SELECTIVE HYDRATION OF OLEIC ACID DERIVATIVES

Abstract

The present invention relates to regio- and stereoselective hydration of oleic acid derivatives, including esters of oleic acid, by action of modified oleate hydratase.

Claims

1. A modified enzyme having fatty acid hydration activity, particularly activity towards hydration of oleic acid derivatives, comprising one of more amino acid substitution(s) at (a) position(s) corresponding to residues selected from 265 and/or 436 and/or 438 and/or 442 in the polypeptide according to SEQ ID NO: 1.

2. A modified enzyme according to claim 1, wherein the oleic acid derivate is a non-natural oleic acid derivative, preferably an oleic acid ester.

3. A modified enzyme according to claim 1, wherein the amino acid substitution is selected from the group consisting of Q265A, T436A, N438A, H442A, and combinations thereof.

4. A modified enzyme according to claim 3, wherein the amino acid substitution is selected from a combination of Q265A/T436A/N438A.

5. A modified enzyme according to claim 1 capable of hydrating an oleic acid derivative with an enantiomeric excess (ee) of at least 98%.

6. A modified enzyme according to claim 1 wherein the hydration activity is at least 2-fold higher compared to a wild-type enzyme.

7. A modified enzyme according to claim 1 capable of catalyzing the hydration of oleic acid derivatives independent of co-factors, preferably independent of FAD or DTT.

8. A process for hydration of oleic acid derivatives using a modified enzyme according to claim 1.

9. A process according to claim 8, wherein the process is conducted in whole cell biotransformation.

10. A process according to claim 8, wherein the modified enzyme is expressed in E. coli.

Description

FIGURES

[0026] FIG. 1. UV-Vis absorption spectra of purified OhyA before (black curve) and after (orange curve) reconstitution of the flavoprotein, as well as after reduction of the FAD cofactor with DTT (blue curves). UV-Vis absorption spectra of wt-OhyA (FIG. 1A). UV-Vis absorption spectra of mutant OhyA Q265A/T436A/N438A (FIG. 1B). For more explanation see text.

[0027] FIG. 2. Conversion of oleamide (FIG. 2A), N-hydroxy oleamide (FIG. 2B), oleyl alcohol (FIG. 2C), OA methyl ester (FIG. 2D), OA ethyl ester (FIG. 2E), OA isopropyl ester (FIG. 2F), OA n-propyl ester (FIG. 2G), OA n-butyl ester (FIG. 2H) by OhyA wild type (WT) and the amino acid exchange variants as whole cell E. coli biocatalysts after over-expression of the enzymes. Control reactions contained either the substrate added to the reaction buffer without cells or the substrate added to an E. coli empty vector control (EVC).

[0028] FIG. 3. Regio- and stereoselective hydration of oleic acid (OA), i.e. substrate (1), and OA derivatives, i.e. substrates (2) to (10), by E. meningoseptica mutant oleate hydratase (OhyA). A whole cell E. coli biocatalyst harboring the over-expressed hydratase was used in the biotransformation assays. For more explanation see text.

[0029] FIG. 4. Conversion of oleic acid, substrate (1), and non-natural oleic acid derived substrates (2) to (10) by OhyA wild type (WT) and OhyA Q265A/T436A/N438A. The reactions were performed for 22 h using a whole cell E. coli biocatalyst upon expressing of the enzymes.

[0030] The following examples are illustrative only and are not intended to limit the scope of the invention in any way.

EXAMPLES

Example 1: General Methods, Strains and Plasmids

[0031] Unless stated otherwise, standard laboratory reagents were obtained from Sigma-Aldrich® (Steinheim, Germany) or Carl Roth GmbH & Co. KG (Karlsruhe, Germany) with the highest purity available. Oleic acid (OA) and esters thereof (methyl, ethyl, i-propyl ester), oleyl alcohol and oleyl amine were purchased from Sigma-Aldrich® (Steinheim, Germany). The OA methyl and ethyl ester and oleyl alcohol were distilled prior to use to a purity >90% according to GC-FID analysis.

[0032] Molecular cloning of the expression vector was performed according to standard procedures (Ausubel et al., Current Protocols in Molecular Biology, 2003). and correct integration of the insert was confirmed by sequencing (LGC Genomics, Berlin, Germany). For gene amplification, Phusion® High Fidelity DNA polymerase (Thermo Fisher Scientific Inc., St. Leon-Rot, Germany) was utilized in accordance with the recommended PCR protocol. A codon-optimized gene variant of OhyA (Elizabethkingia meningoseptica XP_001209325 oleate hydratase) was purchased from DNA2.0 (Menlo Park, Calif.). For expression of recombinant OhyA, a modified pMS470 expression vector, pMS470-HISTEV-OhyA was constructed. For all cloning steps and plasmid replication, E. coli Top10 F′ (F[lacI.sup.q Tn10(tetR)] mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR nupG recA1 araD139 Δ(ara-leu)7697 galU galK rpsL(Str.sup.R) endA1λ.sup.−) from Life technologies (Vienna, Austria) was used. Recombinant OhyA was expressed in E. coli BL21Star™ (DE3) (F- ompT hsdS.sub.B (r.sub.B.sup.−m.sub.B.sup.−) gal dcm rne131 (DE3)) (Life technologies, Vienna, Austria).

[0033] The protein sequence of OhyA was compared to the amino acid sequences in the Hydratase Engineering Database (HyED), in which a total of 2046 sequences are collected. Since OhyA is categorized in homologous family 11 (HFam11) of the HyED, all amino acid sequences from HFam11 were selected for the multiple sequence alignment. Sequences were extracted from the database for a multiple sequence alignment with the Clustal Omega sequence alignment tool using default settings as described in Sievers et al. (Mol. Syst. Biol. 7, 539, 2011), and were visualized with the UGene software. The positions of highly conserved residues which were used for construction of the OhyA mutants are listed in Table 1.

TABLE-US-00001 TABLE 1 multiple sequence alignment of amino acid sequences from HFam11 collected in the hydratase engineering database (HyED), highlighting the conserved residues involved in binding of a carboxylate. Note that H442 is conserved among all but one member, where it is substituted with a Q. For more explanation see text. Elizabethkingia meningoseptica Q265 T436 N438 H442 Methylobacterium extorquens Q257 T428 N431 H435 Methylobacterium sp. MB200 Q257 T428 N431 H435 Bradyrhizobium elkanii Q256 T427 N429 H433 Bradyrhizobium sp DFCI-1 Q256 T427 N429 H433 Stenotrophomonas maltophilia Q261 T432 N434 H438 Stenotrophomonas rhizophila Q261 T432 N434 H438 Sphingobium yanoikuyae Q263 T434 N436 H440 Acinetobacter ursingii Q265 T436 N438 H442 Idiomarina loihiensis Q263 T434 N436 H440 Pseudomonas pelagia Q263 T434 N436 H440 Sphingomonas ssp. NM-1 Q255 T426 N428 H432 Haematobacter missouriensis Q259 T430 N432 H436 Rhodopseudomonas palustris Q260 T431 N433 H437 Aurelmonas altamirensis Q256 T427 N429 H433 Pseudoalteromonas haloplanktis Q255 T426 N428 H432 Paracoccus ssp. 5503 Q257 T428 N430 H434 Paracoccus ssp. 10990 Q257 T428 N430 H434 Paracoccus aminophilus Q253 T424 N426 H430 Marinomonas prodfundimaris Q263 T434 N436 H440 Marinomonas posidonia Q263 T434 N436 H440 Bermanella marisrubri Q258 T429 N431 H435 Pleomorphomonas koreensis Q256 T427 N429 H433 Comamonas testosteroni Q262 T433 N435 H439 Enhydrobacter aerosaccus Q263 T434 N436 Q440 Hyphomonas beringensis Q263 T434 N436 H440 Hyphomonas adhaerens Q263 T434 N436 H440 Gluconobacter oxydans Q260 T431 N433 H437 Mesonia mobilis Q265 T436 N438 H442 Sphingobacterium sp. Ag1 Q265 T436 N438 H442 Flavobacterium psychrophilum Q265 T436 N438 H442 Flavobacterium hibernum Q265 T436 N438 H442 Flavobacterium hydatis Q265 T436 N403 H407 Sulfurospirillum multivorans Q268 T439 N441 H445 Chryseobacterium sp. JM1 Q265 T436 N438 H442 Chryseobacterium taiwanense Q265 T436 N438 H442 Chryseobacterium soli Q265 T436 N438 H442 Chryseobacterium vrystaatense Q265 T436 N438 H442 Halomonas ssp. TD01 Q270 T441 N443 H447 Oleispira antarctica Q245 T416 N418 H422 Marinobacter sp. HL-58 Q270 T441 N443 H447 Marinobacter santoriniensis Q270 T441 N443 H447 Marinobacter excellens Q270 T441 N443 H447 Psychroflexus torquis Q265 T436 N438 H442 Olivibacter sitiensis Q265 T436 N438 H442 Cellulophaga algicola Q265 T436 N438 H442 Myroides odoratimus Q265 T436 N438 H442 Novosphingobium sp. PP1Y Q263 T434 N436 H440 Thalassolituus oleivorans Q263 T434 N436 H440 Frateuria aurantia Q259 T430 N432 H446 Ochrobactrum rhizosphaerae Q260 T431 N433 H447

[0034] For construction of OhyA mutants, the following primers were used according to the manufacturer's manual (Stratagene QuikChange™ site-directed mutagenesis protocol). 25 μL of two separate PCR reactions containing forward and reverse primers, respectively, were prepared (Table 2). After 5 cycling steps, PCR reactions were combined, and PCR was continued for 20 additional cycles according to the manual. Mutated plasmids were verified by DNA sequencing of the coding regions of the constructs.

TABLE-US-00002 TABLE 2 primers used for construction of OhyA mutants. The bold letters indicate the mutated codon. Primer/ SEQ sequence name sequence 5′ to 3′ ID NO: Fw(OhyA_Q265A) GTTTCCGAAGTACAATGC  3 ATATGACACGTTTGTC Rv(OhyA_Q265A) GACAAACGTGTCATATGC  4 ATTGTACTTCGGAAAC Fw(OhyA_T436A) TGGTTGATGAGCTTTGCG  5 TGCAATCGCCAGCCG Rv(OhyA_T436A) CGGCTGGCGATTGCACGC  6 AAAGCTCATCAACCA Fw(OhyA_N438A) GATGAGCTTTACCTGCGC  7 ACGCCAGCCGCATTTCC Rv(OhyA_N438A) GGAAATGCGGCTGGCGTG  8 CGCAGGTAAAGCTCATC Fw(OhyA_H442A) CTGCAATCGCCAGCCGGC  9 CTTCCCGGAGCAGCCGG Rv(OhyA_H442A) CCGGCTGCTCCGGGAAGG 10 CCGGCTGGCGATTGCAG Fw(OhyA_T436A/ GATGAGCTTTGCGTGCGC 11 N438A) ACGCCAGCCGCATTTCC Rv(OhyA_T436A/ GGAAATGCGGCTGGCGTG 12 N438A) CGCACGCAAAGCTCATC

[0035] For purification of the recombinant OhyA, cell pellets were resuspended in 50 mM HEPES, pH 7.4, containing 10 mM imidazole. Cells were lysed by ultrasonication for 4 min with a Sonifier® 250 (Branson, Danbury, Conn.) setting the duty cycle to 80% and the output control to level 8. Cell free extract (CFE) was separated from the total cell lysate (TCL) by centrifugation for 35 min at 48,300×g and 4° C., and was filtered through 0.22 μm filters (Millipore, Bedford, Mass.) prior to loading it onto a pre-equilibrated self-packed Ni-NTA affinity chromatography column (GE Healthcare, United Kingdom). Prior to any further analyses, purified OhyA was incubated with a 10-fold molar excess of FAD over night at 4° C. Unbound FAD was removed from the protein aliquot by buffer exchange via PD-10 desalting columns (GE Healthcare, United Kingdom) according to the recommended protocol.

[0036] UV-Visible (UV-Vis) absorption spectra of wt-OhyA and mutant OhyA Q265A/T436A/N438A were recorded at a spectral range from 250 nm to 1,000 nm on a Specord 205 double-beam spectrophotometer (Analytik Jena AG, Germany) using quartz cuvettes with a path length of 1 cm. Spectral measurements were performed in 50 mM HEPES, pH 7.4, containing 50 mM NaCl. The binding of FAD to purified OhyA was determined by calculating the concentration of the protein based on the ϵ.sub.280 of 111,115 M.sup.−1 cm.sup.−1 for FAD-loaded enzyme, and the previously measured ϵ.sub.480 of 8,074 M.sup.−1 cm.sup.−1 of FAD non-covalently linked to the OhyA structure.

[0037] Free fatty acids were identified and analyzed by gas chromatography-mass spectrometry (GC-MS) and comparison of derived mass fragmentation spectra to authentic standards. A HP-5 column (crosslinked 5% Ph-Me Siloxane; 30 m length, 0.25 mm in diameter and 0.25 μm film thickness) on a Hewlett-Packard 6890 Series II GC equipped with a mass selective detector was used. Sample aliquots of 1 μL were injected in split mode (split ratio 30:1) at 240° C. injector temperature and 290° C. detector temperature with N.sub.2 as carrier at a flow rate set to 36 cm s.sup.−1 in constant flow mode. The temperature program was as follows: 100° C. for 1 min, 15° C. min.sup.−1 to 300° C., hold for 5 min. The total run time was 19.33 min. The mass selective detector was operated in a mass range of 50-400 amu at an electron multiplier voltage of 1765 V. Results were evaluated with the GC-MS Data Analysis software (Agilent Technologies, Austria).

Example 2: Whole Cell Biotransformation of OhyA Mutants Expressed in E. coli

[0038] OhyA was recombinantly expressed in E. coli. First, a pre-culture was inoculated with E. coli BL21 Star (DE3) cells harboring pMS470-HISTEV-OhyA wild type enzyme or variants, and was grown in LB supplemented with 100 μg mL.sup.−1 ampicillin at 28° C. and 130 rpm overnight. Main cultures were inoculated to an OD.sub.600 of 0.1 in auto induction medium (AIM)—Terrific Broth Base including Trace elements (Formedium, UK) containing 100 μg mL.sup.−1 ampicillin. Recombinant protein was expressed at 28° C. and 130 rpm for 22 h. Cells were harvested by centrifugation for 10 min at 4,400×g and 22° C. and were instantly used for whole cell biotransformation or were frozen at −20° C. until protein purification.

[0039] For bioconversion assays of oleic acid (OA) and OA derivatives, i.e. substrates (1) to (10), 50 OD.sub.600 units of thus prepared cells, which are corresponding to a cell dry weight of 50 mg, were resuspended in 50 mM HEPES, pH 6.0, supplemented with 100 mM glucose and 0.2 mM FAD in Pyrex® glass culture tubes (Corning, N.Y.). Biotransformation at 1 mL scale were started by adding substrate to a final concentration of 2 mM from an ethanolic stock solution (100 mM). n-pentadecanoic acid (1 mM) was used as internal standard. The reactions were conducted in the presence of 2% (v/v) of ethanol as co-solvent at 30° C. and shaking at 150 rpm at a defined angle of the Pyrex® tubes (55°). Biotransformation were performed for 22 h or 96 h. The assays were quenched by acidification to pH 2.0 with 0.12 M HCl, and fatty acid derivatives were extracted twice with 2 mL of ethyl acetate while agitating on a Vibrax VXR basic shaker (IKA, Germany) for 30 min. The suspension was centrifuged for 5 min at 2,900×g and 22° C. to improve the separation of the phases. Combined organic phases were concentrated under a N.sub.2 stream. Fatty acid derivatives were silylated with 10 μL of pyridine and 50 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). After incubation for 30 min at 500 rpm, extracts were diluted with 200 μL of ethyl acetate and analyzed by GC-MS.

Example 4: Reduction of the FAD Cofactor and Anaerobic In Vitro Conversions

[0040] For reduction of the enzyme-bound flavin cofactor, wt-OhyA and mutant Q265A/T436A/N438A were diluted in 50 mM HEPES, pH 7.4, containing 50 mM NaCl, 5 mM EDTA, 1 μM 5-deazariboflavin and 4 μM methyl viologen to give an absorption of approximately 0.2 AU at 440 nm. The solution was transferred to a cuvette and oxygen was removed by incubation for at least 2 h in a glove box. Afterwards, the cofactor was reduced by anaerobic photoreduction or chemical reduction with dithiothreitol (DTT). For anaerobic photoreduction, the FAD cofactor was reduced for 150 min by irradiation with a conventional LED (Luminea NC-691; Pearl GmbH, Buggingen, Germany; 10 W, 5400 K). During photoreduction, the cuvette was cooled to 10° C. to compensate for the heat generated by the light source. For chemical reduction, a 100-fold molar excess of DTT was added to the samples. Activity assays with reduced and oxidized OhyA were performed under anaerobic conditions. Assay reactions contained 0.15 mg mL.sup.−1 of reduced or oxidized OhyA and 2 mM of substrates (1), (6) and (7) at a 1 mL scale in 50 mM HEPES, pH 6.0, in presence of 2% (v/v) of ethanol. Conversions of OA, i.e. substrate (1), were incubated for 10 min at 21° C. under manual shaking in a glove box using n-pentadecanoic acid as internal standard. Reactions with OA esters, substrates (6) and (7) were incubated over night at 25° C. and 150 rpm at a 55° angle in Pyrex® glass culture tubes. The reactions were then stopped for GC-MS analyses conducted as described herein.

Example 4: Hydration of OA Derivatives in a Semi-Preparative Scale with E. coli Whole Cells

[0041] OA derivatives, i.e. substrates (3) to (10) were hydrated in a semi-preparative scale. 20-150 mg of non-physiological substrates were converted in 1 mL scale whole cell bioconversions. Each reaction contained 200 mg of E. coli cells in Pyrex® glass culture tubes after over-expression of OhyA Q265A/T436A/N438A, resuspended in 50 mM HEPES, pH 6.0, containing 100 mM glucose and 0.2 mM FAD. Biotransformation were incubated for 96 h at 30° C. and 150 rpm at a defined angle of the Pyrex® tubes (55°). After quenching by acidification to pH 2.0 with 0.12 M HCl, the suspensions were extracted with ethyl acetate (3×2 mL for 30 min) with intermittent centrifugation for 5 min at 2,900×g and 22° C. to improve the phase separation. The organic phases were quantitatively collected and concentrated under a stream of N.sub.2. The results are shown in Table 3.

TABLE-US-00003 TABLE 3 Isolated yield of OA derivatives and hydrated products from E. coli cell extracts. Starting Starting material, Hydrated product, Entry material/mg recovered/mg isolated/g 3 20 4 60 5 20 7.8 6.3 6 80 62.4 5.4 7 80 60.9 10.4 8 80 59.1 1.6 9 60 10 150

[0042] Bioconversions of substrates (1) to (10) were performed with whole E. coli cells—an E. coli empty vector control (EVC) and a biotransformation with cells after over-expression of OhyA are overlaid—with substrate (1) being oleic acid, substrate (3) being oleamide, substrate (4) being N-hydroxy oleamide to be converted into N,10-dihydroxyoctadecanamide, substrate (5) being oleyl alcohol to be converted into 1,10-octadecanediol, substrate (6) being OA methyl ester (methyl oleate) to be converted into 10-hydroxy octadecanoic acid methyl ester, substrate (7) being OA ethyl ester (ethyl oleate) to be converted into 10-hydroxy octadecanoic acid ethyl ester, substrate (8) being OA isopropyl ester (i-propyl oleate) to be converted into 10-hydroxy octadecanoic acid isopropyl ester, substrate (9) being OA n-propyl ester (n-propyl oleate) to be converted into 10-hydroxy octadecanoic acid n-propyl ester, and substrate (10) being OA n-butyl ester (n-butyl oleate) to be converted into 10-hydroxy octadecanoic acid n-butyl ester (not shown). The OA-derived hydroxamic acid, i.e. substrate (4), and the hydrated reaction product were both detected as the respective isocyanates after a Lossen rearrangement occurring under GC-MS analysis conditions. Moreover, conversion of substrate (4) with the E. coli EVC and the strain expressing OhyA led to the unexpected formation of oleamide, i.e. substrate (3), with a subsequent hydration to 10-hydroxy octadecanamide only in OhyA biotransformation. Since the substrate (4) was initially oleamide-free, one must assume that the oleamide was formed by degradation of the substrate (4) in E. coli. Conversion rates using substrates (3) to (10) are shown in FIG. 2. Numbers normalized for biomass of whole cell E. coli biocatalysts are shown in Table 4.

TABLE-US-00004 TABLE 4 Apparent hydration activity normalized for biomass of whole cell E. coli biocatalysts harboring OhyA wild type (WT) and mutant OhyA Q265A/T436A/N438A enzymes for the regio- and stereoselective hydration of oleic acid and derivatives thereof. mU g.sup.−1 CDW OhyA OhyA Abs. conf. e.e. Entry WT Q265A/T436A/N438A at C-10 [%] 1 26.3 ± 0.2  28.4 ± 0.3  R >99 2 — — R >99 3 11.1 ± 0.7  10.7 ± 0.3  R >99 4 3.4 ± 0.7 13.4 ± 2.5  R >99 5 8.4 ± 2.1 15.5 ± 1.0  R >99 6 0.3 ± 0.1 2.5 ± 0.2 R >99 7 0.2 ± 0.1 1.7 ± 0.1 R >99 8 0.1 ± 0.1 1.5 ± 0.1 R >99 9 0.2 ± 0.1 0.9 ± 0.1 R >99 10 0.1 ± 0.1 0.8 ± 0.1 R >99

Example 5: Hydration of OA Derivatives in a Semi-Preparative Scale with Cell Free Extracts (CFE)

[0043] In vitro activity assays were performed with 2 mg of E. coli CFE after recombinant protein expression in Pyrex® glass culture tubes. CFE was incubated with 2 mM substrates (1) to (10) in 1 mL of 50 mM HEPES, pH 6.0, and 2% (v/v) of ethanol. Assays were shaken over night at 25° C. and 150 rpm in the presence of 1 mM n-pentadecanoic acid as internal standard. Conversions were quenched and fatty acids were extracted and derivatized as described in Example 2.

[0044] Anaerobic in vitro hydration reactions of substrate (1) and OA esters, substrates (6) and (7), were performed with purified wt-OhyA and mutant OhyA Q265A/T436A/N438A after reduction of the FAD cofactor. Reactions containing substrate (1) were quenched after 10 min, and reactions containing substrate (6) or (7) were quenched after overnight incubation (not shown). In conversions of substrate (6), we observed a peak at the expected retention time of the hydrated product after incubation with authentic OA standard, wt-OhyA and mutant OhyA Q265A/T436A/N438A only in the case of the variant as proven by GC-MS analysis (not shown).