Sequence‐Based In‐silico Discovery, Characterisation, and Biocatalytic Application of a Set of Imine Reductases

Abstract Imine reductases (IREDs) have recently become a primary focus of research in biocatalysis, complementing other classes of amine‐forming enzymes such as transaminases and amine dehydrogenases. Following in the footsteps of other research groups, we have established a set of IRED biocatalysts by sequence‐based in silico enzyme discovery. In this study, we present basic characterisation data for these novel IREDs and explore their activity and stereoselectivity using a panel of structurally diverse cyclic imines as substrates. Specific activities of >1 U/mg and excellent stereoselectivities (ee>99 %) were observed in many cases, and the enzymes proved surprisingly tolerant towards elevated substrate loadings. Co‐expression of the IREDs with an alcohol dehydrogenase for cofactor regeneration led to whole‐cell biocatalysts capable of efficiently reducing imines at 100 mM initial concentration with no need for the addition of extracellular nicotinamide cofactor. Preparative biotransformations on gram scale using these ‘designer cells’ afforded chiral amines in good yield and excellent optical purity.


Cofactor Preference
The cofactor preference of IREDs A, B, D, F, H, I, and J was assessed by spectrophotometric determination of their activity in the reduction of substrates 1b, 1f, or 1g in the presence of NADPH and NADH (2 mM of either cofactor). Figure S5. Specific activities of various IREDs with NADPH (light blue bars) and NADH (dark blue bars) as cofactor. Error bars represent standard deviations of triplicate experiments. The percentage values shown above the bars are the relative activities of the enzymes with NADH compared to NADPH.

Substrate Scope and Stereoselectivity
Complete substrate scope and selectivity data (conversions after 2 h and after 24 h, and enantiomeric excess after 24 h) are shown in the following tables.

Synthesis of Substrates and Reference Compounds
Imines 1a, 1e, 1g, 1h, 1i, and 1k as well as amines 2a, 2b, and 2e were obtained from commercial suppliers and used as received. The other imines and amines were synthesised following procedures reported in the literature (in some cases with slight modifications). -2,3,4,5-tetrahydropyridine (1b) [CAS 1462-92-6]. [1] To an ice-cooled, stirred solution of N-chlorosuccinimide (NCS; 6.40 g, 47.9 mmol, 1.14 eq.) in anhydrous diethyl ether (90 mL) was added a solution of 2-methylpiperidine (2a; 4.18 g, 42.1 mmol, 1 eq.) in anhydrous diethyl ether (10 mL 7-Methyl-3,4,5,6-tetrahydro-2H-azepine (1c) [CAS 3338-03-2]. [2,3] To a stirred solution of ϵ-caprolactam (4.53 g, 40 mmol, 1 eq.) and triethylamine (5.85 mL, 4.25 g, 42 mmol, 1.05 eq.) in anhydrous diethyl ether (100 mL) under argon atmosphere was added trimethylsilyl chloride (TMSCl; 5.33 mL, 4.56 g, 42 mmol, 1.05 eq.) over 10 min via a syringe. The resulting mixture was heated to gentle reflux on a water bath for 2 h; afterwards, the water bath was removed and the reaction mixture was allowed to cool to room temperature. The white precipitate of triethylammonium chloride that had formed was removed by filtration through a glass frit. The filtrate was transferred to a three-necked round-bottom flask, put under an argon atmosphere and cooled to -50 °C on an EtOH/N2(l) bath. A solution of methyl lithium in anhydrous diethyl ether (25 mL of a 1.6 M solution; 40 mmol, 1 eq.) was added over 15 min via a syringe, the cooling bath was removed, and the reaction mixture was stirred at room temperature overnight, resulting in the formation of a clear, yellow solution. TLC analysis of a 100 µL sample (silica gel 60 F254, EtOAc, KMnO4 staining) indicated complete consumption of ϵ-caprolactam. The reaction was quenched by addition of half-saturated aqueous NH4Cl solution (50 mL), the phases were separated, and the aqueous phase was extracted with dichloromethane (3 × 50 mL). The combined organic phases were dried over MgSO4, filtered, and the solvent was evaporated under reduced pressure to give 5.21 g of a yellowish liquid. Short-path distillation (Kugelrohr) afforded 1c ( [2] 5-Phenyl-3,4-dihydro-2H-pyrrole (1d) . [3] To an ice-cooled, stirred solution of 2-pyrrolidone (3.40 g, 40 mmol, 1 eq.) and triethylamine (5.85 mL, 4.25 g, 42 mmol, 1.05 eq.) in anhydrous diethyl ether (100 mL) under argon atmosphere was added trimethylsilyl chloride (TMSCl; 5.33 mL, 4.56 g, 42 mmol, 1.05 eq.) over 20 min via a syringe. The resulting mixture was heated to gentle reflux on a water bath for 1 h; afterwards, the water bath was removed and the reaction mixture was allowed to cool to room temperature. The white precipitate of triethylammonium chloride that had formed was removed by filtration through a glass frit. The filtrate was transferred to a three-necked round-bottom flask and put under an argon atmosphere. A solution of phenylmagnesium bromide in anhydrous tetrahydrofuran (40 mL of a 1.0 M solution; 40 mmol, 1 eq.) was added over 15 min via a syringe. After the addition was complete, the reaction mixture was heated to reflux for 2 h. The heating source was then removed and the reaction mixture was stirred at room temperature overnight, resulting in the formation of a clear, orange solution. TLC analysis of a 100 µL sample (silica gel 60 F254, EtOAc, UV visualisation, KMnO4 staining) indicated complete consumption of 2-pyrrolidone and formation of a UV-active product. The reaction was quenched by addition of 1 M hydrochloric acid (10 mL) and the resulting mixture was basified (pH 10-11) by addition of 10% (w/v) aqueous NaOH solution (20 mL). The phases were separated and the aqueous phase was diluted with water (30 mL) and extracted with diethyl ether (2 × 50 mL). The combined organic phases were dried over MgSO4, filtered, and the solvent was evaporated under reduced pressure to give 7.63 g of a yellowish liquid. Column chromatography (silica gel 60, hexanes:EtOAc = 3:1) afforded 1d ( (12), 77 (14). The characterisation data are in agreement with literature values. [3] 2-Methyl-3,4-dihydroisoquinolin-2-ium trifluoromethanesulfonate (1f). [4] To a stirred solution of 3,4-dihydroisoquinoline (328 mg, 2.5 mmol, 1 eq.) in dichloromethane (2 mL) was added methyl trifluoromethanesulfonate (285 µL, 427 mg, 1.25 mmol, 1.04 eq.) via a pipette. The reaction mixture was stirred for 1 h, at which time TLC analysis (silica gel 60 F254, EtOAc, UV visualisation) indicated complete consumption of 3,4-dihydroisoquinoline. The solvent was evaporated under reduced pressure, and the oily residue was stirred with 5 mL of pentane for 10 min and afterwards stored in the fridge overnight. The next day, the product had deposited as a pale-yellowish solid, which was collected by filtration through a small glass frit and dried in the desiccator overnight. 1f (724 mg, 98%  5, 49.8, 118.3, 122.5, 124.6, 128.1, 133.3, 136.2, 137.7, 167.1. The characterisation data are in agreement with literature values. [4] 1-Methyl-4,9-dihydro-3H-β-carboline (1j) . [5] To an ice-cooled, stirred solution of tryptamine (2.00 g, 12.5 mmol, 1 eq.) and triethylamine (4.0 mL, 2.90 g, 28.7 mmol, 2.3 eq.) in dichloromethane (40 mL) was added acetyl chloride (976 µL, 1.08 g, 13.8 mmol, 1.1 eq.) dropwise over 2 min via a pipette. The ice bath was then removed and the reaction mixture was stirred for 20 h at room temperature, at which time TLC analysis (silica gel 60 F254, MTBE:MeOH:NH4OH = 90:9:1, UV visualisation) indicated completion of the reaction. The reaction was quenched by addition of water (30 mL), the phases were separated, and the aqueous phase was extracted with dichloromethane (20 mL). The combined organic phases were washed with 1 M hydrochloric acid (20 mL) and water (10 mL), dried over MgSO4, and the solvent was evaporated under reduced pressure to give N- (2-(1H-indol-3-yl)ethyl)acetamide (2.57 g, quant.) as an orange, highly viscous oil.

6-Methyl
A solution of the crude intermediate (368 mg, 182 mmol, 1 eq.) and phosphorous oxychloride (POCl3; 1 mL 1.65 g, 10.7 mmol, 5.9 eq.) in anhydrous acetonitrile (9 mL) under argon atmosphere was heated to 120 °C for 5 min in a glass tube sealed with a Teflon-fitted crimp cap using a Biotage Initiator laboratory microwave instrument. The resulting brown solution was allowed to cool to room temperature and water (10 mL) was carefully added. The resulting solution was concentrated under reduced pressure to remove the acetonitrile before being basified (pH 11-12) by addition of 10 M aqueous NaOH solution (approx. 5 mL). The product was extracted into dichloromethane (3 × 30 mL), the combined extracts were dried over MgSO4, filtered, and the solvent was evaporated under reduced pressure to give 307 mg of an orange liquid. The microwave reaction was repeated with two larger batches of the intermediate (1.11 g, 5.47 mmol each; using 4.94 g, 32.2 mmol each of POCl3 in 12 mL each of acetonitrile) and the crude products thus obtained were combined with the crude product of the first batch to give a combined 1.  (12). The characterisation data are in agreement with literature values. [7] Synthesis of amines 2c, 2d, and 2g-k by NaBH4-reduction of the corresponding imines (General procedure). To a solution of imine 1 (0.2-0.5 mmol, 1 eq.) in methanol (500 µL) in a microcentrifuge tube (2 mL) was added sodium borohydride (38 mg, 1.0 mmol, 2-5 eq.). The vial was closed, shaken briefly to ensure proper mixing of the reagents, opened again (pressure build-up!), and covered with a laboratory tissue. The reaction mixture was allowed to stand at room temperature overnight. The reaction was then quenched by addition of half-saturated aqueous Na2CO3 solution (500 µL) and the product was extracted into ethyl acetate (3 × 500 µL). The combined organic phases were dried over MgSO4, centrifuged (13,000 rpm, 1 min), and the supernatant was evaporated under reduced pressure to give the crude amine 2, which was characterised by GC-MS analysis and used as reference compound without further purification.  (14), 130 (14), 115 (8), 85 (10).
The product was extracted into ethyl acetate (3 × 500 µL), the combined organic phases were dried over MgSO4, centrifuged (13,000 rpm, 1 min), and the supernatant was evaporated under reduced pressure to give 30 mg (41%) of the crude amine 2f as a yellowish liquid, which was characterised by GC-MS analysis and used as reference compound without further purification.

Gene Synthesis and Subcloning
Gene sequences encoding the investigated IREDs were obtained from the Genbank database entries associated with the corresponding protein sequence entries in the UniProt database. Synthetic genes were obtained from Invitrogen (GeneArt ® ; now part of Thermo Fisher Scientific) in the form of linear double-stranded DNA (GeneArt ® Strings™), in which the coding sequence was codon-optimised for expression in E. coli, supplemented with NdeI and XhoI restriction sites at the 5' and 3' ends, respectively, and flanked by a 15-bp "stuffer DNA" at both ends to enable subcloning into a pET28a(+) vector (Novagen). The integrity of the obtained constructs was verified by DNA sequencing (LGC Genomics, Berlin, Germany).
The gene sequence encoding Lb-ADH was obtained from the GenBank database (accession code: AJ544275.1). A synthetic gene was obtained from Invitrogen (GeneArt ® ; now part of Thermo Fisher Scientific) in the form of a 'working plasmid', in which the coding sequence was codonoptimised for expression in E. coli and flanked by NdeI and XhoI restriction sites at the 5' and 3' ends, respectively. The gene was excised and subcloned into a pET21a(+) vector (Novagen) and the integrity of the obtained construct was verified by DNA sequencing (LGC Genomics, Berlin, Germany). For re-cloning into pASK-IBA5plus, the gene was amplified by overhang PCR using the primers Lb-ADH_fw (TATTAAGGTCTCGGCGCCATGAGCAATCGTCTGGAT) and Lb-ADH_rv (GCTACTGGTCTCATATCAATTCTGTGCGGTATAACCACCATCCAC), introducing Eco31I restriction sites at both ends of the gene. The PCR product was restricted using FastDigest Eco31I (Thermo Fisher Scientific) and subcloned into a pASK-IBA5plus vector (IBA Life Sciences).
A non-native C-terminal Asn residue was deleted by mutagenesis PCR using the primers Lb-ADH_N252*_fw (GGTGGTTATACCGCACAGTAATGATATCTAAC) and Lb-ADH_N252*_rv (GTTAGATATCATTACTGTGCGGTATAACCACC) and the integrity of the obtained construct was verified by DNA sequencing (LGC Genomics, Berlin, Germany).
DNA and amino acid sequences of all investigated proteins can be found in a later section of this Electronic Supplementary Information. The plasmids used in the present study are listed in Supplementary Table S5: 50 µg/mL kanamycin) and the resulting culture was incubated at 30 °C and 120 rpm overnight in a closed plastic tube (50 mL). Glycerol stocks were prepared by diluting the cell suspension of the overnight culture (700 µL) with sterile 60% (v/v) aqueous glycerol (300 µL) and were stored at -20 °C (working stock) and -80 °C (backup stock). For expression, LB medium (15 mL, cont. 50 µg/mL kanamycin) was inoculated with 50 µL of cell suspension from a glycerol stock and the resulting culture was incubated at 30 °C and 120 rpm overnight in a closed plastic tube (50 mL). 1 mL of this overnight culture was used to inoculate TB medium (100 mL, cont. 50 µg/mL kanamycin) and the resulting culture was incubated at 30 °C and 120 rpm in a baffled Erlenmeyer flask (300 mL). Samples (1 mL) of the culture were taken at regular intervals and analysed for their optical density at 600 nm (OD600). When the OD600 reached a value of 0.8-1.0 (approx. after 3.5 h), protein expression was induced by the addition of IPTG (1 mM) and incubation was continued at 20 °C and 120 rpm overnight. The culture was transferred to a centrifuge beaker (500 mL) and centrifuged (8,000 rpm, 4 °C, 20 min) to pellet the cells. The supernatant was discarded and the cell pellet was resuspended in potassium phosphate buffer (20 mM, pH 7.0). The cell suspension was transferred to a plastic tube (50 mL), centrifuged again (4,000 rpm, 4 °C, 20 min), and the supernatant was again discarded. The resulting cell pellet (typically 0.8-1.3 g) was stored at -20 °C until use.

Cell disruption:
The cell pellet was thawed and resuspended in Tris-HCl buffer (10 mL/g cell weight; 100 mM, pH 7.5; for preparation of crude cell-free extracts) or HisTrap buffer A (10 mL/g cell weight; potassium phosphate buffer, 100 mM, pH 7.0, 300 mM NaCl; for protein purification). The resulting cell suspension was supplemented with lysozyme (1 mg/mL), incubated at 30 °C for 30 min and cooled to 0 °C on ice. The cells were disrupted by ultrasonication using a Branson Digital Sonifier 250 at 20% amplitude (50 W), 2 s pulse, 4 s pause, for a total pulse time of 2:30 min (75 cycles). The resulting sample was transferred to a centrifuge tube (50 mL) and cell debris was pelleted by centrifugation (16,000 rpm, 4 °C, 20 min). The supernatant was either lyophilised overnight to give a lyophilised, crude cell-free extract or subjected to protein purification by immobilised-metal affinity chromatography (IMAC) as described below.
Purification of IREDs by immobilised-metal affinity chromatography (IMAC): A 5 mL HisTrap FF column (GE Healthcare; Ni 2+ on NTA-modified cross-linked agarose) was equilibrated with HisTrap buffer A (100 mL; potassium phosphate buffer, 100 mM, pH 7.0, 300 mM NaCl). The supernatant obtained by cell disruption of IRED-expressing E. coli BL21(DE3) cells was loaded onto the column using a syringe and a 0.45 µm syringe filter for removal of insoluble matter. The column was then connected to a Biorad BioLogic DuoFlow FPLC system equipped with a BioFrac fraction collector and eluted using the following gradient elution programme while collecting fractions of 10 mL: 50 mL of 95% HisTrap buffer A / 5% HisTrap buffer B (potassium phosphate buffer, 100 mM, pH 7.0, 300 mM NaCl, 500 mM imidazole); 100 mL linear gradient of 95% A / 5% B to 100% B; 50 mL of 100% HisTrap buffer B. Alternatively, a step-elution protocol could be used: 75 mL of 95% HisTrap buffer A / 5% HisTrap buffer B, 75 mL of 85% A / 15% B, 50 mL of 40% A / 60% B (IRED elutes in this step), 50 mL of 100% HisTrap buffer B. The protein-containing fractions were assayed for IRED activity using the photometric method described in the main paper. The active fractions were pooled, concentrated using Sartorius VivaSpin 20 centrifugal filters with a 10 kDa molecular weight cut-off, and desalted using a GE Healthcare PD-10 column and Tris-HCl buffer (100 mM, pH 7.5) as eluent. In the case of IREDs B, E, and N, a buffer with a higher ionic strength (Tris-HCl, 100 mM, pH 7.5, 300 mM NaCl) was used in the desalting step to avoid precipitation of the protein. The desalted protein solution was analysed for protein concentration using the Biorad Bradford protein assay, lyophilised overnight, and stored at -20 °C until use.
A representative FPLC chromatogram is shown in Supplementary Figure   µg/mL ampicillin) and the resulting culture was incubated at 30 °C and 120 rpm overnight in a closed plastic tube (50 mL). Glycerol stocks were prepared by diluting the cell suspension of the overnight culture (700 µL) with sterile 60% (v/v) aqueous glycerol (300 µL) and were stored at -20 °C (working stock) and -80 °C (backup stock). For expression, LB medium (15 mL, cont. 100 µg/mL ampicillin) was inoculated with 50 µL of cell suspension from a glycerol stock and the resulting culture was incubated at 30 °C and 120 rpm overnight in a closed plastic tube (50 mL). 3 mL of this overnight culture were used to inoculate TB medium (300 mL, cont. 100 µg/mL kanamycin and 1 mM MgCl2) and the resulting culture was incubated at 30 °C and 120 rpm in a baffled Erlenmeyer flask (300 mL). Samples (1 mL) of the culture were taken at regular intervals and analysed for their optical density at 600 nm (OD600). When the OD600 reached a value of 0.8-1.0 (approx. after 3.5 h), protein expression was induced by the addition of anhydrotetracyclin (AHT, 0.2 mg/L) and incubation was continued at 20 °C and 120 rpm overnight. The culture was transferred to a centrifuge beaker (500 mL) and centrifuged (8,000 rpm, 4 °C, 20 min) to pellet the cells. The supernatant was discarded and the cell pellet was resuspended in Tris-HCl buffer (100 mM, pH 7.5). The cell suspension was transferred to a plastic tube (50 mL), centrifuged again (4,000 rpm, 4 °C, 20 min), and the supernatant was again discarded. The resulting cell pellet (typically 4.5-5.0 g) was stored at -20 °C until use.
A lyophilised crude cell-free extract was prepared as described above ('Cell disruption').
Co-expression of Lb-ADH and IREDs in E. coli BL21(DE3): LB medium (200 mL, cont. 100 µg/mL ampicillin) was inoculated with 50 µL of cell suspension from a glycerol stock of E. coli BL21(DE3) [pASK-IBA5plus/Lb-ADH] and the resulting culture was used for the preparation of competent cells by the calcium chloride method. 8 The resulting competent cells were transformed with the plasmids encoding the investigated IREDs, using both ampicillin (100 µg/mL) and kanamycin (50 µg/mL) for selection. A single colony from a transformant agar plate was used to inoculate LB medium (15 mL, cont. 50 µg/mL kanamycin) and the resulting culture was incubated at 30 °C and 120 rpm overnight in a closed plastic tube (50 mL). Glycerol stocks were prepared by diluting the cell suspension of the overnight culture (700 µL) with sterile 60% (v/v) aqueous glycerol (300 µL) and were stored at -20 °C (working stock) and -80 °C (backup stock). For expression, LB medium (15 mL, cont. 100 µg/mL ampicillin and 50 µg/mL kanamycin) was inoculated with 50 µL of cell suspension from a glycerol stock and the resulting culture was incubated at 30 °C and 120 rpm overnight in a closed plastic tube (50 mL). 3 mL of this overnight culture were used to inoculate TB medium (300 mL, cont. 100 µg/mL ampicillin, 50 µg/mL kanamycin, and 1 mM MgCl2) and the resulting culture was incubated at 30 °C and 120 rpm in a baffled Erlenmeyer flask (300 mL). Samples (1 mL) of the culture were taken at regular intervals and analysed for their optical density at 600 nm (OD600). When the OD600 reached a value of 0.8-1.0 (approx. after 5 h), protein expression was induced by the addition of IPTG (1 mM) and AHT (0.2 mg/L) and incubation was continued at 20 °C and 120 rpm overnight. The culture was transferred to a centrifuge beaker (500 mL) and centrifuged (8,000 rpm, 4 °C, 20 min) to pellet the cells. The supernatant was discarded and the cell pellet was resuspended in Tris-HCl buffer (20 mL; 100 mM, pH 7.5, 1 mM MgCl2). The cell suspension was transferred to a round-bottom flask, flash-frozen in liquid nitrogen, and lyophilised overnight to afford a dry cell powder (typically 1.0-1.5 g), which was stored at 4 °C until use.

NOTE:
In the DNA sequences, start codons are underlined, stop codons are typeset in lowercase letters, restrictions sites are highlighted by a light blue box, and the 'stuffer DNA' appended at the 5' and 3' ends of the gene (required for the restriction digest) is typeset in grey. In the protein sequences, the N-terminal methionine residue of the native sequence is underlined and the N-terminal elongation containing the His-tag is typeset in grey.

IRED-F (V6KA13)
Native Gene Sequence (Genbank ID AWQW01000146.   Table S7. Pairwise protein sequence identities (above diagonal) and Jukes-Cantorcorrected phylogenetic distances (below diagonal) of IREDs A-N. The comparison is based on a Clustal Omega alignment of the native protein sequences.