Process for the modification of alkanes, fatty acids and fatty alcohols
10975360 · 2021-04-13
Assignee
Inventors
- Martha Sophia Smit (Bloemfontein, ZA)
- Diederik Johannes Opperman (Bloemfontein, ZA)
- Alizé Pennec (Saint Quay Portrieux, FR)
- Jacqueline Van Marwijk (Vanderbijlpark, ZA)
- Mpeyake Jacob Maseme (Virginia, ZA)
- Alberto Perojil Jimenez (Granada, ES)
Cpc classification
C12N9/0071
CHEMISTRY; METALLURGY
C12P17/06
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a process for the microbial in-chain hydroxylation of C12 to C16 fatty acids, alcohols and alkanes at position ω-7, the process including the use of a microorganism expressing a cytochrome P450 monooxygenase CYP505E3 or related fungal cytochrome P450 monooxygenases sharing at least 70% amino acid identity in the production of a hydroxylated product or secondary product. The present invention further relates to a process for the preparation of lactones, esters and polymers by hydroxylation of the corresponding fatty acids, fatty alcohols and alkane precursors by a recombinant cytochrome P450 monooxygenase CYP505E3 or related fungal cytochrome P450 monooxygenases sharing at least 70% amino acid identity.
Claims
1. A process for the microbial in-chain hydroxylation of C12 to C16 fatty acids, alcohols and alkanes at position ω-7, including culturing a microorganism expressing a recombinant cytrochrome P450 (CYP450) monooxygenase that has the amino acid sequence of SEQ ID NO: 1; on a culture medium including an exogenous substrate; and isolating a hydroxylated product or secondary product formed thereof from the medium, wherein the exogenous substrate is selected from the group consisting of C12 to C16 fatty acids, alcohols and alkanes, and wherein the hydroxylated product or secondary product comprises one or more of lactones, esters and polymers.
2. The process of claim 1, wherein the microorganism is a wild-type Aspergillus terreus strain or other wild-type fungal strain expressing a CYP450 monooxygenase having the amino acid sequence of SEQ ID NO: 1.
3. The process of claim 1, wherein the process includes the further step of creating a Cell-Free Extract (CFE) of the cultured microorganism and combining the CFE with a medium containing the exogenous substrate.
4. The process of claim 1, wherein the C12 to C16 fatty acid comprises any one or more of C12 to C16 saturated, unsaturated, straight and branched fatty acids.
5. The process of claim 4, wherein the C12 to C16 fatty acid comprises any one or more of lauric acid, tridecylic acid, myristic acid, pentadecylic acid and palmitic acid.
6. The process of claim 1, wherein the C12 to C16 alcohol comprises any one or more of C12 to C16 saturated, unsaturated, straight and branched alcohols.
7. The process of claim 6, wherein the C12 to C16 alcohol comprises any one or more of lauryl alcohol, tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol and palmitoleyl alcohol.
8. The process of claim 1, wherein the C12 to C16 alkane comprises any one or more of C12 to C16 saturated, unsaturated, branched and unbranched alkanes.
9. The process of claim 8, wherein the C12 to C16 alkane comprises any one or more of dodecane, tridecane, tetradecane, pentadecane and hexadecane.
10. The process of claim 1, wherein the lactone is delta dodecalactone.
11. The process of claim 1, wherein the esters comprise any one or more of heptyl pentanoate, butyl octanoate, heptyl nonanoate, octyl octanoate, hexyl octanoate, and heptyl heptanoate.
12. The process of claim 1, wherein the polymer comprises poly(δ-dodecalactone).
13. The process of claim 1, further comprising using the hydroxylated product, or the secondary product, in the synthesis of a lactone, ester or polymer.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1) The invention will now be described in more detail, by way of example only, with reference to the accompanying figures in which:
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(26) The foregoing and other objects and features and advantages of the present invention will become more apparent from the following description of certain embodiments of the present invention by way of the following non-limiting examples.
DESCRIPTION OF THE INVENTION
(27) The invention will now be described with reference to the following non-limiting experimental examples.
(28) Materials and Methods and General Experimental Procedures
(29) Chemicals and Enzymes
(30) Chemicals and reagents used in these experiments were analytical grade and were, unless otherwise stated, supplied by either Fluka, Sigma-Aldrich or Merck.
(31) Cloning of Enzymes
(32) The gene encoding CYP505E3 from Aspergillus terreus was codon optimized for expression in E. coli and synthesized by Genscript. The gene encoding CYP505A1 was synthesized by GeneArt without any codon optimization, but with common restriction enzyme recognition sites removed. The genes encoding CYP505E3 and CYP505A1 were cloned into the pET28b(+) plasmid. The gene encoding the glucose dehydrogenase (GDH) from Bacillus megaterium (GDH), was cloned into pETDuet (Novagen) multiple cloning site 2 (MC2). The gene encoding horse liver alcohol dehydrogenase (HLADH) was cloned into pET28b(+). For expression of the CYPs, GDH and HLADH, E. coli BL21-Gold(DE3) (Stratagene) was transformed with the relevant plasmids and transformants selected on LB-plates containing 30 μg.Math.ml.sup.−1 kanamycin or 100 μg.Math.ml.sup.−1 ampicillin.
(33) The CYP505E3 gene optimized for expression in Pichia pastoris was synthesized and cloned into the pAO815 vector by GenScript. A combination of chemical and electroporation techniques were used to transform P. pastoris strain KM71 with the plasmid linearized with SalI to yield slow growing (His.sup.+Mut.sup.s) transformants on methanol. The genes encoding amino acid SEQ ID Nos: 2, 3 and 4 were codon optimized for expression in E. coli and synthesized by GenScript. These genes were cloned into the pET28b(+) as well as the pAO815 plasmid. The pET28b(+) plasmids with the genes were cloned into E. coli, but expression was not successful. The genes encoding amino acid SEQ ID Nos: 5 to 7 were synthesized by GenScript without any codon optimization, but with common restriction enzyme recognition sites removed, and also cloned into pAO815. The pAO815 plasmids with all these genes were cloned into P. pastoris KM71 to yield slow growing (His.sup.+Mut.sup.s) transformants on methanol.
(34) Heterologous Expression of Enzymes in E. coli and Preparation of Cell Free Extracts (CFEs) and Whole Cell (WC) Suspensions
(35) Expression of genes in E. coli was performed by using ZYP-5052 auto-induction medium (Table 1) with in the case of the CYP 1 mM 5-aminolevulinic acid and 0.05 mM FeCl.sub.3 added. The CYP505E3 culture was cultivated using baffled flasks and incubated at 20° C. and 180 rpm for 24 h, while the GDH, and HLADH expressing cultures were all cultivated at 25° C. and 200 rpm for 24 h. Cultures were harvested through centrifugation (6 000×g, 10 min) and 1 g (wet weight) resuspended in 2 ml, 5 ml or 10 ml Tris-HCl, Phosphate or MOPS buffer, as indicated, (pH 8, 200 mM) containing 100 mM of glucose and glycerol. CFEs were obtained by a single passage of the suspended cells through a One Shot Cell Disrupter (Constant Systems) at 207 MPa. The soluble fraction was separated from unbroken cells by centrifugation (20 000×g, 20 min).
(36) TABLE-US-00001 TABLE 1 Composition of ZY auto-induction media (Studier, 2005) ZY auto- Stock solutions induction media ZY medium 20x NPS 50x 5052* 50 ml 20x NPS 10 g/l 0.5M (NH.sub.4).sub.2SO.sub.4 250 g/l glycerol 20 ml 50x 5052 tryptone 1M KH.sub.2PO.sub.4 25 g/l glucose 2 ml 1M MgSO.sub.4 5 g/l 1M Na.sub.2HPO.sub.4 100 g/l α-lactose 928 ml ZY-medium yeast extract *5052 final concentrations—0.5% glycerol, 0.05% glucose, 0.2% α-lactose
(37) Heterologous Expression of Enzymes in P. pastoris and Preparation of Whole Cell (WC) Suspensions
(38) Precultures of P. pastoris transformants were prepared in buffered complex glycerol media (BMGY). BMGY contained: 10 g/l yeast extract, 20 g/l peptone, 3.4 g/l yeast nitrogen base, 10 g/l ammonium sulfate, 100 mM potassium phosphate (pH 6), 0.4 mg/l biotin and 1% (v/v) glycerol. Erlenmeyer flasks (500 ml) containing 50 ml BMGY media were inoculated with single colonies of 24 h old YPD-agar cultures of P. pastoris KM71 mut.sup.s3/CYP505E3 and incubated at 30° C. and 225 rpm for 24 h. The cultures were harvested using pre-weighed centrifuge tubes at 3000 g for 5 min and stored on ice until further use.
(39) The main cultures for expression were prepared in buffered complex methanol media (BMMY) containing 10 g/l yeast extract, 20 g/l peptone, 3.4 g/l yeast nitrogen base, 10 g/l ammonium sulfate, 100 mM potassium phosphate (pH 6), 0.4 mg/l biotin and 0.5 or 1% (v/v) methanol. Harvested cells from precultures were resuspended in BMMY media to final concentration of 1 g WC per 40 ml (appx. 25 g/l). The resuspended cultures (100 ml) were transferred to 500 ml Erlenmeyer flasks and incubated at 25° C. and 225 rpm for 24 h. The cultures were then harvested at 3000 g for 5 min using pre-weighed centrifuge tubes.
(40) Analyses
(41) All chemicals were from Sigma-Aldrich and were used without further purification. Methylation of fatty acids was done with trimethylsulfonium hydroxide (TMSH) (1:1 EtOAc extract/TMSH reagent) and fatty acid containing samples analysed on a Restek BPX17 column (60 m×0.25 mm ID×0.25 μm film thickness). Samples containing only unmethylated fatty acids, alkanes, alcohols or lactones were analyzed on Varian FactorFour VF-5 ms column with dimensions: 30 m×0.25 mm×0.25 μm (length×inner diameter×film thickness).
(42) GC-MS analyses were carried out on a Thermo Trace GC ultra chromatograph with DSQ mass spectrometer and standard GC analysis on a Shimadzu GC2010.
(43) CO Difference Spectra
(44) The resuspended cells were further diluted with phosphate buffer in a 1:1 (v/v) ratio to record CO difference spectra. The assay was conducted as described by Choi et al., (2003) using 200 μl of the diluted cells transferred into microtiter strips (Thermo Scientific). Absorbance readings between 400 and 500 nm were measured with a SpectraMax® Microplate Reader (Molecular Devices). The CYP450 concentration was determined using an extinction coefficient of 0.091 nM.sup.−1 ml.sup.−1 (Omura & Sato, 1964) and a pathlength of 0.596 cm. Peak corrections were done and CYP450 concentrations calculated by using the equation A.sub.450−((0.375*A.sub.470)+(0.625*A.sub.438))/(0.091*0.596) (Johnston et al., 2008).
EXPERIMENTS
Experiment 1—Biotransformation of C12, C14 and C16 Fatty Acids and C14 and C16 Fatty Alcohols Using CFE of E. coli Expressing CYP505E3
(45) The biotransformation reaction mixture (BRM) consisted of CYP and GDH CFE suspensions prepared from cells in Tris-HCl buffers (1 g wet weight in 2 ml buffer) mixed in a 1:1 ratio with 0.1 mM NADPH added. Dodecanoic acid, tetradecanoic acid, hexadecanoic acid, 1-tetradecanol and 1-hexadecanol (20 μl of a 255 mM stock solution in DMSO) were added to 40 ml amber glass vials containing 1 ml BRM to give final substrate concentrations of 5 mmol.Math.L.sub.BRM.sup.−1. These vials were placed on an orbital shaker at 20° C., 200 rpm, oscillation amplitude 26 mm. Vials were removed after 24 h for extraction. Biotransformations were stopped by adding 170 ul of 0.5M HCl, extracted with 2×500 μl EtOAc and subjected to GC-MS analysis. A sample of δ-dodecalactone in ethyl acetate (0.1 mM) was also methylated with TMSH and subjected to GC-MS analysis.
(46) GC-MS chromatograms of the methylated samples from the dodecanoic acid biotransformations showed a complex mixture of products (
(47) GC-MS analysis of the methylated samples from the tretradecanoic acid and hexadecanoic acid biotransformations revealed in each case one major product which was different from the products produced by CYP505A1 and identified as the methyl esters of 7-hydroxy tetradecanoic acid (7-OH C14FAME) (
(48) GC-MS analysis of the samples from the tetradecanol and hexadecanol biotransformations did not allow detection of any products formed from hexadecanol, while two products were detected in the samples from the tetradecanol biotransformations by CYP505E3 (
Experiment 2—Biotransformation of C12 Fatty Acid Using CFE of E. coli Expressing CYP505E3 in Different Buffers
(49) The biotransformation reaction mixture (BRM) consisted of CYP and GDH CFE suspensions prepared from cells in Tris-HCl, phosphate and MOPS buffers (1 g wet weight in 5 ml buffer) mixed in a 1:1 ratio with 0.1 mM NADPH added. The final CYP concentrations were 1.1, 1.3 and 1.5 μM in respectively the Tris-HCl, phosphate and MOPS buffers. Dodecanoic acid (20 μl of 255 or 510 mM fatty acid stock solution in DMSO) was added to 40 ml amber glass vials containing 1 ml BRM to give final substrate concentrations of 5 and 10 mmol.Math.L.sub.BRM.sup.−1. These vials were placed on an orbital shaker at 20° C., 200 rpm, oscillation amplitude 26 mm. Vials were removed at specific time intervals for extraction. Biotransformations were stopped by adding 170 ul of 0.5M HCl and extracted with 2×500 μl EtOAc containing 2 mM decanoic as internal standard.
(50) GC-MS analyses of the methylated samples from the dodecanoic acid biotransformations revealed the formation of a complex mixture containing six different products (
Experiment 3—Time Course of Biotransformation of 50 mM C12 Fatty Acid Using CFE of E. coli Expressing CYP505E3 in MOPS Buffer—Samples Analysed with and without Methylation
(51) CYP and GDH CFE suspensions prepared from cells in MOPS buffers (1 g wet weight in 5 ml buffer) were mixed in a 9:1 ratio and diluted with three parts MOPS buffer so that the final CYP concentration in the BRM was 0.2 μM. NADPH (0.1 mM) was added. Dodecanoic acid (20 μl of 2.5 M fatty acid stock solution in DMSO) was added to 1 mL BRM in 40 ml amber glass vials to give a final substrate concentration of 50 mmol.Math.L.sub.BRM.sup.−1. These vials were placed on an orbital shaker at 20° C., 200 rpm, oscillation amplitude 26 mm. Vials were removed at specific time intervals for extraction. Biotransformation were stopped by adding 170 ul of 0.5M HCl and extracted with 2×500 μl EtOAc containing 2 mM decanoic and 2 mM tetradecanol as internal standards.
(52) After extraction aliquots (50 μl) of ethyl acetate extracts containing both decanoic acid and tetradecanol as internal standards were methylated for GC-MS analysis while the rest of the extracts were washed with a Na.sub.2CO.sub.3 (5% w/v) solution to remove the fatty acids. The washed ethyl acetate extracts were also analysed by GC-MS and the δ-dodecalactone and γ-dodecalactone concentrations determined from a standard curve.
(53) The lactone concentrations in the washed ethyl acetate samples were significantly lower (0.6 mmol.Math.L.sub.BRM.sup.−1) than those in the unwashed samples (1.6 mmol.Math.L.sub.BRM.sup.−1) indicating that a large percentage of the hydroxy fatty acids did not close to form the lactone (
Experiment 4—Biotransformation of C12, C14 and C16 Alkanes Using CFE of E. coli Expressing CYP505E3
(54) Biotransformations of dodecane, tetradecane and hexadecane were carried using only CYP505E3 containing CFE prepared from an E. coli cell suspension (1 g wet weight in 10 ml buffer) in MOPS buffer (200 mM, pH 8) containing glucose and glycerol (100 mM each) as well as 0.1 mM NADPH. Alkane substrates (250 μL) were added to 1 mL BRM in 40 ml amber glass vials. These vials were placed on an orbital shaker at 20° C., 200 rpm, oscillation amplitude 26 mm and incubated for 24 h.
(55) GC-MS analysis of the ethyl acetate extracts revealed that dodecane, tetradecane and hexadecane were transformed to give in each case a single product and that all three alkanes were hydroxylated at what can be described as position ω-7 to give respectively 5-dodecanol, 7-tetradecanol and 8-hexadecanol (
Experiment 5—Biotransformation of Dodecanol Using CFE of E. coli Expressing CYP505E3
(56) CYP and GDH CFE suspensions prepared from cells in MOPS buffers (1 g wet weight in 5 ml buffer) were mixed in a 9:1 ratio and diluted with three parts MOPS buffer so that the final CYP concentration in the BRM was 0.2 μM. Dodecanol (40 μL) was added to 1 mL BRM in 40 ml amber glass vials. These vials were placed on an orbital shaker at 25° C., 200 rpm, oscillation amplitude 26 mm. Vials were removed at specific time intervals for extraction. Biotransformation were stopped by adding 170 ul of 0.5M HCl and extracted with 2×500 μl EtOAc containing 2 mM tetradecanol as internal standards. GC-MS revealed the formation of up to 14 mmol.Math.L.sub.BRM.sup.−1 1,5-dodecanediol within 8 h (
Experiment 6—Biotransformation of Dodecanol Using Permeabilized Whole Cells of E. coli Expressing CYP505E3—25 mL Scale
(57) An E. coli culture expressing CYP505E3 was harvested and the cell pellet resuspended in MOPS buffer (200 mM, pH 8) containing 25 mM glucose and 25 mM glycerol to give a final biomass concentration of 60 g.sub.wcw/L. E. coli cells expressing GDH was also harvested and the cell pellet resuspended in MOPS buffer (200 mM, pH 8) to give a final biomass concentration of 30 g.sub.wcw/L. These cell suspensions were treated with 1% wt/v Tween 80 for 10 min at 37° C. to permeabilize the cells. The GDH containing cells were recovered by centrifugation. The CYP containing cell suspension (25 mL) was transferred to a 250 mL Erlenmeyer shake flask and GDH containing cells added to give a final concentration of 1 g.sub.wcw/L of GDH cells. After 10 min the biotransformation reaction was started by the addition of dodecanol (125 μL, 27 mM) and the reaction mixture incubated at 25° C. and 180 rpm. After 15 h GDH containing cells (1 g.sub.wcw/L) as well as glucose (25 mM, 0.3 mL of 2 M solution) and glycerol (25 mM, 0.3 mL of 2 M solution) were added again. The reaction was stopped after 24 h with the addition of ethyl acetate and the total reaction mixture extracted with ethyl acetate (2×25 mL). The final extract (50 mL) contained 17.7 mM dodecanol and 4.5 mM 1,5 dodecanediol.
Experiment 7—Conversion of 1,5-dodecane Diol to δ-dodecalactone and Chiral Analysis of Lactones Produced from Dodecanol and C12 Fatty Acid
(58) To convert 1,5-dodecanediol to δ-dodecalactone 170 μL of an ethyl acetate extract containing 150 mM dodecanol and 5.5 mM 1,5-dodecanediol was placed in a 40 mL vial and the ethyl acetate evaporated by incubating the vial at 100° C. for 5 min. After evaporation of the ethyl acetate the dodecanol/dodecanediol mixture was resuspended in 1 mL of an HLADH expressing culture of E. coli permeabilized with Triton X100 (1% v/v was added and the cell suspension incubated at 37° C. for 10 min). This reaction mixture containing 1 mM of 1,5-dodecanediol and 25 mM of dodecanol was incubated at 30° C. and 180 rpm for 24 h. It was then extracted with ethyl acetate (2×0.5 mL) containing 2 mM undecanol as internal standard and analysed on a VF5 column using a GC with FID detector for quantification and a GC-MS with a Chiraldex G-TA column (30 m×0.25 mm ID) to determine enantioselectivity. The ethyl acetate extract contained 0.2 mM 1,5-dodecanediol and 0.65 mM δ-dodecalactone (R:S ratio 84:16, ee 67%) while the δ-dodecalactone produced from dodecanoic acid in previous experiments using CYP505E3 was an almost racemic mixture (R:S ratio 55:45, ee 10%) (
Experiment 8—Biotransformation of Dodecanol to 1,5-dodecane Diol by a Culture of Wild-Type Aspergillus terreus
(59) Spores of Aspergillus terreus MRC 11081 were inoculated into 100 mL of Potato Dextrose Broth (PDB, 24 g/L) in a 250 mL Erlenmeyer flask, and the flask was incubated for 5 days at 28° C. After this time, 1-dodecanol (1 mL) was added to the culture and incubation continued. Every 24 h triplicate samples (1 mL) of the culture was taken, extracted with ethyl acetate containing 2 mM 1-undecanol (2×0.5 mL) and analysed by Gas Chromatography. The biotransformation reaction was carried out as described above for 6 days.
(60) The ethyl acetate extracts from cultures of the wild-type A. terreus contained a maximum of 4.8 mM 1,5-dodecanediol after 4 days after which the fungus started to consume both the substrate and the product (
Experiment 9—Biotransformation of C12 and C14 Fatty Acids and Fatty Alcohols and C12 Alkane by Different CYP505s Expressed in Pichia pastoris
(61) Different transformants of Pichia pastoris KM71 harbouring the CYP505 genes for SEQ ID Nos 1 to 7 and 9 to 12 were grown and harvested as described above. The harvested cells were resuspended to give 1 g wet cell weight in 40 ml MOPS buffer (200 mM, 100 mM glucose, 100 mM glycerol, pH 8) and 1 ml aliquots of these cell suspensions were transferred to 40 ml amber vials. Dodecane, 1-dodecanol and 1-tetradecanol were added neat to these cell suspensions to give final concentrations of 200 mM in the case of the alcohols and 1 M in the case of dodecane while in the case of dodecanoic acid 20 μl of a 500 mM stock solution (in DMSO) was added to give a final concentration of 10 mM. Transformants harbouring the CYP505 genes for SEQ ID Nos 1 to 4 were tested for the biotransformation of 1-dodecanol, 1-tetradecanol, dodecanoic acid, tetradecanoic acid and dodecane, while those harbouring the CYP505 genes for SEQ ID Nos 5 to 7 and 9 to 12 were only tested for the biotransformation of 1-dodecanol and dodecanoic acid. The biotransformations were incubated at 25° C. and 225 rpm for 24 h. Extractions were performed using ethyl acetate (500 μl×2). The extracts were analyzed using GC/MS.
(62) GC-MS analyses of the methylated samples from the biotransformations of dodecanoic acid by the CYP505s from Aspergillus terreus (CYP505E3, SEQ ID No 1), Aspergillus kawachii (CYP505Ak, SEQ ID No 2), Aspergillus niger (CYP505E1, SEQ ID No 3) and Penicillium expansum (CYP505Pe, SEQ ID No 4) showed that all four these enzymes expressed in P. pastoris produced δ-dodecalactone, 5-hydroxy dodecanoic acid as well as other hydroxylated C12 fatty acids (
(63) GC-MS analyses of unmethylated samples from the biotransformations of dodecanoic acid by the CYP505s from Aspergillus niger (CYP505An, SEQ ID No 5), Penicillium camemberti (CYP505Pc, SEQ ID No 6), Penicillium freii (CYP505Pf, SEQ ID No 7), Aspergillus oryzae (CYP505Ao, SEQ ID No 9), Hypocrea virens (CYP505Hv, SEQ ID No 10), Oidiodendron maius (CYP505Om, SEQ ID No 11) and Setosphaeria turcica (CYP505St, SEQ ID No 11) showed that the first three of these enzymes (SEQ ID Nos 5 to 7) expressed in P. pastoris produced δ-dodecalactone, 5-hydroxy dodecanoic acid as well as other hydroxylated C12 fatty acids while the last four (SEQ ID Nos 9 to 12) produced only sub-terminally hydroxylated C12 fatty acids (
(64) GC-MS analyses of the methylated samples from the biotransformations of tetradecanoic acid by CYP505E3 (SEQ ID No 1), CYP505Ak (SEQ ID No 2), CYP505E1 (SEQ ID No 3) and CYP505Pe (SEQ ID No 4) expressed in P. pastoris showed that all four these enzymes expressed in P. pastoris produced only 7-hydroxy tetradecanoic acid (
(65) No standards were available for the diols that could be produced from 1-dodecanol and diol products had to be identified by comparing the expected fragments (
(66) Only CYP505E3 (SEQ ID No 1), CYP505Ak (SEQ ID No 2), CYP505E1 (SEQ ID No 3) and CYP505Pe (SEQ ID No 4) were tested for the biotransformation of 1-tetradecanol and only CYP505E3 and CYP505Ak gave products which were identified as 1,7- and 1,8-tetradecane diol (
(67) Again only CYP505E3 (SEQ ID No 1), CYP505Ak (SEQ ID No 2), CYP505E1 (SEQ ID No 3) and CYP505Pe (SEQ ID No 4) were tested for the biotransformation of dodecane and all four produced 5-dodecanol (
Experiment 10—Determining the Amino Acid Identity Between CYP505s with ω-7 Hydroxylase Activity
(68) The results obtained with the different CYP505s tested for the hydroxylation of C12, C14 and C16 fatty acids, fatty alcohol and alkanes are summarized in Table 2. The amino acid sequences of the 12 different CYP505s were aligned using the MUSCLE algorithm as applied in the Genious 6.0.6 software package and the calculated amino acid identities are displayed in
(69) TABLE-US-00002 TABLE 2 Summary of ω-7 hydroxylase activity in twelve CYP505s tested ω-7 hydroxylase activity.sup.a Seq (in brackets other hydroxylase activities) ID CYP name Organism C12FA.sup.b C14FA.sup.b C16FA.sup.c C12ol.sup.c C14o.sup.b C12alk.sup.b C14alk.sup.c C16alk.sup.c 1 CYP505E3 A. terreus +++ ++++ ++ ++++ ++ +++ ++ + (++++) (++) (++) 2 CYP505Ak A. kawachii +++ ++++ nd ++++ ++ ++ nd nd (+++) (++) (++) 3 CYP505E1 A. niger ++ +++ nd ++++ − + nd nd (++) (++) 4 CYP505Pe P. expansum + +++ nd ++++ − ++ nd nd (+) (+) 5 CYP505An A. niger +++ nd nd ++++ nd nd nd nd (+++) (+) 6 CYP505Pc P. camemberti ++ nd nd ++ nd nd nd nd (++) 7 CYP505Pf P. freii ++ nd nd +++ nd nd nd nd (+) 8 CYP505A1 F. oxysporum − − − nd − nd nd nd (+++) (++) (++) 9 CYP505Ao A. oryzae − nd nd − nd nd nd nd (+++) (++) 10 CYP505Hv H. virens − nd nd − nd nd nd nd (++) (+++) 11 CYP505Om O. maius − nd nd − nd nd nd nd (+++) (++) 12 CYP505St S. turcica − nd nd − nd nd nd nd (+) (++) .sup.aActivity levels estimated from peak heights of relative abundance (RA) on GC-MS chromatograms ++++ RA > 10 million; +++ 1 million < RA < 10 million; ++ 0.1 million < RA < 1 million; + − 0.01 million < RA < 0.1 million; − no significant activity detected; nd not tested .sup.bAll enzymes except CYP505A1 tested with whole cells of P. pastoris, which was only tested in CFEs of E. coli .sup.cOnly tested with CFE of E. coli
Experiment 11—Biotransformation of C12 and C14 Fatty Acids to δ-dodecalactone by CYP505An Expressed in Pichia pastoris
(70) A transformant of Pichia pastoris KM71 harbouring the gene encoding CYP505An (SEQ ID No 5) was grown and harvested as described above. The harvested cells were resuspended 1 g in 40 ml of MOPS (200 mM, 100 mM glucose, 100 mM glycerol, pH 8) buffer and 12.5 ml was transferred to each of two 500 ml Erlenmeyer flask. One flask was supplemented with 250 μl of a 500 mM dodecanoic acid stock solution (in DMSO) and the other flask with 250 μl of a 500 mM tetradecanoic acid stock solution (in DMSO) to give in each case a final concentration of 10 mM of the fatty acid. The flasks were incubated at 25° C. and 180 rpm for 12 h. Samples (800 μl) were withdrawn after 12 h and 136 μl of 5 M HCl added. These samples were stored for 5-6 h at room temperature to convert hydroxy fatty acids to the lactone. These were then extracted with ethyl acetate (2×800 μl). GC-MS analyses of these extracts showed that δ-dodecalactone was produced from both dodecanoic acid and tetradecanoic acid (
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