Enzymes and materials
Restriction endonucleases, T4 DNA ligase, Pfu DNA polymerase and isopropyl β-D-thiogalactopyranoside (IPTG) were obtained from Fermentas (St. Leon-Rot, Germany). (+)-Valencene and (+)-nootkatone were from Fluka (Buchs, Switzerland). Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (EC 22.214.171.124) and other chemicals, solvents and buffer components were purchased from Sigma-Aldrich (Schnelldorf, Germany).
Molecular biological techniques
General molecular biology manipulations and microbiological experiments were carried out by standard methods . Construction of pT7-camAB – a plasmid based on pET11a (Novagen, Darmstadt, Germany) and harboring the camA (PdR) and camB (Pdx) genes from Pseudomonas putida ATCC 17453 – has been described previously . pT7NS-camAB was prepared by insertion of an NdeI-SpeI linker into pT7-camAB and plasmids for the expression of bacterial P450s were created by amplification of annotated P450-encoding genes from genomic DNA of various bacteria as described in . The plasmid for co-expression of PdR, Pdx and CYP109B1 – pCYP109B1-camAB – was constructed by amplification of the CYP109B1-encoding yjiB-gene [GenBank:CAB13078] from genomic DNA of the Bacillus subtilis strain 168  and by following ligation of the PCR fragment into pT7NS-camAB utilizing the restriction sites for NdeI and SpeI of the NdeI-SpeI linker. pCYP109B1-camAB comprises the genes under control of the IPTG-inducible T7 phage-promoter in an artificial operon in the following order: yjib (CYP109B1), camA (PdR), camB (Pdx).
pET28a-CYP109B1 – a vector for the expression of CYP109B1 alone, without PdR and Pdx – was constructed as follows: The yjiB-gene was amplified from pCYP109B1-camAB with the primers 5'-AATAgctagcATGAATGTGTTAAACCGCCG-3' and 5'-TCGctcgagTTACATTTTCACACGGAAGC-3'. The PCR was performed with Pfu DNA polymerase under the following conditions: 95°C for 4 min, 25 cycles of (95°C for 1 min, 56°C for 30 s, 72°C for 3 min), 72°C for 5 min. The PCR-product was purified, digested with endonucleases NheI and XhoI and inserted into the previously linearized expression vector pET28a(+) (Novagen, Darmstadt, Germany). The resulting DNA-construct encoded for N-terminally His6-tagged CYP109B1 under control of the IPTG-inducible T7 phage-promoter.
pET28a-camA – a vector for the expression of PdR – was constructed by PCR-amplification of the camA-gene from pT7NS-camAB with the primers 5'-GGAATTCCATATGAACGCAAACGAC-3' and 5'-GATCGAATTCTCAGGCACTACTCAG-3'. The PCR was done as described for pET28a-CYP109B1, the PCR-product was purified and digested with endonucleases NdeI and EcoRI and inserted into previously linearized expression vector pET28a(+). The resulting plasmid encoded for N-terminally His6-tagged PdR under control of the IPTG-inducible T7 phage-promoter.
The basic steps for the design of pET28a-camB – which incorporates the camB-gene under control of the IPTG-inducible T7 phage-promoter for expression of N-terminal His-tagged Pdx – were identical to those for construction of pET28a-camA, except for primers. The primers 5'-GGGAATTCCATATGTCTAAAGTAGTGTAT-3' and 5'-CCGGAATTCTTACCATTGCCTATCGGG-3' were used for PCR in this case.
Protein expression and purification
Recombinant CYP109B1 was expressed under the following conditions: E. coli BL21(DE3) cells were transformed with pET28a-CYP109B1 and transformants were selected on LB-agar plates with kanamycin (30 μg ml-1). 400 ml LB supplemented with 30 μg ml-1 of kanamycin were than inoculated with 2 ml overnight culture – grown from a single colony – and grown at 37°C and 180 rpm, until the optical density at 600 nm (OD600) reached approximately 1.0. 100 μM IPTG, 80 μg ml-1 5-aminolevulinic acid and 0.1 μM FeSO4 were added and the culture was grown for another 19 h at 30°C and 140 rpm. Cells were harvested by centrifugation at 10,000 × g for 20 min, the supernatant was discarded and the cell pellet was resuspended in 10 ml purification buffer (50 mM TrisHCl, pH 7.5, 500 mM NaCl, 100 μM phenylmethanesulfonyl fluoride). Cells were lysed by sonification on ice (3× 1,5 min, 1 min intermission), cell debris was removed by centrifugation (35,000 × g, 30 min, 4°C), the soluble protein fraction was recovered and filtered through a 0,2 μm filter.
Recombinant expression of PdR and Pdx was achieved by transformation of E. coli BL21(DE3) with either pET28a-camA or pET28a-camB. Transformants were selected on LB-agar plates with kanamycin (30 μg ml-1). 400 ml LB with kanamycin were inoculated with 2 ml from an overnight culture – grown from a single colony – and grown at 37°C and 180 rpm, until the OD600 reached approximately 0.7. 100 μM IPTG were added and the culture was grown for 17 h at 25°C and 160 rpm. The soluble protein fraction was recovered as described for CYP109B1.
Purification of CYP109B1, PdR and Pdx was done by immobilized metal affinity chromatography (IMAC) with a Talon® resin (7 ml bed volume). Protein lysates were applied to the column, which was pre-equilibrated with 5 column volumes of purification buffer. Non-specifically bound proteins were washed from the column with 4 column volumes of purification buffer with 5 mM imidazol, before the bound protein was eluted with purification buffer containing 100 mM imidazol. 5% Glycerol was added to the eluate and it was dialyzed two times against 2 l of 50 mM TrisHCl, pH 7.5, containing 5% glycerol, 100 μM phenylmethanesulfonyl fluoride and frozen at -20°C until use.
Determination of protein concentration
The P450 expression levels were estimated using the CO-difference spectral assay as described previously [35, 36] using ε450–490 = 91 mM-1 cm-1.
The concentration of PdR was determined as the average of the concentration calculated from each of the three wavelength 378, 454 and 480 nm using extinction coefficients 9.7, 10.0 and 8.5 mM-1 cm-1 .
The concentration of Pdx was determined as the average concentration calculated from the two wavelength 415 and 455 nm using extinction coefficients 11.1 and 10.4 mM-1 cm-1 .
In vitro activity reconstitution of CYP109B1
A reconstituted in vitro system for conversion of (+)-valencene (1) by CYP109B1 was set up as described in  except for addition of catalase. The components were mixed in a 1,5 ml reaction tube, incubated at 30°C for 2 min and 200 μM NADH were added. Absorption at 340 nm was followed spectro-photometrical and NADH-consumption was calculated using ε = 6.22 mM-1 cm-1. For product identification the setup was slightly modified as follows: 1 μM PdR, 10 μM Pdx, 1 μM CYP109B1, 5 units of glucose-6-phosphate dehydrogenase, 4 mM glucose-6-phosphat and 1 mM MgCl2. After incubation, the internal standard (-)-carvone (50 μM) was added, samples were extracted with 400 μl ethyl acetate and the extracts were analyzed by GC/MS.
Construction of whole-cell biocatalysts
E. coli BL21(DE3) was used as a host for the gene expression. The BL21(DE3) cells were correspondingly transformed with plasmids harboring P450-genes and grown in 50 ml of M9 expression-medium, which is based on M9 medium , supplemented with 1% casamino acids, 20 μg ml-1 thymine, 0.1 μM FeSO4, 80 μg ml-1 5-aminolevulinic acid and 50 μg ml-1 carbenicillin. For protein expression the Overnight Express™ Autoinduction System 1 (Novagen, Darmstadt, Germany) was added according to the supplier's manual. The cultivation was carried out for 24 h at 25°C. Cells were collected by centrifugation (2400 × g) and resuspended in 10 ml of aqueous CV2 buffer (50 mM potassium phosphate, pH 7.5, 2% glycerol, 0.1 mM IPTG, 50 μg ml-1 carbenicillin), whereupon the cww was adjusted to 70 g l-1. The substrate was added to a final concentration of 2 mM for the biooxidation assay either directly or from a stock solution dissolved in DMSO to yield a final DMSO concentration in the reaction mixture of 2%. Samples were then split in 2 ml aliquots. For the biphasic systems 10 or 20% (v/v) of an organic solvent (isooctane, n-octane, dodecane or hexadecane) was added to the CV2 buffer. The biotransformation was carried out at 30°C under shaking at 200 rpm for 8 h. After incubation the internal standard (-)-carvone (50 μM) was added and samples were extracted with 1 ml of ethyl acetate for GC or GC/MS analysis. A negative control was performed with E. coli BL21(DE3) transformed with pT7NS-camAB.
For biooxidation reactions on a larger scale, 20 ml of CV2 buffer were set up with 70, 140 or 280 gcww l-1 in round bottom flasks (70 g cww correspond to 18.4 g cdw). (+)-Valencene (1) was added from concentrated stock solutions in DMSO to achieve final concentrations of 2, 3 or 4 mM (+)-valencene (1) and 2% DMSO in the reaction mixture. The reaction was carried out at 30°C for 8 h under stirring with a magnetic stirrer. After incubation the internal standard (-)-carvone (50 μM) was added and samples were extracted with 10 ml ethyl acetate for GC analysis.
E. coli viability during the biotransformation was monitored by taking cell aliquots from the reaction mixtures at certain time points. 40 μl of the aqueous phase were diluted in serial dilutions, plated on Luria broth agar plates containing ampicillin (100 μg ml-1) and after incubation at 30°C for 24 h the grown colonies were counted.
The concentrations of (+)-valencene (1) and its conversion to products was determined by GC analysis using a GC2014 (Shimadzu, Kyoto, Japan) equipped with an Equity-5 column (30 m × 0.25 mm × 0.25 μm, Supelco, Pennsylvania, USA). The injector and detector temperatures were set at 250 and 270°C, respectively. 1 μl of a sample was injected at a split ratio of 4, with helium as carrier gas. The column temperature was maintained at 120°C for 4 min, ramped to 250°C at a rate of 10°C min-1 and held at 250°C for 5 min. For quantitative GC analysis the FID response was calibrated for (+)-valencene (1) and (+)-nootkatone (4). Mixtures of CV2 buffer containing (+)-valencene (1) or (+)-nootkatone (4) in final concentrations of 50 to 2500 μM and (-)-carvone from a 5 mM stock solution in ethanol (final concentration 50 μM) as an internal standard were extracted with 1 ml of ethyl acetate and analyzed as described. The ratio of the area of the substrate to that of the internal standard was plotted against the substrate concentration to give a straight-line calibration plot.
Mass spectra were acquired on a GC/MS-QP2010 (Shimadzu) equipped with a FS-Supreme-5 column (30 m × 0.25 mm × 0.25 μm, Chromatographie Service GmbH, Langerwehe, Germany). The same setup as for the GC-analysis was used. The products were identified by their characteristic mass fragmentation patterns by comparison with mass spectra of authentic reference compounds and by comparison with mass spectra reported elsewhere .
Determination of distribution coefficients
The distribution coefficients (log D) for (+)-valencene (1), trans-nootkatol (3) and (+)-nootkatone (4) were determined by dissolving increasing amounts of the respective compound (final concentration 500 to 5000 μM) in a 50% (v/v) mixture of aqueous CV2 buffer and an organic solvent (isooctane, n-octane, dodecane or hexadecane) in a total volume of 10 ml. After equilibration for 4 h at 25°C under vigorous shaking, phases were separated by centrifugation. 500 μl of each the organic and the aqueous phase were recovered separately and the internal standard (-)-carvone (final concentration 50 μM) was added. The aqueous phase was extracted with 1 ml of ethyl acetate and 500 μl of ethyl acetate were added to the organic phase. Both phases were analyzed quantitatively by GC. Subsequently the log D values were calculated.
Synthesis of cis- (2) and trans-nootkatol (3)
390 mg (+)-nootkatone (4) was dissolved in 2 ml dry diethyl ether and added dropwise under stirring at 0°C on ice to a suspension of 130 mg LiAlH4 in 8 ml dry diethyl ether. After complete conversion, the solution was cooled down to -30°C and 20 ml saturated sodium potassium tartrate solution were added. The solution was allowed to warm to ambient temperature and was stirred for 16 h. The aqueous phase was extracted with diethyl ether (4 × 20 ml). The extracts were combined and dried over anhydrous MgSO4. Solvents were removed at 40°C. The raw product was applied to a silica gel column (ethyl acetate:petroleum ether 1:10). Fractions were analyzed by GC/MS and NMR. Nootkatol was isolated as a mixture of cis- (2) and trans-nootkatol (3) with a ratio of cis:trans of approximately 1:9. Solvents were removed from the nootkatol fraction at 40°C until no change in mass occurred. Nootkatol (2 and 3) (350 mg, 89%) was retained as colorless oil.
1H-NMR (500 MHz, CDCl3, for numbering see figure 1): δ = 0.89 (3 H, d, J 6.9, 4-Me), 0.95 (1 H, br s, 2-OH), 1.00 (3 H, s, 5-Me), 1.21 (1 H, dddd, J 13.9, 12.4, 12.4, 4.3, 8-Hax), 1.37 (1 H, ddd, J 12.7, 12.7, 10.0, 3-Hax), 1.52 (1 H, dqd, J 13.0, 6.8, 2.1, 4-H), 1.71 (3 H, dd, J 1.3, 1.1, 13-Me), 1.77 (1 H, dddd, J 12.3, 2.3, 1.6, 1.3, 8-Heq), 1.78 – 1.84 (1 H, m, 3-Heq), 1.86 (1 H, ddd, J 12.8, 2.7, 2.7, 6-Heq), 2.12 (1 H, ddd, J 14.1, 4.2, 2.6, 9-Heq), 2.21 – 2.28 (1 H, m, 9-Hax), 2.29 – 2.37 (1 H, m, 7-H), 4.23 – 4.27 (1 H, m, 2-Hax), 4.67 – 4.70 (2 H, m, 2× 14-Hax), 5.32 (1 H, ddd, J 2.6, 1.8, 1.8, 1-Hax) ppm.
13C-NMR (500 MHz, CDCl3): δ = 15.4 (4-Me), 18.2 (5-Me), 20.8 (13-Me), 32.4 (C-8), 32.9 (C-6), 37.3 (C-10), 38.2 (C-5), 39.3 (C-4), 40.8 (C-7), 44.6 (C-9), 68.0 (C-2), 108.6 (C-14), 124.3 (C-1), 146.1 (C-10), 150.2 (C-13) ppm.