiled sequence statistics from multiple sequence alignments (MSAs) to aid us in identifying positions for single site saturation9. We first identified homologous sequences to IR-46 using BLAST+10,11 and similar proteins having the same domain architecture as annotated in the INTERPRO 62 dataset12. The collected sequences were filtered to remove protein fragments and clustered at 90% sequence identity using CD-hit13,14. Representatives from each cluster were selected greedily based on length and were clustered further on pairwise sequence similarity using the “Cluster Analysis of Sequences” or CLANS method15. All sequences found to cluster with IR-46 were aligned using Clustal Omega16 and were further curated to remove sequences exhibiting poor sequence overlap (minimum of 80% coverage or better). The resulting collection of sequences were then used to calculate a variety of statistics including: per-position amino acid conservation, probabilities, entropies, compositions, average helical propensities and hydrophobicities. In addition to the calculated sequence statistics, we used the homology model to prioritize positions based on their structural topology, secondary structure and solvent exposure. For simplicity, we divided the IRED model into topological regions and then prioritized residues for exploration based on their calculated sequence and structural properties. For example, one such library contained residues whose sidechain centers of mass were within 8 Å of the model’s pre-existing ligand and had amino acid conservation of less than 90%. A similar criterion was used to target residues near the NADP+ cofactor, with the added constraint of retaining the characteristic Gly-x-Gly-x-x-small motif essential for cofactor binding and recognition. Simpler libraries were also considered; residues belonging to the Rossmann fold or helical bundle that were identified as solvent exposed were targeted for increasing the protein’s stability in solution. Residues that exhibited high surface contact areas upon dimerizing were selected to increase the strength of that interaction. Residues contained within the two flexible loops near the active site (Y142-loop and K242-loop) were targeted exhaustively as those positions may confer significant remodelling on the active site. Eight libraries of 32 mutations each were therefore designed for exploration covering 256 of 296 possible positions. The remaining positions were discounted either because they were far from the active site, buried within the protein’s structure, and/or were heavily conserved across the IRED family. The designed libraries are detailed in Supplementary Table 7.
Supplementary Table 7. Designed single site saturation libraries for Rd1 of evolution.
1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 L116 N263 V146 A124 S240 A223 A25 P29 T117 V265 P147 R125 V241 P227 S26 A49 S118 G266 P148 G160 S274 Q231 P27 G50 G119 L267 G149 G184 A277 T234 A28 Q52 L120 K269 V150 D186 I279 L238 G44 D64 P121 M270 G151 A187 R280 P239 K48 V67 D140 A273 L152 I200 P281 G268 G61 A68 G141 S31 P153 S203 D282 V271 A65 G70 Y142 V32 Q154 S204 L283 E272 Q69 D76 I143 I33 T155 L205 M284 K275 T72 E79
M144 G34 L156 A206 I287 A276 N73 A82 S145 L35 K242 G207 L291 P286 A75 A83 L157 G36 G243 A208 R294 Y290 A78 D85 F158 L37 A244 L209 A303 V295 H98 P103 Y159 M38 A245 H210 L304 A296 E102 Q109 L183 G39 A246 A211 A305 G300 K105 G110 G188 A40 A247 Y212 M307 E301 D106 G126 V189 N57 V248 A213 F308 G306 A107 A128 A190 R58 D249 L214 V310 P221 E129 G135 A191 S59 S250 V215 I311 L285 S132 E170
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L192 A60 G251 A216 R312 A264 G133 L176 Y193 K62 Q252 S217 A41 A224 E137 G177 D194 V89 Y253 E218 A43 E292 D162 H182 L195 C90 A254 I220 T54 E302 A163 I77 G196 V91 T255 A222 V55 A289 D164 V104 L197 V92 S256 L225 L66 D297 A167 V112 L198 D93 V257 A226 A71 K219 K168 A127 A199 A95 S258 F228 A74 Q293 A171 T134 L201 A96 T259 A229 L108 H299 G178 V165 W202 F97 T260 E230 W130 E309 N179 T172 W232 A99 A261 I233 Y138 G278 S180 K174 V236 I100 L262 V237 L173 G298 A185 V175
8 Å of Ligand Binding Site
8 Å of Ligand Binding Site and NADP Cofactor Binding
TYR142 Loop and LYS 242 Loop Near Active Site
TransHelix (188-218) and nearest (4 Å) neighbors
TransHelix (188-218) and nearest (4 Å) neighbors and 9 Buried Rossmann Fold Least Conserved
Remaining C-Bundle Interfacial Residues and C-Bundle Surface positions
Least Conserved Surface Positions in Rossmann Fold
Most Conserved Surface Positions in Rossmann Fold +9 Conserved Buried Positions
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Supplementary Figure 5. Results from round one of evolution. A) Distribution of mutations with FIOP > 1.25 from the primary screen denoted by library. B) Distribution of the mutations based on topological regions identified in the homology model. C) Enumeration of the individual mutations and their FIOPs, coloured by their corresponding library. More than half of the observed beneficial mutations are present in or near the active site.
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Supplementary Figure 6. Homology model of wild-type IR-46 showing residues found to give beneficial FIOP upon mutation in round 1 of evolution as spacefill, highlighted in green. NADP+ is shown as orange sticks.
Rd2 – Recombination of single site saturation variants Of the 5120 possible unique sequence variants from the primary screen, we observed 840 variants at 236 unique positions for a sequence and structure coverage of 16% and 92%, respectively. Of the 840 variants, 52 had a fold increase over parent (FIOP) of 1.25 with most of the beneficial mutations present in or near the active site (Supplementary Figure 5). To recombine these 52 variants, we performed a density based spatial clustering of the corresponding alpha carbons (Cα) using the constructed homology model. This was accomplished by calculating the NxN Euclidean distances between the 3-dimentional coordinate positions of the Cα atoms and performing the density based clustering as implemented in the DBSCAN routine in Python programming language’s Scikit-learn17 package. In doing so, we identified 7 unique clusters of mutations that contained N=4 or more Cα atoms within 6 Å of one another (Supplementary Figure 7). Importantly, the clustering was performed on the dimeric complex, to identify completely those groups of mutations near the interface. These clusters of residues were then used as the basis for library construction with the notion that residues combined close in space may work concertedly to enhance the enzyme’s stability or activity.
180 o
V265
S258
L201
D194 Y142
L37
L37
Y142 D194
L201
V265
S258
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Supplementary Figure 7. Identified clusters of mutations overlaid on the IRED homology model. Density based clustering identified 7 clusters of residues with mutations having a FIOP>1.25 from the primary screen. A residue is considered to be part of a cluster if there are a minimum of N=4 residue position’s Cα atoms within 6 Å of one another. Positions highlighted with the same colour belong to the same cluster. In some cases, a mutation cannot be fit into a cluster, creating singletons (dark blue).
Supplementary Table 8. Combinatorial libraries for Rd2 of evolution.
2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 V92K L37A L37A L120I A60Q G44R A95G G44S
A107H G44S L37V Q154E A75T A107E L120I G44R
L120I A75T G44S K242M L120I G126V A187V G44N
A187V L120I G44R Q252C S142A G126A A187C A60Q
A187C Q154E V92S V257A L198M S180W D194A A75T
L201Y G184S V92L T260I L201F A185P L201F A107H
V215I D194G V92W T260V A226Y D194A V215I L120I
Q231F D194A Q109S G268A G251R D194G Q231F G133Y
K242M L201Y L120I K269V V257A D194W K242M D194G
Q252C V215I V146P S274C T260I L201Y Q252C L198C
S258N S258N D194G S274N A264L K242M S258N L201Y
V265R T260C L201Y M284Y A264G Q52P V265R A261R
M270L T260V L201F M284S G268A T260I D297E V265R
A276R T260I S258N D297E S274C S274N A303D M270L D297E V265R V265R A303S D297E M284A L37Q A276R
L304M M270L M270L A303G L304M V310L S142V S256T
Deconvolution of pairs of mutations that on average contribute to an improvement of the enzymes robustness is an important step in designing libraries for directed evolution. We used Codexis MOSAIC® software to deconvolute these concerted mutations within and between the round two libraries.
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Supplementary Figure 8. Homology model of wild-type (IR-46) showing residues found to give beneficial FIOP when mutated in combination in round 2 of evolution as spacefill, highlighted in cyan. NADP+ is shown as orange sticks.
Rd3 – Recombination of beneficial diversity using MOSAIC® pairwise interaction coefficients. Using the identified beneficial diversity from the previous two rounds, two libraries were designed in Rd3. The chosen library design approach involved maximizing the number of favourable pairwise interactions within each library commensurate with the interaction coefficients calculated using Codexis MOSAIC® software on the retest data from the second round of evolution. In total, 22 out of a potential 30 additional positions were prioritized for mutation (Supplementary Table 9).
Supplementary Table 9. Combinatorial libraries for Rd3 of evolution.
3.1 3.2 G44S G44R Q52E V92L A75T V92K V92W F97V V92S A107H L120I L120I S142V L198M
180 o
A276
V215 Q231
V265
T260
S258
A303 K242
L201
L198
Q154
A187
L120
G133
A107
A75 Q52
G44 L37
V92
A75
Q52 G44 L37
Q154
V92 A107
G133 A187 L120
L198
L201
K242
A303
S258
T260
V265
A276
V215 Q231
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Q154E G251R L198C V257A A226Y T260I K242M T260C A261R S274C V265R A303G A276R A303S D297E A303D L304M g360t
Best MOSAIC® coefficients and predicted pairwise Interactions
Remaining mutations with beneficial MOSAIC® single and pairwise coefficients
As with round two, the MOSAIC® model was used to deconvolute mutations and identify those that most significantly impacted activity. Twenty two mutations were identified as beneficial.
180 o
S274
T260
G251
A303
L198
L120
G126 A107
G44 V92
F97
G44
A107
G126 L120
L198
F97 V92
S274
T260 A303
G251
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Supplementary Figure 9. Homology model of wild-type IRED (IR-46) showing recombined residues to give beneficial FIOP in round 3 of evolution as spacefill, highlighted in magenta. NADP+ is shown as orange sticks.
Scikit Learn DBSCAN clustering script The following Python script was used to spatially cluster beneficial FIOPs on the IRED homology model. The identified clusters were then used as a basis for round two library design. #!/usr/bin/env python
# coding: utf-8
# http://scikit-learn.org/stable/modules/generated/sklearn.cluster.DBSCAN.html
import MDAnalysis
import numpy as np
import sys
import os.path
from sklearn import cluster
def clusterAnalysis(PDB, eps=6.0, min_samples=4):
eps = float(eps)
min_samples=int(min_samples)
prefix = os.path.basename(PDB).split(".")[0]
## Load PDB File
u = MDAnalysis.Universe(PDB)
## Open the output file and write preamble
fid = open(prefix+'_%.1f_%d_clusters.dat' % (eps, min_samples),'w')
fid.write('array set clusters { \n')
## Select only the alpha carbons
protCA=u.select_atoms("name CA and (segid CHNA or segid CHNB)")
## Initialize DB Scan
dbscan = cluster.DBSCAN(eps=eps, min_samples=min_samples)
## Cluster
for ts in u.trajectory:
# Update console output
if ts.frame % 2 == 0:
print("CLUSTERING>> Analyzing frame: %d" % ts.frame)
clusters = dbscan.fit_predict(protCA.positions)
# Write out the index vector to the file
fid.write('%d {' % ts.frame)
clusters.tofile(fid,format="%s",sep=" ")
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fid.write('} \n')
# Write out the corresponding residues
fid.write('resids {')
values = ' '.join(str(v) for v in protCA.resids.tolist())
fid.write(values)
fid.write('} \n')
#Write out the corresponding chains
fid.write('chains {')
values = ' '.join(str(v) for v in protCA.segids.tolist())
fid.write(values)
fid.write('} \n')
fid.write('}')
if __name__ == '__main__':
clusterAnalysis(*sys.argv[1:]) Library sequencing Next Generation Sequencing libraries were prepared directly from the constructed plasmid DNA libraries through PCR of the DNA fragment of interest. The DNA libraries are then nonspecifically cleaved using DNase I (New England Biolabs) and ligated to barcoded adapters with T4 DNA Ligase (New England Biolabs). NGS library size selection was done through a combination of methods- Agencourt AMPure XP’s (Beckman Coulter) magnetic bead-based and PippinHT’s (Sage Science) agarose gel electrophoresis. Quantitative real-time PCR is used to quality control the NGS libraries by quantifying based on adapter-specific PCR. (Library Quantification Kits for Ion Torrent Platforms, Kapa Biosystems) Following the Ion Chef User Guide (Thermo Fisher Scientific), the NGS libraries are loaded onto Ion Chips and sequenced on the Ion S5 XL system18. HTP Protocols for evolution Rd1 Rd1 – Tier 1 Cells generated as described above were re-suspended in 200 μL lysis buffer and lysed by shaking at room temperature for 2 h. The lysis buffer consisted of sodium acetate buffer 200 mM pH 5.6, 1 mg/mL lysozyme, and 500 μg/mL PMBS and 10 U/mL benzonase. After sealing the plates with air-permeable nylon seals, they were shaken vigorously for 2 h at room temperature. Cell debris was pelleted by centrifugation (4000 RPM, 10 min, 4 °C) and the clarified lysate used directly or stored at 4 °C until use (left at 4 °C for no longer than 1h). A 79.3 µL aliquot of aldehyde 3 in DMSO (final conc. 12 mg/mL, 30% DMSO final conc) was added to each well of a Costar deep well 96 plate, followed by addition of 71.4 µL of rac-trans-1 in sodium acetate buffer 200 mM pH 5.0, (1.1 equiv (of wanted enantiomer), final conc. 15.8 mg/mL) followed by the addition of 50 µL of the cofactors stock solution (final conc. 1.55 mg/mL NADP+, 10.7 mg/mL D-glucose, 0.48 mg/mL GDH-CDX-901) in sodium acetate buffer 200 mM pH 5.6) and followed by the addition of clarified lysate (50 µL) using a Biomek FX robotic instrument (Beckman Coulter, Fullerton, CA) to a final reaction volume of 250 µL. The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then shaken for 6 h at 30 °C at 800 RPM in an Infors shaker. Reactions were quenched by the addition of 150 µL of acetic acid 1 M and 200 µL of acetonitrile by a Biomex FX. Plates were resealed, shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 200 µL sample of supernatant was transferred to a shallow well polypropylene plate (NUNC 96) sealed and then analyzed by UPLC using UPLC achiral Method 1.
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Rd1 – Tier 2a Lysis and reaction conditions were identical with the tier 1 screening conditions. Reactions were run in triplicate. Rd1 – Tier 2b Cells generated as described above were re-suspended in 200 μL lysis buffer and lysed by shaking at room temperature for 2 h. The lysis buffer consisted of sodium acetate buffer 200 mM pH 5.6, 1 mg/mL lysozyme, and 500 μg/mL PMBS and 10 U/mL benzonase. After sealing the plates with air-permeable nylon seals, they were shaken vigorously for 2 h at room temperature. Cell debris was pelleted by centrifugation (4000 RPM, 10 min., 4 °C) and the clarified lysate used directly or stored at 4 °C until use (left at 4 °C for no longer than 1h). A 61.5 µL aliquot of aldehyde 3 in DMSO (final conc. 20 mg/mL, 23% DMSO final conc) was added to each well of a Costar deep well 96 plate, followed by addition of 119.0 µL of rac-trans-1 in sodium acetate buffer 200 mM pH 5.0, (1.1 equiv (of wanted enantiomer), final conc. 26.4 mg/mL) followed by the addition of 50 µL of the cofactors stock solution (final conc. 2.6 mg/mL NADP+, 17.8 mg/mL D-glucose, 0.8 mg/mL GDH-CDX-901) in sodium acetate buffer 200 mM pH 5.6) and followed by the addition of clarified lysate (50 µL) using a Biomek FX robotic instrument (Beckman Coulter, Fullerton, CA) to a final reaction volume of 250 µL. The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then shaken for 6 h at 30 °C at 800 RPM in an Infors shaker. Reactions were quenched by the addition of 150 µL of acetic acid 1 M and 200 µL of acetonitrile by a Biomex FX. Plates were resealed, shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 200 µL sample of supernatant was transferred to a shallow well polypropylene plate (NUNC 96) sealed and then analyzed by UPLC using UPLC achiral Method 1. Rd1 – Tier 2c Cells generated as described above were re-suspended in 200 μL lysis buffer and lysed by shaking at room temperature for 2 h. The lysis buffer consisted of sodium acetate buffer 200 mM pH 5.6, 1 mg/mL lysozyme, and 500 μg/mL PMBS and 10 U/mL benzonase. After sealing the plates with air-permeable nylon seals, they were shaken vigorously for 2 h at room temperature. Cell debris was pelleted by centrifugation (4000 RPM, 10 min., 4 °C) and the clarified lysate (120 µL) was transferred into a new Costar deep well 96 plate, lyophilised using the protocol mentioned above and stored at -80 °C until further use (left at -80 °C for no longer than 6 days). Plates were thawed for 30 min and miliQ water (50 µL) was aliquoted to each well of the plate. A 79.3 µL aliquot of aldehyde 3 in DMSO (final conc. 12 mg/mL, 30% DMSO final conc) was added to each well of a Costar deep well 96 plate, followed by addition of 71.4 µL of rac-trans-1 in sodium acetate buffer 200 mM pH 5.0, (1.1 equiv (of wanted enantiomer), final conc. 15.8 mg/mL) followed by the addition of 50 µL of the cofactors stock solution (final conc. 1.55 mg/mL NADP+, 10.7 mg/mL D-glucose, 0.48 mg/mL GDH-CDX-901) in sodium acetate buffer 200 mM pH 5.6) and followed by the addition of clarified lysate (50 µL) using a Biomek FX robotic instrument (Beckman Coulter, Fullerton, CA) to a final reaction volume of 250 µL. The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then shaken for 6 h at 30 °C at 800 RPM in an Infors shaker. Reactions were quenched by the addition of 150 µL of acetic acid 1 M and 200 µL of acetonitrile by a Biomex FX. Plates were resealed, shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 200 µL sample of supernatant was transferred to a shallow well polypropylene plate (NUNC 96) sealed and then analyzed by UPLC using UPLC achiral Method 1. HTP Protocols for evolution Rd2 Rd2 – Tier 1 Cells generated as described above were re-suspended in 200 μL lysis buffer and lysed by shaking at room temperature for 2 h. The lysis buffer consisted of sodium acetate buffer 20 mM pH 5.6, 1 mg/mL lysozyme, and 500 μg/mL PMBS and 10 U/mL benzonase. After sealing the plates with air-permeable nylon seals, they were shaken vigorously for 2 h at room temperature. An additional 200 µL of sodium acetate buffer 200 mM pH 5.6 was added to each well and the plate was shaken for 5 mins. Cell debris was pelleted by centrifugation (4000 RPM, 10 min., 4 °C) and the clarified lysate used directly or stored at 4 °C for use on the same day. A 78.6 µL aliquot of aldehyde 3 in DMSO (final conc. 20.1 mg/mL, 28% DMSO final conc) was added to each well of a Costar deep well 96 plate, followed by addition of 159.9 µL of a stock containing rac-trans-1 (1.05 equiv (of wanted enantiomer), final conc.
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26.4 mg/mL) and cofactor recycling mixture (final conc. 5.1 mg/mL NADP+, 34.4 mg/mL D-glucose, 1.6 mg/mL GDH-CDX-901) in sodium acetate buffer 200 mM pH 5.6). The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then preincubated at 35 °C for 30 min prior to starting the reaction. The reaction was started the by the addition of clarified lysate (20 µL) using a Biomek FX robotic instrument (Beckman Coulter, Fullerton, CA) to a final reaction volume of 259 µL. The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then shaken for 4 h at 35 °C at 800 RPM in an Infors shaker. Reactions were quenched by the addition of 150 µL of acetic acid 1 M and 200 µL of acetonitrile by a Biomex FX. Plates were resealed, shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 200 µL sample of supernatant was transferred to a shallow well polypropylene plate (NUNC 96) sealed and then analyzed by UPLC using UPLC achiral Method 1. Rd2 – Tier 2a Lysis and reaction conditions were identical to the tier 1 screening conditions. Reactions were run in triplicate. Rd2 – Tier 2b Cells generated as described above were re-suspended in 200 μL lysis buffer and lysed by shaking at room temperature for 2 h. The lysis buffer consisted of sodium acetate buffer 20 mM pH 5.6, 1 mg/mL lysozyme, and 500 μg/mL PMBS and 10 U/mL benzonase. After sealing the plates with air-permeable nylon seals, they were shaken vigorously for 2 h at room temperature. An additional 200 µL of sodium acetate buffer 200 mM pH 5.6 was added to each well and the plate was shaken for 5 mins. Cell debris was pelleted by centrifugation (4000 RPM, 10 min., 4 °C) and the clarified lysate used directly or stored at 4 °C for use on the same day. A 78.6 µL aliquot of aldehyde 3 in DMSO (final conc. 20.1 mg/mL, 28% DMSO final conc) was added to each well of a Costar deep well 96 plate, followed by addition of 159.9 µL of a stock containing rac-trans-1 (1.05 equiv (of wanted enantiomer), final conc. 26.4mg/mL) and cofactor recycling mixture (final conc. 5.1 mg/mL NADP+, 34.4 mg/mL D-glucose, 1.6 mg/mL GDH-CDX-901) in sodium acetate buffer 200 mM pH 4.0). The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then preincubated at 35 °C for 30 min prior to starting the reaction. The reaction was started the by the addition of clarified lysate (20 µL) using a Biomek FX robotic instrument (Beckman Coulter, Fullerton, CA) to a final reaction volume of 259 µL. The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then shaken for 4 h at 35 °C at 800 RPM in an Infors shaker. Reactions were quenched by the addition of 150 µL of acetic acid 1 M and 200 µL of acetonitrile by a Biomex FX. Plates were resealed, shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 200 µL sample of supernatant was transferred to a shallow well polypropylene plate (NUNC 96) sealed and then analyzed by UPLC using UPLC achiral Method 1. Rd2 – Tier 2c Cells generated as described above were re-suspended in 200 μL lysis buffer and lysed by shaking at room temperature for 2 h. The lysis buffer consisted of sodium acetate buffer 20 mM pH 5.6, 1 mg/mL lysozyme, and 500 μg/mL PMBS and 10 U/mL benzonase. After sealing the plates with air-permeable nylon seals, they were shaken vigorously for 2 h at room temperature. An additional 200 µL of sodium acetate buffer 200 mM pH 5.6 was added to each well and the plate was shaken for 5 mins. Cell debris was pelleted by centrifugation (4000 RPM, 10 min., 4 °C) and the clarified lysate (20 µL) was transferred into a new Costar deep well 96 plate, lyophilised using the protocol mentioned above and stored at -80 °C until further use (left at -80 °C for no longer than 6 days). Plates were thawed for 30 min. 179.9 µL of a stock containing rac-trans-1 (1.05 equiv (of wanted enantiomer), final conc. 26.4 mg/mL) and cofactor recycling mixture (final conc. 5.1 mg/mL NADP+, 34.4 mg/mL D-glucose, 1.6 mg/mL GDH-CDX-901) in sodium acetate buffer 200 mM pH 5.0) was added. The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and preincubated for 1 h at 40 °C. The reaction was then started by the addition of 78.6 µL aliquot of aldehyde 3 in DMSO (final conc. 20.1 mg/mL, 28% DMSO final conc)) (final reaction volume of 259 µL). The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then shaken for 4 h at 35 °C at 800 RPM in an Infors shaker. Reactions were quenched by the addition of 150 µL of acetic acid 1 M and 200 µL of acetonitrile by a Biomex FX. Plates were resealed, shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 200 µL
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sample of supernatant was transferred to a shallow well polypropylene plate (NUNC 96) sealed and then analyzed by UPLC using UPLC achiral Method 1.
HTP Protocols for evolution Rd3 Rd3 – Tier 1 Cells generated as described above were re-suspended in 200 μL lysis buffer and lysed by shaking at room temperature for 2 h. The lysis buffer consisted of sodium acetate buffer 20 mM pH 5.6, 1 mg/mL lysozyme, and 500 μg/mL PMBS and 10 U/mL benzonase. After sealing the plates with air-permeable nylon seals, they were shaken vigorously for 2 h at room temperature. An additional 200 µL of sodium acetate buffer 20 mM pH 5.6 was added to each well and the plate was shaken for 5 mins. Cell debris was pelleted by centrifugation (4000 RPM, 10 min., 4 °C) and the clarified lysate (40 µL) was transferred into a new Costar deep well 96 plate, lyophilised using the protocol mentioned above and stored at -80 °C until further use (left at -80 °C for no longer than 6 days). Plates were thawed for 30 min. 186.3 µL of a stock containing rac-trans-1 (1.05 equiv (of wanted enantiomer), final conc. 30.5 mg/mL) and cofactor recycling mixture (final conc. 5.7 mg/mL NADP+, 38.6 mg/mL D-glucose, 1.7 mg/mL GDH-CDX-901) in sodium acetate buffer 200 mM pH 4.6) was added. The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and preincubated for 30 min at 30 °C. The reaction was then started by the addition of 78.6 µL aliquot of aldehyde 3 in DMSO (final conc. 24.2 mg/mL, 28% DMSO final conc)) (final reaction volume of 268 µL). The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then shaken for 4 h at 35 °C at 800 RPM in an Infors shaker. Reactions were quenched by the addition of 150 µL of acetic acid 1 M and 200 µL of acetonitrile by a Biomex FX. Plates were resealed, shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 200 µL sample of supernatant was transferred to a shallow well polypropylene plate (NUNC 96) sealed and then analyzed by UPLC using UPLC achiral Method 1. Rd3 – Tier 2a Lysis, lyophilization and reaction conditions were identical with the tier 1 screening conditions. Reactions were run in triplicates. Rd3 – Tier 2b Cells generated as described above were re-suspended in 200 μL lysis buffer and lysed by shaking at room temperature for 2 h. The lysis buffer consisted of sodium acetate buffer 20 mM pH 5.6, 1 mg/mL lysozyme, and 500 μg/mL PMBS and 10 U/mL benzonase. After sealing the plates with air-permeable nylon seals, they were shaken vigorously for 2 h at room temperature. An additional 200 µL of sodium acetate buffer 20 mM pH 5.6 was added to each well and the plate was shaken for 5 mins. Cell debris was pelleted by centrifugation (4000 RPM, 10 min., 4 °C) and the clarified lysate (20 µL) was transferred into a new Costar deep well 96 plate, lyophilised using the protocol mentioned above and stored at -80 °C until further use (left at -80 °C for no longer than 6 days). Plates were thawed for 30 min. 186.4 µL of a stock containing rac-trans-1 (1.05 equiv (of wanted enantiomer), final conc. 35.1 mg/mL) and cofactor recycling mixture (final conc. 6.4 mg/mL NADP+, 44.5 mg/mL D-glucose, 2.0 mg/mL GDH-CDX-901) in sodium acetate buffer 200 mM pH 4.6) was added. The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and preincubated for 120 min at 30 °C. The reaction was then started by the addition of 47.4 µL aliquot of aldehyde 3 in DMSO (final conc. 27.8 mg/mL, 17% DMSO final conc)) (final reaction volume of 234 µL). The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then shaken for 4 h at 35 °C at 800 RPM in an Infors shaker. Reactions were quenched by the addition of 150 µL of acetic acid 1 M and 200 µL of acetonitrile by a Biomex FX. Plates were resealed, shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 200 µL sample of supernatant was transferred to a shallow well polypropylene plate (NUNC 96) sealed and then analyzed by UPLC using UPLC achiral Method 1. Rd3 – Tier 2c Cells generated as described above were re-suspended in 200 μL lysis buffer and lysed by shaking at room temperature for 2 h. The lysis buffer consisted of sodium acetate buffer 20 mM pH 5.6, 1 mg/mL lysozyme, and 500 μg/mL PMBS and 10 U/mL benzonase. After sealing the plates with air-permeable
S26
nylon seals, they were shaken vigorously for 2 h at room temperature. An additional 200 µL of sodium acetate buffer 20 mM pH 5.6 was added to each well and the plate was shaken for 5 mins. Cell debris was pelleted by centrifugation (4000 RPM, 10 min., 4 °C) and the clarified lysate (40 µL) was transferred into a new Costar deep well 96 plate, lyophilised using the protocol mentioned above and stored at -80 °C until further use (left at -80 °C for no longer than 6 days). Plates were thawed for 30 min. 186.4 µL of a stock containing rac-trans-1 (1.05 equiv (of wanted enantiomer), final conc. 35.1 mg/mL) and cofactor recycling mixture (final conc. 6.4 mg/mL NADP+, 44.5 mg/mL D-glucose, 2.0 mg/mL GDH-CDX-901) in sodium acetate buffer 200 mM pH 4.6) was added. The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and preincubated for 120 min at 30 °C. The reaction was then started by the addition of 47.4 µL aliquot of aldehyde 3 in DMSO (final conc. 27.8 mg/mL, 17% DMSO final conc)) (final reaction volume of 234 µL). The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then shaken for 4 h at 35 °C at 800 RPM in an Infors shaker. Reactions were quenched by the addition of 150 µL of acetic acid 1 M and 200 µL of acetonitrile by a Biomex FX. Plates were resealed, shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 200 µL sample of supernatant was transferred to a shallow well polypropylene plate (NUNC 96) sealed and then analyzed by UPLC using UPLC achiral Method 1.
Rd3 – Tier 2d Cells generated as described above were re-suspended in 200 μL lysis buffer and lysed by shaking at room temperature for 2 h. The lysis buffer consisted of sodium acetate buffer 20 mM pH 5.6, 1 mg/mL lysozyme, and 500 μg/mL PMBS and 10 U/mL benzonase. After sealing the plates with air-permeable nylon seals, they were shaken vigorously for 2 h at room temperature. An additional 200 µL of sodium acetate buffer 20 mM pH 5.6 was added to each well and the plate was shaken for 5 mins. Cell debris was pelleted by centrifugation (4000 RPM, 10 min., 4 °C) and the clarified lysate (20 µL) was transferred into a new Costar deep well 96 plate, lyophilised using the protocol mentioned above and stored at -80 °C until further use (left at -80 °C for no longer than 6 days). Plates were thawed for 30 min. 186.4 µL of a stock containing rac-trans-1 (1.05 equiv (of wanted enantiomer), final conc. 35.1 mg/mL) and cofactor recycling mixture (final conc. 6.4 mg/mL NADP+, 44.5 mg/mL D-glucose, 2.0 mg/mL GDH-CDX-901) in sodium acetate buffer 200 mM pH 4.2) was added. The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and preincubated for 30 min at 30 °C. The reaction was then started by the addition of 47.4 µL aliquot of aldehyde 3 in DMSO (final conc. 27.8 mg/mL, 17% DMSO final conc)) (final reaction volume of 234 µL). The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then shaken for 4 h at 35 °C at 800 RPM in an Infors shaker. Reactions were quenched by the addition of 150 µL of acetic acid 1 M and 200 µL of acetonitrile by a Biomex FX. Plates were resealed, shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 200 µL sample of supernatant was transferred to a shallow well polypropylene plate (NUNC 96) sealed and then analyzed by UPLC using UPLC achiral Method 1. Rd3 – Tier 2e Cells generated as described above were re-suspended in 200 μL lysis buffer and lysed by shaking at room temperature for 2 h. The lysis buffer consisted of sodium acetate buffer 20 mM pH 5.6, 1 mg/mL lysozyme, and 500 μg/mL PMBS and 10 U/mL benzonase. After sealing the plates with air-permeable nylon seals, they were shaken vigorously for 2 h at room temperature. An additional 200 µL of sodium acetate buffer 20 mM pH 5.6 was added to each well and the plate was shaken for 5 mins. Cell debris was pelleted by centrifugation (4000 RPM, 10 min., 4 °C) and the clarified lysate (40 µL) was transferred into a new Costar deep well 96 plate, lyophilised using the protocol mentioned above and stored at -80 °C until further use (left at -80 °C for no longer than 6 days). Plates were thawed for 30 min. 186.4 µL of a stock containing rac-trans-1 (1.05 equiv (of wanted enantiomer), final conc. 35.1 mg/mL) and cofactor recycling mixture (final conc. 6.4 mg/mL NADP+, 44.5 mg/mL D-glucose, 2.0 mg/mL GDH-CDX-901) in sodium acetate buffer 200 mM pH 4.2) was added. The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and preincubated for 30 min at 30 °C. The reaction was then started by the addition of 47.4 µL aliquot of aldehyde 3 in DMSO (final conc. 27.8 mg/mL, 17% DMSO final conc)) (final reaction volume of 234 µL). The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then shaken for 4 h at 35 °C at 800 RPM in an Infors shaker. Reactions were quenched by the addition of 150 µL of acetic acid 1 M and 200 µL of acetonitrile by a Biomex FX. Plates were resealed, shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 200 µL
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sample of supernatant was transferred to a shallow well polypropylene plate (NUNC 96) sealed and then analyzed by UPLC using UPLC achiral Method 1. Rd3 – Tier 2f Cells generated as described above were re-suspended in 200 μL lysis buffer and lysed by shaking at room temperature for 2 h. The lysis buffer consisted of sodium acetate buffer 20 mM pH 5.6, 1 mg/mL lysozyme, and 500 μg/mL PMBS and 10 U/mL benzonase. After sealing the plates with air-permeable nylon seals, they were shaken vigorously for 2 h at room temperature. An additional 200 µL of sodium acetate buffer 20 mM pH 5.6 was added to each well and the plate was shaken for 5 mins. Cell debris was pelleted by centrifugation (4000 RPM, 10 min., 4 °C) and the clarified lysate (20 µL) was transferred into a new Costar deep well 96 plate, lyophilised using the protocol mentioned above and stored at -80 °C until further use (left at -80 °C for no longer than 6 days). Plates were thawed for 30 min. 186.4 µL of a stock containing rac-trans-1 (0.525 equiv (of wanted enantiomer), final conc. 17.5 mg/mL) and cofactor recycling mixture (final conc. 6.4 mg/mL NADP+, 44.5 mg/mL D-glucose, 2.0 mg/mL GDH-CDX-901) in sodium acetate buffer 200 mM pH 4.6) was added. The reaction was then started by the addition of 47.4 µL aliquot of aldehyde 3 in DMSO (final conc. 27.8 mg/mL, 17% DMSO final conc)) (final reaction volume of 234 µL). The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then shaken for 4 h at 35 °C at 800 RPM in an Infors shaker. Reactions were quenched by the addition of 150 µL of acetic acid 1 M and 200 µL of acetonitrile by a Biomex FX. Plates were resealed, shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 200 µL sample of supernatant was transferred to a shallow well polypropylene plate (NUNC 96) sealed and then analyzed by UPLC using UPLC achiral Method 1. Rd3 – Tier 2g Cells generated as described above were re-suspended in 200 μL lysis buffer and lysed by shaking at room temperature for 2 h. The lysis buffer consisted of sodium acetate buffer 20 mM pH 5.6, 1 mg/mL lysozyme, and 500 μg/mL PMBS and 10 U/mL benzonase. After sealing the plates with air-permeable nylon seals, they were shaken vigorously for 2 h at room temperature. An additional 200 µL of sodium acetate buffer 20 mM pH 5.6 was added to each well and the plate was shaken for 5 mins. Cell debris was pelleted by centrifugation (4000 RPM, 10 min., 4 °C) and the clarified lysate (40 µL) was transferred into a new Costar deep well 96 plate, lyophilised using the protocol mentioned above and stored at -80 °C until further use (left at -80 °C for no longer than 6 days). Plates were thawed for 30 min. 186.4 µL of a stock containing rac-trans-1 (0.525 equiv (of wanted enantiomer), final conc. 17.5 mg/mL) and cofactor recycling mixture (final conc. 6.4 mg/mL NADP+, 44.5 mg/mL D-glucose, 2.0 mg/mL GDH-CDX-901) in sodium acetate buffer 200 mM pH 4.6) was added. The reaction was then started by the addition of 47.4 µL aliquot of aldehyde 3 in DMSO (final conc. 27.8 mg/mL, 17% DMSO final conc)) (final reaction volume of 234 µL). The plates were heat-sealed with aluminum/polypropylene laminate heat seal tape at 165 °C for 2.5 seconds and then shaken for 4 h at 35 °C at 800 RPM in an Infors shaker. Reactions were quenched by the addition of 150 µL of acetic acid 1 M and 200 µL of acetonitrile by a Biomex FX. Plates were resealed, shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 200 µL sample of supernatant was transferred to a shallow well polypropylene plate (NUNC 96) sealed and then analyzed by UPLC using UPLC achiral Method 1.
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Top variants identified after each evolution round
Supplementary Table 10. Top variants identified after Rd1 of evolution, using tier 2 conditions, as catalysts in the model reaction 3→4. [a]
Entry Variant Mutation over WT FIOP[b] Tier 1 Tier 2a[c] Tier 2b[c] Tier 2c[c] 1 WT 1 1 1 1 2 M1 Y142S; 46.4 37.4 20.0 249.8 3 Y142V; 7.9 16.1 10.0 142.1 4 Y142R; 19.4 13.0 6.2 96.1 5 L201Y; 12.5 9.4 8.2 139.6 6 Y142T; 5.2 7.7 6.1 73.4 7 L201F; 5.7 5.9 3.4 49.8
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[a] Reaction conditions detailed in section HTP Protocols for evolution Rd1. [b] FIOP – Fold increase over the parent. [c] Average of triplicates.
Backbone for next round, Selected for detailed investigation
8 D194G; 9.0 4.9 1.7 70.4 9 L37A; 4.6 4.3 2.2 28.6 10 S258N; 3.7 4.0 2.3 48.1 11 V265R; 2.6 3.5 2.3 16.6 12 V92W; 2.2 3.3 1.9 38.5 13 L120I; 2.1 3.1 2.2 17.7 14 S274C; 2.6 2.9 1.6 29.6 15 A75T; 1.9 2.9 1.2 3.1 16 A187V; 2.2 2.8 1.4 3.2 17 A187C; 1.9 2.7 1.7 1.7 18 L304M; 2.0 2.7 1.6 39.4 19 M270L; 1.8 2.6 1.8 4.9 20 Q154E; 1.7 2.5 1.6 2.8 21 V92S; 1.9 2.5 1.7 2.2
S30
Supplementary Table 11. Top variants identified after Rd2 of evolution, as catalysts in reaction 3→4.[a]
Entry Variant Mutations over M1 FIOP[b] Conv. [%] [d] e.e. [%] (1R,2S)-4
Tier 1 Tier 2a[c] Tier 2b[c] Tier 2c[d] Tier 2a[c]
1 M1 - 1 1 1 - [e] n.d. 2 L37A;L201Y;S258N;V265R; 13.5 13.7 28.4 - [e] 97.6 3 L37A;L201Y; 13.6 11.9 27.7 - [e] 99.1 4 L37Q;A187C;L201F;Q231F;K242M;S258N;A303D; 14.5 15.3 25.9 0.06 99.6 5 S4T;L37A;V92W;L120I;L201F;V265R; 11.6 10.0 25.7 - [e] 99.2 6 L37Q;L120I;Q231F;K242M;Q252C;S258N;V265R;D297E;A303D; 15.0 14.1 24.4 0.06 98.1 7 M2 L37Q;A187V;L201F;V215I;Q231F;S258N; 11.1 13.5 24.3 0.07 99.7 8 L37V;V92L;L120I;L201Y; 12.3 10.0 23.9 - [e] 99.0 9 L37Q;A187V;L201F;V215I;Q231F;S258N;V265R; 13.9 13.6 23.0 0.07 99.2 10 L37Q;L201F;Q231F;Q252C;A303D; 12.7 12.9 22.8 - [e] 99.9 11 G44R;A75T;A107H;L120I;L198C;L201Y; 11.9 13.4 18.2 - [e] 99.9 12 L37Q;L120I;S142V;A187V;L201F;K242M;S258N;D297E; 12.3 14.1 18.0 - [e] 99.9 13 L37A;G44S;V92W;L201Y; 9.9 11.0 17.3 - [e] 98.8 14 V92K;A107H;L201Y;V215I;Q231F;K242M;A276R; 11.7 13.6 17.1 - [e] 98.1 15 G44R;G133Y;L198C;L201Y;V265R;A276R; 13.0 12.0 16.5 - [e] 99.9 16 G44R;A75T;L198C;L201Y;S256T;A261R;V265R; 12.6 10.8 16.4 - [e] 99.9 17 L37A;Q154E;L201Y;S258N;T260V; 14.0 19.4 14.2 - [e] 98.7 18 L37A;A75T;Q154E;L201Y;S258N; 17.9 18.7 13.0 - [e] 98.9 19 L37A;A75T;Q154E;L201Y;T260C; 13.3 18.7 12.4 - [e] 98.3 20 L37A;L201Y;T260I;V265R; 13.8 17.3 12.3 - [e] 97.6 21 L37A;G44S;Q52E;L120I;Q154E;L201Y;T260V; 15.6 19.7 12.0 - [e] 97.6
[a] Reaction conditions detailed in section HTP Protocols for evolution Rd2. [b] FIOP – Fold increase over the parent. [c] Average of triplicates. [d] Conversion rather than FIOP given since no detectable conversion was observed for positive controls. [e] not detectable.
Backbone for next round, Selected for detailed investigation.
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Supplementary Table 12. Top variants identified after Rd3 of evolution, as catalysts in the model reaction 3→4.[a]
Entry Variant Mutations over M2 Tier 1 Tier 2a Tier 2b Tier 2c Tier 2d Tier 2e Tier 2f Tier 2g
conv [%][c]
FIOP [b,c]
conv [%][c]
FIOP [b,c]
conv [%][d]
conv [%][d]
conv [%][d]
conv [%][d]
conv [%][f]
e.e. [%]
conv [%][f]
e.e. [%]
1 M2 1.0 1.0 -[f] -[f] -[f] -[f] 34.0 n.d 43.4 n.d.
2 M3 G44R;V92K;F97V;L198M;T260C;A303D; 78.0 156.1 82.9 76.5 25.1 42.0 36.6 51.6 48.6 98.4 48.9 95.3
3 V92K;F97V;A107H;L198M;G251R;T260C;A303D; 75.4 147.6 81.8 75.8 22.8 34.8 36.9 54.6 45.1 98.2 48.9 95.6
4 G44R;V92K;A107H;L198M;T260C;S274C; 72.7 145.6 81.1 74.6 29.1 44.4 33.6 56.5 44.8 97.9 49.7 93.4
5 G44R;V92K;F97V;L198M;T260I;S274C; 76.9 154.0 80.6 74.4 28.9 45.4 34.4 55.9 44.9 98.3 48.4 94.5
6 G44R;V92K;A107H;L198M;T260C;S274C;A303S; 71.9 144.0 80.1 73.8 29.8 43.7 44.8 51.9 44.9 98.2 49.1 95.0
7 V92K;F97V;L198M;T260I;S274C; 72.3 144.8 78.7 72.5 18.7 31.9 27.6 47.2 44.2 98.7 49.5 96.2
8 G44R;V92K;L198M;T260C;S274C;A303D; 73.2 134.5 77.8 73.1 22.1 33.1 33.4 46.7 45.9 96.5 50.9 93.4
9 V92K;F97V;L120I;G126A;L198M;G251R;T260C;S274C; 70.5 141.3 76.7 70.8 19.7 34.2 38.8 57.1 44.4 98.1 49.1 93.8
10 V92K;F97V;L198M;T260C; 71.1 130.8 76.4 71.5 14.4 24.7 17.0 38.1 44.3 98.9 49.2 98.0
11 G44R;V92K;F97V;L198M;S274C;A303S; 75.1 138.0 76.2 71.7 23.3 40.2 33.0 53.0 44.4 99.2 49.8 97.0
12 G44R;V92K;F97V;L198M;T260C;A303S; 77.3 101.1 75.8 110.1 25.3 42.2 37.1 52.6 45.2 98.6 44.9 97.3
13 G44R;V92K;L198M;G251R;T260C;A303D; 75.3 112.4 75.5 109.9 21.6 36.5 16.3 40.3 45.7 97.9 44.9 94.3
14 V92K;A107H;L198M;G251R;T260C;A303S; 76.3 142.4 74.9 69.3 16.3 28.1 18.2 36.5 44.3 99.2 48.8 97.4
15 G44R;V92K;F97V;L120I;L198M;T260C; 75.9 99.3 74.8 98.8 15.7 31.8 22.2 47.0 45.4 98.2 45.0 96.9
16 V92K;F97V;L198M;T260I;A303S; 71.7 131.9 74.6 70.3 15.4 26.8 16.3 39.0 44.4 99.4 49.2 98.5
17 V92K;A107H;L198M;T260C;S274C; 73.5 126.9 74.1 68.3 19.5 31.8 20.4 39.3 43.9 98.2 50.4 94.7
18 V92K;F97V;L198M;T260C;A303S; 76.6 100.3 73.7 104.1 14.9 26.9 23.7 44.0 45.0 99.0 45.2 97.6
19 V92K;L198M;T260C;S274C;A303S; 69.4 127.6 72.9 67.3 13.1 21.2 19.5 36.0 43.5 98.6 49.2 96.3 [a] Reaction conditions detailed in section HTP Protocols for evolution Rd3. [b] FIOP – Fold increase over parent. [c] Average of triplicates. [d] Conversion (amine to product) rather than FIOP was used as the key selection criterion since no activity was observed for the positive controls. [e] 0.5 equivalents of rac-trans-1 used (in respect to wanted enantiomer). Conversion rather than FIOP used to highlight how close conversion is to the theoretical maximum of 50%. [f] not detectable.
Backbone for next round, Selected for detailed investigation.
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Selection of the M1 variant Engineered imine reductases described in Supplementary Table 10 were evaluated at preparative scale in sodium acetate buffer 200 mM pH 5.6 as follows. The enzymes were expressed and lyophilised powder generated. Reactions were run using EasyMax, a controlled laboratory reactor capable of running up to 2 reactions in parallel and sample automatically. By running reactions at 1000 mg scale of 3, to a final reaction volume of 35 mL this allowed us to select the best variant. Rac-trans-1 (1.2 g) was dissolved in 26.2 mL 200 mM sodium acetate buffer pH 5.6. This was followed by the addition and dissolution of D-glucose (1.18 g) and NADP+ (0.13 g). Aldehyde 3, 1.0 g dissolved in DMSO (3.7 mL) was then added followed directly by 5 mL freshly prepared enzyme stock for each of the 3 imine reductase variants selected (stock solution contained: imine reductase lyophilised powder (0.5 g) in 5 mL 200 mM sodium acetate buffer pH 5.6, GDH-CDX-901 (40 mg)) All reactions were run at 30 °C and 300 rpm stirring for 1.5 h after which the reaction mixture was solubilized by the addition of 1M acetic acid: acetonitrile 1:2. Samples were then analyzed, using HPLC Method 2, and results summarized in Supplementary Table 13.
Supplementary Table 13. High scale validation of the top 3 enzyme variants, from the Rd1 evolution selected after running tier 2 conditions, as catalysts in the model reaction 4→5.a
Entry Variant Mutations (backbone IR-46) Conv [%][a] e.e. [%] (1S,2R)-5
1 Y142V; 60.2 ND 2 L201Y; 36.6 ND 3 M1 Y142S; 82.6 ND aReaction conditions: 3 1.0 g, 1 equiv, 28.6 mg/mL) in 3.7 mL DMSO, rac-trans-1 (1.2 g, 2.1 equiv), IRED variants (50% wt/wt), D-glucose (1.18 g, 2 equiv), NADP+ (0.13 g, 0.05 equiv), GDH-CDX-901 (40 mg, 4% wt/wt), DMSO (18%), 30 °C, 4 h, 200 mM sodium acetate buffer pH 4.6, to a final volume of 35 mL. Selection of the M2 variant Engineered imine reductases described in Supplementary Table 11 were evaluated at preparative scale in sodium acetate buffer 200 mM pH 4.6 as follows. The enzymes were expressed and lyophilised powder generated. Reactions were run using AmigoChem, a controlled laboratory equipment capable of running up to 10 reactions in parallel and sample automatically. By running reactions at 0.2 g scale of 3, to a final reaction volume of 10 mL this allowed us to easily select the best variant. Rac-trans-1 (2.0 g) was dissolved in 48.0 mL 200 mM sodium acetate buffer pH 4.6. This solution was used to solubilize D-glucose (1.9 g), NADP+ (0.2 g) and GDH-CDX-901 (64 mg). 6.4 mL of the mixture was added to the reaction tube. Enzyme stock solutions were freshly prepared for each of the 6 imine reductase variants selected in 200 mM sodium acetate buffer pH 4.6 and 400 µL of the stock solution were added to the reaction tube to a final concentration of 10% (v/v). DMSO (2 mL) was added followed by 1 mL solution of aldehyde 3 (0.2 g) in 4 x 250 µL batches at 10 min intervals to obtain a substrate loading of 20 g/L. The addition of the first batch of 3 marked the start of the reaction. All reactions were run at 30 °C and 1000 rpm stirring for 3 h after which the whole reaction mixture was solubilized by the addition of 1 mL acetic acid. 100 µL samples were the taken and diluted in 600 µL 1M acetic acid: acetonitrile 1:2. Samples were then analyzed, using HPLC Method 2, and results summarized in Supplementary Table 14.
Supplementary Table 14. High scale validation of the top 7 enzyme variants, from the Rd2 evolution selected after running tier 2 conditions, as catalysts in the model reaction 3→4.a
Entry Variant Mutations (backbone M1) Conv [%][a] e.e. [%] (1S,2R)-4
1 L37A; L201Y; 0.5 ND 2 L37Q; A187C; L201F; Q231F; K242M; S258N; A303D 72.4 99.8 3 M2 L37Q; A187V; L201F; V215I; Q231F; S258N; 82.3 99.8 4 G44R; A75T; A107H; L120I; L198C; L201Y; 10.9 ND 5 G44R; G133Y; L198C; L201Y; V265R; A276R; 2.9 ND 6 Y142S; 2.7 ND aReaction conditions: 3 (0.2 g, 1 equiv, 20.0 mg/mL) in 3 mL DMSO, rac-trans-1 (0.2 g, 2.1 equiv), IRED variants (10% wt/wt), D-glucose (0.2 g, 2 equiv), NADP+ (26 mg, 0.05 equiv), GDH-CDX-901 (8 mg, 4% wt/wt), DMSO (30%), 30 °C, 3 h, 200 mM sodium acetate buffer pH 4.5, to a final volume of 10 mL.
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Selection of the M3 variant Engineered imine reductases described in Supplementary Table 12 were evaluated at preparative scale in sodium acetate buffer 200 mM pH 4.6 as follows. The enzymes were expressed and lyophilised powder generated. Reactions were run using AmigoChem, a controlled laboratory equipment capable of running up to 10 reactions in parallel and sample automatically. By running reactions at 0.25 g scale of 3, to a final reaction volume of 10 mL this allowed us to easily select the best variant. Reactions were run in two separate runs of 4 reactions each. Rac-trans-1 (1.5 g) was dissolved in 40 mL 200 mM sodium acetate buffer pH 4.6. This solution was used to solubilize D-glucose (1.5 g), NADP+ (0.16 g) and GDH-CDX-901 (12 mg). 8 mL of the mixture was added to each reaction tube. Enzyme stock solutions (12.5 mg/mL) were freshly prepared for each of the 8 imine reductase variants selected in 200 mM sodium acetate buffer pH 4.6 and 200 µL of the stock solution were added to the reaction tube. 1.82 mL of a previously obtained solution of aldehyde 3 (137.2 mg/mL) was added to obtain a substrate loading of 25 g/L of 3. The addition of the 3 marked the start of the reaction. All reactions were run at 30 °C and 500 rpm stirring for 4 h with the first 3 h being monitored by the AmigoChem. Due to the nature of the AmigoChem set up sample points were taken with a slight shift in time between them. For Amigochem samples 100 µL of reaction sample were mixed with 300 µL of acetic acid (1M): acetonitrile 3:4. For the final sample after 4 h the whole reaction mixture was solubilized by the addition of 20 mL acetic acid: acetonitrile 3:4. Samples were then analyzed, using HPLC Method 2, and results summarized in Supplementary Table 15.
Supplementary Table 15. High scale validation of the top 8 enzyme variants, from the Rd3 evolution selected after running tier 2 conditions, as catalysts in the model reaction 3→4.a
Entry Variant Mutations (backbone M2) Conv. [%] e.e. [%] (1S,2R)-4
1 M3 G44R; V92K; F97V; L198M; T260C; A303D; 73.8 99.7 2 G44R; V92K; F97V; L198M; T260I; S274C; 60.7 99.5 3 V92K; F97V; L198M; T260I; S274C; 47.7 99.6 4 G44R; V92K; A107H; L198M; T260C; S274C; A303S; 59.1 99.6 5 G44R; V92K; F97V; L198M; S274C; A303S 60.1 99.7 6 V92K; F97V; L198M; T260I; A303S; 50.3 99.8 7 G44R; V92K; F97V; L198M; T260I; S274C; 64.6 99.5 8 G44R; V92K; F97V; L198M; T260C; A303S; 63.2 99.5 aReaction conditions: 3 (0.25 g, 1 equiv, 25.0 mg/mL) in 1.8 mL DMSO, rac-trans-1 (0.3 g, 2.1 equiv), IRED variants (1% wt/wt), D-glucose (0.3 g, 2 equiv), NADP+ (32 mg, 0.05 equiv), GDH-CDX-901 (2.5 mg, 1% wt/wt), DMSO (18%), 30 °C, 4 h, 200 mM sodium acetate buffer pH 4.6, to a final volume of 10 mL. The selected M3 variant exhibits 13 mutations from that of wild type IR-46. Three mutations are located within the active site: Y142S, L201F and L198M, which likely contribute to stabilization substrate binding. While L201F and L198M may form non-specific hydrophobic interactions with the substrate, Y142S may supply a hydrogen bond, as the Y142G variant exhibits no appreciable activity, and the Y142T variant is nearly as active as Y142S. Three mutations were introduced near the cofactor binding region: L37Q, V92K and S258N, with the amide groups from L37Q and S258N presumably forming a network of hydrogen bonding interactions with the nicotinamide, and V92K interacting with the negatively charged phosphate groups. These mutations may help to position the cofactor for facile hydride transfer. Three mutations were also introduced into the Rossman fold but do not have direct interactions with the cofactor. Mutation G44R, potentially helps to stabilize the alpha helix contained in the canonical βαβ fold that is responsible for recognition of the cofactor. The other two mutations, F97V and A187V, are at the interface between two adjoining helices and likely increase the local packing order of the adjacent residues between those helices. Two mutations, V215I and Q231F were introduced at the interfaces between the two subunits of the IRED homodimer. V215I is positioned at the beginning of the transhelix with its Cα-Cβ vector pointing at the symmetric subunits’ transhelix. Like F97V and A187V, this mutation likely increases the local packing order of the residues adjoining position 215, leading to an increase in the stability of the homodimer. The role of Q231F, however, is less clear, but this residue mutation presumably forms a non-specific hydrophobic interaction with the neighbouring subunits’ residues. Lastly, two mutations were introduced adjacent to the active site, T260C and A303D, but it is difficult to deduce their role in stabilizing the enzyme based on the D10 structural model. A reasonable hypothesis is that A303D may form a specific interaction with the substrate given its position in the flexible loop adjacent to the active site.
Specific Activity measurements for IR-46, M1, M2 and M3 Purified imine reductase variants were assayed for activity at a concentration of 3 µM (IR-46) or 1 µM (M1, M2, M3) by measuring initial rates of consumption of NADPH by absorbance change at 340 nm in 200 mM KPi buffer
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pH 7.0, 10% DMSO, 1 mM NADPH, 1 mM aldehyde 3, and either 4.75 mM or 63 mM rac-trans-1. Specific activity was calculated from Beer-Lambert’s law using an extinction coefficient change of 6300 M-1cm-1. Amine stocks were adjusted to pH 7.0 with HCl prior to addition. Half-life measurements for IR-46, M1, M2 and M3 Purified imine reductase variants were incubated at 33 µM (IR-46) or 10 µM (M1, M2, M3) in 200 mM NaOAc buffer pH 5.0 containing 30% DMSO at 30 °C for the indicated time before the remaining activity was measured by diluting 10 fold into 1 mM rac-trans-1, 1 mM aldehyde 3, 1 mM NADPH in 200 mM KPi buffer pH 7.0, 11% DMSO (final) and initial rates were calculated by absorbance change at 340 nm as above.
Supplementary Figure 10. % Activity remaining after incubation in 200 mM NaOAc buffer pH 5.0 containing 30% DMSO at 30 °C for IR-46 (open circles), M1 (filled circles), M2 (open squares) and M3 (filled squares). Solid line represents fit to single exponential decay (y = Ae-kt).
Supplementary Table 16. Half-life values derived from single exponential fit to data in Supplementary Figure 10.
Variant half-life [h] WT (IR-46) 0.042 M1 0.93 M2 24 M3 >200a aNo measurable loss of activity after 170 h. Melting temperature (Tm) measurements for IR-46, M1, M2 and M3 Melting temperatures were determined by the thermofluor method. Purified imine reductase variants were incubated in 200 mM KPi buffer pH 7.0 together with 5x final concentration of SYPRO® Orange dye (from 5000x DMSO stock) at a total volume of 10 ul in a MicroAmp™ Optical 384-Well PCR Reaction Plate. The plate was heated from 30 °C to 95 °C at 0.05 °C/s while monitoring fluorescence in a Applied Biosystems QuantStudio 12K Flex RT-PCR machine. Data was analysed using Applied Biosystems Protein Thermal Shift™ software version 1.3.
Time (h)
0
% a
ctiv
ity re
mai
ning
0
20
40
60
80
100
WT
M1
M2
M3
0.001 0.01 0.1 1 10 100
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Supplementary Figure 11. Melting curve first derivative for IR-46 (20 µM, magenta), M1 (20 µM, blue), M2 (10 µM, green) and M3 (10 µM red) in 200 mM potassium phosphate buffer pH 7. Monitored by Sypro Orange fluorescence. Data represents mean of 4 replicates, error bars are ± 1 SD.
Supplementary Table 17. Tm values for Imine reductase variants.
Variant Tm [°C] (StDev)[a] WT 41.3 (0.3) M1 47.6 (0.3) M2 57.3 (0.3) M3 70.0 (0.1)
a Mean and standard deviation of Tm for 4 replicates.
Selected scale-up examples Preparation of (1R,2S)-4 using IR-46 (WT) as catalyst Aqueous potassium phosphate buffer pH 6.5 100 mM (K2HPO4/KH2PO4) (160 mL) and sodium acetate 100 mM (NaOAc/AcOH) buffer pH 5.6 (245 mL) were added to a 1 L CLR conditioned at 30 °C and equipped with an overhead stirrer. D-glucose (9.9 g, 3.07 equiv), nicotinamide adenine dinucleotide phosphate (NADP+) (0.8 g, 0.05 equiv) and D-glucose dehydrogenase GDH-CDX-901 (0.4 g, 7.4% wt/wt) were then added to the CLR and contents stirred for 20 min at 30 °C until D-glucose dissolved. Next solid rac-trans-1 (8.5 g, 2.60 equiv) and crude 3 in DMSO (5.4 g, 17.7 mmol final volume 42 mL) were added. The reaction was started by the addition of lyophilised cell lysate of imine reductase (IR-46) solution in 4 portions (24.5 g in 73 mL potassium phosphate buffer 100 mM, pH 6.5). Reaction was monitored by HPLC until it stalled (~4.5 h from the first addition of IR-46) at which point reaction was then quenched by adding glacial acetic acid (30 mL) with a dropping funnel over 10 min to a pH of 3.8. Conversion to the product 4 (4 with respect to 3) indicated 85.0% a/a. Precipitated enzyme was then filtered through cotton wool and precipitate washed with additional neat acetic acid (21 mL) and potassium phosphate buffer pH 6.5 (14 mL). Reaction crude was then cooled to 10 °C and 26% NaCl solution (35 mL) added drop wise over 30 min and contents stirred for 60 min. The product dihydrochloride salt precipitate 4 was then collected by filtration and washed with cold water (2 x 21 mL). The solid was then dried in vacuum oven at 40 °C for 20 h to afford (1R,2S)-4 (3.8 g, 42.9% yield, 98.6% purity, >99.9% e.e.). 1H NMR (400 MHz, CD3OD, 300K): δ 8.06 (d, J = 8.3 Hz, 2H), 7.68 (d, J=8.3 Hz, 2H), 7.30 (t, J=7.6 Hz, 2H), 7.16-7.25 (m, 3H), 4.41 (s, 2H), 3.53 (br d, J = 11.5 Hz, 2H), 3.18 (br d, J = 6.1 Hz, 2H), 3.11 (br t, J = 11.5 Hz, 2H), 2.98-3.04 (m, 1H), 2.56-2.64 (m, 1H), 2.10-2.22 (m, 1H), 2.10 (br d, J = 14.4 Hz, 2H), 1.3-1.76 (m, 3H), 1.61 (s, 9H), 1.38 (q, J = 7.4 Hz, 1H) ppm. 13C NMR (101 MHz, CD3OD, 300K): δ 166.6 (1C), 139.5 (1C), 135.0 (1C), 134.8 (1C), 132.8 (2C), 131.2
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(2C), 129.9 (2C), 128.2 (1C), 127.6 (2C), 83.0 (1C), 61.3 (1C), 53.6 (1C), 53.1 (2C), 39.9 (1C), 32.5 (1C), 28.5 (3C), 28.3 (2C), 22.6 (1C), 13.6 (1C) ppm. HRMS (ESI) m/z [M+H]+: molecular ion calculated for C27H37N2O2: 421.2850, found 421.2854 (free base).
Preparation of (1R,2S)-4 using M1 as catalyst Rac-trans-1 (7.2 g, 2.1 equiv), D-glucose (6.8 g, 2.01 equiv), nicotinamide adenine dinucleotide phosphate (NADP+) (0.7 g, 0.06 equiv) and GDH-CDX-901 (0.35 g, 6.1% wt/wt) were added to a 1 L CLR conditioned at 30 °C and equipped with an overhead stirrer. Sodium acetate (NaOAc/AcOH) buffer 100 mM pH 5.0 (473 mL) and DMSO (123 mL) were added and contents stirred at 400 rpm and 30°C for 5 min. M1 lyophilised powder (3.5 g, 60.9% wt/wt) was then added and mixture stirred for additional 10 min. The reaction was started by the addition of aldehyde 3 in DMSO (5.7 g in 35 mL DMSO) dropwise over 10 min. Reaction was monitored by HPLC until it stalled (2.0 h from the addition of M1) at which point reaction was then quenched by adding glacial acetic acid (70 mL) with a dropping funnel over 10 min to a pH of 3.8. Conversion to the product 4 (4 with respect to 3) indicated 87.8% a/a. Celite (21 g, 3.65 wt) was then added to the mixture and contents stirred for 30 min. The resulting mixture was filtered twice using a Buchner funnel at 50 mbar and 150 mbar vacuum. Filtrate was charged back in the CLR and cooled down to 5 °C and stirred at 300 rpm. Brine 26% (35 mL) was then added drop wise over 25 min and contents stirred for additional 30 min. Mixture was then filtered cold using a Buchner funnel. The product dihydrochloride salt precipitate 4 was washed with cold water (2 x 14 mL). The solid was then dried in vacuum oven at 40 °C over the weekend to afford (1R,2S)-4 (6.4 g, 68.0% yield, >99.9% purity, >99.9% e.e.).
Preparation of (1R,2S)-4 using M2 as catalyst Rac-trans-1 (6.2 g, 2.04 equiv), D-glucose (5.8 g, 1.94 equiv), NADP+ (0.66 g, 0.05 equiv) were added to a 400 mL EasyMax reactor conditioned at 30 °C and equipped with an overhead stirrer. Sodium acetate (NaOAc/AcOH) buffer 100 mM, pH 5.0 (216 mL) and DMSO (54 mL) were added and contents stirred at 300 rpm and 30 °C for 30 min. M2 (0.6 g, 12.0 wt/wt) and GDH-CDX-901 (0.2 g, 4.7% wt/wt) were added and mixture stirred for additional 10 min. Reaction was started by the addition of aldehyde 3 in DMSO (5.0 g in DMSO, 30 mL final volume) dropwise over 30 min using a dropping funnel. Reaction was monitored by HPLC until it stalled (~2.5 h from the addition of M2) at which point reaction was then quenched by adding glacial acetic acid (30 mL) with a dropping funnel. Conversion to the product 4 (4 with respect to 3) indicated 87.6% a/a. Celite 545 (1.7 g, 0.34 wt/wt) was then added to the mixture and contents stirred for 1 h. The resulting mixture was then filtered through glass fiber filter paper using a Buchner funnel. Filtrate was charged back in the EasyMax and cooled down to 5 °C and stirred at 300 rpm. Solid NaCl (7.8 g, 1.54 wt) was then added in portions and contents stirred overnight. Mixture was then filtered cold using a Buchner funnel. The product dihydrochloride salt precipitate was washed with cold water (2 x 15 mL) and acetone (15 mL ). The solid was then dried in vacuum oven at 40 °C over night to afford (1R,2S)-4 (5.6 g, 68.5% yield, 99.3% purity, 99.4% e.e.) as a white solid.
Preparation of (1R,2S)-5 using M3 as catalyst Rac-trans-1 (23.8 g 2.38 equiv), D-glucose (14.1 g, 1.42 equiv) and NADP+ (1.0 g, 0.23 equiv) were added to a 1 L CLR conditioned to 30 °C and equipped with an overhead stirrer. Sodium acetate (NaOAc/AcOH) buffer 100 mM, pH 5.0 (700 mL) was then added and contents stirred at 300 rpm and 30 °C for 30 min. Next GDH-CDX-901 (0.2 g, 1.2% wt/wt) and lyophilised powder of M3 (0.2 g, 1.2% wt/wt) were added and contents stirred for additional 10 min. Reaction was started by the addition of aldehyde 3 in DMSO (16.6 g in DMSO, final volume 100 mL) dropwise over 60 min. The reaction was stirred for approximately 5 h at which point it was quenched by adding glacial acetic acid (60 mL) with a dropping funnel. Conversion to the product 4 (4 with respect to 3 only) indicated 91.4% a/a. Celite 545 (10.0 g, 0.59 wt) was added and suspension stirred for 1 h at 30 °C. Reaction mixture was then filtered through glass fibre filter paper to remove the precipitated enzyme. Next day the filtrate was charged into a clean CLR and then cooled to 5 °C. Brine 26% (44 mL) was added during 1 h using a dropping funnel and slurry allowed to stir for additional 1 h. Mixture was then filtered through glass fibre filter paper using a Buchner funnel. The solid was then washed with cold water (2 x 50 mL) and then transferred into a 400 mL EasyMax system and reslurried with acetone (200 mL) and stirred for 1 h at 5 °C followed by filtration though glass fibre filter paper. Solid was then washed with cold acetone (2 x 50 mL). Wet cake was dried under vacuum for 30 min and then placed into oven to dry at 25 °C for 2 h, followed by 40 °C drying over the weekend to afford (1R,2S)-4 as a white solid (19.6 g, 72.2% yield, >99.9% purity, 99.7% e.e.).
S37
Kilogram make of (1R,2S)-4 using M3 as catalyst Three scale up batches were run in order to test the robustness of M3 variant, according to the described protocol.
Distilled water (4 L) was added to a 20 L CLR followed by sodium acetate trihydrate (93.2 g) and acetic acid (40.8 mL). Further water (10 L) was added and stirred for at least 10 min before checking the pH of the resulting 100 mM Na-acetate buffer (pH indicated 4.6). To the prepared buffer rac-trans-1 (476.0 g, 2.4 equiv), D-glucose (280.0 g, 1.43 equiv), nicotinamide adenine dinucleotide phosphate disodium salt (NADP+) (20.5 g) and the mixture was stirred at 30 °C for 44 min to give a clear solution (Supplementary Figure 12, A). Lyophilised clarified cell lysate of IRED M3 (0.012 wt, 4.0 g) and GDH- CGX-901 (0.012 wt, 4.0 g) were added to give a thin suspension and stirred for 10 min. Aldehyde 3 in DMSO (329.2 g, in ~2.34 L final volume) was added at 30°C over 1 h and stirred for 4 h before analysing by HPLC (Supplementary Figure 12, B). The reaction mixture was then quenched with neat acetic acid (1.2 L) at 30 °C over at least 10 min and then stirred for at least 10 min. Celite 545 filtering agent (200.2 g) was then added and stirred for 1h. Conversion to the product 4 (4 with respect to 3) indicated 95.6% a/a.The precipitated enzyme and Celite was then filtered over 8 min on a ca 24 cm PTFE mini filter fitted with glass fibre filter paper and Whatman No 113 wet strengthened filter paper (raised side up), using a 20 L Buchner flask as a receiver. The filtrate was then transferred to a 20 L CLR via PTFE suck-up line fitted with a 1 micron in-line filter (Supplementary Figure 12, C). Solution was then cooled to 6-7 °C before 26%wt/v brine (880 mL) was added over at least 60 min and the resulting suspension stirred for at least 1 h at 5 °C (Supplementary Figure 12, D). Slurry was then filtered on a V2 filter dryer equipped with 10 µm Hastelloy C22 poroplate filter. CLR was then cleaned sequentially with water (2 x 1L) and acetone (1 x 4 L, 2 x 1 L) and contents filtered on the V2 filter drier to recover all the product precipitated on the reaction vessel. The product was then dried using a positive pressure of nitrogen and then dried under vacuum (50 mbar) at 20 °C for 2 h then the temperature was increased to 40 °C to constant probe temperature to afford (1R,2S)-4 as a white solid (452.1 g; yield 84.4%, 100.0% purity, 99.7% e.e.) (Supplementary Figure 12, D).
Supplementary Figure 12. M3 batch scale up (329.1 g). From left to right A: reaction mixture containing rac-trans-1, NADP+ and buffer, and in front GDH and M3; B: reaction mixture upon the aldehyde 3 addition; C: reaction crude after quench and enzyme removal; D: product (1R,2S)-4 precipitation after brine addition; E: (1R,2S)-4 harvesting.
A B C D E
S38
Redox-neutral enzymatic cascade conversion of 2 to (1R,2S)-4
NADP+ NADPH
N
NH
O
O
2HCl
(1R,2S)-4
3
N
O
OO
rac-trans-1
NH3+
NH3+
SO42-
N
O
OHO
2
KRED
IRED
Selection of the KRED catalyst To identify KREDs capable of catalysing the oxidation of 2 to 3 in the redox-neutral cascade, a panel of over 400 KREDs was screened directly for the cascade reaction using an IRED previously evolved for the reductive amination (M1). The most promising hits (affording (1S,2R)-4 in up to 27% conversion and >99.5% e.e. in the cascade reaction from this screen) were used to optimise reaction conditions in plates and using the Integrity10/AmigoChem platform (details not shown). Screening KREDs for the cascade reaction Costar deep-96 well plates containing clarified KRED lysate (100 µL) were prepared according to the methods described above for panel screening. To these plates was added IRED stock (95.6 µL) containing IRED M1 lyopholised lysate (26 mg/mL, final concentration 10 mg/mL) and NADP+ (6.5 mg/mL, final concentration 2.5 mg/mL) in 100 mM sodium phosphate buffer (pH 5.6). Amine stock (104 µL) containing racemic 1 (30 mg/mL, final concentration 12.5 mg/mL) in 100 mM sodium acetate buffer (pH 5.6) was then added. Reactions when initiated by addition of alcohol 2 stock to each well (50 µL, 50 mg/mL 2 in DMSO, final concentration 10 mg/mL 2, 10 v/v% DMSO). The reaction plate was then sealed using an aluminium/polypropylene laminate heat seal tape and incubated at 30 °C with shaking at 800 RPM in an Infors MultiTron shaker. After incubation for 16 h, reactions were quenched by addition of 1M acetic acid (200 µL) and acetonitrile (300 µL) to each well of the reaction plate. The plate was sealed as described above and shaken at room temperature for 5 min before centrifugation (4000 RPM, 10 min). Supernatant (200 µL) was transferred from each well to a flat-bottom Nunc 96-well plate which was sealed before analysis by UPLC.
Preparation of (1R,2S)-4 using M3/KRED as catalysts Alcohol 2 (5.0 g, 16.37 mmol) was added to a 1 L CLR conditioned at 30 °C and equipped with an overhead stirrer. DMSO (50 mL) and potassium phosphate buffer (K2HPO4/KH2PO4) 100 mM pH 7.0 (200 mL) and were added and contents stirred at 300 rpm and 30°C for 5 min. Rac-trans-1 (6.26 g, 17.19 mmol, 2.1 equiv) was then added to the CLR using a dropping funnel. To a separate Schott bottle KRED lyophilised powder (5.0 g, 100% wt/wt), M3 lyophilised powder (1.0 g, 20.0% wt/wt) and NADP+ (0.025 wt, 0.128 g, 0.01 equiv) were added over potassium phosphate buffer (K2HPO4/KH2PO4) 100 mM pH 7.0 (200 mL) and contents added into the CLR over 1 min using a dropping funnel. Schott bottle was further washed with potassium phosphate buffer (K2HPO4/KH2PO4) 100 mM pH 7.0 (50 mL) and contents added into the CLR using a dropping funnel. After 24 h glacial acetic acid (25 mL) was added and enzyme removed by filtration. Brine (25 %, 35 mL) was then added and mixture cooled at 5 °C and stirred for 2 h. Product (1R,2S)-4 was then filtered through the filter paper and the precipitate washed with chilled water (cooled at a temperature below 4 °C) (4 x 12.5 mL) and acetone (2 x 10 mL), dried in the oven overnight at 40°C±3°C under 20-100 mbar pressure to afford (1R,2S)-4 as a white powder (3.9 g, 48.3% yield, 97.9% HPLC purity a/a, >99.7% e.e.).
S39
Sequence listing IR-46 Organism: Saccharothrix espanaensis
NCBI Reference Sequence: WP_015102218.1
DNA sequence ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGTCACGGAAGCGTCCCCGGCCCCGGTGTCGGTTATCGGTCTGGGTCTGATGGGTGCGGCTCTGGCTGGTGCCTACCTGAAAGCGGGCCATCAGACCACGGTGTGGAACCGTAGTGCGGGCAAAGCTGATGCGCTGGTTGCTCAAGGTGCGACCAATGCGGCCGACATTGCCGAAGCAGTTGCAGCTTCCGATGTCCTGGTGGTTTGCGTCGTGGACTATGCGGCCTTTCACGCAATTCTGGAACCGGTGAAAGATGCCCTGCAGGGCAAAGTGATCGTTAACCTGACCTCAGGTCTGCCGGATGATGCGCGCGGTGCAGCTGAATGGGCAAGCGGCACGGGTGCAGAATATCTGGATGGTTACATCATGAGCGTCCCGCCGGGCGTGGGTCTGCCGCAAACCCTGCTGTTTTACGGCGGTGATGCCGACGTTTTCGCAAAACATGAAGCTACGCTGAAAGTCCTGGGCGGTAATTCAATTCATCTGGGTGCTGATGCGGGTGTGGCCGCACTGTATGACCTGGGTCTGCTGGCGATCCTGTGGAGCTCTCTGGCCGGTGCACTGCATGCTTACGCGCTGGTTGCCTCGGAAAAAATTCCGGCAGCTGCGCTGGCCCCGTTTGCAGAACAGTGGATCACCCACGTTGTCCTGCCGTCAGTCAAAGGCGCCGCAGCTGCGGTGGATTCGGGTCAATATGCGACGAGTGTGTCCACCACCGCTCTGAATGCCGTGGGTCTGGGTAAAATGGTTGAAGCGAGCAAAGCCGCAGGCATTCGTCCGGATCTGATGCTGCCGATCAAAGCCTATCTGGAACAGCGTGTTGCGGATGGTCATGGTGAAGAAGCCCTGGCAGGCATGTTCGAAGTTATTCGTTCTCCGGAACGCTAA
Amino acid sequence MGSSHHHHHHSSGLVPRGSHMVTEASPAPVSVIGLGLMGAALAGAYLKAGHQTTVWNRSAGKADALVAQGATNAADIAEAVAASDVLVVCVVDYAAFHAILEPVKDALQGKVIVNLTSGLPDDARGAAEWASGTGAEYLDGYIMSVPPGVGLPQTLLFYGGDADVFAKHEATLKVLGGNSIHLGADAGVAALYDLGLLAILWSSLAGALHAYALVASEKIPAAALAPFAEQWITHVVLPSVKGAAAAVDSGQYATSVSTTALNAVGLGKMVEASKAAGIRPDLMLPIKAYLEQRVADGHGEEALAGMFEVIRSPER
M1 DNA sequence
ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGTCACGGAAGCGTCCCCGGCCCCGGTGTCGGTTATCGGTCTGGGTCTGATGGGTGCGGCTCTGGCTGGTGCCTACCTGAAAGCGGGCCATCAGACCACGGTGTGGAACCGTAGTGCGGGCAAAGCTGATGCGCTGGTTGCTCAAGGTGCGACCAATGCGGCCGACATTGCCGAAGCAGTTGCAGCTTCCGATGTCCTGGTGGTTTGCGTCGTGGACTATGCGGCCTTTCACGCAATTCTGGAACCGGTGAAAGATGCCCTGCAGGGCAAAGTGATCGTTAACCTGACCTCAGGTCTGCCGGATGATGCGCGCGGTGCAGCTGAATGGGCAAGCGGCACGGGTGCAGAATATCTGGATGGTAGTATCATGAGCGTCCCGCCGGGCGTGGGTCTGCCGCAAACCCTGCTGTTTTACGGCGGTGATGCCGACGTTTTCGCAAAACATGAAGCTACGCTGAAAGTCCTGGGCGGTAATTCAATTCATCTGGGTGCTGATGCGGGTGTGGCCGCACTGTATGACCTGGGTCTGCTGGCGATCCTGTGGAGCTCTCTGGCCGGTGCACTGCATGCTTACGCGCTGGTTGCCTCGGAAAAAATTCCGGCAGCTGCGCTGGCCCCGTTTGCAGAACAGTGGATCACCCACGTTGTCCTGCCGTCAGTCAAAGGCGCCGCAGCTGCGGTGGATTCGGGTCAATATGCGACGAGTGTGTCCACCACCGCTCTGAATGCCGTGGGTCTGGGTAAAATGGTTGAAGCGAGCAAAGCCGCAGGCATTCGTCCGGATCTGATGCTGCCGATCAAAGCCTATCTGGAACAGCGTGTTGCGGATGGTCATGGTGAAGAAGCCCTGGCAGGCATGTTCGAAGTTATTCGTTCTCCGGAACGCTAA
Amino acid sequence
MGSSHHHHHHSSGLVPRGSHMVTEASPAPVSVIGLGLMGAALAGAYLKAGHQTTVWNRSAGKADALVAQGATNAADIAEAVAASDVLVVCVVDYAAFHAILEPVKDALQGKVIVNLTSGLPDDARGAAEWASGTGAEYLDGSIMSVPPGVGLPQTLLFYGGDADVFAKHEATLKVLGGNSIHLGADAGVAALYDLGLLAILWSSLAGALHAYALVASEKIPAAALAPFAEQWITHVVLPSVKGAAAAVDSGQYATSVSTTALNAVGLGKMVEASKAAGIRPDLMLPIKAYLEQRVADGHGEEALAGMFEVIRSPER
M2 DNA sequence
S40
ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGTCACGGAAGCGTCCCCGGCCCCGGTGTCGGTTATCGGTCTGGGTCAGATGGGTGCGGCTCTGGCTGGTGCCTACCTGAAAGCGGGCCATCAGACCACGGTGTGGAACCGTAGTGCGGGCAAAGCTGATGCGCTGGTTGCTCAAGGTGCGACCAATGCGGCCGACATTGCCGAAGCAGTTGCAGCTTCCGATGTCCTGGTGGTTTGCGTCGTGGACTATGCCGCCTTTCACGCAATTCTGGAACCGGTGAAAGATGCCCTGCAGGGCAAAGTGATCGTTAACCTGACCTCAGGTCTGCCGGATGATGCGCGCGGTGCAGCTGAATGGGCAAGCGGCACGGGTGCAGAATATCTGGATGGTAGTATCATGAGCGTCCCGCCGGGCGTGGGTCTGCCGCAAACCCTGCTGTTTTACGGCGGTGATGCCGACGTTTTCGCAAAACATGAAGCTACGCTGAAAGTCCTGGGCGGTAATTCAATTCATCTGGGTGCTGATGTTGGTGTGGCCGCACTGTATGATCTGGGTCTGCTGGCGATCTTCTGGAGCTCTCTGGCCGGTGCACTGCATGCTTACGCGCTGATTGCCTCGGAAAAAATTCCGGCAGCTGCGCTGGCCCCGTTTGCAGAATTTTGGATCACCCACGTTGTCCTGCCGTCAGTCAAAGGCGCCGCAGCTGCGGTGGATTCGGGTCAATATGCGACGAGTGTGAACACCACCGCTCTGAATGCCGTGGGTCTGGGTAAAATGGTTGAAGCGAGCAAAGCCGCAGGCATTCGTCCGGATCTGATGCTGCCGATCAAAGCCTATCTGGAACAGCGTGTTGCGGATGGTCATGGTGAAGAAGCCCTGGCAGGCATGTTCGAAGTTATTCGTTCTCCGGAACGCTAA
Amino acid sequence
MGSSHHHHHHSSGLVPRGSHMVTEASPAPVSVIGLGQMGAALAGAYLKAGHQTTVWNRSAGKADALVAQGATNAADIAEAVAASDVLVVCVVDYAAFHAILEPVKDALQGKVIVNLTSGLPDDARGAAEWASGTGAEYLDGSIMSVPPGVGLPQTLLFYGGDADVFAKHEATLKVLGGNSIHLGADVGVAALYDLGLLAIFWSSLAGALHAYALIASEKIPAAALAPFAEFWITHVVLPSVKGAAAAVDSGQYATSVNTTALNAVGLGKMVEASKAAGIRPDLMLPIKAYLEQRVADGHGEEALAGMFEVIRSPER
M3 DNA sequence
ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGTCACGGAAGCGTCCCCGGCCCCGGTGTCGGTTATCGGTCTGGGTCAGATGGGTGCGGCTCTGGCTCGTGCCTACCTGAAAGCGGGCCATCAGACCACGGTGTGGAACCGTAGTGCGGGCAAAGCTGATGCGCTGGTTGCTCAAGGTGCGACCAATGCGGCCGACATTGCCGAAGCAGTTGCAGCTTCCGATGTCCTGGTGGTTTGCGTCAAGGACTATGCCGCCGTTCACGCAATTCTGGAACCGGTGAAAGATGCCCTGCAGGGCAAAGTGATCGTTAACCTGACCTCAGGTCTGCCGGATGATGCGCGCGGTGCAGCTGAATGGGCAAGCGGCACGGGTGCAGAATATCTGGATGGTAGTATCATGAGCGTCCCGCCGGGCGTGGGTCTGCCGCAAACCCTGCTGTTTTACGGCGGTGATGCCGACGTTTTCGCAAAACATGAAGCTACGCTGAAAGTCCTGGGCGGTAATTCAATTCATCTGGGTGCTGATGTTGGTGTGGCCGCACTGTATGATCTGGGTCTGATGGCGATCTTCTGGAGCTCTCTGGCCGGTGCACTGCATGCTTACGCGCTGATTGCCTCGGAAAAAATTCCGGCAGCTGCGCTGGCCCCGTTTGCAGAATTTTGGATCACCCACGTTGTCCTGCCGTCAGTCAAAGGCGCCGCAGCTGCGGTGGATTCGGGTCAATATGCGACGAGTGTGAACACCTGTGCTCTGAATGCCGTGGGTCTGGGTAAAATGGTTGAAGCGAGCAAAGCCGCAGGCATTCGTCCGGATCTGATGCTGCCGATCAAAGCCTATCTGGAACAGCGTGTTGCGGATGGTCATGGTGAAGAAGACCTGGCAGGCATGTTCGAAGTTATTCGTTCTCCGGAACGCTAA
Amino acid sequence
MGSSHHHHHHSSGLVPRGSHMVTEASPAPVSVIGLGQMGAALARAYLKAGHQTTVWNRSAGKADALVAQGATNAADIAEAVAASDVLVVCVKDYAAVHAILEPVKDALQGKVIVNLTSGLPDDARGAAEWASGTGAEYLDGSIMSVPPGVGLPQTLLFYGGDADVFAKHEATLKVLGGNSIHLGADVGVAALYDLGLMAIFWSSLAGALHAYALIASEKIPAAALAPFAEFWITHVVLPSVKGAAAAVDSGQYATSVNTCALNAVGLGKMVEASKAAGIRPDLMLPIKAYLEQRVADGHGEEDLAGMFEVIRSPER
KRED
Organism: Lactobacillus coleohominis 101-4-CHN
NCBI Reference Sequence: WP_006915881.1
UniProtKB - C7XTX6 (C7XTX6_9LACO)
DNA sequence
ATGGGCGACTACAAGGACGACGATGACAAGGGTCATCATCACCATCATCACGAAAACCTGTATTTTCAGGGAATGGCAGTAAAGGGCAAGGTAGTAGTAATTACTGGCGCAAGCAGCGGCATTGGCGAAGCAACTGCAAAGCTGCTGGCAGCAAACGGCGCAATGGTAATGCTGGGCGCACGCCGCGAAGATCGCCTGTACAAGATTGCAAAC
S41
GAAATTAACGTAAACGGCGGCCGCGCAGATTACCGCACTGTAGATGTAACTAAGCCGGAAGAAGTAGAAGCACTGGTAAAGGCAGCACAGGATAGCTTCGGCCAGGTAGATGTAATTTTCAACAACGCAGGCATTATGCCGAACAGCCCGATGAGCGCAGTACGCACTGATGAATGGAACAAGATGATTGATGTAAACATTAAGGGCGTACTGAACGGCATTGCAGCAGTAATGCCGATTTTCACTGCACAGAAGCACGGCCACATTATTACTACTAGCAGCGTAGCAGGCCTGAAGAACTACGTAGGCAGCGGCGTATACGGCGCAACTAAGTTCGCAGTAAAGAACGCAATGGAAGTAACTCGCATGGAAAGCGCAAACGAAGGCACTAACATTCGCACTACTACTCTGTACCCGGCAGCAATTAACACTGAACTGCTGGATCACATTGGCGATGAACAGACTGCAAGCAACATGAAGAACTTCTACAAGCAGCACGGCATTAGCCCGGATGCAATTGCACGCGTAGTAAACTTCGCAATTGATCAGCCGGAAGATGTAGATATTAGCGAGTTCACTATTTACCCGACTAACCAGGCATAA
Amino acid sequence
MGDYKDDDDKGHHHHHHENLYFQGMAVKGKVVVITGASSGIGEATAKLLAANGAMVMLGARREDRLYKIANEINVNGGRADYRTVDVTKPEEVEALVKAAQDSFGQVDVIFNNAGIMPNSPMSAVRTDEWNKMIDVNIKGVLNGIAAVMPIFTAQKHGHIITTSSVAGLKNYVGSGVYGATKFAVKNAMEVTRMESANEGTNIRTTTLYPAAINTELLDHIGDEQTASNMKNFYKQHGISPDAIARVVNFAIDQPEDVDISEFTIYPTNQA
UPLC analysis and selected chromatograms
Method 1: High-throughput analytical UPLC method to determine conversion of aldehyde 3 to amine 4
Conversion of the aldehyde 3 to the amine 4 was determined using an Agilent 1290 UPLC equipped with a Waters XSelect CSH column installed (2.1 x 30mm, 2.5 µm) 0.05 % Trifluoroacetic Acid in water (mobile phase A) and 0.05 % Trifluoroacetic Acid in Acetonitrile (mobile phase B) at flowrate of 2 mL/min at a column temperature of 60 °C. The run time was 3 min with gradient table as below. Beginning from a 99:1 ratio of A:B, the method followed a 0.3 minute hold, followed by a 0.4 minute gradient to 92:8 A:B, followed by a 0.2 minute gradient to 89:11 A:B, followed by a 0.6 minute gradient to 72:28 A:B, followed by a 0.5 minute gradient to 5:95 A:B, followed by a 0.3 minute hold then a 0.2 minute purge gradient to 99:1 A:B , and finally a 0.5 minute hold at 99.1 A:B. Compound elution was monitored at 220 nm.
Method 2: Achiral analytical HPLC method to determine conversion of aldehyde 3 to amine 4
Impurity profile and conversion of the aldehyde 3 to the amine 4 was determined using an Agilent HPLC equipped with an Agilent Bonus RP column (150 mm x 4.6 mm, 3.5 μm) column. 0.05 % Trifluoroacetic Acid in water (mobile phase A) and 0.05 % Trifluoroacetic Acid in Acetonitrile (mobile phase B) at flowrate of 1 mL/min at a column temperature of 40 °C. The run time was 20 min with gradient table as below and a post run time of 8 min. Beginning from a 95:5 ratio of A:B, the method followed a 12 minute gradient to 77:23 ratio of A:B, followed by a 6 minute gradient to 5:95 A:B, followed by 2 min hold to 5:95 A:B and then immediate change to 95:5 A:B. Compound elution was monitored at 220 nm.
Method 3: Chiral normal phase HPLC analytical method to determine enantiomeric excess of 4
Enantiomeric excess of the amine product 4 was determined using an Agilent 1290 UPLC equipped with a ChiralPak AD-H (250 x 4.6 mm, 5.0 µm) using a gradient of 0.1% DEA in hexanes (mobile phase A) and 0.1% DEA in EtOH and MeOH (1:1, v/v) (mobile phase B) at a flow rate of 1.0 mL/min at a column temperature of 40 °C. Beginning from a 90:10 ratio of A:B, the method followed a 15 minute hold. Compound elution was monitored at 200 nm.
Method 4: Chiral reverse phase UPLC analytical method to determine enantiomeric excess of 4
S42
Enantiomeric excess of the amine product 4 was determined using an Agilent 1290 UPLC equipped with a ChiralPak AD-RH (150 x 2.1 mm, 5 µm) and guard column using isocratic mixture of 20 mM sodium tetraborate decahydrate pH 9.0 (mobile phase A) and methanol (mobile phase B) (10:90 v/v), online mixing, at a flor rate of 0.45 mL/min at a column temperature of 45 °C. Compound elution was monitored at 230 nm.
Selected UPLC Chromatograms Example of overlaid chromatograms run using Method 1.
Example of chromatogram runs using Method 2.
(1R,2S)-4
(1R,2S)-4
S43
Example of a chromatogram run of (1R,2S)-5 using Method 3.
Example of a chromatogram run using Method 4.
(1R,2S)-4
(1S,2R)-4
(1R,2S)-4(1S,2R)-4
S44
SUL chromatogram
(1R,2S)-4(1S,2R)-4
S45
(1R,2S)-4
(1S,2R)-4
S46
NMR of aldehyde (3) and rac-trans-1 mixture in the absence of enzyme In order to show that the reductive amination is catalysed by the IRED following experiment has been conducted. 1 M solutions of d4-AcOH and d3-NaOAc were prepared in D2O. These 1 M solutions were then combined (254 µ 1 M d3-NaOAc and 255 µL 1 M d4-AcOH) with further D2O (4500 µL) to prepare deuterated 100 mM sodium acetate buffer pH 4.6. Rac-trans-1 (11.9 mg, 32.6 µmol) was then weighed into a vial before addition of deuterated 100 mM sodium acetate buffer pH 4.6 (1.63 mL). Aldehyde 3 (14.0 mg, 46.14 µmol) was dissolved in DMSO-d6 (600 µL) and a 200 µL aliquot of this solution added to an NMR tube. An 800 µL aliquot of the solution containing rac-trans-1 in deuterated 100 mM sodium acetate buffer pH 4.6 was then added to the NMR tube to create a solution containing 3 (4.6 mg, 15.16 µmol, 1.0 equiv) and rac-trans-1 (5.8 mg, 15.91 µmol, 1.1 equiv). The NMR tube was then vortexed, and the sample analysed by 1H NMR. After incubation at room temperature for 5 h, the sample was analysed by 1H NMR again. The spectra of this mixed sample are shown in Fig. S13 and Fig. S14 and show no presence of the imine as judged by NMR.
Supplementary Figure 13. 1H NMR (400 MHz) of Aldehyde (3), rac-trans-1, and a mixture of aldehyde (3) and rac-trans-1 before and after incubation at room temperature for 5 h. All spectra were recorded in deuterated 100 mM sodium acetate buffer (pH 4.6).
rac-trans-1
Aldehyde (3) + rac-trans-1 [5 h]
Aldehyde (3)
Aldehyde (3) + rac-trans-1 [0 h]
S47
Supplementary Figure 14. 1H NMR (400 MHz) of aldehyde (3), rac-trans-1, and a mixture of aldehyde (3) and rac-trans-1 before and after incubation at room temperature for 5 h – expansion between 10.5 and 5 ppm. All spectra were recorded in deuterated 100 mM sodium acetate buffer (pH 4.6) containing 20% DMSO-d6.
rac-trans-1
Aldehyde (3) + rac-trans-1 [5 h]
Aldehyde (3)
Aldehyde (3) + rac-trans-1 [0 h]
S48
NMR spectra
1H NMR (400 MHz, D2O) and 13C NMR (101 MHz, D2O) spectra of rac-trans-1
NH2
NH2
H2SO4
1H NMR (D2O, 400 MHz, 300K): δ 7.36 (t, J = 7.7 Hz, 2H), 7.28 (t, J = 7.7 Hz, 1H), 7.19 (d, J = 7.7 Hz, 2H), 2.86 (dt, J = 7.9, 4.0 Hz, 1H), 2.43 (ddd, J=10.2, 6.6, 3.6 Hz, 1H), 1.44 (ddd, J=10.5, 6.9, 4.5 Hz, 1H), 1.32 (q, J=7.5 Hz, 1H). 13C NMR (DMSO-d6, 101MHz): δ 138.7 (1C), 128.8 (2C), 126.9 (1C), 126.4 (2C), 30.6 (1C), 20.8 (1C), 12.5 (1C) ppm
MG103201 SKF-T-385-B.010.001.1r.esp
10 9 8 7 6 5 4 3 2 1 0Chemical Shift (ppm)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Nor
mal
ized
Inte
nsity
2.131.001.005.16
7.37
7.36 7.
207.
18
4.75
2.88
2.87
2.86
2.85
2.44
2.43
2.43
2.42 1.45 1.
45 1.44
1.33
1.31
1.29
MG103201 SKF-T-385-B.011.001.1r.esp
150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Nor
mal
ized
Inte
nsity
138.
73
128.
8112
6.38
30.6
3 20.8
2
12.5
1
S49
1H NMR (400 MHz, DMSO-d6) and 13C NMR (101 MHz, DMSO-d6) spectra of (1R,2S)-1
NH3
(R)
(S)
(R) COO
OH
MG103199 GSK234753D.010.001.1r.esp
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Nor
mal
ized
Inte
nsity
1.001.000.980.970.9914.04
7.40
7.39
7.26
7.25
7.24 7.
077.
06
4.67
2.65
2.64 2.64
2.63
2.62
2.61
2.18
2.17
2.17 1.28 1.
27 1.27
1.26
1.25
1.07
1.05
1.03
MG103199 GSK234753D.011.001.1r.esp
180 160 140 120 100 80 60 40 20 0Chemical Shift (ppm)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Nor
mal
ized
Inte
nsity
175.
14
142.
9614
0.23
128.
2712
7.51
126.
3512
5.97
73.3
3
39.9
339
.72
39.5
139
.30
39.0
9
31.6
4
21.8
0
14.3
9
S50
1H NMR (400 MHz, DMSO-d6) and 13C NMR (101 MHz, DMSO-d6) spectra of 3
O
O
N
O
MG103200 N44464-94-C1.010.001.1r.esp
12 11 10 9 8 7 6 5 4 3 2 1 0Chemical Shift (ppm)
0
0.05
0.10
0.15
Nor
mal
ized
Inte
nsity
11.992.142.141.092.112.162.162.131.00
9.58 7.
867.
83
7.41
7.39
3.51
3.31 2.
692.
68 2.65
2.50
2.50
2.50
2.08
2.06 2.06
1.82
1.81
1.53
1.52
1.51
1.48
1.48
MG103200 N44464-94-C1.011.001.1r.esp
220 200 180 160 140 120 100 80 60 40 20 0 -20Chemical Shift (ppm)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Nor
mal
ized
Inte
nsity
204.
66
164.
84
143.
78
129.
9612
8.91
128.
67
80.4
5
61.7
3
51.9
746
.82
39.9
339
.72
39.5
139
.30
39.0
938
.88
27.7
625
.00
S51
1H NMR (400 MHz, CD3-OD) and 13C NMR (101 MHz, CD3-OD) spectra of (1R,2S)-4
N
NH
O
O
2HCl.
MG103765.010.001.1r_1H.esp
9 8 7 6 5 4 3 2 1 0Chemical Shift (ppm)
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
Nor
mal
ized
Inte
nsity
1.3812.443.391.035.190.381.882.285.052.012.00
8.07 8.
05
7.69
7.67
7.33
7.31
7.29 7.
207.
18
4.82
4.41
3.55 3.52
3.32
3.31
3.31
3.31
3.19
3.18
3.14 3.
113.
01 2.12 2.09
1.71
1.61
1.41
1.39
1.38
1.36 1.
19
MG103765.010.001.1r_13C.esp
180 160 140 120 100 80 60 40 20 0Chemical Shift (ppm)
-0.005
0
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
Nor
mal
ized
Inte
nsity
166.
55
139.
4513
5.00
132.
7913
1.20
129.
8912
7.63
83.0
4
61.2
7 53.0
7 49.8
2
49.7
949
.58
49.3
649
.15
49.1
248
.94
48.7
248
.51
48.4
848
.34
48.2
839
.91
32.4
628
.50
28.2
722
.63
13.6
1
S52
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