REGIOSELECTIVE HYDROXYLATION OF ISOPHORONE

Abstract

The present invention relates to novel process for the production of ketoisophorone via biocatalytic conversion of isophorone, in particular a one-pot biocatalytic system for conversion of α-isophorone in a two-step oxidation process, with a first oxidation being catalyzed by a heme containing oxidoreductase such as a cytochrome P450 monooxygenase followed by another oxidation which can be either a chemical reaction or a biocatalytic reaction, in particular wherein the oxidation is catalyzed by an NAD(P) or NADP(H)-dependent oxidoreductase. The invention further provides polypeptides and nucleic acid sequences coding for cytochrome P450 monooxygenase with modified (higher) substrate selectivity, total turnover numbers and/or (re)activity compared to the wild-type enzyme. Ketoisophorone is useful as building block in the synthesis of vitamins and carotenoids.

Claims

1. A one-pot biocatalytic process for the conversion of α-isophorone into ketoisophorone (KIP) comprising the enzymatic steps of: (a) conversion of α-isophorone into 4-hydroxy-α-isophorone (HIP) with a conversion rate of at least 80% via catalytic action of a P450 monooxygenase selected from a polypeptide having at least 35% identity to SEQ ID NO:1 or a polypeptide having at least 62% identity to SEQ ID NO:3, said P450 monooxygenase comprising one or more mutation(s) on a position corresponding to position(s) 96, 87, 244, 247, and/or combinations thereof in P450cam-RhFRed P450 monooxygenase according to SEQ ID NO:3, and wherein the total turnover number is increased by at least 2-fold compared to the respective non-modified P450 monooxygenase under incubation conditions of pH of from 4.0 to 10.0 for 1 to 48 h with optionally isolation of HIP from the reaction mixture; and (b) conversion of HIP into KIP via catalytic action of an enzyme having alcohol dehydrogenase or carbonyl reductase activity, preferably an alcohol dehydrogenase from Candida magnoliae or a carbonyl reductase from Sporobolomyces salmonicolor, said suitable conditions include incubation at pH of from 4.0 to 10.0 for 1 to 48 h with optionally isolation of KIP from the reaction mixture.

2. A process according to claim 1, wherein the biocatalytic process for the conversion of α-isophorone to 4-hydroxy-α-isophorone (HIP) and/or the conversion of HIP to ketoisophorone (KIP) is carried out in the presence of (a) a co-substrate selected from the group consisting of glucose, isopropanol and phosphite with regards to conversion of α-isophorone to HIP; or (b) a co-substrate selected from the group consisting of acetone, chloroacetone, ethyl acetoacetate, ethyl levulinate, chloroacetone and ethyl acetoacetate with regards to conversion of HIP to KIP.

3. A process according to claim 1, wherein the enantiomeric excess towards the (R)-configuration of HIP and/or KIP generated via the biocatalytic process is at least 50% based on the total amount of HIP and/or KIP.

4. A process according to claim 1 which is performed at a temperature about 30° C. to 40° C.

5. A modified P450 monooxygenase for use in a process according to claim 1 comprising one or more mutation(s) on a position corresponding to position(s) 96, 87, 244, 247, and/or combinations thereof in P450cam-RhFRed P450 monooxygenase according to SEQ ID NO:3, wherein: (a) the introduced amino acid on a position corresponding to position 244 are selected from the group consisting of alanine, asparagine, serine, glycine, isoleucine, cysteine, tyrosine and histidine; and/or (b) the introduced amino acid on a position corresponding to position 247 are selected from the group consisting of lysine, phenylalanine, isoleucine and serine; and/or (c) the introduced amino acid on a position corresponding to position 87 is tryptophan; and/or (d) the introduced amino acid on a position corresponding to position 97 is phenylalanine.

6. A modified P450 monooxygenase according to claim 5, wherein: (a) the introduced amino acid on a position corresponding to position 244 is selected from alanine and/or (b) the introduced amino acid on a position corresponding to position 247 is selected from lysine; and/or (c) the introduced amino acid on a position corresponding to position 87 is tryptophan; and/or (d) the introduced amino acid on a position corresponding to position 97 is phenylalanine.

7. A modified P450 monooxygenase according to claim 5, comprising combinations of amino acid substitutions on a position corresponding to position(s) 244 and 247 in P450cam-RhFRed P450 monooxygenase according to SEQ ID NO:3 which are selected from L244A-V247 and L244A-V247L.

8. A polynucleotide sequence comprising a DNA sequence coding for a P450 monooxygenase according to claim 5.

9. A host cell wherein a P450 monooxygenase according to claim 5 is expressed, said host being selected from the group consisting of bacteria, fungi, yeasts or plant or animal cells, more preferably selected from Escherichia, Streptomyces, Bacillus, Rhodococcus, Pseudomonas, Saccharomyces, Aspergillus, Pichia, Hansenula or Yarrowia, even more preferably selected from Escherichia coli, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus ruber, Rhodococcus equi, Pseudomonas putida, Saccharomyces cerevisiae, Aspergillus niger, Pichia pastoris, Hansenula polymorpha or Yarrowia lipolytica, most preferably selected from Escherichia coli B, in particular E. coli BL21 (DE3) or other derivatives or E. coli K-12.

10. A process for the production of vitamin E, comprising the step of biocatalytic conversion of α-isophorone into KIP according to claim 1.

Description

[0078] The present invention is now described in greater detail with reference to FIGS. 1 to 9 and the following examples. The work leading to this invention has received funding from the European Union (EU) project ROBOX (grant agreement no 635734) under EU's Horizon 2020 Programme Research and Innovation actions H2020-LEIT BIO-2014-1.

[0079] FIG. 1: FIG. 1A shows the proposed one-pot double allylic oxidation, wherein α-isophorone (α-IP, 1) is in a first step oxidized via enzymatic action of a cytochrome P450 enzyme (P450) leading to 4-hydroxy-α-isophorone (HIP, 6), which might be further oxidized via enzymatic action of aldehyde dehydrogenase (ADH) to 2,6,6-trimethylcyclohex-2-ene-1,4-dione (KIP, 3). For further explanation see in the text. Possible products of the P450 oxidation of α-IP (1) are HIP (6), HIMP (7) and IPO (8) shown in FIG. 1B. The regeneration of CM-ADH10 catalyzing the oxidation from (R)-HIP to KIP using different co-substrates such as acetone (9a), chloroacetone (9b), ethyl acetoacetate (9c) and ethyl levulinate (9d) leading to the respective reduction products 10a-d, wherein “R” defines the respective substituent to result in the different co-substrates 9a-d is shown in FIG. 1C. Whole-cell double oxidation cascade of α-IP to KIP using E. coli co-expressing P450-WAL and Cm-ADH10 is shown in FIG. 1D. For more explanation, see text.

[0080] FIG. 2: Comparison of TTN as shown of the x-axis for HIP (black bars) and HMIP (grey bars) obtained for P450cam-RhFRed pooled variants shown on the y-axis. For further explanation see in the text.

[0081] FIG. 3: Comparison of TTN as shown of the x-axis for HIP (black bars) and HMIP (grey bars) obtained for library D (mutated locations) shown on the y-axis. The data is based on the wild-type P450cam-RhFRed carrying the substitution Y96F. The introduction of F87W in said background together with L244A-V247L led to more than a 6-fold improvement of the TTN values with respect to L244A-V247L mutant. For further explanation see in the text.

[0082] FIG. 4: Bar chart showing conversion values obtained with different HIP concentration. Reaction set-up: 1 mg/mL purified Cm-ADH10, 0.25 mM NADP+ and 0.5% v/v ethyl acetoacetate as co-substrate for cofactor regeneration (30° C., 24 h). Product concentrations (mM) are given above each bar. Conversion of HIP in % is shown on the y-axis, HIP concentration in mM is shown on the x-axis. For further explanation see in the text.

[0083] FIG. 5A: Effect of buffer concentration and pH on Cm-ADH10 activity. Reaction set-up: 1 mg/mL purified Cm-ADH10, 0.25 mM NADP+ and 0.5% v/v ethyl acetoacetate as co-substrate for cofactor regeneration (30° C., 4.5 h). For further explanation see in the text.

[0084] FIG. 5B: Effect of the pH reached after the P450-catalyzed reaction on Cm-ADH10 performance. KPi buffer concentration used in the first step is given on the x-axis (“1”=50+100 mM KC; “2”=100 mM; “3”=200 mM; “4”=300 mM). Black bars: HIP conversion after direct addition of Cm-ADH10 (the pH measured after the first step is indicated above). Grey bars: HIP conversion when the supernatant of the first reaction was titrated to pH 8.0 before Cm-ADH10 addition. For more explanation, see text.

[0085] FIG. 6: Optimization of the allylic oxidation of α-IP catalyzed by P450cam-RhFRed-WAL. FIG. 6A shows the effect of buffer concentration. Reaction set-up: 200 mg mL.sup.−1 wet cells resuspended in KPi buffer pH 8.0 (concentration given on the x-axis), 10 mg mL.sup.−1 glucose, 20 mM α-IP, 2% DMSO, 20° C., 1 mL final volume in deep-well plate, 24 h reaction time. FIG. 6B shows effect of temperature. Reaction set-up: same as above, exception being the buffer adopted (200 mM KP, pH 8.0) and substrate concentration (15 mM) The x-axis shows the temperature (up to 40° C.), the y-axis shows α-IP conversion in %.

[0086] FIG. 7: Optimization of the oxidation of (R)-HIP catalyzed by Cm-ADH10. The y-axis shows HIP conversion in %. FIG. 7A shows the effect of different co-substrates. Reaction set up: 100 mM KPi buffer pH 7.5, 1 mg mL.sup.−1 Cm-ADH10, 0.25 mM NADP+, 0.5% v/v co-substrate, 40 mM (R)-HIP, 30° C. FIG. 7B shows the effect of pH as indicated on the x-axis. Reaction set up: 100 mM indicated buffer, 1 mg mL.sup.−1 Cm-ADH10, 0.25 mM NADP+, 0.5% v/v chloroacetone, 40 mM (R)-HIP, 30° C., 4 h. FIG. 7C shows the effect of temperature as indicated in ° C. on the x-axis. Reaction set up: 200 mM KPi buffer pH 8.0, 1 mg mL.sup.−1 Cm-ADH10, 0.25 mM NADP+, 0.5% v/v co-substrate, 40 mM (R)-HIP, 24 h. For further details see text or FIG. 1C.

[0087] FIG. 8: Effect of buffer concentration and pH on Cm-ADH10 activity. Reaction set-up: 1 mg/mL purified Cm-ADH10, 0.25 mM NADP+ and 0.5% v/v ethyl acetoacetate as co-substrate for cofactor regeneration (30° C., 4.5 h). HIP-conversion in % is shown on the y-axis, the concentration of KPi buffer in mM is shown on the x-axis.

[0088] FIG. 9: Time-course experiment for the one-pot two-step double allylic oxidation of α-IP. Reaction set up: 200 mg mL.sup.−1 wet cells resuspended in 200 mM KPi buffer pH 8.0, 10 mg mL.sup.−1 glucose, 10 mM α-IP, 2% DMSO (first step, 28° C.), then addition of 20% v/v Cm-ADH10 cell-free extract and 1.6 eq. of chloroacetone to the supernatant of the first reaction (second step, 40° C.). The arrow indicates the time of addition. Composition in % is shown on the y-axis, Times per h is shown on the x-axis.

EXAMPLE 1: GENERAL METHODOLOGY

[0089] All chemicals solvents, and carbon monoxide for CO difference spectroscopy used were of analytical grade and purchased from Sigma Aldrich (Poole, Dorset, UK) or BOC gases (Guildford, UK). Competent cells and enzymes were received from New England Biolabs (NEB). M9 minimal salts (5×) were purchased from Sigma-Aldrich, reconstituted by stirring the recommended amount of powder in water and sterilized by autoclaving. 40% glucose (w/v), antibiotics 1000×, 1 M MgSO.sub.4, 1 M CaCl.sub.2), and 25% (w/v) FeCL were prepared in dH.sub.2O and filter sterilized through a 0.2 μm syringe filter.

[0090] Inverse PCR reactions carried out using Eppendorf Mastercycler Gradient thermal cyclers according to NEB guidelines, followed by DpnI digestion before carrying out ligation reaction for 1 h at 25° C. with T4 DNA ligase and polynucleotide kinase, according to the manufacturers instruction. NEB 5-alpha competent E. coli (high efficiency) were then transformed according to the manufacturer instruction and sequence verified by plasmid sequencing. Expression plasmids were generated by standard restriction cloning. P450cam-RhFRed site-directed mutants were made starting from variants in the previously developed libraries (see Example 2) using the appropriate primers shown in Table 1. Primer synthesis and DNA sequencing were performed by Eurofins Genomics. An Avril restriction site was added to P450cam-RhFRed by PCR using the primers “17 for modified” and “AvrII rev” and the sequence cloned in pCDF-1b using NcoI and Avril restriction sites. For the two-vector strategy for coexpression (or ADHs expression trials), 55CR and Cm-ADH10 were cloned into pET28a vector, using NdeI and XhoI restriction sites that were introduced by PR using primers “NdeI Sporo for” and “XhoI Sporo rev” or “NdeI ADH10” for and “XhoI ADH10 rev”, respectively. Plasmid carrying genes encoding for the selected ADHs were kindly provided by c-LEcta GmbH, Leipzig, Germany.

TABLE-US-00001 TABLE 1. Oligonucleotides used for inverse PCR  reactions. Mismatching bases are underlined. Primer SEQ sequence ID Primer 5′-3′ NO: F87W for, GAGTGCCCGTGGA 9 Tm = 57° C. TCCCTCGTGaaGC F87W rev, GCTGGAAAAGT 10 Tm = 57° C. GGCGGTAATC L244A for, AGGATGTGTGGCGC 11 Tm = 59° C. GTTACTGGTCGGC L244A rev, CTTGGCTTCGTCA 12 Tm = 61° C. CTGGTGATCG L244A-V247L for, AGGATGTGTGGCGCG 13 Tm = 63° C. TTACTGCTCGGCGGC CTGGATAC AvrII rev, TAGTCTCCTAGGTCAG 14 Tm = 65° C. AGTCGCAGGGCCAGCc AflII rev, TCGTCTCTTAAGTCAG 15 Tm = 65° C. AGTCGCAGGGCCAGCC T7 for modified, TAATACGACTCACTAT 16 Tm = 62° C. AGGGAGACCACAACGG NdeI ADH10 for, ACGTAGCATATGACGA 17 Tm = 63° C. CTACTTCAAACGCGCT TGTC NcoI CmADH for, ATGCTACCATGGGGAT 18 Tm = 63° C. GACGACTACTTCAAAC GCGCTTGTC AflII CmADH rev, ATGTATCTTAAGCAAT 19 Tm = 60° C. CAAGCCATTGTCGACC AC XhoI ADH10 rev, ACGTCACTCGAGTTAA 20 Tm = 60° C. GCAATCAAGCCATTGT CGACCAC AvrII ADH10 rev, AGTCAGCCTAGGTTAA 21 Tm = 60° C. GCAATCAAGCCATTGT CGACCAC NdeI Sporo for, CTGGATCATATGGCCAA 22 Tm = 60° C. AATCGATAATGCCGTG XhoI Sporo rev, TGCATGCTCGAGTTAG 23 Tm = 61° C. GCTGTTTCGCTACCAA CCAGG

[0091] Chiral normal HPLC for measurement of enzymatic activity was carried out on an Agilent System (Santa Clara, Calif., USA) equipped with a G4225A degasser, G1311A quaternary pump, a G1329A well plate autosampler unit, a G1315B diode array detector and a G1316A thermostatted column compartment. Separation of (S)- and (R)-HIP was carried out using a CHIRALPAK® AS-H column (5 μm particle size, 4.6 mm diameter×250 mm; Daicel Chemical Industries Ltd) operating in isocratic mode with 80% hexane and 20% isopropanol for 18 min at 25C. Injection volume was 5NL and chromatograms were monitored at 254 nm. Retention times were as follow: KIP 9.5 min, (S)-HIP 10.9 min α-IP 12.4, (R)-HIP 13.3 min. Automated GC analysis was performed on an Agilent 6850 GC (Agilent, Santa Clara, Calif., USA) with aflame ionization detector (FID) equipped with an Agilent HP-1 column (30 m length, 0.32 mm inner diameter, 0.25 μm film thickness, Agilent, Santa Clara, Calif., USA). 2 μL of property diluted sample was injected at a split ratio 10:1. The inlet temperature was set at 200° C., detector temperature at 250° C. and pressure maintained at 6.8 psi. The following method was applied: initial temperature 50° C.; 10° C./min to 220, hold 2 min. The corresponding retention times were: α-IP 7.4 min, KIP 7.6, possible KIP-reduction by-product 7.8 min and HIP 9.7 min. Calibration curves of decane vs 6 or 3 were constructed in order to calculate TTNs or conversion values, respectively.

[0092] Protein activity measurement of the P450 BM3 mutant libraries is described in more details in Examples 1 of WO2013160365. The wild-type P450 BM3 is shown in SEQ ID NO:3, with the encoding nucleic acid sequence shown in SEQ ID NO:4.

[0093] Whole cell P450 concentration measurement was performed on a plate reader (Tecan Infinite 200 series, Männedorf, CH) according to Kelly et al. (Beilstein J. Org. Chem. 2015, 11, 1713-1720) and for cell lysates on a Cary 50 UV/Visible spectrophotometer (Agilent Technologies, Santa Clara, Calif., USA) according to the protocol by Omura and Sato (J. Biol. Chem. 1964, 239, 2370-2378). NMR spectra were recorded on a Bruker Avance 400 spectrometer (400.1 MHz for 1H and 100.6 MHz for 13C) in deuterated chloroform.

[0094] Preparation of (R)-HIP and (S)-HIP were prepared according to Hennig et. al. (Tetrahedron: Asymmetry 2000, 11, 1849-1858). Benzeneruthenium(II) chloride dimer (10.5 mg) and (1S,2R)-2-Amino-1,2-diphenylethanol (17.7 mg) were dissolved in 5 mL of isopropanol and the red solution stirred for 30 min at 80° C. Afterwards, 36.5 mL of isopropanol were added and the solution cooled to 28° C. Then, ketoisophorone (631.6 μL) and NaOH (2 mL, 0.1 M in isopropanol) were added to give a darker solution and the reaction followed until completion (ca. 3 hours). The reaction mixture was then filtered through Celite and the filtrate evaporated to afford a black oily residue. The product (S)-HIP was then purified by silica gel chromatography. The column was successively eluted with cyclohexane containing 20, 50 and 70% ethyl acetate and then solvents evaporated to afford a dark green oily residue (253.4 mg, 40% isolated yield). For the preparation of (R)-HIP, the same procedure was followed, but (1R,2)-2-Amino-1,2-diphenylethanol was used.

[0095] Expression and purification of proteins was as follows: plasmids (pET-14b, pCDF-1b or pET-28a) carrying genes encoding P450cam-RhFRed variants were stored at 4° C. Chemically competent E. coli BL21 (DE3) cells were transformed by heat shock according to manufacturer instructions. Transformants were plated on LB agar with added antibiotic (150 μg/ml ampicillin or 50 μg/ml for spectinomycin and kanamycin) and grown at 37° C. for 16 hours. Single colonies were picked from plates to prepare starter cultures in LB medium supplemented with antibiotic. After 16 hours, expression cultures were inoculated 1/100 using LB starter cultures and, to guarantee proper aeration, cultures volume was no more than 25% of the total conical flask volume. P450 expression in minimal medium was carried out according to Kelly et al. (supra): 1×M9 salts solution were supplemented with 0.4% glucose, 0.05% of FeCl.sub.3, 1 mM MgSO.sub.4, 1 mM CaCl.sub.2) and cells were grown with vigorous shaking (200 rpm) at 37° C. until an OD600 of 0.8 was reached. At this stage, isopropyl-D-1-thiogalactopyranoside (IPTG, 0.4 mM) was added to induce protein expression, along with ALA (0.5 mM) supplementation. Protein expression was carried out at 20° C. with shaking at 200 rpm for 20 h. The use of complex media (e.g., LB, TB and auto-induction media) resulted in the expression of the protein in the insoluble fraction or soluble inactive protein (data not shown).

[0096] Similarly, production of Cm-ADH10 and 55CR was carried out following a protocol similar to that for P450cam-RhFRed expression, the exception being the medium used (TB instead of M9) and the need for 5-ALA supplementation (not added). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was employed to confirm protein expression. Recombinant Cm-ADH10 was obtained by inoculating 1 L of Terrific broth containing 50 μg/mL kanamycin with transformed cell E. coli BL21 (DE3) and incubated until an OD600 of 0.6; protein production was induced with 0.4 mM IPTG at 20° C. and production was kept for 16 h. Cells were harvested by centrifugation (2831 g, 20 min, 4° C.) and kept at −80° C. until purification. Cells from 1 L of culture were suspended in 50 mL of 20 mM Tris-HCl, pH 7.5. The crude extract was prepared by sonication 5 min, 70% amplitude, 5 s on/off. Cell debris was removed by ultracentrifugation at 31,000 g for 45 min, 4° C. The supernatant was filtrated using a 0.45 um filter. The cell-free extract was applied to a 5 mL HisTrap FF column using an AKTA Pure (GE healthcare) at 1 ml/min at 4° C. and a 20 mM imidazole solution in 20 mM Tris-HCl buffer, pH 7.5. The column was washed with 4-6 CV of a 50 mM imidazole solution in 20 mM Tris-HC buffer, pH 7.5. Pure enzyme was eluted using 3 CV of a 500 mM imidazole solution in 20 mM Tris-HC buffer, pH 7.5. Enzyme was concentrated and excess of imidazole was removed with a concentrator Amicon® Ultra concentrator (10,000 NMWL; Millipore) at 4000 g. Typically, the enzyme was diluted to 10 mg/mL in 50 mM KPi buffer (pH 7.5) to carry out biotransformation trials and kinetic measurements, whereas for crystallographic studies, concentrated aliquots (1-1.5 mL) were treated with thrombin (0.5 U, 4° C., overnight shacking) and cleaved 6×-His Tag was removed by gel filtration in a Superdex 200 10/30 GL. Pure protein, as judged by UV chromatogram and SDS-PAGE was concentrated to 77 mg/mL and stored at −80° C.

[0097] For screening and characterization of ADHs, a panel of alcohol dehydrogenases enzymes as freeze-dried cell free extracts in 96-deep well plates were kindly supplied by c-LEcta GmbH (Leipzig, Germany). For a first screening, each freeze-dried extract was resuspended in 50 μL of 50 mM sodium phosphate buffer pH=7.2, 100 mM KC, and reaction carried out on a 500 μL scale with both 10 mM (R)- and (S)-HIP, 0.5 mM of both NAD+ and NADP+ and acetone 5% v/v (30° C.).

[0098] Then, best hits were chosen for a further screen on a 500 μL scale with 10 mM (R)-HIP, 0.5 mM NADP+ and acetone 5% v/v. Reaction mixtures were extracted with methyl-tert-butyl ether (MTBE), vortexed for 30 s and centrifuged 5 minutes before removing the organic layer, which was then transferred to fresh tubes containing MgSO.sub.4. Finally, 250 μL of the MTBE extract was taken for chiral normal phase HPLC analysis.

[0099] For protein crystallization and docking experiments, native Cm-ADH10 was crystallized at 293 K using the sitting-drop vapour diffusion technique at 20° C. Equal volumes of 12 mg/mL Cm-ADH10 in 20 mM Tris-HCl at pH 7.5 and reservoir solution were mixed. The reservoir solution contained 40% polyethylene glycol (PEG) 200 in 0.1 M MES at pH 6.5 (w/v). Initial conditions were screened using the JSCG, PEG and ammonium sulphate screens (Qiagen) in 96-well sitting drop trays. X-Ray diffraction data were collected at the ID23-2 beamline of the European Synchrotron Radiation Facility in Grenoble, France (ESRF). The images were integrated and scaled using MOSFLM (Battye et al., Acta Crystallogr. Sect. D Biol. Crystallogr. 2011, 67, 271-281). Intensities were merged and converted to amplitudes with Aimless (Evans and Murshudov, Acta Crystallogr. Sect. D Biol. Crystallogr. 2013, 69, 1204-1214) and other software of the CCP4 Suite (Winn et al., Acta Crystallogr. Sect. D Biol. Crystallogr. 2011, 67, 235-242). The structure was solved with MOLREP (Vagin and Teplyakov, J. Appl. Cryst 1997, 30, 1022-1025) and the coordinates of the dehydrogenase/reductase from Sinorhizobium meliloti 1021 (PDB: 3V2G) as the search model. AutoDock Vina (Trott and Olson, supra) was used to run ligand-receptor docking calculation of (R)-HIP on Cm-ADH10. Structures were prepared with DockPrep (Lang et al., Rna 2009, 1219-1230) in Chimera. The best model yielded a score of −5.6 kcal/mol, a rmsd (l.b) of 0.0 and a rmsd (u.b.) of 0.0.

Example 2: Screening and Improvement of P450cam-RhFRed Variants

[0100] A plasmid library encoding 96 P450cam-RhFRed mutants generated in a previous work (Kelly et al., Beilstein J. Org. Chem. 2015, 11, 1713-1720) were purified from bacterial glycerol stocks. P450cam-RhFRed variants were produced targeting 7 pairs of residues by saturation mutagenesis using NDT codon degeneracy. This resulted in the production of seven libraries: A to F (see Table 2). In order to cluster these variants, 100 ng of each plasmid purified were added to one of five pools according to the mutations introduced, the exceptions being variants from library E, F and G that were pooled together because the reduced size of these libraries (giving five pooled libraries in total). These plasmids preparations were then transformed into E. coli NEB-5-alpha competent cells and plasmids purified again for transformation into E. coli BL21 (DE3) for expression. FIG. 2 shows the measured biocatalytic performance expressed in terms of the total TTN, that is the ratio between total product concentration (in μM) and catalyst concentration (in μM), calculated over 24 h. Finally, the enantiomeric excess values (ee) were also determined by comparison with synthesized standards (see Table 2). The P450-catalysed single oxidation of α-IP may lead to (at least) three major products (FIG. 1B): HIP (6), the regioisomeric allyic oxidation product (3-(hydroxymethyl)-5,5-dimethylcyclohex-2-en-1-one, HMIP, 7) and isophorone oxide (2,3-epoxy-3,5,5-trimethyl-1-cyclohexanone, IPO, 8). Product 7 was identified after scale up and NMR analysis (not shown). The formation of isophorone oxide was not observed and (R)-HIP was the preferred product (FIG. 1B), with library D (mutations at residues L244-V247) showing the highest TTNs in the desired α-IP allytic oxidation. Notably, no activity was detected for mutants in pool C (mutations at residues M184-T185).

TABLE-US-00002 TABLE 2 Possible products of the P450 oxidation of α-IP and comparison of (R)-HIP ee values for the different pooled mutation libraries, “nd” means “not detectable”. Pool Position of mutation (R) HIP ee value EFG G248-T252; V295-D297; I395-V396 77% D L244-V247 94% C M184-T185 n.d. B F98-T101 48% A F87-F96 >99% 

[0101] These initial results drove the next step of the investigation towards the single-clone level analysis of mutants at positions L244-V247.

[0102] Expression cultures were centrifuged (2831 g, 20 min, 4° C.) and the pellet resuspended to 230 mg/mL (wet weight) in the appropriate biotransformation buffer (50 mM sodium phosphate buffer, pH=7.2, 100 mM KCl).

[0103] Biotransformations were carried out in 48 well plates on a 1 ml scale, with 880 μL of re-suspended cells, 100 μL of 100 mg/mL glucose and the appropriate final concentration of substrate typically added from concentrated stocks in DMSO (2% v/v final concentration). Plates were sealed with a gas permeable membrane and reaction carried out with 250 rpm orbital shaking for 24 h. Reaction mixtures were extracted with 1 mL of methyl-tert-butyl ether (MTBE) with 1 mM decane (as internal standard) vortexed for 30 s and centrifuged 5 minutes before removing the organic layer, which was then transferred to fresh tubes containing MgSO.sub.4. Finally, 250 μL of the MTBE extract was taken for GC analysis and diluted when necessary. In order to determine the ee %, the samples were also analyzed by chiral normal phase HPLC (see Ex. 1).

[0104] Analysis of biotransformation products (FIG. 3) demonstrated that positions 244 and 247 greatly affect TTNs of α-IP. Interestingly, the majority of mutants bearing the bulky aromatic phenylalanine residue show not only reduced TTNs but also a decrease in the regioselectivity of the reaction, with the formation of HIP and HMIP in approximately equal amounts by mutants L244Y-V247F and L244I-V247F. Given the observed positive effect of the substitution of L244 with small or apolar residues, the mutation L244A was also introduced, considering the effect of L244A mutation on substrate acceptance, regio- and enantioselectivity. The primers for said mutagenesis reactions are listed in Table 1. The protocol is described in Example 1. The protein and DNA sequences of P450cam-RhFRed wild-type enzyme carrying the amino acid substitution Y96F are shown in SEQ ID NO:1 and 2, respectively.

[0105] This mutation was studied either in presence of the wild-type V247 or V247L, leading to good TTNs values. The two mutants L244A-V247 and L244A-V247L showed the best TTN values (94±9 and 83±11, respectively) and were selected for further engineering. Furthermore, the bulky tryptophan was introduced in place of phenylalanine at position 87. As shown in Table 3, the introduction of F87W in the L244A-V247L background led to more than a 6-fold improvement of TTNs values with respect to L244A-V247L mutant, with no HMIP detected and an ee of 99% (R)-HIP, suggesting a better orienting effect of this biocatalyst towards the highly reactive iron-oxo species. Finally, the best improved variant (Y96F-F87W-L244A-V247L, now termed P450-WAL) was selected for subsequent optimization experiments for the designed cascade reaction (see Example 1) The results are shown in Table 3.

TABLE-US-00003 TABLE 3 (R)-HIP ee values obtained for different mutations from library D. Note that the strain already carries the Y96F mutation. Mutant (R) HIP ee value L244N-V247 94% L244S-V247L 99% L244N-V247L 99% L244G-V247L 99% L244I-V247L 98% L244C-V247L 98% L244I-V247F 95% L244Y-V247F 93% L244N-V247F 98% L244H-V247F 99% L244G-V247F 96% L244I-V247I 98% L244I-V247S 59% L244A-V247L 97% L244A-V247 84% F87W-L244A-V247 82% F87-L244A-V247L 99%

Example 3: Screening and Characterization of HIP-Oxidizing ADHs

[0106] A screening kit of 116 ADHs from very diverse organisms and with a broad range of accepted substrates was provided by c-LEcta. In order to identify suitable biocatalysts for the oxidation of (R)-HIP, the panel was screened against this substrate by HPLC (see Example 1). Several enzymes (>80%) showed almost no activity, due to the steric hindrance of the substrate, however, among the positive hits, two were selected for having the highest activity: Cm-ADH10 from Candida magnoliae (GeneBank accession no. AGA42262.1; SEQ ID NO:5) and the NADPH-dependent carbonyl reductase (55CR) from Sporobolomyces salmonicolor (UniProt accession no. Q9UUN9; SEQ ID NO:7). Notably, both enzymes accept NADP(H) as cofactor, which is desirable to create a self-sufficient cascade with respect to the cofactor. The corresponding genes were cloned into a pET28a vector to carry out expression trials (see Example 1), which revealed good expression levels for Cm-ADH10. Unfortunately, expression of 55CR was very low (not shown). The respective nucleotide acid sequences are shown in SEQ ID NO:6 and 8, respectively.

[0107] To understand better the catalytic properties of Cm-ADH10, we solved the crystal structure of its complex with NADP+ to a resolution of 1.6 Å (see Example 1; data not shown). After soaking the crystal with a solution of (R)-HIP (10 mM), no substrate was bound to the crystal. However, after docking (AutoDock Vina), the most favored calculation for the bound ligand (R)-HIP confirms that the cavity can accommodate the substrate at a distance and geometry that would favor the transfer of a hydride from the chiral carbon on the 4R-hydroxy moiety to C4N of NADP+. In this position, the 4-hydroxy group would be in the right geometry to interact with the catalytically important residues 5144 and Y157, while the rest of the molecule of 4R-HIP is possibly stabilized by interactions with residues H149 and Y189 in the entrance of the active site. Coincidentally, this area in the crystal structure is occupied by a tetrad of water molecules that have been reported to be bound to the Tyr-OH and Lys side chain, thus mimicking substrate and ribose hydroxyl group positions.

[0108] Preliminary biotransformations by varying (R)-HIP concentration from 10 to 100 mM were carried out, using 1 mg mL.sup.−1 purified Cm-ADH10 with a constant NADP+ concentration (0.25 mM) and 0.5% v/v ethyl acetoacetate as co-substrate for cofactor regeneration by the same ADH acting as a dual-functional enzyme. With this setup, conversions ranged from 92% with 10 mM substrate, down to 48% conversion with 100 mM substrate (see FIG. 4). Thus, Cm-ADH10 lends itself to being applied in the designed bi-enzymatic cascade.

Example 4: Optimization of Buffer Concentration, pH, and Temperature

[0109] Initially, we tried to combine the two selected biocatalysts in a one-pot, two-step format, adding 1 mg mL.sup.−1 of purified Cm-ADH10, cofactor and co-substrate (see above) directly to the supernatant of the whole-cell P450 reaction carried out with 200 mg mL.sup.−1 wet cell load in 50 mM sodium phosphate buffer pH 7.2, 100 mM KC (indicated as standard biotransformation buffer) and 10 mg mL.sup.−1 glucose for cofactor regeneration by E. coli metabolism. Unexpectedly, we obtained very low conversion values for the ADH catalyzed reaction, starting from just 10 mM α-IP in the first step. We reasoned that potential inhibitors for the second step could originate from the growth of the whole-cell biocatalysts in the M9 minimal medium supplemented with glucose (see FIG. 5A). The pH of the supernatant resulting from the first oxidation step carried out in phosphate buffer with different concentration was measured (FIG. 5B). Surprisingly, the pH dropped by 1.5 units when the standard biotransformation buffer was used, and even with a 300 mM potassium phosphate (KPi) buffer the pH decrease was significant. Nevertheless, by increasing the capacity of the buffer employed in the first step, higher conversions where observed in the second one. Eventually, more than a 2-fold increase in HIP conversion was observed when P450 catalyzed reaction carried out in standard biotransformation buffer were titrated to pH 8.0 before the addition of Cm-ADH10. A similar trend was observed when cells grown in M9 medium where simply resuspended in buffer without any addition of substrate or glucose, suggesting that the observed pH decrease is linked to the metabolism of cells grown to high density (OD600—5.0-6.5). Thus, in order to establish a one-pot two-step process there is a need for a careful optimization of the each individual step before combination.

[0110] Next, we proceeded with the parallel optimization of the two oxidative steps with respect to buffer concentration, pH and temperature. For the P450 allylic oxidation step, KPi buffer (pH 8.0) was chosen for the unique K+ binding site of P450cam, which displays higher stability and superior camphor binding in presence of K+ ions. The effect of buffer concentration was examined, along with the temperature optimum. As shown in FIG. 6A, conversion improved with increasing buffer concentration up to 200-300 mM, and by comparing biotransformations carried out in 50 mM KPi without KC, with KCL or in 100 mM KPi, it can be concluded that buffer capacity seems to have a greater effect on conversion than K+ concentration. Eventually, 200 mM KPi buffer was chosen to investigate the effect of temperature on conversion, finding the optimum at 28° C. (FIG. 6B).

[0111] With respect to the Cm-ADH10 alcohol oxidation, different co-substrates were selected and tested in order to exploit the biocatalyst as a dual-functional enzyme capable of performing the whole substrate oxidation-cofactor regeneration cycle (FIG. 7A). Besides testing acetone (9a), we have also included activated ketones such as chloroacetone (9b), ethyl acetoacetate (9c) and ethyl levulinate (9d), since their reduction (and hence cofactor regeneration) is favored by an additional intramolecular hydrogen bond between the newly formed alcohol functional group and the electronegative moiety.

[0112] Chloroacetone and ethyl acetoacetate proved to be superior and, even if the latter performed better during the first six hours of reaction, the former pushed the conversion to 84% with 40 mM substrate after 24 h (vs. 67% with ethyl acetoacetate). Therefore, chloroacetone was employed as hydrogen acceptor in all the subsequent optimization steps. The pH effect on the reaction outcome (FIG. 7B) partially explains our initial unsuccessful attempts to combine the two enzymes: in fact, enzyme activity drops below pH 6.0, with an optimum pH range between 7.0 and 9.0. Moreover, the buffer system employed in the P450 catalyzed reaction (200 mM KPi) displayed perfect compatibility with the second step (FIG. 8). Finally, we turned our attention to temperature optimization, finding the highest conversion at 40° C. (FIG. 7C).

[0113] With these optimized conditions in hand, we moved on to the envisaged bi-enzymatic cascade concept to carry out scale-up experiments for product isolation and characterization.

Example 5: Double Oxidation from α-Isophorone to Ketoisophorone

[0114] In order to further simplify the entire process, the oxidation of (R)-HIP was accomplished using a concentrated cell-free extract (CFE) of E. coli overexpressing Cm-ADH10, thus avoiding the expensive protein purification step. The course of the reaction under the optimized conditions was followed over a total reaction time of 50 h, showing that the P450 catalyzed allylic oxidation of 10 mM α-IP reached 94±2% conversion in 18 h, whereas the second step was much faster, reaching complete conversion over 6 h (FIG. 9). Remarkably, the addition of NADP+ for the second step was found to be unnecessary to achieve the highest conversion, further reducing process costs. Some degree of overoxidation of HIP to KIP was observed during the first step.

[0115] Given these results, we have also attempted to co-express the P450cam-RhFRed-WAL variant with Cm-ADH10 in the same host with a two-vector system to carry out the whole-cell double oxidation of α-IP (FIG. 1D). After 24 h, the conversion reached an average value of 732%, approximately 20% lower than for the one-pot two-step process. However, the co-expression of the two enzymes and cofactor preferences make this reaction redox-balanced, avoiding addition of a CFE and co-substrate. In fact, P450 expression level was reduced 3-fold in the “designer” microorganism (6.0 μM for the two-step process vs. 1.9 μM for the whole-cell process), which accounted for the residual α-IP (not shown).