UNSPECIFIC PEROXYGENASE ENZYME VARIANTS FOR SELECTIVE FATTY ACID EPOXIDATION OR HYDROXYLATION

Abstract

The invention relates to a recombinant Marasmius rotula unspecific peroxygenase (rMroURO) and two mutants thereof, wherein said mutants show enhanced selectivity towards either the epoxidation or the (sub)terminal ω/(ω-1)-hydroxylation of unsaturated fatty acids. The invention also refers to the use of these enzyme variants for the specific epoxidation or hydroxylation of fatty acids such as oleic acid, linoleic acid and/or alpha-linolenic acid.

Claims

1. A recombinant peroxygenase enzyme that comprises the amino acid sequence shown in SEQ ID NO: 1, wherein said enzyme is encoded by a polynucleotide sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 9, preferably by the polynucleotide sequence shown in SEQ ID NO: 9.

2. The enzyme according to claim 1, which consists of the amino acid sequence shown in SEQ ID NO: 1.

3. The enzyme according to any of claim 1 or 2, which comprises the amino acid substitution I153T.

4. The enzyme according to any of claim 1 or 2, which comprises the amino acid substitutions I153F and S156F.

5. A polynucleotide sequence that encodes the enzyme according to any of claims 1 to 4.

6. A genetic construct that comprises the polynucleotide sequence according to claim 5.

7. A host cell that comprises the polynucleotide sequence according to claim 5 or the genetic construct according to claim 6.

8. The host cell according to claim 7 which is an Escherichia coli cell.

9. Use of the host cell according to any of claim 7 or 8 for producing the enzyme according to any of claims 1 to 4.

10. Use of the enzyme according to any of claim 1 or 2 for the epoxidation and hydroxylation of unsaturated fatty acids.

11. Use of the enzyme according to claim 3 for the epoxidation of unsaturated fatty acids.

12. Use of the enzyme according to claim 11, wherein the unsaturated fatty acids are polyunsaturated fatty acids.

13. Use of the enzyme according to claim 4 for the terminal and sub-terminal hydroxylation of unsaturated fatty acids.

14. Use according to any of claims 10 to 13, wherein the unsaturated fatty acids are selected from the list consisting of: oleic acid, linoleic acid, alpha-linolenic acid or any combination thereof.

Description

DESCRIPTION OF THE DRAWINGS

[0064] FIG. 1. Chromatogram that shows the products (and remaining substrate, underlined) of the reaction of rMroUPO (0.2 μM) with oleic acid (cis-9-octadecenoic acid; 0.1 μM) and H.sub.2O.sub.2 (2,500 μM), performed in 50 mM phosphate, pH 5.5, at 30° C. for 30 min, analyzed by gas chromatography-mass spectrometry.

[0065] FIG. 2. Chromatogram that shows the products (and remaining substrate, underlined) of the reaction of rMroUPO I153T mutated variant (0.2 μM) with oleic acid (cis-9-octadecenoic acid; 0.1 μM) and H.sub.2O.sub.2 (2,500 μM), performed in 50 mM phosphate, pH 5.5, at 30° C. for 30 min, analyzed by gas chromatography-mass spectrometry.

[0066] FIG. 3. Chromatogram that shows the products (and remaining substrate, underlined) of the reaction of rMroUPO I153F/S156F mutated variant (0.2 μM) with oleic acid (cis-9-octadecenoic acid; 0.1 μM) and H.sub.2O.sub.2 (2,500 μM), performed in 50 mM phosphate, pH 5.5, at 30° C. for 30 min, analyzed by gas chromatography-mass spectrometry.

[0067] FIG. 4. Chromatogram that shows the products (and remaining substrate, underlined) of the reaction of rMroUPO (0.6 μM) with linoleic acid (cis,cis-9,12-octadecadienoic acid; 0.1 μM) and H.sub.2O.sub.2 (1,250 μM), performed in 50 mM phosphate, pH 5.5, at 30° C. for 30 min, analyzed by gas chromatography-mass spectrometry.

[0068] FIG. 5. Chromatogram that shows the products (and remaining substrate, underlined) of the reaction of rMroUPO I153T mutated variant (0.6 μM) with linoleic acid (cis,cis-9,12-octadecadienoic acid; 0.1 μM) and H.sub.2O.sub.2 (1,250 μM), performed in 50 mM phosphate, pH 5.5, at 30° C. for 30 min, analyzed by gas chromatography-mass spectrometry.

[0069] FIG. 6. Chromatogram that shows the products (and remaining substrate, underlined) of the reaction of rMroUPO (0.2 μM) with α-linolenic acid (cis,cis,cis-9,12,15-octadecatrienoic acid; 0.1 μM) and H.sub.2O.sub.2 (1,250 μM), performed in 50 mM phosphate, pH 5.5, at 30° C. for 30 min, analyzed by gas chromatography-mass spectrometry.

[0070] FIG. 7. Chromatogram that shows the products (and remaining substrate, underlined) of the reaction of rMroUPO I153T mutated variant (0.2 μM) with α-linolenic acid (cis,cis,cis-9,12,15-octadecatrienoic acid; 0.1 μM) and H.sub.2O.sub.2 (1,250 μM), performed in 50 mM phosphate, pH 5.5, at 30° C. for 30 min, analyzed by gas chromatography-mass spectrometry.

[0071] FIG. 8. Purification of rMroUPO heterologously expressed in E. coli as soluble active enzyme. (A) Mono-S chromatography showing 280 nm (dashed line) and 410 nm (continuous line) absorbance profiles and NaCl gradient (dotted line). (B) Sodium dodecylsulfate-polyacrylamide gel electrophoresis of purified rMroUPO (lane b) compared with molecular-mass standards (lane a).

EXAMPLES

Example 1: Recombinant MroUPO Enzyme (rMroUPO) and its Mutated Variants

[0072] To investigate substrate epoxidation, the most frequent positions of oleic acid at the MroUPO heme-access channel were predicted by inspection of the crystal structure (Protein Data Bank entries 5FUJ and 5FUK). Then, mutations in neighbor residues were designed aiming at modulating the enzyme epoxidation vs hydroxylation ratio. Both a recombinant MroUPO (rMroUPO) and mutated variants thereof were expressed in Escherichia coli as active enzymes, and their action on oleic and other fatty acids was investigated by gas chromatography-mass spectrometry.

[0073] A recombinant MroUPO (rMroUPO) and its amino acid substitution I153S, I153T, I153V and I153F/S156F mutants were expressed as soluble recombinant proteins in E. coli. In short, cells were lysed by addition of lysozyme and sonication, and debris was removed from the soluble fraction by ultracentrifugation. Three chromatographic steps—two anionic at pH 7.0 and one final cationic at pH 4.0—were used to obtain pure enzymes. The last purification step yielded electrophoretically homogeneous enzyme (as illustrated for rMroUPO in FIG. 8).

[0074] The rMroUPO substitution mutants were prepared using the Expand Long Template PCR kit from Roche (Basel, Switzerland) for site-directed mutagenesis. PCR reactions were run using the following DNA oligos harboring the desired mismatches (underlined nucleotides in bold triplets): i) I153S mutation: 5′-CCGATTTAACTGCGACTCCGCTCTTCAGAATCTG-3′ (SEQ ID NO: 5); ii) I153T mutation: 5′-CCGATTTAACTGCGACTACCGCTCTTCAGAATCTG-3′ (SEQ ID NO: 6); iii) I153V mutation: 5′-CCGATTTAACTGCGACTTCCGCTCTTCAGAATCTG-3′ (SEQ ID NO: 7); and iv) I153F/S156F mutation: 5′-CCGATTTAACTGCGACTTCCGCTCTTGAATCTGCG-3′ (SEQ ID NO: 8), along with their reverse complementary counterparts.

[0075] The PCR reactions (50 μl volume) were carried out in an Eppendorf (Hamburg, Germany) Mastercycler pro-S using 30 ng of template DNA, 500 μM each dNTP, 125 ng forward and reverse primers, 5 units of Expand Long Template PCR System polymerase mix (Roche), and the manufacturer buffer. Reaction conditions included: i) initial denaturation step of 1 min at 95° C.; ii) 22 cycles of 30 s at 95° C., 30 s at 60° C., and 7 min at 68° C., each; and iii) final elongation step of 7 min at 68° C. The mutated upo genes were expressed in E. coli as described above.

Example 2: Oleic Acid Reactions with rMroUPO Mutated Variants

[0076] Simple (I153S, I153T and I153V) and double (I153F/S156F) mutations-which could potentially improve or abolish, respectively, the epoxidation ability of MroUPO-were experimentally introduced by site-directed mutagenesis and the mutated genes were transformed into E. coli as explained in Example 1. However, only I153T and I153F/S156F could be obtained as soluble active enzymes.

[0077] Interestingly, a small modification of the channel shape in the I153T variant increased the ratio between stearic acid epoxide and its additionally hydroxylated derivatives. A fully opposite effect was attained with the double I153F/S156F variant that completely abolished the MroUPO ability to epoxidize oleic acid.

Example 3: Reaction of the rMroUPO with Oleic Acid (Cis-9-Octadecenoic Acid)

[0078] rMroUPO (SEQ ID NO: 1) was expressed using the protocol described in Example 1, after transformation of the pET23b plasmid harbouring the mroupo gene into Escherichia coli BL21 C41 cells. The said enzyme was obtained as a soluble active protein, and purified to electrophoretic homogeneity through several ion-exchange chromatographic steps.

[0079] The rMroUPO (0.2 μM) was incubated with oleic acid (100 μM) and H.sub.2O.sub.2 (2,500 μM) at 30° C. in 50 mM phosphate, pH 5.5, for 30 min. Oleic acid had been previously dissolved in acetone so that the final acetone concentration attained 20% (v/v) in the embodiment. After the incubation time, the products were recovered by liquid-liquid extraction with t-butyl-methyl-ether. The organic solvent was removed under N.sub.2 current. N,O-Bis-(trimethylsilyl)trifluoroacetamide was employed to prepare the trimethylsilyl derivatives of the compounds to be separated by gas chromatography and identified by mass spectrometry.

[0080] Chromatographic analyses were carried out in a gas chromatograph coupled to a mass-spectrometry detector. The column used was a fused-silica DB-5HT capillary column (30 m×0.25 mm internal diameter x 0.1 μm film thickness). Oven was heated from 120° C. (1 min) to 300° C. (5 min) at 5° C..Math.min. Injection was performed at 300° C. and transfer line was maintained at 300° C. Compounds were identified by comparison of their mass spectra with those of authentic standards and those from the NIST and Wiley libraries as well as by mass fragmentography. Quantification of the products was carried out by integrating the total ion peak areas, using external standard curves of the same or closely related compounds.

[0081] A chromatogram of the products obtained in the embodiment is illustrated in FIG. 1. rMroUPO produces the epoxide between C.sub.9-C.sub.10, as well as the (ω-7) and (ω-1) hydroxylated derivatives of the said epoxide, labelled as (ω-7)-OH and (ω-1)-OH, respectively. Conversion of the substrate (oleic acid) into substrates attained 94%, being 40% of the products only epoxides and 57% other (hydroxylated) epoxides. Therefore, the selectivity towards only epoxidation is of 0.67 (estimated as the ratio between only epoxides and other oxygenation products).

Example 4: Reaction of the rMroUPO I153T Mutated Variant with Oleic Acid (Cis-9-Octadecenoic Acid)

[0082] The rMroUPO I153T mutated variant (SEQ ID NO: 2), in which isoleucine 153 was replaced by threonine, was constructed by site-directed mutagenesis, in a PCR reaction in which the following oligos—complementary to the regions of the gene to be mutated, but bearing the desired mismatches—were used as primers: 5′-CCGATTTAACTGCGACTACCCGCTCTTCAGAATCTG-3′ (SEQ ID NO: 6), along with its reverse complementary counterpart. The PCR reactions were performed in a thermocycler, adding 30 ng of template DNA—plasmid pET23b with the mroupo gene as an insert-, 500 μM each dNTP, 125 ng primers, 5 units of polymerase mix, and buffer to a final volume of 50 μl. Reaction conditions were as follows: i) initial denaturation step of 1 min at 95° C.; ii) 22 cycles of 30 s at 95° C., 30 s at 60° C., and 7 min at 68° C., each; and iii) final elongation step of 7 min at 68° C. PCR products were treated with restriction enzyme Dpnl at 37° C. for 1 h in order to degrade parental (non-mutated) DNA.

[0083] The mutated DNA was transformed into Escherichia coli DH5a cells to propagate the DNA obtained. Mutation was confirmed by sequencing the gene encoding the protein using the T7 promoter primers.

[0084] Expression and purification of the enzyme was carried out as described in Example 3 for rMroUPO.

[0085] Reactions with oleic acid and identification and quantification of the products were conducted as explained in Example 3.

[0086] As depicted in FIG. 2, reaction of the rMroUPO I153T mutated variant with oleic acid gave rise to the same products than rMroUPO, although with very different relative abundances. Conversion into products reached 85%, constituting only epoxides the 72% and other (hydroxylated) epoxide derivatives 17%. Therefore, the selectivity towards only epoxidation (defined in Example 3) attained a 2.67 value.

Example 5: Reaction of the rMroUPO I153F/S156F Mutated Variant with Oleic Acid (Cis-9-Octadecenoic Acid)

[0087] The rMroUPO I153F/S156F mutated variant (SEQ ID NO: 3), in which isoleucine 153 and serine 156 were both replaced by phenylalanine residues, was constructed by site-directed mutagenesis, in a PCR reaction in which the following oligos—complementary to the region of the gene to be mutated, but bearing the desired mismatches—were used as primers: 5′-CCGATTTAACTGCGACTTTCCGCTCTTTCGAATCTGCG-3′ (SEQ ID NO: 8), along with its reverse complementary counterpart. PCR reaction and sequencing of the mutated variant were carried out as described in Example 4. Expression, purification of the enzyme, reactions with oleic acid and GC-MS identification and quantification of the products were conducted as in Example 3.

[0088] FIG. 3 depicts a chromatogram of the products obtained, which were restricted to ω-1 and ω hydroxylated, keto and carboxylic species derived from oleic acid, all resulting from the hydroxylation (and re-hydroxylation) of the oleic acid. No traces of epoxidized species were detected. Therefore, the selectivity of this MroUPO I153F/S156F mutated variant is total towards hydroxylation.

Example 6: Reaction of the rMroUPO with Linoleic Acid (Cis,Cis-9,12-Octadecadienoic Acid)

[0089] Expression and purification of rMroUPO (SEQ ID NO: 1) were carried out as described in Example 3.

[0090] An embodiment was designed in which rMroUPO (0.6 μM) was incubated with linoleic acid (100 μM, previously dissolved in acetone so that the final acetone concentration in the embodiment was of 20%) and H.sub.2O.sub.2 (1,250 μM) at 30° C. in 50 mM phosphate, pH 5.5, for 30 min. Identification and quantification of the products was conducted as detailed in Example 3.

[0091] FIG. 4 illustrates the chromatogram of the products of the said embodiment. The main products were di-epoxides (anti and syn isomers) and hydroxylated mono- and di-epoxides. Conversion attained 98%, of which 21% were only epoxides, and 79% hydroxylated epoxides. Thus, the selectivity towards only epoxidation was of 0.27.

Example 7: Reaction of the rMroUPO I153T Mutated Variant with Linoleic Acid (Cis,Cis-9,12-Octadecadienoic Acid)

[0092] Construction of the rMroUPO I153T mutated variant (SEQ ID NO: 2) was carried out as described in Example 4. Expression and purification of the mutant was as detailed in Example 3.

[0093] Reactions were performed as in Example 6. Identification and quantification of the products is described in Example 3.

[0094] FIG. 5 depicts a chromatogram of the products obtained in the embodiment. The main products were di-epoxides (anti and syn isomers) and hydroxylated mono- and di-epoxides. Conversion attained 93%, of which 42% were only epoxides, and 58% hydroxylated epoxides. Thus, the selectivity towards only epoxidation was of 0.72, which represents an improvement in the epoxidation selectivity of this variant compared to the rMroUPO (SEQ ID NO: 1).

Example 8: Reaction of the rMroUPO with α-Linolenic Acid (Cis,Cis,Cis-9,12,15-Octadecatrienoic Acid)

[0095] Expression and purification of rMroUPO (SEQ ID NO: 1) was carried out as described in Example 3.

[0096] Reactions were performed in an embodiment consisting of rMroUPO (0.2 μM), incubated with α-linolenic acid (100 μM) and H.sub.2O.sub.2 (1,250 μM) at 30° C., in 50 mM phosphate, pH 5.5, for 30 min. Extraction and separation of the reaction products was carried out as described in Example 3. Since no available commercial standards of epoxidized α-linolenic acid were available, they were chemically synthesised as follows: a solution of peracetic acid (1.8 mmol, 3.6 equiv) and NaOAc (0.7 mmol; 1.4 equiv) was added to α-linolenic acid (0.5 mmol) using a syringe pump at 0° C. for 1 h. The mixture was stirred at 0° C. for an additional h. Products were recovered by liquid-liquid extraction with t-butyl-methyl-ether, resulting in a mixture of mono- and di-epoxides. Identification of the compounds was made by comparison of the mass spectra with those of the synthesised standards and by mass fragmentography. Quantification was carried out as detailed in Example 3.

[0097] FIG. 6 illustrates a chromatogram of the products obtained in the reaction. The main products included: di-epoxides, other epoxidized derivatives and (ω-7)-OH mono-epoxides. Conversion reached 98%, of which only epoxides were 44%, while other epoxides represented the 56%. Therefore, the selectivity towards only epoxidation was of 0.79.

Example 9: Reaction of the rMroUPO I153T Mutated Variant with α-Linolenic Acid (Cis,Cis,Cis-9,12,15-Octadecatrienoic Acid)

[0098] Construction of the rMroUPO I153T mutated variant (SEQ ID NO: 2), expression and purification of the mutant was carried out as described in Example 4.

[0099] Reactions and analyses of the products were performed as in Example 8. FIG. 7 illustrates a chromatogram of the reaction products, among which single epoxides, di-epoxides and other epoxidized derivatives. Conversion of the substrate attained 97%, of which 92% (of which 88% are di-epoxides) represented only epoxides and a mere 8% other epoxide derivatives. Therefore, the selectivity towards only epoxidation reached 11.50.