REGIO- AND STEREOSELECTIVE HYDROXY LATION OF ALPHA- AND BETA-

IONONE

Background of the Invention

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 β-ionone are norisoprenoids which possess characteristic organoleptic properties. Interestingly, 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.

The chemical synthesis of hydroxylated variants of ionones still poses a challenge due to the formation of a variety of side products. Therefore microbial and enzymatic

biotransformation for selective hydroxylation of isoprenoids (sometimes called

terpernoids) has gained importance over the years. Microbial biotransformation of o ionone using different Streptomyces strains, fungi like Aspergillus Niger, immobilized cells of Nicotium tabacum, cultured cells of Caragana chamlagu has been reported. Summary of the Invention

It is known that 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. With respect to present invention, 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. In particular, 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 β-ionone.

Inventors have found that the above object is surprisingly achieved by several novel P450 BM3 mutants which are engineered by rational design or evolved by random mutagenesis and showed an increased activity.

Therefore, 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 structures of possible hydroxylated diastereomers are shown in Figure 1 . An overview of the amino acid positions in the active site where the mutations are present is shown in Figure 2.

Detailed Description of the Invention

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.

Table 1 Sequence of wild type P450 BM3

(Upper Line Amino Acid Seq. SEQ ID NO: 1; bottom line Nucleic Acid Seq. SEQ ID NO: 2)

Met Thr lie Lys Glu Met Pro Gin Pro Lys Thr Phe Gly Glu Leu Lys Asn Leu Pro Leu 20

ATG ACA ATT AAA GAA ATG CCT CAG CCA AAA ACG TTT GGA GAG CTT AAA AAT TTA CCG TTA 754

Leu Asn Thr Asp Lys Pro Val Gin Ala Leu Met Lys lie Ala Asp Glu Leu Gly Glu lie 40

TTA AAC ACA GAT AAA CCG GTT CAA GCT TTG ATG AAA ATT GCG GAT GAA TTA GGA GAA ATC 814

Phe Lys Phe Glu Ala Pro Gly Arg Val Thr Arg Tyr Leu Ser Ser Gin Arg Leu lie Lys 60 TTT AAA TTC GAG GCG CCT GGT CGT GTA ACG CGC TAC TTA TCA AGT CAG CGT CTA ATT AAA 874

Glu Ala Cys Asp Glu Ser Arg Phe Asp Lys Asn Leu Ser Gin Ala Leu Lys Phe Val Arg 80 GAA GCA TGC GAT GAA TCA CGC TTT GAT AAA AAC TTA AGT CAA GCG CTT AAA TTT GTA CGT 934 Asp Phe Ala Gly Asp Gly Leu Phe Thr Ser Trp Thr His Glu Lys Asn Trp Lys Lys Ala 100 GAT TTT GCA GGA GAC GGG TTA TTT ACA AGC TGG ACG CAT GAA AAA AAT TGG AAA AAA GCG 994

His Asn lie Leu Leu Pro Ser Phe Ser Gin Gin Ala Met Lys Gly Tyr His Ala Met Met 120

CAT AAT ATC TTA CTT CCA AGC TTC AGT CAG CAG GCA ATG AAA GGC TAT CAT GCG ATG ATG 1054

Val Asp lie Ala Val Gin Leu Val Gin Lys Trp Glu Arg Leu Asn Ala Asp Glu His lie 140

GTC GAT ATC GCC GTG CAG CTT GTT CAA AAG TGG GAG CGT CTA AAT GCA GAT GAG CAT ATT 1114

Glu Val Pro Glu Asp Met Thr Arg Leu Thr Leu Asp Thr lie Gly Leu Cys Gly Phe Asn 160 GAA GTA CCG GAA GAC ATG ACA CGT TTA ACG CTT GAT ACA ATT GGT CTT TGC GGC TTT AAC 1174

Tyr Arg Phe Asn Ser Phe Tyr Arg Asp Gin Pro His Pro Phe lie Thr Ser Met Val Arg 180 TAT CGC TTT AAC AGC TTT TAC CGA GAT CAG CCT CAT CCA TTT ATT ACA AGT ATG GTC CGT 1234 Ala Leu Asp Glu Ala Met Asn Lys Leu Gin Arg Ala Asn Pro Asp Asp Pro Ala Tyr Asp 200 GCA CTG GAT GAA GCA ATG AAC AAG CTG CAG CGA GCA AAT CCA GAC GAC CCA GCT TAT GAT 1294 Glu Asn Lys Arg Gin Phe Gin Glu Asp lie Lys Val Met Asn Asp Leu Val Asp Lys lie 220

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

ATT GCA GAT CGC AAA GCA AGC GGT GAA CAA AGC GAT GAT TTA TTA ACG CAT ATG CTA AAC 1414

Gly Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn lie Arg Tyr Gin lie lie 260

GGA AAA GAT CCA GAA ACG GGT GAG CCG CTT GAT GAC GAG AAC ATT CGC TAT CAA ATT ATT 1474

Thr Phe Leu lie Ala Gly His Glu Thr Thr Ser Gly Leu Leu Ser Phe Ala Leu Tyr Phe 280

ACA TTC TTA ATT GCG GGA CAC GAA ACA ACA AGT GGT CTT TTA TCA TTT GCG CTG TAT TTC 1534

Leu Val Lys Asn Pro His Val Leu Gin Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val 300

TTA GTG AAA AAT CCA CAT GTA TTA CAA AAA GCA GCA GAA GAA GCA GCA CGA GTT CTA GTA 1594

Asp Pro Val Pro Ser Tyr Lys Gin Val Lys Gin Leu Lys Tyr Val Gly Met Val Leu Asn 320

GAT CCT GTT CCA AGC TAC AAA CAA GTC AAA CAG CTT AAA TAT GTC GGC ATG GTC TTA AAC 1654

Glu Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala Lys Glu Asp Thr 340

GAA GCG CTG CGC TTA TGG CCA ACT GCT CCT GCG TTT TCC CTA TAT GCA AAA GAA GAT ACG 1714

Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp Glu Leu Met Val Leu lie Pro Gin 360

GTG CTT GGA GGA GAA TAT CCT TTA GAA AAA GGC GAC GAA CTA ATG GTT CTG ATT CCT CAG 1774

Leu His Arg Asp Lys Thr lie Trp Gly Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe 380

CTT CAC CGT GAT AAA ACA ATT TGG GGA GAC GAT GTG GAA GAG TTC CGT CCA GAG CGT TTT 1834

Glu Asn Pro Ser Ala lie Pro Gin His Ala Phe Lys Pro Phe Gly Asn Gly Gin Arg Ala 400

GAA AAT CCA AGT GCG ATT CCG CAG CAT GCG TTT AAA CCG TTT GGA AAC GGT CAG CGT GCG 1894

Cys lie Gly Gin Gin Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly Met Met Leu Lys 420

TGT ATC GGT CAG CAG TTC GCT CTT CAT GAA GCA ACG CTG GTA CTT GGT ATG ATG CTA AAA 1954

His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu Asp lie Lys Glu Thr Leu Thr Leu 440

CAC TTT GAC TTT GAA GAT CAT ACA AAC TAC GAG CTC GAT ATT AAA GAA ACT TTA ACG TTA 2014

Lys Pro Glu Gly Phe Val Val Lys Ala Lys Ser Lys Lys lie Pro Leu Gly Gly lie Pro 460

AAA CCT GAA GGC TTT GTG GTA AAA GCA AAA TCG AAA AAA ATT CCG CTT GGC GGT ATT CCT 2074

Ser Pro Ser Thr Glu Gin Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn 480

TCA CCT AGC ACT GAA CAG TCT GCT AAA AAA GTA CGC AAA AAG GCA GAA AAC GCT CAT AAT 2134

Thr Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr Ala Arg Asp 500

ACG CCG CTG CTT GTG CTA TAC GGT TCA AAT ATG GGA ACA GCT GAA GGA ACG GCG CGT GAT 2194

Leu Ala Asp lie Ala Met Ser Lys Gly Phe Ala Pro Gin Val Ala Thr Leu Asp Ser His 520

TTA GCA GAT ATT GCA ATG AGC AAA GGA TTT GCA CCG CAG GTC GCA ACG CTT GAT TCA CAC 2254

Ala Gly Asn Leu Pro Arg Glu Gly Ala Val Leu lie Val Thr Ala Ser Tyr Asn Gly His 540

GCC GGA AAT CTT CCG CGC GAA GGA GCT GTA TTA ATT GTA ACG GCG TCT TAT AAC GGT CAT 2314

Pro Pro Asp Asn Ala Lys Gin Phe Val Asp Trp Leu Asp Gin Ala Ser Ala Asp Glu Val 560

CCG CCT GAT AAC GCA AAG CAA TTT GTC GAC TGG TTA GAC CAA GCG TCT GCT GAT GAA GTA 2374

Lys Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr Thr Tyr Gin 580

AAA GGC GTT CGC TAC TCC GTA TTT GGA TGC GGC GAT AAA AAC TGG GCT ACT ACG TAT CAA 2434

Lys Val Pro Ala Phe lie Asp Glu Thr Leu Ala Ala Lys Gly Ala Glu Asn lie Ala Asp 600

AAA GTG CCT GCT TTT ATC GAT GAA ACG CTT GCC GCT AAA GGG GCA GAA AAC ATC GCT GAC 2494

Arg Gly Glu Ala Asp Ala Ser Asp Asp Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His 620

CGC GGT GAA GCA GAT GCA AGC GAC GAC TTT GAA GGC ACA TAT GAA GAA TGG CGT GAA CAT 2554

Met Trp Ser Asp Val Ala Ala Tyr Phe Asn Leu Asp lie Glu Asn Ser Glu Asp Asn Lys 640

ATG TGG AGT GAC GTA GCA GCC TAC TTT AAC CTC GAC ATT GAA AAC AGT GAA GAT AAT AAA 2614

Ser Thr Leu Ser Leu Gin Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala Lys Met His 660

TCT ACT CTT TCA CTT CAA TTT GTC GAC AGC GCC GCG GAT ATG CCG CTT GCG AAA ATG CAC 2674

Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu Leu Gin Gin Pro Gly Ser Ala Arg 680

GGT GCG TTT TCA ACG AAC GTC GTA GCA AGC AAA GAA CTT CAA CAG CCA GGC AGT GCA CGA 2734

Ser Thr Arg His Leu Glu lie Glu Leu Pro Lys Glu Ala Ser Tyr Gin Glu Gly Asp His 700

AGC ACG CGA CAT CTT GAA ATT GAA CTT CCA AAA GAA GCT TCT TAT CAA GAA GGA GAT CAT 2794

Leu Gly Val lie Pro Arg Asn Tyr Glu Gly lie Val Asn Arg Val Thr Ala Arg Phe Gly 720

TTA GGT GTT ATT CCT CGC AAC TAT GAA GGA ATA GTA AAC CGT GTA ACA GCA AGG TTC GGC 2854

Leu Asp Ala Ser Gin Gin lie Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala His Leu Pro 740

CTA GAT GCA TCA CAG CAA ATC CGT CTG GAA GCA GAA GAA GAA AAA TTA GCT CAT TTG CCA 2914 Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gin Tyr Val Glu Leu Gin Asp Pro Val 760

CTC GCT AAA ACA GTA TCC GTA GAA GAG CTT CTG CAA TAC GTG GAG CTT CAA GAT CCT GTT 2974

Thr Arg Thr Gin Leu Arg Ala Met Ala Ala Lys Thr Val Cys Pro Pro His Lys Val Glu 780

ACG CGC ACG CAG CTT CGC GCA ATG GCT GCT AAA ACG GTC TGC CCG CCG CAT AAA GTA GAG 3034

Leu Glu Ala Leu Leu Glu Lys Gin Ala Tyr Lys Glu Gin Val Leu Ala Lys Arg Leu Thr 800

CTT GAA GCC TTG CTT GAA AAG CAA GCC TAC AAA GAA CAA GTG CTG GCA AAA CGT TTA ACA 3094

Met Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser Glu Phe lie Ala 820

ATG CTT GAA CTG CTT GAA AAA TAC CCG GCG TGT GAA ATG AAA TTC AGC GAA TTT ATC GCC 3154

Leu Leu Pro Ser lie Arg Pro Arg Tyr Tyr Ser lie Ser Ser Ser Pro Arg Val Asp Glu 840

CTT CTG CCA AGC ATA CGC CCG CGC TAT TAC TCG ATT TCT TCA TCA CCT CGT GTC GAT GAA 3214

Lys Gin Ala Ser lie Thr Val Ser Val Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu 860

AAA CAA GCA AGC ATC ACG GTC AGC GTT GTC TCA GGA GAA GCG TGG AGC GGA TAT GGA GAA 3274

Tyr Lys Gly lie Ala Ser Asn Tyr Leu Ala Glu Leu Gin Glu Gly Asp Thr lie Thr Cys 880

TAT AAA GGA ATT GCG TCG AAC TAT CTT GCC GAG CTG CAA GAA GGA GAT ACG ATT ACG TGC 3334

Phe lie Ser Thr Pro Gin Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu Thr Pro Leu lie 900

TTT ATT TCC ACA CCG CAG TCA GAA TTT ACG CTG CCA AAA GAC CCT GAA ACG CCG CTT ATC 3394

Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly Phe Val Gin Ala Arg Lys Gin 920

ATG GTC GGA CCG GGA ACA GGC GTC GCG CCG TTT AGA GGC TTT GTG CAG GCG CGC AAA CAG 3454

Leu Lys Glu Gin Gly Gin Ser Leu Gly Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro 940

CTA AAA GAA CAA GGA CAG TCA CTT GGA GAA GCA CAT TTA TAC TTC GGC TGC CGT TCA CCT 3514

His Glu Asp Tyr Leu Tyr Gin Glu Glu Leu Glu Asn Ala Gin Ser Glu Gly lie lie Thr 960

CAT GAA GAC TAT CTG TAT CAA GAA GAG CTT GAA AAC GCC CAA AGC GAA GGC ATC ATT ACG 3574

Leu His Thr Ala Phe Ser Arg Met Pro Asn Gin Pro Lys Thr Tyr Val Gin His Val Met 980

CTT CAT ACC GCT TTT TCT CGC ATG CCA AAT CAG CCG AAA ACA TAC GTT CAG CAC GTA ATG 3634

Glu Gin Asp Gly Lys Lys Leu lie Glu Leu Leu Asp Gin Gly Ala His Phe Tyr lie Cys 1000

GAA CAA GAC GGC AAG AAA TTG ATT GAA CTT CTT GAT CAA GGA GCG CAC TTC TAT ATT TGC 3694

Gly Asp Gly Ser Gin Met Ala Pro Ala Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp 1020

GGA GAC GGA AGC CAA ATG GCA CCT GCC GTT GAA GCA ACG CTT ATG AAA AGC TAT GCT GAC 3754

Val His Gin Val Ser Glu Ala Asp Ala Arg Leu Trp Leu Gin Gin Leu Glu Glu Lys Gly 1040

GTT CAC CAA GTG AGT GAA GCA GAC GCT CGC TTA TGG CTG CAG CAG CTA GAA GAA AAA GGC 3814

Arg Tyr Ala Lys Asp Val Trp Ala Gly End 1050

CGA TAC GCA AAA GAC GTG TGG GCT GGG TAA 3844

Particularly preferred monooxygenase mutants of this type are those which have at least one of the following mono- or polyamino acid substitutions:

1 ) R74G

2) F87V

3) L188Q

4) E267V

5) G415S

6) Mutation pattern as shown in Table 2 for P450 BM3 mutants MT33, MT35, MT36, MT68, MT78, MT80, MT90

and functional equivalents thereof. Table 2

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.

In this context, "functional equivalents" or analogs of the 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

monooxygenases according to the invention. Preferred 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. 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 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.

Preferably, 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.

In addition to the artificial regulatory sequences, 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. However, 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. 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 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. Particular preference is given to using inducible promoters, for example light- and in particular temperature-inducible promoters, such as the PrP1 promoter. In principle, all natural promoters with their regulatory sequences can be used. In addition, 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. Thus, 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". In addition, 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. To this end, 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

Interscience (1987). For expression in a suitable host organism, 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

abovementioned nucleic acids in the expression system in question. 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.

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

monooxygenase from the culture. If desired, 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.

To isolate the recombinant protein, it is particularly advantageous to use 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).

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.

These 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

a2) incubating a substrate-containing reaction medium with an enzyme according to the invention, and b) isolating the hydroxylation product formed or a secondary product thereof from the medium.

The present invention is now described in greater detail with reference to figures 1 to 6 and the following examples. Examples

Figure Legends

Figure 1 : Structures of α-ionone enantiomers and their 3 ^' hydroxylated products. A: {6R)- a-ionone B: f6S)-a-ionone C: (3S,6f?j-OH -a-ionone D: (3fl,6Sj-OH-a-ionone E: (3R,6R)- OH -a-ionone F: (3 S,6S,)-OH -a-ionone C &D are the cis isomers and E & F are the trans isomers

Figure 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 α-ionone by (A) MT80 (B) MT33. (C) MT44. The identification of the peaks: (1 ) cis-3-ΟΗ-α ionone t _R 9.84 (2) trans-3-ΟΗ-α 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 α-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 α-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.

Materials and Methods

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

Netherlands. All other chemicals were of analytical grade and purchased from Sigma unless otherwise mentioned. Restriction endonucleases were obtained from Westburg, Pfu polymerase and isopropyl β-D-thiogalactopyranoside (IPTG) were obtained from Fermentas. Enzymes and plasmids

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

mutagenesis protocol using the forward primers mentioned in Table 3.The reverse primers were exactly complementary to the forward primers. After mutagenesis, the presence of right mutations were verified by DNA sequencing (Service XS, Leiden, The

Netherlands). The whole gene was subcloned to pET28a+ vector using BamYW and EcoR\ sites and later transformed to BL21 (DE3) cells for expression.

Table 3: Forward primers used for site directed mutagenesis

Target sites Oligonucleotide sequences

74 5'-GATAAAAACTTAAGTCAAGAWCTTAAATTTGTACGT-3' (D)

82 5'-GTCAAGCGCTTAAATTTGTTCGCGATTTTTGGGGAGACGGG-3' (W)

87 5'-GCAGGAGACGGGTTA XACTAGTTGGACGCAT-3' ^a

437 5'-CTACGAGCTCGATATTAAAAACACTGAGACGTTAAAACCTGAAGGCTTTGTGG-3' (N)

5'-CTACGAGCTCGATATTAAAAGCACTGAGACGTTAAAACCTGAAGGCTTTGTGG- 3'

(S)

5'-CTACGAGCTCGATATTAAAACCACTGAGACGTTAAAACCTGAAGGCTTTGTGG-3' (T) ^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.

Expression and Purification of P450 BM3 mutants

For screening of products of racemic a-ionone hydroxylation, 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 μg/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

resuspended in 20 mL 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. 3 mL of Ni-NTA slurry 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.

Analysis of biotransformation of g-ionone by GC-MS

For the biotransformation reaction, 200 nM of cytosolic fraction was incubated with 500 μΜ of racemic oionone in a final volume of 250 μί. The reaction was initiated by the addition of 25 μί 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. After addition of 10 μΙ_ 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.

The extracts were analyzed using a Hewlett-Packard 6890 series with AT AS

Programmable Injector 2.1. Samples were separated on a DB-5 GC-column (30m x 0.25cm). Ionization was done by electron impact (El) at 70 eV with the mass selective detector 5973. The injector temperature was maintained at 250 ^° C.The temperature program for the GC was as follows: 2min. stationary at 60°C, ramp at 20°C min ^"1 to 280°C and maintained at 280 ^° C for 4min. For incubations with individual enantiomers, same conditions were used as described above.

Determination of total turnover numbers of MT80 and MT33

200 nM of purified enzymes (MT80 and MT33) was incubated with 1 mM of (f?)- or (S)- enantiomers (2 % methanol) in a total volume of 500 μί. The reaction was initiated by the addition of 50 μί NADPH regenerating system as mentioned in section 2.4. The reaction was performed in duplicate in 1 .5 mL eppendorf tubes at 24 ^° C for 12 hours with shaking. After this incubation time, benzoxyresorufin-O-dealkylation was used to check the enzyme inactivation to ensure completion of the reaction. Briefly, an aliquot of the incubation (62.5 μί) was mixed with Kpi buffer to a final volume of 125 μί. Benzoxyresorufin (25 μί) was added in the assay buffer at a final concentration of 10 μΜ.

100 μΜ of NADPH regenerating system was added to the reaction mixture and the reaction was monitored on a micro-plate reader. No increase in resorufin fluorescence was observed indicating that the enzyme was completely inactive. The samples were then extracted and analyzed by GC-MS as described above. The same procedure was repeated for both MT80 and MT33 at a higher enzyme concentration (1 μΜ) and higher substrate concentration (5 mM containing 2% methanol). Total turnover numbers were calculated as nmol of product formed per nmol enzyme by using the calibration curve of (3S,6S)-OH-a-ionone (R ² =0.9982) with concentrations ranging from 0.1 mM to 1 mM. Results

Screening of the P450 BM3 library with racemic a- ionone

As shown in Figure 3, 2 distinct product profiles were observed: mutants that converted racemic a-ionone predominantly to trans-3-OH-oionone and mutants which produced almost equal amounts of both cis and trans hydroxy enantiomers. The retention time (t _R ) of trans-3-OH-oionone and cis-3-OH-oionone were 9.84 and 9.92 minutes respectively based on the reference hydroxy compounds. Mutants MT80, MT78, MT68 converted racemic α-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 α-ionone to products other than the desired ones with low activity. The product profile of the mutants tested with racemic α-ionone is shown in Figure 4.

Table 4: Product profiles and conversion of substrate by selected BM3 mutants showing high selectivity to 3 -OH product

Mutant % cis -OH- a- ionone % trans-OH-a- ionone Substrate

conversion(%) ^a

MT80 6.2 90 80

MT78 6 87 76

MT68 10 87.1 60

MT33 48 50 65

MT35 40 48 67

MT36 38.9 48.2 83

a Conversions in % were measured with 200 nM P450, 500 μΜ racemic α-ionone after 1 hour. Substrate conversion and product distribution were calculated based on the peak area ratio between α-ionone and the internal standard, before and after reactions. Error rates are not more than 10%. The amino acid substitutions in the above mentioned mutants with respect to wildtype CYP102A1 are summarized in Table 2. It was also seen that some of the mutants converted racemic α-ionone to 3-oxo-a-ionone and an unidentified over oxidation product in addition to trans-3-OH and cis-3-ΟΗ-α- ionone. Representative gas chromatogram of this product profile is shown in Figure 3C. 3- oxo-a-ionone has been recently shown to be a phagostimulant and also has commercial value because of its fragrance. Incubation with individual enantiomers

As seen from the screening of the mutants with racemic a-ionone, 3 mutants MT33, MT35, MT36 were aselective producing 2 peaks on achiral GC analysis. In order to elucidate which particular hydroxy diastereomer was formed, these three mutants were incubated with individual enantiomers (6f?) and (6S). Interestingly, all three of them turned out to be stereoselective with individual enantiomer incubations producing (3S,6S)-OH-a- ionone with (6S)-enantiomer and (3S,6f?)-OH-oionone with (6f?)-enantiomer. Among these 3 mutants, 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. The fact that the P450 BM3 mutants MT80 and MT33 exhibit different stereo selectivities towards hydroxylation of (f?)-and (S)-enantiomers, makes them highly useful for industrial biotechnological applications. In order to assess the catalytic capacity of these enzymes, the total turnover numbers of MT80 and MT33 for each of the stereoselective

hydroxylation reaction was determined.

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). Similarly MT33 can also catalyze the formation of (3S, 6f?)-OH-oionone with high turnover numbers of about 3000. For both MT80 and MT33 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.

Table 5: Stereo selective hydroxylation of (/7)-a-ionone and (S)-a-ionone by engineered BM3

Hydroxylated Diastereomeric Total turnover

Mutant Substrate

product formed excess (d.e. %) number ³

(R)- a- ionone 3R,6R 91 3500

MT80

(S)- a- ionone 3S,6S 95 4000

(R)- a- ionone 3S,6R 90 3000

MT33

(S)- a- ionone 3S,6S 91 3800

a Total turnover numbers are reported as nmol product per nmol enzyme. The values are calculated from 2 independent experiments with ± 5% error.

Based on the above results, the tested BM3 mutants were able to catalyze selectively, the formation of 3 out of the 4 possible hydroxy diastereomers. In particular the preferred BM3 mutants MT33 and MT80 show the following properties (Figure 9).

• Regio and stereoselective hydroxylation of optically active oionone

• 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.