WO2013076258A2 - Mutants p450 bm3 et leur utilisation pour l'hydroxylation régio-et stéréosélective d'αlpha- et βeta-ionone - Google Patents

Mutants p450 bm3 et leur utilisation pour l'hydroxylation régio-et stéréosélective d'αlpha- et βeta-ionone Download PDF

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WO2013076258A2
WO2013076258A2 PCT/EP2012/073490 EP2012073490W WO2013076258A2 WO 2013076258 A2 WO2013076258 A2 WO 2013076258A2 EP 2012073490 W EP2012073490 W EP 2012073490W WO 2013076258 A2 WO2013076258 A2 WO 2013076258A2
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leu
ionone
ala
glu
gaa
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Jan COMMANDEUR
M. Venkataraman H.
Nico VERMEULEN
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Dsm Ip Assets B.V.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12N9/0042—NADPH-cytochrome P450 reductase (1.6.2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/02—Oxidoreductases acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12Y106/02004—NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
    • C12Y114/14001—Unspecific monooxygenase (1.14.14.1)

Definitions

  • the present invention relates to novel P450 BM3 monooxygenase with modified substrate specificity, nucleic acid sequences coding therefore, expression constructs and vectors comprising these sequences, microorganisms transformed therewith, processes for the microbiological hydroxylation of isoprenoids, in particular processes for the hydroxylation of alpha- and beta-ionones.
  • lonones are a class of norisoprenoids which are widely used in the flavor and fragrance industry as well as building blocks for many chemicals.
  • Especially a- and ⁇ -ionone are norisoprenoids which possess characteristic organoleptic properties.
  • the individual enantiomers of oionone possess different odors, thereby making them important in the fragrance industry.
  • the hydroxylated variants of oionone are also of commercial interest. For example, 3-hydroxy-oionone is used an intermediate for total synthesis of lutein and its stereoisomers.
  • cytochrome P450 CYP102A1 from Bacillus megaterium commonly referred to as the P450 BM3 has good perspectives for catalyzing oxidation reactions of industrial significance.
  • the fact that the heme and reductase domain of P450 BM3 are fused into a single polypeptide makes the electron transfer very efficient.
  • wild type P450 BM3 exhibits a very low hydroxylation activity towards ionone hydroxylation. It is therefore an object of the present invention to make available novel P450 BM3 monooxygenase having modified substrate specificity or modified substrate profile.
  • mutants of the BM3 monooxygenase are to be provided which, in comparison with the non-mutated wild-type enzyme show a selective hydroxylation of isoprenoids, in particular norisoprenoids like a- and ⁇ -ionone.
  • the present invention relates to isolated polypeptides having monooxygenase activity which have at least one functional mutation, in particular amino acid substitution, in at least one of the sequence regions 47, 64, 74, 81 , 82, 87, 143, 188, 198, 267, 285, 415, 437 when compared to the wild type sequence of P450 BM3.
  • the present invention also relates to isolated polynucleotides encoding the polypeptides of the present invention, nucleic acid constructs, recombinant expression vectors, and recombinant host cells comprising the polynucleotides, and to methods of producing the polypeptides.
  • the monooxygenase according to the invention derives from P450 BM3 wild type having an amino acid sequence according to SEQ ID NO:1 (upper line of Table 1 ), which has at least one functional mutation.
  • Val Asp lie Ala Val Gin Leu Val Gin Lys Trp Glu Arg Leu Asn Ala Asp Glu His lie 140
  • GAA AAC AAG CGC CAG TTT CAA GAA GAT ATC AAG GTG ATG AAC GAC CTA GTA GAT AAA ATT 1354 lie Ala Asp Arg Lys Ala Ser Gly Glu Gin Ser Asp Asp Leu Leu Thr His Met Leu Asn 240
  • Glu Asn Pro Ser Ala lie Pro Gin His Ala Phe Lys Pro Phe Gly Asn Gly Gin Arg Ala 400
  • Leu Ala Asp lie Ala Met Ser Lys Gly Phe Ala Pro Gin Val Ala Thr Leu Asp Ser His 520
  • Lys Val Pro Ala Phe lie Asp Glu Thr Leu Ala Ala Lys Gly Ala Glu Asn lie Ala Asp 600
  • Tyr Lys Gly lie Ala Ser Asn Tyr Leu Ala Glu Leu Gin Glu Gly Asp Thr lie Thr Cys 880
  • Particularly preferred monooxygenase mutants of this type are those which have at least one of the following mono- or polyamino acid substitutions:
  • the number indicates the position of the mutation; the original amino acid is indicated before the number and the newly introduced amino acid after the number.
  • mutants which are disclosed specifically are mutants differing there from which furthermore have the desired substrate specificity with respect to at least one of the hydroxylation reactions described above, i.e., for example, for selective hydroxylation of ionones.
  • “Functional equivalents” are also to be understood as meaning in accordance with the invention mutants which exhibit, in at least one of the abovementioned sequence positions, an amino acid substitution other than the one mentioned specifically, but still lead to a mutant which, like the mutant which has been mentioned specifically, show a "modified substrate profile" with respect to the wild-type enzyme and catalyze at least one of the abovementioned hydroxylation reactions.
  • Functional equivalence exists in particular also in the case where the modifications in the substrate profile correspond qualitatively, i.e. where, for example, the same substrates are converted, but at different rates.
  • “Functional equivalents” also encompass the mutants which can be obtained by one or more additional amino acid additions, substitutions, deletions and/or inversions, it being possible for the abovementioned additional modifications to occur in any sequence position as long as they give rise to a mutant with a modified substrate profile in the above sense.
  • the invention also relates to nucleic acid sequences coding for one of the
  • nucleic acid sequences are derived from SEQ ID NO:2 (Table 2), which have at least one nucleotide substitution which leads to one of the functional amino acid mutations described above.
  • the invention moreover relates to functional analogs of the nucleic acids obtained by addition, substitution, insertion and/or deletion of individual or multiple nucleotides, which furthermore code for a monooxygenase having the desired substrate specificity.
  • the invention also encompasses those nucleic acid sequences which comprise so-called silent mutations or which are modified in comparison with a specifically mentioned sequence in accordance with the codon usage of a specific origin or host organism, and naturally occurring variants of such nucleic acid sequences.
  • the invention also encompasses modifications of the nucleic acid sequences obtained by degeneration of the genetic code (i.e. without any changes in the corresponding amino acid sequence) or conservative nucleotide substitution (i.e.
  • sequences modified by nucleotide addition, insertion, inversion or deletion which sequences encode a monooxygenase according to the invention having a "modified substrate profile", and the corresponding complementary sequences.
  • the invention furthermore relates to expression constructs comprising a nucleic acid sequence encoding a mutant according to the invention under the genetic control of regulatory nucleic acid sequences; and vectors comprising at least one of these expression constructs.
  • the constructs according to the invention encompass a promoter 5'-upstream of the encoding sequence in question and a terminator sequence 3'-downstream, and, optionally, further customary regulatory elements, and, in each case operatively linked with the encoding sequence.
  • Operative linkage is to be understood as meaning the sequential arrangement of promoter, encoding sequence, terminator and, if appropriate, other regulatory elements in such a manner that each of the regulatory elements can fulfill its intended function on expression of the encoding sequence.
  • operatively linkable sequences are targeting sequences, or else translation enhancers, enhancers, polyadenylation signals and the like.
  • Further regulatory elements encompass selectable markers, amplification signals, replication origins and the like.
  • the natural regulatory sequence can still be present upstream of the actual structural gene. If desired, this natural regulation may be switched off by genetic modification, and the expression of the genes may be enhanced or lowered.
  • the gene construct may also be simpler in construction, i.e. no additional regulatory signals are inserted upstream of the structural gene and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated in such a way that regulation no longer takes place and the gene expression is increased or reduced.
  • One or more copies of the nucleic acid sequences may be present in the gene construct.
  • promoters examples include cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, l-PR or l-PL promoter, which are advantageously employed in Gram-negative bacteria; and Gram-positive promoters amy and SP02, the yeast promoters ADC1 , MFa, Ac, P-60, CYC1 , GAPDH or the plant promoters CaMV/35S, SSU, OCS, Iib4, usp, STLS1 , B33, nos or the ubiquitin or phaseolin promoter.
  • inducible promoters for example light- and in particular temperature-inducible promoters, such as the PrP1 promoter.
  • inducible promoters for example light- and in particular temperature-inducible promoters, such as the PrP1 promoter.
  • all natural promoters with their regulatory sequences can be used.
  • synthetic promoters may also be used in an advantageous fashion.
  • the abovementioned regulatory sequences are intended to allow the targeted expression of the nucleic acid sequences and of protein expression. Depending on the host organism, this may mean, for example, that the gene is expressed or over expressed only after induction has taken place, or that it is expressed and/or over expressed immediately.
  • the regulatory sequences or factors can preferably have a positive effect on expression and in this manner increase or reduce the latter.
  • an enhancement of the regulatory elements may advantageously take place at the transcriptional level by using strong transcription signals such as promoters and/or "enhancers".
  • translation may also be enhanced by improving, for example, mRNA stability.
  • An expression cassette is generated by fusing a suitable promoter with a suitable monooxygenase nucleotide sequence and a terminator signal or polyadenylation signal.
  • customary recombination and cloning techniques are used as they are described, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley
  • the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which allows optimal gene expression in the host.
  • Vectors are well known to the skilled worker and can be found, for example, in "Cloning Vectors" (Pouwels P. H. et al., Ed., Elsevier, Amsterdam- New York-Oxford, 1985). Vectors are to be understood as meaning not only plasmids, but all other vectors known to the skilled worker such as, for example, phages, viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, plasmids, cosmids, and linear or circular DNA. These vectors can be replicated autonomously in the host organism or chromosomally.
  • the vectors according to the invention allow the generation of recombinant
  • microorganisms which are transformed, for example, with at least one vector according to the invention and which can be employed for producing the mutants.
  • the above-described recombinant constructs according to the invention are advantageously introduced into a suitable host system and expressed. It is preferred to use usual cloning and transfection methods known to the skilled worker in order to bring about expression of the
  • Suitable systems are described, for example, in current protocols in molecular biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997.
  • Suitable host organisms are, in principle, all organisms which allow expression of the nucleic acids according to the invention, their allelic variants, and their functional equivalents or derivatives. Host organisms are to be understood as meaning, for example, bacteria, fungi, yeasts or plant or animal cells.
  • Preferred organisms are bacteria such as those of the genera Escherichia, such as, for example, Escherichia coli, Streptomyces, Bacillus or Pseudomonas, eukaryotic microorganisms such as Saccharomyces cerevisiae, Aspergillus, and higher eukaryotic cells from animals or plants.
  • Escherichia such as, for example, Escherichia coli, Streptomyces, Bacillus or Pseudomonas
  • eukaryotic microorganisms such as Saccharomyces cerevisiae, Aspergillus, and higher eukaryotic cells from animals or plants.
  • the invention furthermore provides a process for preparing a monooxygenase according to the invention, which comprises cultivating a monooxygenase-producing microorganism, if appropriate inducing the expression of the monooxygenase, and isolating the
  • the monooxygenase according to the invention can thus also be produced on an industrial scale.
  • the microorganism can be cultivated and fermented by known methods.
  • Bacteria for example, can be grown in a TB or LB medium and at 20-40°C. and a pH of 6-9. Suitable cultivation conditions are described in detail in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), for example.
  • vector systems or oligonucleotides which extend the cDNA by certain nucleotide sequences and thus code for modified polypeptides or fusion proteins which serve to simplify purification.
  • Suitable modifications of this type are, for example, so-called “tags” which act as anchors, such as, for example, the modification known as hexa-histidine anchor, or epitopes which can be recognized as antigens by antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press).
  • tags which act as anchors, such as, for example, the modification known as hexa-histidine anchor, or epitopes which can be recognized as antigens by antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press).
  • anchors can be used to attach the proteins to a solid support such as, for example, a polymer matrix, which can, for example, be packed into a chromatography column, or to a micro titer plate or to another support.
  • a solid support such as, for example, a polymer matrix, which can, for example, be packed into a chromatography column, or to a micro titer plate or to another support.
  • anchors can also at the same time be used to recognize the proteins. It is also possible to use for recognition of the proteins conventional markers such as fluorescent dyes, enzyme markers which form a detectable reaction product after reaction with a substrate, or radioactive markers, alone or in combination with the anchors for derivatizing the proteins.
  • the invention moreover relates to a process for the microbiological oxidation of isoprenoids, in particular hydroxylation of oionone according to the above definition, which comprises
  • a1 culturing a recombinant microorganism according to the above definition in a culture medium, in the presence of an exogenous (added) substrate or an intermediately formed substrate, which substrate can be hydrolyzed by the monooxygenase according to the invention, or
  • Figure 1 Structures of ⁇ -ionone enantiomers and their 3 ' hydroxylated products.
  • FIG. 2 Active site of P450 BM3 (PDB 1 BU7).
  • the heme group is shown in blue.
  • the amino acid residues where the mutations are present are depicted in stick format.
  • Figure 3 Gas chromatograms of bio transformation products of racemic ⁇ -ionone by (A) MT80 (B) MT33. (C) MT44. The identification of the peaks: (1 ) cis-3- ⁇ - ⁇ ionone t R 9.84 (2) trans-3- ⁇ - ⁇ ionone t R 9.92 (3) 3-oxo-a ionone t R 10.02 (4) unknown product t R 10.82 (Refer Figure 7 for fragmentation pattern)
  • Figure 4 Product profile and conversion of racemic ⁇ -ionone by BM3 mutants depicting C3 hydroxylation.
  • Figure 5 Representative gas chromatograms showing opposite stereo selectivity (A) (6fl)-a-ionone by MT33 and (B) (6S)-a-ionone by MT33
  • Figure 6 Docked binding poses of a-ionone in the BM3 mutants MT80 (A,B) and MT33 (C,D).
  • the (f?)-enantiomer of ⁇ -ionone is shown in green, the (S)-enantiomer in magenta.
  • the positions where hydroxylation occurs are marked in balls.
  • the carbonyl oxygen atom form hydrogen bonds with the hydroxyl group of Ser72.
  • Trans-hydrogens are shown in dark green and dark purple balls for the (f?)-enantiomer and the (S)-enantiomer, respectively.
  • Cis-hydrogens are shown in grey balls.
  • Picture C shows a docking pose where the (f?)-enantiomer is likely to be subjected to cis-hydroxylation.
  • Racemic a-ionone, enantiomers (R)-and (S)-a-ionone and the hydroxy diastereomers (3S,6f?)-OH-a-ionone and (3S,6S)-OH-a-ionone were obtained from DSM, The
  • P450 BM3 mutants M01 , M02, M05 and M1 1 have been described previously [van Vugt- Lussenburg, B.M., et al., Identification of critical residues in novel drug metabolizing mutants of cytochrome P450 BM3 using random mutagenesis. J Med Chem, 2007, 50(3): p. 455-61].
  • Other mutants according to the invention were made by site directed mutagenesis using M01 or M1 1 as template. In general, the mutations were introduced in the corresponding templates in pBluescript II KS(+) vector by the QuickChange
  • the whole gene was subcloned to pET28a+ vector using BamYW and EcoR ⁇ sites and later transformed to BL21 (DE3) cells for expression.
  • a XXX re pe resents the codon that was used to introduce the specific mutation at position 87.
  • the following codons were use: Ala, GCC; Arg, CGG; Cys, UGC; Gin, CAG; Glu, GAG; Gly, GGG; His, CAC; He, AUC; Leu, CUG; Lys, AAG; Met, AUG; Phe, UUC; Pro, CCC; Thr, ACC; Trp, UGG; Tyr, UAC.
  • cytosolic fractions containing 200 nM of P450 BM3 mutants were used. Mutants MT80 and MT33 were grown on a large scale and purified as follows: 600 mL terrific broth medium with 30 ⁇ g/mL kanamycin was inoculated with 15 mL of overnight culture. The cells were grown at 37 °C and 175 rpm until the OD 6 oo reached 0.6. The protein expression was then induced by the addition of 0.6 mM IPTG. The temperature was lowered to 20 °C and 0.5 mM of heme precursor delta aminolevulinic acid was added. Expression was allowed to proceed for 18h. Cells were harvested by centrifugation (4600 g, 4 °C, 20 min) and the cell pellet was
  • KPi-glycerol buffer 100 mM potassium phosphate [KPi] pH 7.4, 10% glycerol, 0.5 mM EDTA, and 0.25 mM DTT.
  • Cells were disrupted using a French press (1000 psi, 3 repeats), and the cytosolic fraction was separated from the membrane fraction by ultracentrifugation of the lysate (120,000 g, 4 °C, 60 min). Then the enzymes were purified using Ni-NTA agarose (Sigma). To prevent aspecific binding, 1 mM histidine was added to the cytosolic fraction.
  • Ni-NTA slurry 3 mL was added to 20 mL of cytosol and the mixture was equilibrated at 4 °C for 2 hours.
  • the Ni-NTA agarose was retained in a polypropylene tube with porous disc (Pierce, Rockford,USA) and was washed 4 times with 4 mL Kpi-glycerol buffer containing 2 mM histidine.
  • the P450 was eluted in 10 mL Kpi-glycerol containing 200 mM histidine.
  • the histidine was subsequently removed by repeated washing with Kpi-glycerol buffer in Vivaspin 20 filtration tube (10,000 MWCO PES, Sartorius) at 4000 g until the histidine concentration was below 250 nM.
  • cytosolic fraction 200 nM was incubated with 500 ⁇ of racemic oionone in a final volume of 250 ⁇ .
  • the reaction was initiated by the addition of 25 ⁇ NADPH regeneration system containing 5 mM NADPH, 50 mM glucose- 6-phosphate and 20 units of glucose-phosphate dehydrogenase.
  • the reaction was allowed to proceed for 1 hour and then stopped by placing on ice.
  • 10 ⁇ _ of 5 mM carbazole as internal standard samples were extracted by vortexing with 2 mL of ethyl acetate for 30 sec. Subsequently samples were centrifuged at 4000 g for 5 minutes to separate phases. About 1 mL of the organic layer was then transferred to GC vials for analysis.
  • Mutants MT80, MT78, MT68 converted racemic ⁇ -ionone mainly to trans-3-OH-a-ionone (>85% e.e.), whereas mutants MT33, MT35, MT36 converted them to almost equal amounts of trans-3-OH-a-ionone and cis-3- OH-a-ionone. Mutants exhibiting high selectivity are shown in Table 4. Other mutants converted ⁇ -ionone to products other than the desired ones with low activity. The product profile of the mutants tested with racemic ⁇ -ionone is shown in Figure 4.
  • mutant MT33 showed the highest diastereoselectivity (>90%) ( Figure 5) and was therefore chosen for determination of the total turnover number. So far with respect to the hydroxylation of optically active oionone by P450s, mutant MT33 is the first engineered P450 BM3 to be reported for the production of one of the cis-3-OH products i.e. (3S, 6f?) with high stereoselectivity. Additionally, among mutants that produced predominantly trans-3-OH-oionone, MT80 was selected for incubation with individual enantiomers as it exhibited the highest selectivity as well as activity. MT80 catalyzed the formation of the (3S,6S)-OH-a-ionone and (3f?,6f?)-a-ionone with (6S) and (6f?)- enantiomers respectively with remarkable selectivity.
  • An optimal biocatalyst ideally should be able to catalyze the formation of desired product in a highly selective (regio, enantio or stereo) with high catalytic turnovers.
  • MT80 catalyzes the formation of (3S, 6S)-OH-a-ionone and (3f?,6f?)-OH-a-ionone with turnover numbers in the range of 3500-4000 (Table 5).
  • MT33 can also catalyze the formation of (3S, 6f?)-OH-oionone with high turnover numbers of about 3000.
  • these reactions are carried out with high selectivity to the desired product without the formation of any side products making them ideal for industrial use for synthesis of fine chemicals.
  • the tested BM3 mutants were able to catalyze selectively, the formation of 3 out of the 4 possible hydroxy diastereomers.
  • the preferred BM3 mutants MT33 and MT80 show the following properties ( Figure 9).
  • P450 BM3 mutant MT80 catalyzes the formation of trans-3-hydroxy-oionone with both enantiomers (> 90% d.e) while MT33 exhibits opposite stereoselectivity
  • Another mutant MT90 which also showed an aselective product profile with racemic-o ionone, was tested with individual enantiomers.
  • This mutant produced the other cis diastereomer, (3f?,6S)-OH-oionone with 40% selectivity (Figure 8). Even though this mutant shows only moderate selectivity for the formation of this product, it could be a good starting point to improve the stereoselectivity by directed evolution.

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Abstract

L'hydroxylation sélective est une réaction critique dans la synthèse de produits chimiques fins tels que les isoprénoïdes hydroxylés. La présente invention concerne des mutants P450 BM3 de cytochromes pouvant exécuter une hydroxylation régio- et stéréosélective d'un α- et β-ionone. L'invention concerne des polypeptides isolés ayant une activité oxydative et des polynucléotides isolés codant pour ces polypeptides. La présente invention concerne également des constructions d'acides nucléiques, des vecteurs et des cellules hôtes incluant les polynucléotides, ainsi que des procédés permettant l'hydroxylation microbiologique d'isoprénoïdes, en particulier des ionones.
PCT/EP2012/073490 2011-11-23 2012-11-23 Mutants p450 bm3 et leur utilisation pour l'hydroxylation régio-et stéréosélective d'αlpha- et βeta-ionone WO2013076258A2 (fr)

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CN111699248A (zh) * 2018-01-31 2020-09-22 密执安大学评议会 用于合成噻吩并吡啶化合物的混合二硫键缀合物的生物催化剂和方法
JP2021511800A (ja) * 2018-01-31 2021-05-13 ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン 生体触媒およびチエノピリジン化合物の混合ジスルフィド結合体の合成方法
EP3746551A4 (fr) * 2018-01-31 2021-11-03 The Regents Of The University Of Michigan Biocatalyseur et procédés de synthèse de conjugués disulfure mixtes de composés de thiénopyridine
JP7485367B2 (ja) 2018-01-31 2024-05-16 ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン 生体触媒およびチエノピリジン化合物の混合ジスルフィド結合体の合成方法
CN111699248B (zh) * 2018-01-31 2024-05-24 密执安大学评议会 用于合成噻吩并吡啶化合物的混合二硫键缀合物的生物催化剂和方法
EP3858986A1 (fr) * 2020-02-03 2021-08-04 Bayer Aktiengesellschaft Variantes p450 bm3 de monooxygénase pour l'hydroxylation c19 des stéroïdes
WO2021156200A1 (fr) * 2020-02-03 2021-08-12 Bayer Aktiengesellschaft Variants de la mono-oxygénase p450 bm3 pour l'hydroxylation en c19 de stéroïdes

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