Classifications

• • 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
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/02—Oxygen as only ring hetero atoms
  • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
• • 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 cytochrome P450 monooxygenase with modified (higher) substrate selectivity and/or (re)activity towards aromatic hydroxylation of benzenes.
• the invention provides nucleic acid sequences coding for the novel enzymes, expression constructs and vectors comprising these sequences, transformed microorganisms expressing these novel enzymes, and processes for the stereoselective hydroxylation of substituted benzenes useful as building blocks in vitamin E synthesis.
• Regioselective hydroxylation of aromatic rings leading to enantiopure products is of high interest for producing precursors of pharmaceuticals, flavors or vitamins.
• the demand for phenol and alkylphenols as feedstock for resins, plastics and bisphenol-A is constantly increasing.
• Substituted benzenes are also used as building blocks such as e.g. in vitamin E synthesis, wherein 2,3,6-trimethylphenol is converted via oxidation and catalytic hydrogenation into trimethylhydroquinone (TMHQ), in important intermediate in vitamin E synthesis (for more detail see e.g. Ullmann's Encyclopedia of Industrial Chemistry, 6 th , completely revised edition, Volume 38, Wiley-VCH, 2002). Improving the known vitamin E synthesis is advantageous in order to save costs and resources.
• TMHQ trimethylhydroquinone
• Direct hydroxylation with enzymes can be an attractive alternative reducing reaction and purification steps, as well as waste generation and process energy demands.
• Cytochrome P450 monooxygenases CYPs
• CYPs Cytochrome P450 monooxygenases
• P450 BM3 is a water-soluble enzyme of 1 18 kDa.
• P450 BM3 contains its heme and reductase domains on a single polypeptide chain.
• a model has been proposed for regioselective aromatic hydroxylation for substituted benzenes catalyzed by P450 BM3 (Whitehouse et al. , ChemBioChem 2010, 1 1, 2549-2556).
• these enzymes have so far hardly been used for
• enzymes such as toluene 4-monooxygenase suffer from low activity (k ca t 36 min "1 ), stability (four-protein complex) as well as selectivity (20% aromatic and 80% benzylic hydroxylation) using p-xylene as substrate.
• Stereoselective aromatic ring hydroxylation can be furthermore interesting in the synthesis of vitamin E.
• novel biocatalysts to be used in industrial processes which are capable of direct aromatic hydroxylation of substituted benzenes, in particular hydroxylation of p-xylene or pseudocumene, into the respective alkylphenol, wherein the biocatalyst is modified in such a way that it shows increased enzymatic activity, coupling efficiency, product stability and stereoselectivity.
• novel P450 mutants which show an increased activity of up to 30 times towards aromatic hydroxylation of methylbenzenes, in particular p-xylene or pseudocumene, compared to the activity of a wild-type P450. Furthermore, the coupling efficiency and the selectivity for the production of alkylphenols could be increased.
• the inventors have identified at least 5 amino acid positions which are involved in the specific enzyme activity, coupling efficiency and/or selectivity of the enzyme. In particular, these at least 5 positions are highly important for aromatic hydroxylation of a given substrate.
• the present invention provides isolated polynucleotides encoding the mutant polypeptides, i.e.
• P450 enzymes of the present invention, nucleic acid constructs, recombinant expression vectors, and recombinant host cells comprising the polynucleotides, and to methods of producing the polypeptides.
• Novel polynucleotides, polypeptides and recombinant host cells can be used in a process for producing alkylphenols from substituted benzenes, e.g., production of 2,5-DMP from p-xylene.
• Useful substrates include p-xylene, m-xylene, o- xylene, toluene or pseudocumene. Preferred is the catalysis of pseudocumenes or p-xylene using the novel enzymes.
• the present invention is directed to a modified enzyme having P450 monooxygenase activity, comprising at least one mutation, in particular amino acid substitution, wherein the at least one mutation leads to an increase in the monooxygenase activity and/or selectivity and/or coupling efficiency, i.e. is said to be a functional mutation, and wherein the at least one mutation is at one or more amino acid positions selected from the group consisting of amino acid positions corresponding to positions 47, 51 , 87, 330, 401 and combinations thereof of the amino acid sequence of Bacillus megaterium P450 BM3 as shown in SEQ ID NO:2, wherein SEQ ID NO:2 shows the wild-type sequence.
• the corresponding polynucleotide encoding the wild-type P450 BM3 is shown in SEQ ID NO: 1 (for both sequences, see Figure 4).
• the wild- type P450 BM3 according to SEQ ID NO:2 is mutated wherein the mutated or modified enzyme comprises a mutation in one or more of the amino acid residues R47, Y51 , F87, A330 and/or 1401 .
• Particularly preferred are amino acid substitutions but also other forms of mutations known to the skilled person are possible.
• the modified/mutated P450 monooxygenase of the present invention comprises combinations of mutations, in particular amino acid substitutions. Such combinations may include three of the positions mentioned above, e.g. a substitution of amino acid residues corresponding to amino acids R47/Y51 /I401 on SEQ ID NO:2, more preferably a combination of substitutions R47S/Y51W/I401M or R47E/Y51W/I401M.
• the mutated enzyme comprises a
• the modified enzyme according to the present invention preferably contains at least 1 , at least 2, at least 3, at least 4 or at least 5 mutations, e.g.
• substitutions on one of the above-identified positions when compared with the amino acid sequence of the corresponding non-modified monooxygenase as exemplified by SEQ ID NO:2.
• the respective positions corresponding to amino acid residues shown in SEQ ID NO:2 are listed in Table 1 .
• monooxygenase in particular a mutant P450 BM3 are used in a process for direct aromatic ring hydroxylation of substituted benzenes, such as preferably p- xylene or pseudocumene.
• Particularly useful amino acid substitutions are the ones selected from R47S, R47E, Y51W, I401M or the mutation pattern shown in Table 1 for mutants M1 , M2, M3, and functional equivalents thereof.
• Residue F87 proofed to be essential for efficient coupling and aromatic hydroxylation, in particular with p-xylene or pseudocumene as substrate. It is also possible to substitute the original amino acids on the above-mentioned positions by other functional equivalent amino acids.
• 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 the same properties with respect to the wild -type enzyme and catalyze at least one of the abovementioned hydroxylation reactions.
• “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 profile in the above sense.
• mutant enzymes in particular the recombinant P450 BM3 mutants, show increased specific activity in the hydroxylation of a given substrate such as e.g. p-xylene or pseudocumene, increased coupling efficiency, and increased
• the activity increase is in the range of about at least 5, 10, 20-fold but can be up to 30-fold and more, such as e.g. obtained with mutant M2 catalyzing the aromatic hydroxylation of p-xylene or pseudocumene in comparison to the wild-type enzyme.
• this could be increased to more than about 40, 50%, such as 60 and even 70%, compared to the wild-type enzyme.
• aromatic hydroxylation of p-xylene or pseudocumene using a modified enzyme according to the present invention the selectivity for 2,5 DMP can be increased to above 98% or more, such as even 100%, compared to a process using wild-type P450 BM3.
• the term "specific activity” or "activity” with regards to enzymes means its catalytic activity, i.e. its ability to catalyze formation of a product from a given substrate.
• the specific activity defines the amount of substrate consumed and/or product produced in a given time period and per defined amount of protein at a defined temperature.
• specific activity is expressed in ⁇ substrate consumed or product formed per min per mg of protein.
• the hydroxylation of methylated benzenes is catalyzed by mutant M2, leading to 30-fold increased activity compared to wild-type P450-BM3, excellent coupling
• enzyme As used herein, the terms "enzyme”, “P450 monooxygenase” or “cytochrome P450 monooxygenase” are used interchangeably herein in connection with the description of the present invention.
• a wild-type P450 monooxygenase may include any P450 monooxygenase including enzymes isolated from microorganisms, yeast or mammals that are used as starting point for designing mutants with increased activity, increased coupling efficiency and/or (enantio)selectivity according to the present invention.
• the wild -type enzyme is selected from the P450
• Rhodopseudomonas Rhodococcus, Delftia, Saccharopolyspora, Comamonas, Burkholderia, Cupriavidus, Variovorax, Fusarium, Gibberella, Aspergillus or Amycolatopsis, more preferably selected from Bacillus megaterium, Bacillus subtilis, Bacillus licheniformis, Bacillus weihenstephanensis, Bacillus cereus, Bacillus anthracis, Burkholderia sp. 383, Erythrobacter litoralis, Geobacillus sp.
• Y412MC10 Herpetosiphon aurantiacus, Ralstonia eutropha, Ralstonia pickettii, Ralstonia metallidurans, Bradyrhizobium japonicum, Azorhizobium caulinodans, Streptomyces avermitilis, Rhodopseudomonas palustris, Rhodococcus ruber, Rhodococcus sp.
• NCIMB 9784 Delftia acidovorans, Saccharopolyspora erythraea, Comamonas testosteroni, Burkholderia mallei, Cupriavidus taiwanensis,
• Variovorax paradoxus Fusarium oxysporum, zeae, Gibberella moniliformis, Aspergillus fumigatus or Amycolatopsis orientalis, most preferably Bacillus megaterium, Bacillus subtilis, Bacillus licheniformis, Bacillus
• weihenstephanensis Bacillus cereus, Bacillus anthracis, in particular Bacillus megaterium or Bacillus subtilis, such as Bacillus megaterium (P450 BM3) shown in SEQ ID NO:2 or homologous sequences thereof showing the same enzymatic activity.
• An example of a useful mammalian enzyme is the human CYP2D6.
• Wild-type in the context of the present invention may include both P450 monooxygenase sequences derivable from nature as well as variants of synthetic P450 enzymes.
• wild-type P450 and “non-modified P450” are used interchangeably herein.
• a “mutant”, “mutant enzyme”, or “mutant P450 enzyme”, such as P450 BM3 enzyme, may include any variant derivable from a given wild-type enzyme/ P450 (according to the above definition) according to the teachings of the present invention and being more active/ efficient in coupling/selective than the respective wild-type enzyme.
• mutants may also be generated, e.g. , by designing synthetic genes, and/or produced by in vitro (cell-free) translation. For testing of specific activity, mutants may be (over-)expressed by methods known to those skilled in the art. Different test assays are available, such as e.g. pNCA-assays, 4-AAP-assay or pNTP-assay which are all known to the skilled person.
• test assays are available, such as e.g. pNCA-assays, 4-AAP-assay or pNTP-assay which are all known to the skilled person.
• mutant P450 enzyme and
• modified P450 enzyme are used interchangeably herein.
• the invention also relates to nucleic acid sequences coding for one of the monooxygenases according to the invention.
• Preferred nucleic acid sequences are derived from SEQ ID NO:1 or homologous sequences thereof, 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
• 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. the corresponding amino acid is replaced by another amino acid of the same charge, size, polarity and/or solubility), and sequences modified by nucleotide addition, insertion, inversion or deletion, which sequences encode a
• 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. Examples of 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 with their regulatory sequences can be used.
• synthetic promoters may also be used in an advantageous fashion. The skilled person will know which and how the promoters are to be used. Examples of suitable promoters are: 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 examples of suitable promoters, 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
• 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, lib4, 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.
• 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 overexpressed only after induction has taken place, or that it is expressed and/or overexpressed immediately.
• 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.
• Suitable cloning techniques 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 Interscience (1987).
• 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 person and include, 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 (micro)organism or chromosomally.
• the vectors according to the invention allow the generation of recombinant (micro)organisms 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.
• the skilled person knows about preferred cloning and transfection methods in order to bring about expression of the abovementioned nucleic acids in the expression system in question. Suitable systems are known to the skilled person.
• nucleic acids encoding the mutant monooxygenases according to the present invention can be done in any host system, including
• microorganisms which allows expression of the nucleic acids according to the invention, their allelic variants, and their functional equivalents or derivatives.
• suitable host (micro)organisms are 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, Pichia pastoris, Hansenula polymorpha or Yarrowia lipolytica 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 host, e.g. microorganism, if appropriate inducing the expression of the monooxygenase, and isolating the monooxygenase from the culture. If desired, the monooxygenase according to the invention can thus also be produced on an industrial scale.
• a monooxygenase-producing host e.g. microorganism
• the microorganism can be cultivated and fermented by known methods.
• Bacteria for example, can be grown in a TB or LB medium at 20 to 40° C and a pH of 6 to 9. Suitable cultivation conditions are described in detail in e.g.
• Fermentation media may further contain suitable carbon substrates.
• suitable substrates may include but are not limited to monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from
• the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.
• the fermentation of the microorganism can be performed as batch, fed-batch or as continuous process.
• the use of so-called “resting cells”, i.e. cells that are not growing any further, is also within the scope of the present invention.
• 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).
• These 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.
• 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.
• a process for direct aromatic hydroxylation of substituted benzenes comprising:
• a1 culturing a recombinant host, e.g. 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 modified monooxygenase according to the invention, or a2) incubating a substrate-containing reaction medium with an isolated modified enzyme according to the invention, and optionally
• a concentration in the range of about 10 to 100 mM and 1 to 1000 mM might be workable.
• reaction conditions to be used with a mutant enzyme of the present invention Isolation of the product including e.g. 2,5 DMP or TMHQ is also known.
• the products formed by a process according to the present invention i.e. using a modified P450 enzyme in a aromatic hydroxylation reaction with one of the herein-mentioned substrates, in particular
• pseudocumene or p-xylene can be used in industrial processes, e.g. production of polymers or synthesis of vitamin E wherein these products serve as building blocks which result upon further conversion /rearrangement steps to vitamin E.
• any substituted benzene can be used for aromatic hydroxylation reaction catalyzed by the modified enzyme according to the present invention.
• a suitable substrate is selected from methylated or methoxylated benzenes, wherein the methylation includes 1 , 2, 3, or all C-atoms of the benzene ring.
• Examples are toluene, p-/o-/m-xylene, pseudocumene, o-/m-/p- cresol, bromo-/chloro- or nitrobenzenes such as, e.g., p-/m-/o-nitrotoluene or bromoanisole.
• Possible hydroxylation steps are depicted in Figure 5, wherein the mutant enzyme can be used for step I and/or II, i.e. for introduction of 2 hydroxyl-groups.
• Substrates useful in vitamin E synthesis are for example p-xylene (hydroxylated to 2,5 DMP) or pseudocumene (hydroxylated to 2,3,5 or 2,3,6-tremethylphenol), as well as m-xylene, o-xylene or toluene in a process described above using one of the mutated enzymes, wherein the use of M2 is particularly useful.
• a preferred reaction is the hydroxylation of p-xylene or pseudocumene, in particular catalyzed by M2.
• the mutated enzyme can catalyze the first and second hydroxylation, preferably the first hydroxylation reaction (see Figure 5).
• vitamin E is used herein as a generic descriptor for all tocol and tocotrienol derivatives exhibiting qualitatively the biological activity of a- tocopherol (l UPAC-I UB Recommendation 1981 , Eur. J. Biochem. 123, 473-475, 1982).
• Figure 1 Gas chromatograms show the hydroxylation of p-xylene by P450 BM3 wild-type (3), M1 (4) and M2 (5).
• As standards 2,5-dimethylphenol (2) as well as 4-Methylbenzylalcohol (1 ) were employed with an identical GC-program.
• the retention time is blotted on the x-axis [min]
• the FID signal is blotted on the y- axis. Further explanations are given in the text.
• Figure 2 Kinetic characterization of wild-type P450 BM3 ( A ), variants M1 ( ⁇ ) and M2 ( ⁇ ) for determination of k ca t and K M with p-xylene as substrate. All measurements were performed in triplicate with less than 10% standard deviation. Fitting of kinetic parameters was achieved using Origin 7.0 software and Hill equation fitting (OriginLab Corporation, Northampton, MA, USA). The amount of p-xylene is blotted on the x-axis [mM], the productivity is blotted on the y-axis [nmol 2,5-DMP*nmol "1 P450*min "1 ] . For further explanation see the text.
• Figure 3 Gas chromatograms show the hydroxylation of toluene (1 ), m-xylene (2) and o-xylene (3) by P450 BM3 variant M2 (R47S/Y51W/I401 M). All potential products (a- and aromatic hydroxylation) were available as standards and employed with an identical GC-program (data not shown). The retention time is blotted on the x-axis [min], the FID signal is blotted on the y-axis. For further explanation see the text.
• Figure 4 Nucleotide sequences (bottom line) and amino acids sequence (upper line) of the wild-type cytochrome P450 BM3 monooxygenases, corresponding to SEQ I D NO: 1 and 2, respectively (see text) .
• Step 5A Hydroxylation of p-xylene to 2, 5 dimethylphenol (Step I ) and further to 2,5 dimethylhydroquinone (Step II ), wherein Step I and II are hydroxylations which might be catalyzed by P450.
• Step I and II are hydroxylations which might be catalyzed by P450.
• Step I Hydroxylation of pseudocumene to 2,3,5 trimethylphenol (Step I) and further to 2, 3,5 trimethylhydroquinone (Step II ), wherein Step I and II are hydroxylations which might be catalyzed by P450.
• Step II Hydroxylation of pseudocumene to 2,3,5 trimethylphenol (Step I) and further to 2, 3,5 trimethylhydroquinone (Step II ), wherein Step I and II are hydroxylations which might be catalyzed by P450.
• Step II Hydroxylation of pseudocumene to 2,3,5 trimethylphenol (Step II) and further to 2, 3,5 trimethylhydroquinone (Step II ), wherein Step I and II are hydroxylations which might be catalyzed by P450.
• Example 1 General methodology
• the 3.1 kb P450 BM3 gene from Bacillus megaterium (CYP102A1 ) was cloned into the 2 kb pET22b-derived pALXtreme-1 a plasmid (Blanusa et al. , Anal. Biochem 406, 141 -146, 201 1 ).
• the pALXtreme-1 a plasmid was transformed into chemical competent E. coli BL21 Gold (DE3)-derived E. coli laql Q1 Gold (DE3) cells.
• Preparation of chemical competent cells was achieved following a standard protocol generating 1 0 8 clones per ⁇ pUC19 (Inoue et al. , Gene 1990, 96, 23-28). All plasmid
• Oligonucleotides were designed for optimal PCR product yields according to recommendations of user's manual from Stratagene's QuikChange Site-directed mutagenesis Kit (Stratagene, La Jolla, CA, USA).
• a standard PCR set-up contained 20 ng plasmid DNA, 5 U Phusion DNA polymerase, 1 x Phusion DNA polymerase buffer, 0.2 mM dNTP mix and 0.4 ⁇ of forward and reverse primer (Table 2). All PCRs were performed in thin wall PCR tubes (Saarstedt, Nuembrecht, Germany) and using an Eppendorf Mastercycler proS (Hamburg, Germany).
• the cycling protocol was performed according to Stratagene's QuikChange Site-directed mutagenesis Kit manual instruction (Stratagene, La Jolla, CA, USA). Temperatures and incubation times during each PCR cycle were adjusted to provider's recommendation for Phusion DNA polymerase (New England Biolabs, Frankfurt, Germany). Annealing temperature during each PCR cycle was adjusted to 55 °C for 30 s. Successful PCR amplifications were verified on 0.8 % agarose gel according to a standard protocol (Maniatis et al. , 1982 1 - 545). Digestion of template DNA was achieved by addition of 20 U Dpnl to 50 ⁇ PCR mixture followed by incubation at 37° C for 3 h.
• the PCR products were transformed without further purification into chemically competent E . coii Gold (DE3) laql Q1 cells using a heat shock transformation protocol (Hanahan et al. , Methods in enzymology 1991 , 204, 63-1 13). Recovered cells were plated and incubated overnight at 37° C on LB agar plates containing 100 Mg/ml kanamycin. Obtained colonies were transferred with sterile toothpicks into Greiner BioOne 96-well microtiterplates (MTP) (Frickenhausen, Germany) pre-filled with 100 ⁇ LB media and 100 Mg/ml kanamycin.
• MTP Greiner BioOne 96-well microtiterplates
• Table 1 Comparison of the amino acids in the wild-type P450-BM3 (WT) and in mutant enzymes (M1 to M4), wherein the amino acid substitution is indicated. The positions are according to the ones in SEQ ID NO:2. WT M1 M2 M3 M4
• frozen cell pellets were resuspended in 10 ml 50mM KPi buffer pH 7.8 prior to disruption in an Avestin EmulsiFlex-C3 high-pressure homogenizer (Ottawa, ON, Canada) by applying three cycles of 1500 bar pressure.
• Cell debris was removed by centrifuging homogenized samples for 30 min at 16000 g and 4° C in a Sorvall RC-6 Plus ultracentrifuge (Thermo Scientific, Rockford, IL, USA). Removal of small insoluble particles was achieved by pressing the supernatant through a 0.45 ⁇ membrane filter.
• the filtered cell lysate was loaded on a Kronlab TAC15/ 125PE5-AB-2 column (YMC Europe GmbH, Dinslaken, Germany) prefilled with Toyopearl DEAE 650S anion exchange matrix (Tosoh Bioscience, Stuttgart, Germany) .
• An AKTAprime Plus chromatography system with UV- detection (GE Healthcare, Muenchen, Germany) was used for purification and collection of eluted protein samples.
• a standard protocol was applied to purify P450 BM3 wild-type as well as the variants M1 and M2 (Schwaneberg et al. , J. Chromatogr. , A 1999, 848, 149-159. ).
• Eluted protein samples were collected and purity was estimated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fractions containing active and approximately 90% pure protein, were pooled in an Amicon Ultra-4 centrifugation tube (30 kDa cutoff; Millipore, Schwalbach, Germany) and concentrated to a final volume of 2 ml. Concentrated protein samples were desalted and equilibrated in 50 mM KPi pH 7.5 buffer using a PD-10 gel-filtration column (GE Healthcare, Muenchen, Germany).
• Lyophilized P450 BM3 variants were resuspended in 50 mM KPi pH 7.5 buffer and the concentration of active P450 BM3 was assayed applying a standard CO quantification protocol for P450 monooxygenases (Omura & Sato, J. Biol. Chem. 1964, 239, 2379-2385).
• Kinetic characterization (KM, k cat ) of wild type P450 BM3 and improved variants was performed using the 4-AAP assay for quantification of 2,5-DMP.
• variant M1 showed a low activity for p-xylene (k ca t 68 min "1 ; 45% coupling efficiency), producing 2, 5-DMP with high selectivity (>98%).
• Variant M1 R47S/Y51W
• variant M1 showed more than a 7-fold higher product formation (k ca t 500 min "1 2,5-DMP) compared to the wild-type P450 BM3.
• variant M1 showed a remarkable catalytic performance since the coupling efficiency increased by 20% (total coupling efficiency of 54%) accompanied by a slightly decreased K M (from 7.9 to 7.1 mM).
• Lower coupling efficiencies were also achieved when using o-xylene as well as m-xylene as substrates (12 and 29%).
• Variant M2 (R47S/Y51W/I401M) showed an up to 5-fold improved activity compared to M1 .
• Purified M2 displayed in total a 30-fold higher catalytic activity (kcat 1953 min "1 ) than the wild-type P450 BM3 as well as a 46% increased coupling efficiency (in total 65%) as well as high selectivity (>98%) for 2, 5-DMP production while the K M is reduced by 15% to 6.1 mM.
• the high k ca t value makes the M2 variant an efficient monooxygenase catalyst (k e n: 320.2 mM "1 *min "1 ), as depicted in Figure 2.

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