FIELD OF THE INVENTION

[0001 ] The present invention relates to the field of biocatalysts and more specifically to the field of monooxygenases. More specifically, the present invention relates to methods for functionalizing 1 ,8-cineole and polynucleotides, polypeptides, host cells and vectors for doing the same. The invention also relates to biohydroxylated 1 ,8-cineole, which gives rise to 2-hydroxycineole.

BACKGROUND OF THE INVENTION

[0002] 1 ,8-cineole (1 ,3,3-Trimethyl-2-oxabicyclo[2,2,2]octane) is a chemically stable saturated ditertiary ether. It is a bicyclic monoterpenoid and a major component of many essential oils, including eucalyptus oil. It possesses antimicrobial properties and a camphor-like scent. 1 ,8-cineole is an abundant natural resource, especially from the essential oil of several species of the genus Eucalyptus and from turpentine as a by-product of commercial pine oil production.

[0003] Because 1 ,8-cineole is relatively chemically inert as there are no activated C-H bonds in the molecule, it is a compound of small economic significance. It requires to be functionalised before it is suitable for further modification such as esterification, etc. The product of functionalizing 1 ,8-cineole such as oxidised or hydroxylated-cineole derivatives are chiral compounds that can serve for example, as building blocks and precursors for uses in organic chemistry. 1 ,8-cineole and its functionalised forms have applications for example in the manufacture of pharmaceuticals, insecticides, insect repellents, fragrances, cleaning products, and as flavouring in the food industry.

[0004] There are a number of chemical processes for functionalising 1 ,8-cineole but each of these has its disadvantages. For example, chemical techniques may be used to hydroxylate 1 ,8-cineole but involves a number of steps, often using harsh chemical conditions. Chemical techniques also result in a mixture of isomers of hydroxylated 1 ,8-cineole and it is challenging to purify a specific isomer. Accordingly, stereospecific introduction of functional groups such as hydroxyl-group to non- activated carbon atoms remains a challenge in synthetic organic chemistry. Examples of processes for producing functionalised 1 ,8-cineole using organic chemistry processes include multi-step syntheses using hot aqueous acids, multiple oxidative treatments, bromination, peracid treatments and other harsh or even hazardous chemical techniques. Free-radical reactions such as photochlorination yield a complex mixture of products. These chemical processes generally require the use of harsh reaction conditions and there is little to no control over the stereoisomers created during the chemical reaction. Furthermore, these chemical processes are usually complex and multi-stepped.

[0005] An alternative way of functionalising 1 ,8-cineole is through biological transformation such as by enzymes. Biotransformation of 1 ,8-cineole has been reported to occur in a number of organisms. For example, several alcohol and ketone metabolites such as 2-exo-hydroxy-1 ,8-cineole, (±)-3-encfo-hydroxy-1 ,8-cineole, (±)-3- exo-hydroxy-1 ,8-cineole, 2-oxoA ,8-cineole and 3-oxo-1 ,8-cineole were identified in rabbit urine.

[0006] Biotransformation of 1 ,8-cineole can also occur within bacterial cells. Bacterial 1 ,8-cineole metabolism was first discovered in the late 1970s when the isolation of Pseudomonas flava capable of growing on 1 ,8-cineole as a sole source of carbon was reported. Subsequently, 1 ,8-cineole biohydroxylation has also been observed in several other bacterial species including Rhodococcus C1 , Bacillus cereus, Citrobacter braakii, Novosphingobium subterranea and Sphingomonas capsulata.

[0007] However, only one enzyme involved in the bacterial 1 ,8-cineole- hydroxylation has been identified, isolated and characterised. It was shown that the initial oxidation in the gram-negative bacterium C. braakii was catalysed by a P450 designated P450 _C i _n with the aid of electron transport partners to yield (1 )-6β- Hydroxy-1 ,8-cineole. [0008] Cytochrome P450s are a diverse group of oxidative hemoproteins that catalyse an enormous range of reactions including the introduction of an oxygen atom into inactivated carbon-hydrogen bonds. Sequences and nomenclature information are available for more than 1 1500 different P450 proteins originating from all kingdoms of life including more than 1 100 bacterial P450s. Several of these P450s are involved in monoterpenoid oxidation, including P450 _ca m isolated from Pseudomonas putida grown on (1 R)-(+)-camphor.

[0009] There are a number of advantages associated with biohydroxylation of 1 ,8- cineole. For example, the number of steps in the process is generally reduced, the reaction conditions are usually mild and enzymes usually display stereoselectivity or stereospecificity with regards to the reaction products.

[0010] There is a need for enzymes capable of biohydroxylating 1 ,8-cineole in order to functionalise this chemically inert molecule. In particular, the stereoselectivity or stereospecificity of the enzyme results in different stereoisomers of hydroxycineole.

SUMMARY OF THE INVENTION

[001 1 ] Functionalizing 1 ,8-cineole by chemical methods presently available requires multiple and complicated steps to achieve the end product, which may be hydroxycineole. The use of biocatalysts and more specifically the use of monooxygenases can reduce the number of steps required to functionalize 1 ,8- cineole.

[0012] One aspect of the invention provides an isolated polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof selected from the group comprising:

(i) a sequence of nucleotides as provided in SEQ ID NO: 2, 32 or 34, or variant thereof;

(ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in SEQ ID NO: 1 , 31 or 33 or a biologically active fragment or equivalent thereof; (iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 75% similar to SEQ ID NO: 1 , 31 or 33 or a biologically active fragment or equivalent thereof;

(iv) a sequence of nucleotides comprising at least SEQ ID NO: 27 or SEQ ID

NO: 28 or the reverse complements thereof, wherein the sequence comprises a portion of a polynucleotide as provided in SEQ ID NO: 2 or a biologically active fragment or equivalent thereof or a sequence of nucleotides comprising at least SEQ ID NO: 35 or SEQ ID NO: 36 or the reverse complements thereof, wherein the sequence comprises a portion of a polynucleotide as provided in SEQ ID NO: 32, or a biologically active fragment or equivalent thereof or a sequence of nucleotides comprising at least SEQ ID NO: 37 or SEQ ID NO: 38 or the reverse complements thereof, wherein the sequence comprises a portion of a polynucleotide as provided in SEQ ID NO: 34, or a biologically active fragment or equivalent thereof; and

(v) a sequence complementary to any one of (i) to (iv).

[0013] The present invention is based in part on the identification and characterisation of Sphingobium yanoikuyae polypeptides that are capable of functionalizing 1 ,8-cineole.

[0014] The P450 monooxygenases of the present invention demonstrate stereoselectivity in that they functionalize 1 ,8-cineole preferably in the 2-position to produce the product 2-hydroxy cineole. Accordingly, in one embodiment the main product of 1 ,8-cineole hydroxylation is 2-hydroxy cineole.

[0015] In another aspect there is provided substantially purified and/or recombinant polypeptides having P450 monooxygenase activity or biologically active fragments or equivalents thereof wherein said polypeptides hydroxylate or functionalize 1 ,8-cineole in the 2-position of 1 ,8-cineole or oxidises a hydroxy-1 ,8- cineole such as 6-hydroxy-1 ,8-cineole in the 2-position to form oxo-hydroxy-1 ,8- cineole, wherein the polypeptides comprise amino acid sequences which are at least 75% similar to SEQ ID NO: 1 or 31 .

[0016] In another embodiment the substantially purified and/or recombinant polypeptide is selected from the group comprising:

(i) an amino acid sequence as provided in SEQ ID NO: 1 or 31 ;

(ii) an amino acid sequence encoded by a polynucleotide which encodes a

P450 monooxygenase as hereinbefore described; and

(iii) biologically active fragment or equivalent thereof.

[0017] In another aspect the invention provides a vector comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof as herein described.

[0018] In yet another aspect the invention provides a host cell comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof as herein described, and/or at least one plasm id comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof as herein described.

[0019] In one embodiment, the host cell expresses any one of the P450 monooxygenases of the present invention and facilitates the whole-cell biotransformation or functionalization of 1 ,8-cineole to 2-hydroxy-cineole or oxidises a hydroxy-1 ,8-cineole such as 6-hydroxy-1 ,8-cineole in the 2-position to form oxo- hydroxy-1 ,8-cineole. The host cell may comprise one, two or three of the P450 monooxygenases of the present invention and expresses one, two or three of the P450 monooxygenases. In another embodiment, the host cell of the present invention is capable of metabolising further cineole-derivatives including but not limited to oxo-hydroxy-1 ,8-cineole, hydroxy-2,6-oxo-1 ,8-cineole from a hydroxyl 1 ,8 cineole such as (1 R)-6p-hydroxy-1 ,8-cineole. [0020] In a further aspect the invention provides a host cell comprising a polynucleotide or vector as hereinbefore described and further comprising an electron transport partner such as but not limited to a ferredoxin (FdX) and/or a ferredoxin reductase (FdR). The electron transport partners such as FdX and/or the FdR may be expressed on separate vectors.

[0021 ] In another aspect of the invention, there is provided a method of expressing a monooxygenase which functionalizes 1 ,8-cineole to a 2 hydroxy cineole, said method comprising:

(i) providing a vector comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof which functionalizes 1 ,8 cineole to a 2-hydroxy cineole;

(ii) providing another vector comprising a polynucleotide which encodes a first electron transport partner or a biologically active fragment or equivalent thereof; and

(iii) optionally providing a further vector comprising a polynucleotide which encodes another electron transport partner or a biologically active fragment or equivalent thereof; and

(iv) inserting at least two of the vectors into a host cell to express the monooxygenase.

[0022] The Applicants have found a method which enables reliable expression of a monooxygenase which can hydroxylate and functionalize 1 ,8-cineole to produce a 2 hydroxy cineole. This method explicitly expresses the monooxygenase and the electron transport partners on separate vectors.

[0023] In another aspect of the invention there is provided a method of functionalizing 1 ,8 cineole said method comprising contacting 1 ,8 cineole with an effective amount of a monooxygenase polypeptide as hereinbefore described to produce 2-hydroxycineole. The monooxygenase polypeptide which contacts 1 ,8- cineole may be any one of the polypeptides of P450 _B ui , P450 _B u2 or P450 _B u3 as hereinbefore described.

[0024] In one embodiment the invention provides a method of functionalizing 1 ,8 cineole said method comprising

(i) transforming a host cell with a polynucleotide or vector, which encodes any one of the P450 monooxygenase polypeptides of the present invention as hereinbefore described;

(ii) culturing the host cell under conditions suitable to express the polypeptide encoded by the polynucleotide;

(iii) exposing the host cell and the polypeptide to a substrate of 1 ,8-cineole.

[0025] A further aspect of the invention provides an isolated polynucleotide which encodes a ferredoxin or a biologically active fragment or equivalent thereof selected from the group comprising:

(i) a sequence of nucleotides which is,

(a) at least 50% similar to FdX1 (SEQ ID NO: 4); or

(b) at least 59% similar to FdX2 (SEQ ID NO: 6); or

(c) at least 50% similar to FdX3 (SEQ ID NO: 8); or

(d) at least 50% similar to FdX4 (SEQ ID NO: 10); or

(e) at least 46% similar to FdX5 (SEQ ID NO: 12); or

(f) at least 84% similar to FdX6 (SEQ ID NO: 14); or

(g) at least 98% similar to FdX7 (SEQ ID NO: 16) (ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in any one of SEQ ID NO: 3, 5, 7, 9, 1 1 , 13 and 15, or a biologically active fragment or equivalent thereof;

(iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is,

(a) at least 71 % similar to FdX1 (SEQ ID NO: 3); or

(b) at least 99% similar to FdX4 (SEQ ID NO: 9); or

(c) at least 67% similar to FdX5 (SEQ ID NO: 1 1 );

(iv) a sequence complementary to any one of (i) to (iii); and

(v) a biologically active fragment or equivalent and/or variant thereof. 6] In another aspect the invention provides an isolated polynucleotide which encodes a ferredoxin reductase or a biologically active fragment or equivalent thereof selected from the group comprising:

(i) a sequence of nucleotides which is,

(a) at least 86% similar to FdR1 (SEQ ID NO: 18); or

(b) at least 71 % similar to FdR3 (SEQ ID NO: 22); or

(c) at least 59% similar to FdR4 (SEQ ID NO: 24); or

(d) at least 67% similar to FdF s (SEQ ID NO: 26);

(ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in any one of SEQ ID NO: 17, 19, 21 , 23 and 25, or a biologically active fragment or equivalent thereof;

(iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is, (a) at least 98% similar to FdR4 (SEQ ID NO: 23); or

(b) at least 99% similar to FdF s SEQ ID NO: 25);

(iv) a sequence complementary to any one of (i) to (iii); and

(v) a biologically active fragment or equivalent and/or variant thereof.

[0027] In a further aspect the invention provides a vector comprising at least one polynucleotide encoding a ferredoxin reductase as hereinbefore described.

FIGURES

[0028] Figure 1 shows a Coomassie-stained SDS-PAGE (4-12% Bis-Tris, MES running buffer) of PF (purified fraction) 1 and 2 after IEX (ion-exchange) and GF (gel filtration). Lane 1 : Molecular weight marker. Lane 2: PF 1 after IEX. Lane 3 and 4: PF 1 after GF. Lane 5: PF 2 after IEX. Lane 3 and 4: PF 2 after GF.

[0029] Figure 2 shows SDS-PAGE analysis of purified P450 proteins. Figure 2A lane 1 : molecular weight markers, lane 2: purified P450 _B ui protein, lane 2: P450 _B u3 protein. Figure 2B lane 1 : molecular weight markers, lane 2: purified P450 _B u2 protein.

[0030] Figure 3 shows absorbance spectra of (A) P450 _BU i , (B) P450 _BU2 and (C) P450 _B u3 in 50 mM Tris, pH 7.4, of the oxidised (full black line), oxidised in the presence of 1 ,8-cineole (full gray line), reduced by the addition of sodium dithionite in the presence of 1 ,8-cineole (broken black line) and reduced and in complex with CO in the presence of 1 ,8-cineole (broken grey line) forms. All three P450s show the characteristic absorbance peaks and shifts.

[0031 ] Figure 4A shows a schematic representing the combination of the three expression vectors transformed into E. coli. Conversion of 1 ,8-cineole to 2- hydroxycineole using the various combinations of P450 _BU i and ferredoxins found in the new strain of S. yanoikuyae. [0032] Figure 4B shows a bar graph representing the efficiency of hydroxylating 1 ,8-cineole by E. coli transformed with each combination of P450 _B ui with S. yanoikuyae ferredoxin FdX1 , FdX2, FdX3, FdX4, FdX5, FdX6 or FdX7. The negative control is E. coli with "empty vectors". The error bars represent one standard deviation from the mean.

[0033] Figure 5A shows a schematic representation of the plasm ids transformed into E. coli. B: Conversion of 1 ,8-cineole to 2-hydroxycineole using the various combinations of P450 _B ui and ferredoxin reductases found in the S. yanoikuyae of the present invention.

[0034] Figure 5B shows a bar graph representing the efficiency of hydroxylating 1 ,8-cineole by E. coli transformed with each combination of P450 _B ui with S. yanoikuyae ferredoxin reductase FdR1 , FdR2, FdR3, FdR4 or FdR4s. The negative control is E coli with all three "empty vectors". The error bars represent standard deviation from the mean.

[0035] Figure 6 shows a bar graph representing the efficiency of each combination of P450 _B ui with any one of S. yanoikuyae ferredoxin FdX1 , FdX2, FdX3, FdX4, FdX5, FdX6 and FdX7 and any one of S. yanoikuyae ferredoxin reductase FdR1 , FdR2, FdR3, FdR4 and FdR4s and its efficiency at hydroxylating 1 ,8-cineole to 2-hydroxycineole. The negative control is E coli with all three "empty vectors". The error bars represent one standard deviation from the mean.

[0036] Figure 7A shows the rates of hydroxy-cineole production by various combinations of P450 _BU i with S. yanoikuyae ferredoxin reductases and ferredoxins. 1 ,8-cineole was added to the cell at 0 hours, 3 hours and 6 hours. The error bars represent one standard deviation from the mean.

[0037] Figure 8 show the rate of production of 2-hydroxycineole using recombinant P450 _BU i in combination with FdR3 and FdX2 in a bioreactor.

[0038] Figure 9 shows the rate of production of 2-hydroxycineole using recombinant P450 _BU2 in combination with FdR3 and FdX2 in a bioreactor when cultured at 30°C. [0039] Figure 10 shows the rate of production of 2-hydroxycineole using recombinant P450 _B u3 in combination with FdR3 and FdX2 in a bioreactor when cultured at 30°C.

[0040] Figure 1 1 shows the rate of production of 2-hydroxycineole using recombinant P450 _B u3 in combination with FdR3 and FdX2 in a bioreactor when cultured at 20°C.

[0041 ] Figure 12 shows production of cineole derivatives in a bioreactor using P450 _B ui-FdR3-FdX2 at 30°C using Terrific broth as the growth medium. The substrate used was (1 R)-6B-hydroxy-1 ,8-cineole. Samples of the culture were taken just prior to and just after addition of the substrate and when the culture was ended. "Oxo-1 ,8-cineole" = (1 R)-2-oxo-1 ,8-cineole; "Hydroxy-1 ,8-cineole" = (1 R)-6B-hydroxy- 1 ,8-cineole and "Oxo-hydroxy-1 ,8-cineole" = 2-hydroxy-6-oxo-1 ,8-cineole or 6- hydroxy-2 -oxo-1 ,8-cineole.

[0042] Figure 13 shows GC MS data showing production of hydroxy-1 ,8-cineole using P450 _B ui , P450 _BU 2, P450 _BU 3 at 30°C using Terrific broth. Peak with a retention time of 2.76 minutes = cineole; Peak with a retention time of 4.29 minutes = hydroxy- 1 ,8-cineole; peak at 4.72-4.73 min is possibly indole extracted from the medium.

[0043] Figure 14 shows the polynucleotide and polypeptide sequences of P450 _B ui , P450 _B u2 and P450 _BU 3, as well as the T7 sequencing primers suitable amplifying the cloned sequences from pCDFDuet and the oligonucleotide primers for PCR amplifying P450 _BU i , P450 _BU 2 and P450 _BU3 from messenger RNA.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Functionalizing 1 ,8-cineole by chemical methods presently available requires multiple and complicated steps to achieve the end product, which may be hydroxycineole. Biological transformation of 1 ,8-cineole can substantially simplify the process.

[0045] Accordingly in a first aspect the invention provides an isolated polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof selected from the group comprising:

(i) a sequence of nucleotides as provided in SEQ ID NO: 2, 32 or 34, or variant thereof;

(ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in SEQ ID NO: 1 , 31 or 33 or a biologically active fragment or equivalent thereof;

(iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 75% similar to SEQ ID NO: 1 , 31 or 33 or a biologically active fragment or equivalent thereof;

(iv) a sequence of nucleotides comprising at least SEQ ID NO: 27 or SEQ ID

NO: 28 or the reverse complements thereof, wherein the sequence comprises a portion of a polynucleotide as provided in SEQ ID NO: 2 or a biologically active fragment or equivalent thereof or a sequence of nucleotides comprising at least SEQ ID NO: 35 or SEQ ID NO: 36 or the reverse complements thereof, wherein the sequence comprises a portion of a polynucleotide as provided in SEQ ID NO: 32, or a biologically active fragment or equivalent thereof or a sequence of nucleotides comprising at least SEQ ID NO: 37 or SEQ ID NO: 38 or the reverse complements thereof, wherein the sequence comprises a portion of a polynucleotide as provided in SEQ ID NO: 34, or a biologically active fragment or equivalent thereof; and (v) a sequence complementary to any one of (i) to (iv).

[0046] Cytochrome P450 proteins are a diverse family of proteins containing a heme cofactor and therefore are classified as hemoproteins. P450 proteins use a variety of small and large molecules as substrates in enzymatic reactions. P450 proteins have been identified in all domains of life, such as but not limited to animals, plants, fungi, protists, bacteria, archaea, and even in viruses.

[0047] Monooxygenases are enzymes that incorporate one hydroxyl group into its substrate. The phrase "monooxygenase activity" refers to an enzyme's ability to incorporate a hydroxyl group into its substrate.

[0048] The present invention is based in part on the identification and characterisation of Sphingobium yanoikuyae polypeptides that are capable of functionalizing 1 ,8-cineole. Without being limited by theory, in one example these isolated polypeptides were N-terminally sequenced using an Applied Biosystems Procise sequencer using solid support (glass fibre disc) and following traditional Edman degradation techniques. The amino acid sequences of these polypeptides were then used to infer the possible encoding polynucleotide sequences based on the sequenced genome of S. yanoikuyae.

[0049] Only one other enzyme has been identified and involved in bacterial 1 ,8- cineole-hydroxylation. The enzyme P450 _cin from C. braakii with the aid of electron transport partners yielded (1 R)-6p-Hydroxy-1 ,8-cineole. The present inventors have now found a novel source of enzymes that are capable of 1 ,8-cineole-hydroxylation as well as novel enzymes capable of hydroxylating 1 ,8-cineole to provide 2- hydroxycineole.

[0050] Sphingobium sp. is a relatively common soil bacterium although S. yanoikuyae was originally isolated from a clinical sample. The Sphingobium yanoikuyae of the present invention was isolated from activated sludge and in part selected on the basis of its ability to metabolise 1 ,8-cineole as the sole source of carbon. The skilled addressee would appreciate that activated sludge is a diverse microbial culture. It is known that Sphingobium species are capable of degrading a variety of chemicals in the environment such as aromatic and chloroaromatic compounds, phenols like nonylphenol and pentachlorophenol, herbicides such as (RS)-2-(4-chloro-2-methylphenoxy) propionic acid and hexachlorocyclohexane, and polycyclic aromatic hydrocarbons. Furthermore, it is also known that different strains of S. yanoikuyae are capable of utilizing different carbon sources for energy production, e.g. strain XLDN2-5 is able to metabolise carbazole and strain B1 is able to metabolise biphenyl, naphthalene, phenanthrene, toluene, and m-/p-xylene as sole sources of carbon energy. Therefore the selection of S. yanoikuyae as a source of a monooxygenase which is capable of hydroxylating 1 ,8-cineole is surprising.

[0051 ] These polynucleotides were found to encode proteins which presented with the typical Soret absorbance of a P450 protein, that is an absorbance maxima of 416 - 417 nm in the absence of its substrate. The absorbance maxima shift to a lower range of wavelengths of 392 - 396 nm upon binding to their substrates 1 ,8-cineole. Accordingly, the polynucleotides encode a P450 protein, preferably a P450 monooxygenase.

[0052] The P450 proteins of the present invention have been further demonstrated to preferentially bind 1 ,8-cineole. Four other related substrates, 2- adamantanone, β-ionone, (1 S)-(-)-camphor and (1 R)-(+)-camphor were also tested for binding to the P450 monooxygenases of the present invention and none of them bound to any significant extent, except for weak binding by β-ionone.

[0053] When 1 ,8-cineole is added to a recombinant E. coli culture expressing the P450 proteins of the present invention an intermediate metabolic product was isolated and identified (1 S)-2a-hydroxy-1 ,8-cineole.. Accordingly, the P450 proteins of the present invention were classified as P450 monooxygenases.

[0054] 1 ,8-Cineole has low chemical reactivity since there are no activated C-H bonds in the molecule. The regioselective hydroxylation of such structure remains a challenge in organic synthesis and studies on the chemistry of this monoterpenoid is mostly related to the cleavage of the ether bridge to obtain p-menthane derivatives (Boggiato et al., 1987; Liu and Rosazza, 1990). Known chemical processes for functionalising 1 ,8-cineole, such as by chemical techniques, generally require the use of harsh reaction conditions and there is little or no control over the stereoisomer created during the chemical reaction. As a result, significant time and costs are required downstream to purify the desired isomer from the reaction product mixture.

[0055] Microbial transformation of 1 ,8-cineole has a number of advantages including mild reaction conditions, biodegradable reagents, etc. Another main advantage of biotransformation is the stereoselectivity or stereospecificity of enzymes. Stereoselectivity of an enzyme refers to a property of the enzyme wherein during biotransformation, a single reactant is transformed into an unequal mixture of stereoisomers during the non-stereospecific transformation of the reactant. Stereospecificity of an enzyme refers to a property of an enzyme wherein during biotransformation, a single reactant is transformed to a single stereoisomer. This means that biotransformation produces only specific stereoisomers and the process is predictable. The isomerism of the functionalised groups is important for further processing of the product. For example, in certain circumstances, a specific stereoisomer is desired.

[0056] The P450 monooxygenases of the present invention demonstrate stereoselectivity in that they functionalize 1 ,8-cineole preferably in the 2-position to produce the product 2-hydroxy cineole. Accordingly, in one embodiment the main product of 1 ,8-cineole hydroxylation is 2-hydroxy cineole. The P450 monooxygenases of the present invention are also able to utilise (1 R)-6B-hydroxy- 1 ,8-cineole as its substrate and functionalize (1 R)-6B-hydroxy-1 ,8-cineole to oxo- hydroxy-1 ,8-cineole thereby producing a difunctionalized 1 ,8 cineole. Hence the invention allows for functionalization at additional positions of the compound for any hydroxylated cineole. Applicants have exemplified functionalization of (1 R)-6B- hydroxy-1 ,8-cineole to provide difunctionalized 1 ,8 cineole. However, any 1 ,8 cineole may be used as a substrate that can be further functionalized. For instance, any one of 2-hydroxy-1 ,8 cineole, 3-hydroxy-1 ,8 cineole, 5-hydroxy-1 ,8 cineole, 6-hydroxy-1 ,8 cineole, 7-hydroxy-1 ,8 cineole, 9-hydroxy-1 ,8 cineole, or 10-hydroxy-1 ,8 cineole may be further functionalized to provide a multifunctionalized 1 ,8 cineole being functionalized at least at one additional position of the compound. [0057] The polynucleotides of the present invention are not required to encode for the full length P450 monooxygenase polypeptides, rather, they may encode biologically active fragments or equivalents thereof.

[0058] The polynucleotides of the present invention may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. Such polynucleotides encoding P450 monooxygenases may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al.), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0059] The phrase "biologically active fragment" when used to refer to a polypeptide refers to a portion of a larger molecule, which retains the same activity as the polypeptides of the present invention.

[0060] The term "equivalent" when used herein refers to a molecule which is different but retains the same activity as the polypeptides of the present invention. For example, two polypeptides may have different polypeptide sequences but both polypeptides are capable of functionalizing or biohydroxylating 1 ,8-cineole to 2- hydroxycineole or functionalizing or biohydroxylating (1 R)-6B-hydroxy-1 ,8-cineole to oxo-hydroxy-1 ,8-cineole. Similarly, two polynucleotides may have different sequences but encode for polypeptides having the same activity as the polypeptides of the present invention and hence provide equivalents. The polypeptides encoded by these polynucleotides are therefore capable of functionalizing or biohydroxylating 1 ,8-cineole to 2-hydroxycineole or functionalizing or biohydroxylating (1 R)-6B- hydroxy-1 ,8-cineole to oxo-hydroxy-1 ,8-cineole.

[0061 ] To assess whether a polypeptide is a biologically active fragment or equivalent of the P450 monooxygenases of the present invention, a substrate-binding assay based on observing the spectral absorbance shift when a P450 monooxygenase binds to its substrate may be used. To assess whether a polypeptide is a biologically active fragment or equivalent of the electron transport partners such as but not limited to ferredoxins or ferredoxin reductases of the present invention, the polypeptide may be co-expressed with the P450 monooxygenase of the present invention, and the rate of functionalization or biohydroxylation of 1 ,8-cineole may be measured to determine if there is an increase in the rate of functionalization or biohydroxylation.

[0062] The phrase "variant" when used to refer to a polynucleotide refers to a sequence of a polynucleotide that still comprises the information for translation into the same protein. Degeneracy in the genetic code or codons is one way in which a variant of the polynucleotides of the present invention still comprises the information for translation into the same polypeptide.

[0063] To assess whether a polynucleotide is a variant of a polynucleotide of the present invention, the polynucleotide can be translated into a polypeptide in silico by reference to a codon degeneracy table, then comparing the sequence of the resultant polypeptide to the sequences of the polypeptides of the present invention. Alternatively, a polynucleotide can be cloned into an expression vector and transformed into a host cell. The host cell can then be cultured in the presence of 1 ,8-cineole and assessed for its ability to functionalize 1 ,8-cineole to 2a- hydroxycineole.

[0064] The term "functionalize", "functionalizing" or "functionalization" as used herein refers to modifying a compound such as by hydroxylation, biohydroxylation, or oxidation to add a functional group to the compound.

[0065] The polynucleotides of the present invention encode for P450 monooxygenases that display stereoselectivity when they functionalize 1 ,8-cineole to 2-hydroxy cineole, and were further characterised to be (1 S)-2a-hydroxy-1 ,8-cineole as determined preferably by chiral gas chromatography (GC), GC-MS, nuclear magnetic resonance (NMR) and optical rotation measurements. The analysis also determined that the hydroxy-cineole is not (1 R)-6p-hydroxy-1 ,8-cineole or its enantiomer (1 S)-2p-hydroxy-1 ,8-cineole.

[0066] In this aspect, the polynucleotides of the P450 monooxygenases of the present invention are defined in SEQ ID NO: 2, 32 or 34. The polynucleotides may comprise polynucleotides that encode polypeptides according to SEQ ID NO: 1 , 31 or 33. The P450 monooxygenase defined according to the polynucleotide of SEQ ID NO: 2 and the polypeptide of SEQ ID NO: 1 is herein referred to as P450 _B ui - The P450 monooxygenase defined according to the polynucleotide of SEQ ID NO: 32 and the polypeptide of SEQ ID NO: 31 is herein referred to as P450 _BU2 . The P450 monooxygenase defined according to the polynucleotide of SEQ ID NO: 34 and the polypeptide of SEQ ID NO: 33 is herein referred to as P450 _B u3-

[0067] For the purpose of simplicity, the P450 monooxygenases of the present invention will be referred to as P450 _B ui , P450 _BU 2 or P450 _BU 3- Where P450 _BU i , P450 _BU 2 or P450 _BU 3 are referred to, these include the biologically active fragments and equivalents of their polynucleotide sequences and the variants of their polypeptide sequences.

[0068] The nucleotides encoding the STOP codon of the P450 monooxygenases of the present invention have been substituted from TGA to TAA to optimise protein translation in E. coli. Further codon optimisation is possible.

[0069] Furthermore, the polynucleotide may encode a polypeptide that is at least 75% similar to the polypeptide of SEQ ID NO: 1 , 31 or 33. More preferably, the polynucleotide encodes a polypeptide that is at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of SEQ ID NO: 1 , 31 or 33.

[0070] This aspect of the invention further provides oligonucleotide primers according to SEQ ID NOs: 27, 28, 35, 36, 37 or 38, which may be used to obtain polynucleotides that encode polypeptides comprising polypeptide sequences according to SEQ ID NO: 1 , 31 or 33 or a biologically active fragment or equivalent thereof. These oligonucleotides may be used to obtain polynucleotides as hereinbefore described using a method such as but not limited to polymerase chain reaction (PCR). The oligonucleotide primers may also comprise restriction enzyme sites for Ndel (5 ATATG-3') and Xhol (5'-CTCGAG-3') to facilitate molecular cloning of the polynucleotides into a vector. It should be appreciated that these restriction enzyme sites are not always required for cloning into a vector as the polynucleotides may be cloned first into a cloning vector without the use of any restriction enzymes, e.g. using a TA cloning technique, before transferring into the final vector. Furthermore, the restriction enzyme sites may be substituted for any other appropriate restriction enzyme sites and the choice of restriction enzyme is in part dependent on the vector to be cloned into.

[0071 ] This aspect of the invention further provides polynucleotide sequences that are complementary to the polynucleotide sequences of the present invention as hereinbefore described.

[0072] The present aspect of the invention also provides variants of the polynucleotides of the present invention as hereinbefore described.

[0073] The polynucleotides of the present invention may be recombinantly expressed to produce the P450 monooxygenase they respectively encode. These enzymes can then be used for biotransforming or biohydroxylating 1 ,8-cineole to 2- hydroxycineole, preferably (1 S)-2a-hydroxy-1 ,8-cineole.

[0074] The term "isolated" and "substantially purified" refers to a molecule that is substantially free of its natural environment. For instance, an isolated polynucleotide is substantially free of cellular material or other proteins from the cell or tissue source from which it was derived; or at least 45-80% (w/w) pure; or at least 80-90% (w/w) pure; or at least 90-95% pure; or at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.

[0075] The term "polynucleotide" refers to a nucleic acid fragment that encodes a specific protein. The polynucleotide may include regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. [0076] The term "encodes" refers to a nucleic acid or polynucleotide which comprises the information for translation into a specific protein.

[0077] The term "hydroxylates" as used herein refers to the addition of a chemical process that introduces a hydroxyl group (-OH) into an organic compound.

[0078] The term ,8-cineole" as used herein refers to the chemical compound which has the lUPAC name 1 ,3,3-trimethyl-2-oxabicyclo[2,2,2]octane. The naming of the compound and numbering of the structure used herein is the same as those of Azerad et al., 2014. The numbering of the carbon atoms used herein and according to Azerad et al. is as follows:

[0079] In another embodiment the invention provides isolated polynucleotides as hereinbefore described, wherein the polynucleotides encode P450 monooxygenases or biologically active fragments or equivalents thereof, wherein said P450 monooxygenases hydroxylate 1 ,8-cineole to form 2-hydroxycineole.

[0080] In this embodiment, the isolated polynucleotides of the present invention encode P450 monooxygenases or biologically active fragments or equivalents thereof that are able to functionalize 1 ,8-cineole to produce 2-hydroxycineole. Analysis of the biotransformation product of the P450 monooxygenase of the present invention using NMR, GC-MS and optical rotation measurements demonstrated that the major product is (1 S)-2a-hydroxy-1 ,8-cineole or oxidises 6-hydroxy-1 ,8-cineole to form oxo- hydroxy-1 ,8-cineole.

[0081 ] The term "peptide" or "polypeptide" or "protein" will be used interchangeably and will refer to a sequence of contiguous amino acids. [0082] The term "amino acid" will refer to the basic chemical structural unit of a protein or polypeptide.

[0083] The term "recombinant" when used to refer to polynucleotides or polypeptides refers to polynucleotides or polypeptides formed by laboratory methods of genetic recombination (such as molecular cloning). Recombinant expression of a polynucleotide refers to the laboratory manipulation of the polynucleotide that allows the expression of the polynucleotide, for example in a host cell.

[0084] The terms "similar" or "similarity" when used to refer to a polynucleotide or polypeptide, refer to the overall similarity between the two sequences. A number of computer algorithms are available for performing sequence alignments and for calculating the percentage similarity between two sequences, e.g. BLAST (Altschul et al, 1997). A sequence that is 100% similar to a second sequence is identical to the second sequence. The term "sequence identity" refers to the amount of nucleotides/amino acids which match exactly between two different sequences. In calculating sequence identity, gaps are not counted and the measurement is relational to the shorter of the two sequences.

[0085] The term "complementary" is used to describe the relationship between nucleotide bases that are capable to hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. When the term is used to refer to polynucleotides, it is referring to the percentage of the number of nucleotide bases between the two polynucleotide sequences that are complementary. When two polynucleotide sequences are complementary, preferably at least 50% of their nucleotide bases are complementary. More preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of their nucleotide bases are complementary.

[0086] In another aspect there is provided substantially purified and/or recombinant polypeptides having P450 monooxygenase activity or biologically active fragments or equivalents thereof wherein said polypeptides hydroxylate 1 ,8-cineole in the 2-position of 1 ,8-cineole or oxidises 6-hydroxy-1 ,8-cineole in the 2-position to form oxo-hydroxy-1 ,8-cineole, wherein the polypeptides comprise amino acid sequences which are at least 75% similar to SEQ ID NO: 1 or 31 .

[0087] In this aspect of the invention, the polypeptides or biologically active fragments or equivalents thereof that are able to functionalize or hydroxylate 1 ,8- cineole in the 2-position are any one of P450 _B ui or P450 _B u2 and the polypeptide sequences are at least 75% similar to the polypeptide sequences according to SEQ ID NO: 1 or 31 , respectively. More preferably, the polypeptide is at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of SEQ ID NO: 1 or 31 .

[0088] In another embodiment the substantially purified and/or recombinant polypeptide is selected from the group comprising:

(i) an amino acid sequence as provided in SEQ ID NO: 1 or 31 ;

(ii) an amino acid sequence encoded by a polynucleotide which encodes a

P450 monooxygenase as hereinbefore described; and biologically active fragment or equivalent thereof.

[0089] In this embodiment, the polypeptide comprises the polypeptide sequence of P450 _B ui according to SEQ ID NO: 1 , or the polypeptide sequence of P450 _B u2 according to SEQ ID NO: 31.

[0090] The polypeptides may be encoded by any of the polynucleotides or variants which encode P450 monooxygenases or biologically active fragments or equivalents thereof as hereinbefore described. [0091 ] The P450 monooxygenases P450 _B ui , P450 _B u2 and P450 _B u3 are able to functionalize or biohydroxylate 1 ,8-cineole in relatively mild conditions, i.e. conditions that are suitable for the growth of microorganisms such as E. coli. Furthermore, the recombinant expression of P450 _B ui , P450 _BU 2 or P450 _BU 3 does not negatively impact the biological function and growth of the host cell, which may be E. coli in this instance.

[0092] P450 _BU i , P450 _BU 2 and P450 _BU 3 have the typical Soret absorbance of a P450 protein, that is an absorbance maxima of 416 - 417 nm in the absence of its substrate. This absorbance maxima shift to a lower range of wavelengths of 392 - 396 nm upon binding to their substrate 1 ,8-cineole.

[0093] P450 _B ui and P450 _BU 2 were further demonstrated to preferentially bind 1 ,8- cineole. Five other related substrates, 2-adamantanone, β-ionone, (1 S)-(-)-camphor, toluene and (1 R)-(+)-camphor were also tested for binding to the P450 monooxygenases of the present invention and none of these bound to any significant extent, except for weak binding by β-ionone. The binding affinity of P450 _BU i and P450 _BU 2 for 1 ,8-cineole was found to be about 20 μΜ, which increased by a factor of two in the presence of 0.2 M potassium chloride. The binding affinity of P450 _BU 3 for 1 ,8-cineole was found to be about 8 μΜ, which increased by a factor of 1 .5 in the presence of 0.2 M potassium chloride.

[0094] In another aspect the invention provides a vector comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof as herein described.

[0095] The vector may be an expression vector, which comprises promoter elements that enhance the expression of the polynucleotide. Alternatively, the vector may be a cloning vector, which contains features that allow for the convenient insertion or removal of a DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments together. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use, e.g. an expression vector. It should be appreciated that the choice of vector is dependent on the intended purpose. Classes of cloning vectors include plasm ids, cosmids, bacteriophages, bacterial artificial chromosomes, yeast artificial chromosomes, human artificial chromosomes etc. Non-limiting specific examples of cloning vectors include Promega pGEM-T Easy, Life Technology TOPO- TA, pUC-19, pCR2.1 , etc.

[0096] The terms "plasm id" and "vector" are used interchangeably and refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0097] In an embodiment the invention provides a vector as hereinbefore described wherein the vector is an expression vector.

[0098] An expression vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. The vector is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.

[0099] In this embodiment the expression vectors may be part of the Duet Vectors from Novagen. This vector system is preferred based on compatibility of the origins of replication and antibiotics resistance when multiple vectors are transformed into a single Escherichia coli cell. The pETDuet vector carries the ColE1 replicon with ampicillin resistance, the pRSFDuet vector carries the RSF1030 replicon with kanamycin resistance and the pCDFDuet vector carries the CloDF13 replicon with streptomycin resistance. However, this is particularly useful for E. coli. Should other host cells be used, any alternative vectors can be adopted which complement the host cell selected. Accordingly, co-transformation of all three vectors into a single E. coli cell results in the expression of all three genes encoded by each vector as well as resistance to ampicillin, kanamycin and streptomycin. This allows the selection of transformed E. coli cells which express all three vectors. This also means that the transformation and antibiotics selection can be carried out for all three vectors simultaneously rather than in a step-wise fashion, i.e. there is no need to transform E. coli with a pETDuet vector and selecting for ampicillin resistance, followed by transforming with a pRSFDuet vector and selecting for kanamycin resistance and then transforming with a pCDFDuet vector and selecting for streptomycin resistance.

[0100] In one embodiment, all three specific pRSFDuet, PETDuet and pCDFDuet vectors may be used and contain a coding sequence for a six (6) histidine tag 5' to the multi-cloning site. Accordingly, the expressed protein comprises an N-terminal 6x histidine tag that facilitates purification of the expressed protein using an affinity purification column such as a nickel column. It should be readily appreciated that a his-tag is not required if purification of the expressed proteins is not required.

[0101 ] In this embodiment, the expression vector may also be pET28a. Other non-limiting examples of expression vectors include pcDNA3.1 , pDEST vector systems, pET vector systems, etc. The skilled addressee would readily appreciate that any expression vector that is suitable for co-expressing multiple proteins in a host cell is suitable.

[0102] In yet another aspect the invention provides a host cell comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof as herein described, and/or at least one plasm id comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof as herein described.

[0103] In one embodiment, the host cell expresses any one of the P450 monooxygenases of the present invention and facilitates the whole-cell biotransformation or functionalization of 1 ,8-cineole to 2-hydroxy-cineole. The host cell may comprise one, two or three of the P450 monooxygenases of the present invention and expresses one, two or three of the P450 monooxygenases. [0104] The term "host cell" refers to those transformable cells capable of growth in culture and expressing a desired polynucleotide and/or polypeptide. While the preferred host cells of this invention are bacterial cells such as E. coli, other microorganism cells may be used, for example, other bacterial cells, yeast cells, fungal cells, insect cells, vertebrate cells, etc.

[0105] In an preferred embodiment, the host cell of the present invention is not capable of metabolising 1 ,8-cineole without the polypeptides or polynucleotides of the present invention. Accordingly, the host cell may be unable to further metabolise the 2-hydroxycineole eventually into water and carbon and therefore the functionalised intermediate products of the biotransformation may be purified.

[0106] In another embodiment, the host cell of the present invention is capable of metabolising further cineole-derivatives from (1 R)-6p-hydroxy-1 ,8-cineole including but not limited to oxo-hydroxy-1 ,8-cineole, hydroxy-2,6-oxo-1 ,8-cineole or 6-oxo-2- hydroxy-1 ,8-cineole. By providing a suitable substrate having a hydroxylation site, other than at the 2 position of 1 ,8-cineole, the monooxygenases of the present invention are capable of further functionalising at other positions of the substrate. Accordingly, any hydroxylated 1 ,8 cineole may be used and functionalized to provide a multifunctionalized 1 ,8 cineole.

[0107] In a further aspect the invention provides a host cell comprising a polynucleotide or vector as hereinbefore described and further comprising an electron transport partner such as but not limited to a ferredoxin (FdX) and/or a ferredoxin reductase (FdR). The electron transport partners such as FdX and/or the FdR may be expressed on separate vectors. A number of other enzymes may act as electron transport partners. In some bacterial systems, the most commonly used electron transport partners are FdX/FdR. In some systems, Flavodoxin reductase and a Flavodoxin may be used instead. A review of other electron transport partners may be found in Hannemann F et al (2007) Biochimica et Biophysica Acta 1770 330-344.

[0108] P450 proteins are usually associated with one or more electron transport partners such as but not limited to ferredoxins and ferredoxin reductases in a biochemical pathway. [0109] In one embodiment the ferredoxins serve as electron carriers between the corresponding NADH or NADPH-dependent ferredoxin reductases and the P450 protein. Accordingly, the host cell of the present invention may further comprise an electron transport partner such as a ferredoxin (Fdx) and/or a ferredoxin reductase (FdR), wherein the electron transport partner such as a FdR and/or the FdX may be derived from the host cell or may be introduced into the host cell. The FdX and/or FdR may additionally be recombinantly over-expressed. Preferably P450 _B ui , P450 _B u2 or P450 _B u3 are capable of functionalizing 1 ,8-cineole when coupled with electron transport partners. Accordingly, whole-cell biotransformation of 1 ,8-cineole with P450 _B ui , P450 _B U2, or P450 _B u3 is possible with surrogate electron transport partners. Preferably, the surrogate electron transport partners are derived from E. coli.

[01 10] In an embodiment the invention provides a host cell as hereinbefore described, further comprising a heterologous electron transport partner such as a ferredoxin and/or a heterologous ferredoxin reductase.

[01 1 1 ] The ferredoxin and/or ferredoxin reductases may also be heterologous to the host cell. For instance, the ferredoxin and/or ferredoxin reductases may be recombinantly introduced into a host organism that does not normally express that electron transport partner such as a ferredoxin and/or ferredoxin reductases.

[01 12] In a further embodiment the invention provides a host cell as hereinbefore described wherein the electron transport partner such as a ferredoxin and/or the ferredoxin reductase are from Sphingobium and more preferably from Sphingobium yanoikuyae.

[01 13] In one embodiment the ferredoxin and/or the ferredoxin reductase may be derived from Sphingobium yanoikuyae. It is demonstrated that P450 _B ui , P450 _B u2 or P450 _BU 3's efficiency at functionalizing 1 ,8-cineole may be improved when P450 _BU i is co-expressed with a ferredoxin and/or ferredoxin reductase from S. yanoikuyae. Furthermore, it is likely that ferredoxin and/or ferredoxin reductases from other Sphingobium species may also be used. The ferredoxins and ferredoxin reductases are most preferably derived from S. yanoikuyae as herein described. [01 14] In order to introduce a heterologous electron transport partner such as a ferredoxin and/or ferredoxin reductase into the host cell of the present invention, it is not necessary to transfer the protein per se, rather, the polynucleotide encoding the electron transport partner such as a ferredoxin and/or ferredoxin reductase can be introduced into the host cell in a manner that allows transcription of the polynucleotide and subsequent translation into a polypeptide. For example, the polynucleotide encoding FdR and/or FdX may be cloned into a vector or expression vector then recombinantly introduced into the host cell separately to the vector, which expresses the monooxygenases, such as but not limited to, P450 _B ui , P450 _B u2 or P450 _B u3-

[01 15] In another aspect of the invention, there is provided a method of expressing a monooxygenase which functionalizes 1 ,8-cineole, said method comprising,

(i) providing a vector comprising at least one polynucleotide which encodes a P450 monooxygenase or a biologically active fragment or equivalent thereof which functionalizes 1 ,8 cineole;

(ii) providing another vector comprising a polynucleotide which encodes a first electron transport partner or a biologically active fragment or equivalent thereof; and

(iii) providing a further vector comprising a polynucleotide which encodes another electron transport partner or a biologically active fragment or equivalent thereof; and

(iv) inserting the vectors into a host cell to express the monooxygenase.

[01 16] The Applicants have found a method which enables reliable expression of a monooxygenase which can hydroxylate and functionalize 1 ,8-cineole. This method explicitly expresses the monooxygenase and the electron transport partners on separate vectors.

[01 17] In one embodiment the electron transport partners are FdX and/or FdR. The Applicants have found advantages and greater efficiencies and effectiveness in expressing these components separately to provide a monooxygenase which can hydroxylate or functionalize 1 ,8-cineole. The second electron transport partner is expressed on the third vector. Preferably, all three vectors are inserted into the host cell and express the monooxygenase and electron partners individually and separately from three separate vectors.

[01 18] In one embodiment, the monooxygenase, and electron transport partners such as FdX and/or FdR are as hereinbefore described. However, the method of separately expressing a monooxygenase, and the electron transport partners such as a FdX and FdR on expression vectors may be applied to any monooxygenase system. The introduction of the vectors expressing the monooxygenase, and the electron transport partners such as FdX and/or FdR into a host cell may be separately or simultaneously introduced by methods available to the skilled addressee.

[01 19] In one preferred embodiment, the electron transport partners are FdX and FdR.

[0120] In a further embodiment the invention provides a host cell as hereinbefore described or a vector or a method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin (Fdx) is encoded by a polynucleotide selected from the group comprising:

(i) a sequence of nucleotides or variant thereof, which is,

(a) at least 50% similar to FdX1 (SEQ ID NO: 4); or

(b) at least 59% similar to FdX2 (SEQ ID NO: 6); or

(c) at least 50% similar to FdX3 (SEQ ID NO: 8); or

(d) at least 50% similar to FdX4 (SEQ ID NO: 10); or at least 46% similar to FdX5 (SEQ ID NO: 12);

(f) at least 84% similar to FdX6 (SEQ ID NO: 14); or (g) at least 98% similar to FdX7 (SEQ ID NO: 16)

(ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in any one of FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), FdX6 (SEQ ID NO: 13) and FdX7 (SEQ ID NO 15) or a biologically active fragment or equivalent thereof;

(iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is,

(a) at least 71 % similar to FdX1 (SEQ ID NO: 3); or

(b) at least 67% similar to FdX5 (SEQ ID NO: 1 1 );

(iv) a sequence complementary to any one of (i) to (iii); and

(v) a biologically active fragment or equivalent or variant thereof.

[0121 ] In this embodiment, the host cell or the vector or a method of expressing a monooxygenase comprises a heterologous ferredoxin polynucleotide from S. yanoikuyae wherein the ferredoxin is any one of ferredoxin 1 (FdX1 ), ferredoxin 2(FdX2), ferredoxin 3(FdX3), ferredoxin 4(FdX4), ferredoxin 5(FdX5), ferredoxin 6(FdX6) or ferredoxin 7(FdX7), as defined by SEQ ID NO: 4, 6, 8, 10, 12, 14 and 16, respectively.

[0122] The host cell or the vector may also comprise a heterologous ferredoxin polypeptide from S. yanoikuyae wherein the ferredoxin is any one of ferredoxin 1 (FdX1 ), ferredoxin 2(FdX2), ferredoxin 3(FdX3), ferredoxin 4(FdX4), ferredoxin 5(FdX5), ferredoxin 6(FdX6) or ferredoxin 7(FdX7), as defined by SEQ ID NO: 3, 5, 7, 9, 1 1 , 13 and 15, respectively.

[0123] The polynucleotide encoding the ferredoxin 1 (FdX1 ) may be at least 50% similar to SEQ ID NO: 4. More preferably, the polynucleotide encodes a polypeptide that is at least 55%, at least 60%, at least 65%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 4.

[0124] The polynucleotide encoding the ferredoxin 2 (FdX2) may be at least 59% similar to SEQ ID NO: 6. More preferably, the polynucleotide encodes a polypeptide that is at least 60%, at least 65%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 6.

[0125] The polynucleotide encoding the ferredoxin 3 (FdX3) may be at least 50% similar to SEQ ID NO: 8. More preferably, the polynucleotide encodes a polypeptide that is at least 55%, at least 60%, at least 65%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 8.

[0126] The polynucleotide encoding the ferredoxin 4 (FdX4) may be at least 50% similar to SEQ ID NO: 10. More preferably, the polynucleotide encodes a polypeptide that is at least 55%, at least 60%, at least 65%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 10.

[0127] The polynucleotide encoding the ferredoxin 5 (FdX5) may be at least 46% similar to SEQ ID NO: 12. More preferably, the polynucleotide encodes a polypeptide that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 12.

[0128] The polynucleotide encoding the ferredoxin 6 (FdX6) may be at least 84% similar to SEQ ID NO: 14. More preferably, the polynucleotide encodes a polypeptide that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 14.

[0129] The polynucleotide encoding the ferredoxin 7 (FdX7) may be at least 98% similar to SEQ ID NO: 16. More preferably, the polynucleotide encodes a polypeptide that is at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 16.

[0130] The polynucleotides encoding the ferredoxin 1 (FdX1 ) polypeptide of the present invention may encode a polypeptide that is at least 71 % similar to SEQ ID NO: 3. More preferably, the polynucleotide encodes a polypeptide that is at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 3. [0131 ] The polynucleotides encoding the ferredoxin 5 (FdX5) polypeptide of the present invention may encode a polypeptide that is at least 67% similar to SEQ ID NO: 1 1. More preferably, the polynucleotide encodes a polypeptide that is at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 1 1.

[0132] The heterologous ferredoxin may also be encoded by a polynucleotide sequence complimentary to any of the polynucleotides encoding a ferredoxin as hereinbefore described. The heterologous ferredoxin may also be encoded by a polynucleotide sequence complimentary to a polynucleotide encoding a ferredoxin polypeptide as hereinbefore described.

[0133] The polynucleotide may not encode for the entire ferredoxin polynucleotide or polypeptide as hereinbefore described. It may encode for a biologically active fragment or equivalent or variant thereof.

[0134] In another preferred embodiment the invention provides a host cell or vector or a method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin is encoded by a polynucleotide selected from the group comprising:

(i) a sequence of nucleotides which is,

(a) at least 50% similar to FdX1 (SEQ ID NO: 4); or

(b) at least 59% similar to FdX2 (SEQ ID NO: 6); or

(c) at least 50% similar to FdX3 (SEQ ID NO: 8); or

(d) at least 50% similar to FdX4 (SEQ ID NO: 10); or (e) at least 46% similar to FdX5 (SEQ ID NO: 12); or

(f) at least 84% similar to FdX6 (SEQ ID NO: 14); a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in any one of FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), and FdX6 (SEQ ID NO: 13), or a biologically active fragment or equivalent thereof; a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence,

(a) at least 71 % similar to FdX1 (SEQ ID NO: 3); or

(b) at least 67% similar to FdX5 (SEQ ID NO: 1 1 ); a sequence complementary to any one of (i) to (iii); and a biologically active fragment or equivalent or variant thereof.

[0135] In this embodiment, the host cell or vector or a method of expressing a monooxygenase comprises a heterologous ferredoxin polynucleotide from S. yanoikuyae wherein the ferredoxin is any one of ferredoxin 1 (FdX1 ), ferredoxin 2(FdX2), ferredoxin 3(FdX3), ferredoxin 4(FdX4), ferredoxin 5(FdX5), or ferredoxin 6(FdX6) , as defined by SEQ ID NO: 4, 6, 8, 10, 12 or 14, respectively, or a variant thereof.

[0136] The host cell or vector or a method of expressing a monooxygenase may also comprise a heterologous ferredoxin polypeptide from S. yanoikuyae wherein the ferredoxin is any one of ferredoxin 1 , ferredoxin 2, ferredoxin 3, ferredoxin 4, ferredoxin 5 or ferredoxin 6, as defined by SEQ ID NO: 3, 5, 7, 9, 1 1 and 13, respectively, or a biologically active fragment or equivalent thereof.

[0137] The heterologous ferredoxin may also be encoded by a polynucleotide sequence complimentary to any of the polynucleotides encoding a ferredoxin as hereinbefore described. The heterologous ferredoxin may also be encoded by a polynucleotide sequence complimentary to a polynucleotide encoding a ferredoxin polypeptide as hereinbefore described.

[01 38] The polynucleotide may not encode for the entire ferredoxin polynucleotide or polypeptide as hereinbefore described. It may encode for a biologically active fragment or equivalent or variant thereof.

[01 39] In yet another embodiment the invention provides a host cell or a vector or a method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin is encoded by a polynucleotide encoding an amino acid sequence provided in FdX2 (SEQ ID NO: 5). The polynucleotide is preferably SEQ ID NO: 6.

[0140] It has been demonstrated that FdX2 facilitates an effective functionalization by P450 _B ui out of the seven ferredoxins hereinbefore described. FdX2 has also demonstrated effective functionalization of 1 ,8-cineole when in combination with

[0141 ] In a further aspect the invention provides a host cell or vector or a method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin reductase (FdR) as hereinbefore described is encoded by a polynucleotide selected from the group comprising:

(i) a sequence of nucleotides which is, at least 86% similar to FdR1 (SEQ ID NO: 18);

(b) FdR2 (SEQ ID NO: 20); or

(c) at least 71 % similar to FdR3 (SEQ ID NO: 22); or at least 59% similar to FdR4 (SEQ ID NO: 24); at least 67% similar to FdR4s (SEQ ID NO: 26); (ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in any one of FdR1 (SEQ ID NO: 17), FdR2 (SEQ ID NO: 19), FdR3 (SEQ ID NO: 21 ), FdR4 (SEQ ID NO: 23) and FdR4s (SEQ ID NO: 25), or a biologically active fragment or equivalent thereof;

(iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is,

(a) at least 98% similar to FdR4 (SEQ ID NO: 23); or

(b) at least 99% similar to FdR4s SEQ ID NO: 25);

(iv) a sequence complementary to any one of (i) to (iii); and

(v) a biologically active fragment or equivalent or variant thereof.

[0142] In this aspect of the invention, the host cell or vector or a method of expressing a monooxygenase as hereinbefore described may further comprise a heterologous ferredoxin reductase from S. yanoikuyae wherein the ferredoxin reductase is any one of ferredoxin reductase 1 (FdR1 ), ferredoxin reductase 2 (FdR2), ferredoxin reductase 3 (FdR3), ferredoxin reductase 4 (FdR4) or ferredoxin reductase 4s (FdR4s), as defined by SEQ ID NO: 18, 20, 22, 24 and 26, respectively.

[0143] The host cell or vector or a method of expressing a monooxygenase may also comprise a heterologous ferredoxin reductase polypeptide from S. yanoikuyae wherein the ferredoxin reductase is any one of ferredoxin reductase 1 (FdR1 ), ferredoxin reductase 2 (FdR2), ferredoxin reductase 3 (FdR3), ferredoxin reductase 4 (FdR4) or ferredoxin reductase 4s (FdR4s), as defined by SEQ ID NO: 17, 19, 21 , 23 and 25, respectively.

[0144] The polynucleotide encoding the ferredoxin reductase 1 (FdR1 ) may be at least 86% similar to SEQ ID NO: 18. More preferably, the polynucleotide encodes a polypeptide that is at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 18.

[0145] The polynucleotide encoding the ferredoxin reductase 3 (FdR3) may be at least 71 % similar to SEQ ID NO: 22. More preferably, the polynucleotide encodes a polypeptide that is at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 22.

[0146] The polynucleotide encoding the ferredoxin reductase 4 (FdR4) may be at least 59% similar to SEQ ID NO: 24. More preferably, the polynucleotide encodes a polypeptide that is at least 60%, at least 65%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 24.

[0147] The polynucleotide encoding the ferredoxin reductase 4s (FdR4s) may be at least 67% similar to SEQ ID NO: 26. More preferably, the polynucleotide encodes a polypeptide that is at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to the polypeptide of any one of the polypeptides according to SEQ ID NO: 26. [0148] The polynucleotides encoding the ferredoxin reductase 4 (FdR4) polypeptide of the present invention may encode a polypeptide that is at least 98% similar to SEQ ID NO: 23. More preferably, the polynucleotide encodes a polypeptide that is at least 99% similar to the polypeptide according to SEQ ID NO: 23.

[0149] The polynucleotides encoding the ferredoxin reductase 4s (FdR4s) polypeptide of the present invention may encode a polypeptide that is at least 99% similar to SEQ ID NO: 25.

[0150] The heterologous ferredoxin reductase may also be encoded by a polynucleotide sequence complimentary to any of the polynucleotides encoding a ferredoxin reductase as hereinbefore described. The heterologous ferredoxin reductase may also be encoded by a polynucleotide sequence complimentary to a polynucleotide encoding a ferredoxin reductase polypeptide as hereinbefore described.

[0151 ] The polynucleotide may not encode for the entire ferredoxin reductase polynucleotide or polypeptide as hereinbefore described. It may encode for a biologically active fragment or equivalent or variant thereof.

[0152] Accordingly, the host cell or vector or a method of expressing a monooxygenase as hereinbefore described may comprise a heterologous ferredoxin, a heterologous ferredoxin reductase or a combination thereof.

[0153] In a further embodiment the invention provides a host cell or vector or a method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin reductase is encoded by a polynucleotide selected from the group comprising:

(i) a sequence of nucleotides which is,

(a) FdR2 (SEQ ID NO: 20); or

(b) at least 71 % similar to FdR3 (SEQ ID NO: 22); or (c) at least 67% similar to FdR4s (SEQ ID NO: 26);

(ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in any one of FdR2 (SEQ ID NO: 19), FdR3 (SEQ ID NO: 21 ), and FdR4s (SEQ ID NO: 25), or a biologically active fragment or equivalent thereof;

(iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is at least 99% similar to FdR4s SEQ ID NO: 25);

(iv) a sequence complementary to any one of (i) to (iii); and

(v) a biologically active fragment or equivalent or variant thereof.

[0154] In this embodiment of the invention, the host cell or vector or the method of expressing a monooxygenase as hereinbefore described may further comprise a heterologous ferredoxin reductase polynucleotide from S. yanoikuyae wherein the ferredoxin reductase is any one of ferredoxin reductase 2, ferredoxin reductase 3 or ferredoxin reductase 4s, as defined by SEQ ID NO: 20, 22 and 26, respectively.

[0155] The host cell or vector or the method of expressing a monooxygenase may also comprise heterologous ferredoxin reductase polypeptide from S. yanoikuyae wherein the ferredoxin reductase is any one of ferredoxin reductase 2, ferredoxin reductase 3 or ferredoxin reductase 4s, as defined by SEQ ID NO: 19, 21 and 25, respectively.

[0156] The heterologous ferredoxin reductase may also be encoded by a polynucleotide sequence complimentary to any of the polynucleotides encoding a ferredoxin reductase as hereinbefore described. The heterologous ferredoxin reductase may also be encoded by a polynucleotide sequence complimentary to a polynucleotide encoding a ferredoxin reductase polypeptide as hereinbefore described. [0157] The polynucleotide may not encode for the entire ferredoxin reductase polynucleotide or polypeptide as hereinbefore described. It may encode for a biologically active fragment or equivalent or variant thereof.

[0158] In another embodiment the invention provides a host cell or vector or a method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin reductase is encoded by a polynucleotide which encodes an amino acid sequence provided in FdR3 (SEQ ID NO: 21 ). The polynucleotide is preferably SEQ ID NO: 22.

[0159] As described, the electron transport partners such as the FdXs and FdRs of the present invention may be used in combination to facilitate and improve the biohydroxylation or functionalization of 1 ,8-cineole by the monooxygenases of the present invention. The electron transport partners such as but not limited to FdXs and the FdRs are ideally expressed by separate vectors in a host cell.

[0160] In yet another embodiment the invention provides a host cell or vector or method of expressing a monooxygenase as hereinbefore described wherein the ferredoxin reductase is encoded by a polynucleotide provided in FdR3 (SEQ ID NO: 22) and the ferredoxin is encoded by a polynucleotide provided in FdX2 (SEQ ID NO: 6).

[0161 ] Experimental data demonstrated that the combination of P450 _B ui with FdR3 and FdX2 provided the highest efficiency of 1 ,8-cineole hydroxylation. The efficiency of the hydroxylation is higher than P450 _B ui with FdR3 or FdX2 alone.

[0162] In another aspect the invention provides a host cell or vector as hereinbefore described which expresses a ferredoxin having an amino acid sequence selected from the group comprising FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), FdX6 (SEQ ID NO: 13) and FdX7 (SEQ ID NO 15) or a biologically active fragment or equivalent or variant thereof.

[0163] The host cell or vector of the present invention expresses a heterologous ferredoxin polypeptide from S. yanoikuyae. The ferredoxin may be one of FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), FdX6 (SEQ ID NO: 13) and FdX7 (SEQ ID NO 15) or a biologically active fragment or equivalent or variant thereof.

[0164] In an aspect the invention provides a host cell or vector as hereinbefore described which expresses a ferredoxin reductase polypeptide having a sequence selected from the group comprising FdR1 (SEQ ID NO: 17), FdR2 (SEQ ID NO: 19), FdR3 (SEQ ID NO: 21 ), FdR4 (SEQ ID NO: 23) and FdR4s (SEQ ID NO: 25) or a biologically active fragment or equivalent or variant thereof.

[0165] In this aspect of the invention, the host cell or vector expresses a heterologous ferredoxin reductase from S. yanoikuyae. The ferredoxin reductase may be one of FdR1 (SEQ ID NO: 17), FdR2 (SEQ ID NO: 19), FdR3 (SEQ ID NO: 21 ), FdR4 (SEQ ID NO: 23) and FdR4s (SEQ ID NO: 25) or a biologically active fragment or equivalent or variant thereof.

[0166] In yet another embodiment the invention provides a host cell as hereinbefore described which expresses a ferredoxin having a polypeptide sequence selected from the group comprising FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), FdX6 (SEQ ID NO: 13) and FdX7 (SEQ ID NO 15) or a biologically active fragment or equivalent thereof and a ferredoxin reductase having a polypeptide sequence selected from the group comprising FdR1 (SEQ ID NO: 17), FdR2 (SEQ ID NO: 19), FdR3 (SEQ ID NO: 21 ), FdR4 (SEQ ID NO: 23) and FdR4s (SEQ ID NO: 25) or a biologically active fragment or equivalent thereof, or a combination of a FdR and a FdX thereof.

[0167] In a further preferred embodiment the invention provides a host cell or vector as hereinbefore described which expresses a ferredoxin having a polypeptide sequence provided in FdX2 (SEQ ID NO: 5) and a ferredoxin reductase having a polypeptide sequence provided in FdR3 (SEQ ID NO: 21 ). The polynucleotide encoding the FdX2 is preferably SEQ ID NO: 6 and the polynucleotide encoding the FdR3 is preferably SEQ ID NO: 22. [0168] It has now been found that a host cell expressing the combination of FdX2 and FdR3 polypeptides, facilitates efficient 1 ,8-cineole hydroxylation or functionalization when expressed in combination with any one of P450 _B ui , P450 _B u2 or P450 _B u3- Furthermore, this combination of electron transport partners such as FdR, FdX and a monooxygenase of the present invention facilitates efficient functionalization of (1 R)-6p-hydroxy-1 ,8-cineole to oxo-hydroxy-1 ,8-cineole to provide a difunctionalized compound

[0169] In an embodiment the invention provides a host cell that is an E. coli cell. E. coli is not normally capable of metabolising or functionalizing 1 ,8-cineole. Accordingly, it also lacks the metabolic pathways that further metabolise 2- hydroxycineole eventually to water and carbon dioxide. This allows the functionalised intermediate products to be isolated.

[0170] In another aspect of the invention there is provided a method of functionalizing 1 ,8 cineole said method comprising contacting 1 ,8 cineole with an effective amount of a monooxygenase polypeptide as hereinbefore described to produce 2-hydroxycineole. The monooxygenase polypeptide which contacts 1 ,8- cineole may be any one of the polypeptides of P450 _B ui , P450 _B u2 or P450 _B u3 as hereinbefore described.

[0171 ] The term "effective amount" when used to refer to the polypeptides of the present invention, refers to a quantity of the polypeptides such that measurable amounts of 1 ,8-cineole hydroxylation or functionalization can be observed. The functionalization may be observed as hydroxylation or oxidation.

[0172] In an embodiment the present invention provides a method of producing 2- hydroxycineole said method comprising hydroxylating or functionalizing 1 ,8-cineole in the presence of the host cell as hereinbefore described.

[0173] This embodiment of the invention provides a method of whole-cell functionalization or biohydroxylation of 1 ,8-cineole using the host cell as hereinbefore described. [0174] In one embodiment the invention provides a method of functionalizing 1 ,8 cineole said method comprising

(i) transforming a host cell with a polynucleotide or vector, which encodes any one of the P450 monooxygenase polypeptides of the present invention as hereinbefore described;

(ii) culturing the host cell under conditions suitable to express the polypeptide encoded by the polynucleotide;

(iii) exposing the host cell and the polypeptide to a substrate of 1 ,8-cineole.

[0175] The techniques of transforming a suitable host cell with a polynucleotide of the present invention and culturing the host cells are well known in the field. Non- limiting examples of transforming bacterial host cells include electroporation, cationic liposome formulations such as Lipofectamine®, calcium phosphate transfection, the use of chemically competent cells, etc. As an example, chemically competent E. coli BL21 (DE3) can be transformed with the P450 _B ui -expressing vector P450 _B ui- pET28a(+), then cultured in a 500 ml of TB media at 30°C and agitated at 180 rpm until the OD ₆ oo reached 0.6 to 0.8. The culture can then be induced with 1 mM IPTG and at the same time 1 ,8-cineole is added to the culture. The recombinant host cell may then biohydroxylate 1 ,8-cineole to 2-hydroxycineole, which can then be isolated from the culture supernatant using ethyl acetate.

[0176] The substrate of 1 ,8 cineole may be a non-hydroxylated or hydroxylated form of 1 ,8 cineole. Where the 1 ,8 cineole is a hydroxylated 1 ,8 cineole the substrate may be further functionalized using the monooxygenases of the present invention selected from any one of P450 _B ui , P450 _B u2 or P450 _BU 3 as hereinbefore described. The substrate may be 6 - hydroxy-1 ,8-cineole and functionalizing using the monooxygenases of the present invention may result in a difunctionalized cineole preferably to produce oxo-hydroxy cineole.

[0177] In an embodiment the invention provides a method of functionalizing 1 ,8 cineole wherein the host cell expresses a polypeptide having: (i) an amino acid sequence as provided in SEQ ID NO: 1 , 31 or 33; or

(ii) an amino acid sequence encoded by a polynucleotide of a P450 monooxygenase as hereinbefore described; or

(iii) a biologically active fragment or equivalent thereof.

[0178] In this embodiment, the method of functionalizing or biohydroxylating 1 ,8- cineole uses a host cell that expresses any one of the P450 monooxygenases of the present invention as hereinbefore described according to the polypeptide of SEQ ID NO: 1 , 31 or 33 or a polypeptide encoded by any of the P450 monooxygenase polynucleotides as hereinbefore described or biologically active fragments or equivalents or variant thereof.

[0179] Preferably the method monofunctionalizes a substrate of 1 ,8 cineole to produce 2 hydroxycineole.

[0180] Another aspect of the invention provides a method of functionalizing 1 ,8 cineole further including conducting the functionalization or hydroxylation in the presence of an electron transport partner.

[0181 ] It would be readily appreciated that a suitable electron transport partner protein is a protein that can interact with the monooxygenase of the present invention and act as an electron carrier to the monooxygenase.

[0182] The method of the present invention may be performed as a whole-cell biotransformation, wherein the host cell expresses at least one of P450 _B ui , P450 _B u2 or P450 _B u3- In another embodiment, the host cell is transformed to express the monooxygenases, and the electron transport partners of the present invention together. The monooxygenases, and the electron transport partners of the present invention may be expressed from a single vector or from separate vectors. Alternatively, the method may be carried out in the absence of a host cell.

[0183] Preferably the electron transport partners are Fdx or FdR. More preferably, they are selected from the FdX and the FdR as hereinbefore described. [0184] In a preferred embodiment, the invention provides a method of producing 2-hydroxycineole wherein the electron transport partner is a ferredoxin (FdX) and/or a ferredoxin reductase (FdR).

[0185] In another preferred embodiment the invention provides a method of producing 2-hydroxycineole wherein the electron transport partner is a heterologous ferredoxin (FdX) and/or a heterologous ferredoxin reductase (FdR).

[0186] In this embodiment, the ferredoxin and/or ferredoxin reductase is heterologous as compared to the P450 monooxygenase of the present invention.

[0187] In another preferred embodiment the invention provides a method of producing 2-hydroxycineole wherein the ferredoxin (FdX) comprises an amino acid sequence according to any one of FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), FdX6 (SEQ ID NO: 13), FdX7 (SEQ ID NO: 15) and a biologically active fragment or equivalent or variant thereof.

[0188] In this preferred embodiment, the ferredoxin or a biologically active fragment or equivalent thereof is a S. yanoikuyae ferredoxin.

[0189] In another preferred embodiment the invention provides a method of producing 2-hydroxycineole wherein the ferredoxin (FdX) comprises an amino acid sequence as provided in any one of FdX1 (SEQ ID NO: 3), FdX2 (SEQ ID NO: 5), FdX3 (SEQ ID NO: 7), FdX4 (SEQ ID NO: 9), FdX5 (SEQ ID NO: 1 1 ), and FdX6 (SEQ ID NO: 15) and a biologically active fragment or equivalent thereof.

[0190] In another prefer embodiment the invention provides a method of functionalizing 1 ,8 cineole wherein the ferredoxin (FdX) comprises an amino acid sequence according to FdX2 (SEQ ID NO: 5).

[0191 ] In another embodiment the invention provides a method of functionalizing 1 ,8 cineole wherein the ferredoxin reductase (FdR) comprises an amino acid sequence according to any one of FdR1 (SEQ ID NO: 17), FdR2 (SEQ ID NO: 19), FdR3 (SEQ ID NO: 21 ), FdR4 (SEQ ID NO: 23), FdR4s (SEQ ID NO: 25) or a biologically active fragment or equivalent thereof as hereinbefore described.

[0192] In this embodiment, the ferredoxin reductase or a biologically active fragment or equivalent thereof is a S. yanoikuyae ferredoxin reductase.

[0193] In another embodiment the invention provides a method of functionalizing 1 ,8 cineole wherein the ferredoxin reductase (FdR) comprises an amino acid sequence according to any one of FdR2 (SEQ ID NO: 19), FdR3 (SEQ ID NO: 21 ), FdR4s (SEQ ID NO: 25) or a biologically active fragment or equivalent thereof as hereinbefore described.

[0194] In another preferred embodiment the invention provides a method as hereinbefore described wherein the ferredoxin reductase (FdR) comprises an amino acid sequence according to FdR3 (SEQ ID NO: 21 ) or a biologically active fragment or equivalent thereof as hereinbefore described.

[0195] The method of functionalizing 1 ,8 cineole proves a means to prepare 2- hydroxycineole from 1 ,8 cineole or stereospecifically target functionalization at the 2 position of 1 ,8 cineole.

[0196] In a further aspect the invention provides a 2-hydroxycineole prepared by the methods as hereinbefore described.

[0197] The method of the present invention may be carried out at an experimental or research scale. Alternatively, it may also be scaled up, e.g. in a bioreactor. The bioreactor may be static or agitated, it may also employ permanent or semipermanent growth chambers. Alternatively, the bioreactor may be disposable. It should be readily appreciated that the choice of bioreactor and the requirements for up-scaling is dependent in part on the host cell used for biotransformation. Alternatively, if the biotransformation is performed in the absence of a host cell, the up-scaling may be performed in a bioreactor suitable for such a reaction. [0198] A further aspect of the invention provides an isolated polynucleotide which encodes a ferredoxin or a biologically active fragment or equivalent thereof selected from the group comprising:

(i) a sequence of nucleotides which is,

(a) at least 50% similar to FdX1 (SEQ ID NO: 4); or

(b) at least 59% similar to FdX2 (SEQ ID NO: 6); or

(c) at least 50% similar to FdX3 (SEQ ID NO: 8); or

(d) at least 50% similar to FdX4 (SEQ ID NO: 10); or

(e) at least 46% similar to FdX5 (SEQ ID NO: 12); or

(f) at least 84% similar to FdX6 (SEQ ID NO: 14); or

(g) at least 98% similar to FdX7 (SEQ ID NO: 16)

(ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in any one of SEQ ID NO: 3, 5, 7, 9, 1 1 , 13 and 15, or a biologically active fragment or equivalent thereof;

(iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is,

(a) at least 71 % similar to FdX1 (SEQ ID NO: 3); or

(b) at least 99% similar to FdX4 (SEQ ID NO: 9); or

(c) at least 67% similar to FdX5 (SEQ ID NO: 1 1 );

(iv) a sequence complementary to any one of (i) to (iii); and

(v) a biologically active fragment or equivalent and/or variant thereof. [0199] In this aspect, the polynucleotide encodes a S. yanoikuyae ferredoxin or a biologically active fragment or equivalent thereof.

[0200] After identifying the P450 monooxygenase as hereinbefore described, the sequenced genome of S. yanoikuyae was further analysed to identify potential electron transporter partners for the P450 monooxygenase of the present invention.

[0201 ] To that end, seven different ferredoxins were identified and named ferredoxin 1 to 7. The ability of each of these ferredoxins to function as an electron carrier protein for the P450 monooxygenases of the present invention was verified by co-expressing the ferredoxin with the P450 _B ui , P450 _B u2 or P450 _B u3 as hereinbefore described, in a host cell and measuring the ability of the host cell to hydroxylate 1 ,8- cineole. In these experiments, the other electron transport partners were provided by the host cell, E. coli. Six of the seven ferredoxins were able to function as electron carrier proteins for the P450 _B ui , P450 _BU 2 or P450 _BU 3.

[0202] The START and STOP codons within the polynucleotides of the ferredoxins of the present invention were codon modified for better translation in E. coli. Specifically, the following codon changes were made to the ferredoxin polynucleotides:

[0203] In an embodiment the invention provides a substantially purified and/or recombinant polypeptide having electron carrier activity or a biologically active fragment or equivalent thereof, wherein the polypeptide comprises an amino acid sequence, which is: at least 71 % similar to FdX1 (SEQ ID NO: 3);

(b) FdX4 (SEQ ID NO: 9); or

(c) at least 67% similar to FdX5 (SEQ ID NO: 1 1 ).

[0204] In a further aspect the invention provides a vector as hereinbefore described comprising at least one polynucleotide encoding a ferredoxin as hereinbefore described.

[0205] In an embodiment the invention provides that the vector as hereinbefore described comprising a polynucleotide encoding a ferredoxin is an expression vector.

[0206] The polynucleotides encoding the ferredoxins of the present invention were cloned into expression vectors to allow recombinant expression of the ferredoxin within a host cell. The expression vector used was the pRSFDuet from Novagen, however any other expression vectors as hereinbefore described may be suitable.

[0207] In another aspect the invention provides an isolated polynucleotide which encodes a ferredoxin reductase or a biologically active fragment or equivalent thereof selected from the group comprising:

(i) a sequence of nucleotides which is,

(a) at least 86% similar to FdR1 (SEQ ID NO: 18); or

(b) at least 71 % similar to FdR3 (SEQ ID NO: 22); or

(c) at least 59% similar to FdR4 (SEQ ID NO: 24); or

(d) at least 67% similar to FdR4s (SEQ ID NO: 26); (ii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence as provided in any one of SEQ ID NO: 17, 19, 21 , 23 and 25, or a biologically active fragment or equivalent thereof;

(iii) a sequence of nucleotides encoding a polypeptide comprising an amino acid sequence which is,

(a) at least 98% similar to FdR4 (SEQ ID NO: 23); or

(b) at least 99% similar to FdR4s SEQ ID NO: 25);

(iv) a sequence complementary to any one of (i) to (iii); and

(v) a biologically active fragment or equivalent and/or variant thereof.

[0208] In addition to the seven ferredoxins identified, five ferredoxin reductases were also identified.

[0209] The ability of each of these ferredoxin reductases to function as an electron transport partner or an electron carrier protein for the monooxygenases of the present invention was verified by co-expressing the ferredoxin reductases with the P450 _B ui , P450 _B u2 or P450 _B u3 monooxygenase in a host cell and measuring the ability of the host cell to hydroxylate 1 ,8-cineole. In these experiments, the electron transport partner(s) were provided by the host cell, E. coli. All five ferredoxin reductases were able to function as electron carrier proteins for the P450 _B ui , P450 _B u2 or P450 _B u3-

[0210] The START and STOP codons within the polynucleotides of the ferredoxin reductases of the present invention were codon modified for better translation in E. coli. Specifically, the following codon changes were made to the ferredoxin reductases polynucleotides: Gene Name START Codon STOP Codon

Substitution Substitution

FdR1 No change TGA to TAA

FdR2 No change No change

FdR3 No change No change

FdR4 No change TGA to TAA

FdR4s No change TGA to TAA

[021 1 ] In an embodiment the invention provides a substantially purified and/or recombinant polypeptide having electron carrier activity or a biologically active fragment or equivalent thereof, wherein the polypeptide comprises an amino acid sequence, which is:

(a) at least 98% similar to FdR4 (SEQ ID NO: 23); or

(b) at least 99% similar to FdR4s SEQ ID NO: 25).

[0212] In a further aspect the invention provides a vector comprising at least one polynucleotide encoding a ferredoxin reductase as hereinbefore described.

[0213] In an embodiment the invention provides that the vector comprising a polynucleotide encoding a ferredoxin reductase is an expression vector.

[0214] The polynucleotides encoding the ferredoxin reductases of the present invention were cloned into expression vectors to allow recombinant expression of the ferredoxin reductase within a host cell. Preferably, the expression vector used is the pETDuet expression vector from Novagen, however any other expression vectors may be suitable.

[0215] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0216] Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof.

[0217] The present invention will now be more fully described by reference to the following non-limiting Examples.

EXAMPLES

General Materials and Methods DM2:

(416S) (Rasmussen et al., 2005) (supplemented with required carbon sources) Modified terrific broth:

TB; 12 g L ^"1 tryptone, 24 g L ^"1 yeast extract, 4 ml L ^"1 glycerol, 9.4 g L ^"1 K ₂ HP0 ₄ and 2.2 g L ^"1 KH ₂ P0 ₄ )

Luria Bertani broth:

(LB; 10 g L ^"1 tryptone, 5 g L ^"1 yeast extract, 10 g L ^"1 NaCI)

DM3 seed medium:

13.33 g/L Potassium di-hydrogen orthophosphate (KH ₂ P0 ₄ )

4.0 g/L Di-ammonium hydrogen orthophosphate

((NH ₄ ) ₂ HP0 ₄ )

1 .7 g/L Citric acid (monohydrate)

Before autoclaving the pH was adjusted to pH 7 using 2 M NaOH.

Added separately after autoclaving/sterilisation:

Glucose added to a final concentration of 25 g/L (stock solution = 660 g/L).

2.5 mL/L 1 M Magnesium sulphate heptahydrate

(MgSO ₄ .7H ₂ O)

5 mL/L Trace metal solution (see below for formulation)

0.13 mL/L 0.1 M Thiamine hydrochloride

Antibiotics as required

Composition of Trace metal solution (per litre):

CuSO ₄ .5H ₂ O 2.00 g

Nal 0.08 g

MnSO ₄ .H ₂ O 3.00 g

NaMoO ₄ .2H ₂ O 0.20 g

H3BO3 0.02 g CoCI ₂ .6H ₂ 0 0.50 g

ZnCI ₂ 7.00 g

FeS0 ₄ .7H ₂ 0 22.0 g

CaS0 ₄ .2H ₂ 0 0.50 g

Sulphuric acid 1 ml_

DM3 bioreactor medium:

10.64 g/L Potassium di-hydrogen orthophosphate (KH ₂ P0 ₄ )

4.0 g/L Di-ammonium hydrogen orthophosphate

((NH ₄ ) ₂ HP0 ₄ )

1 .7 g/L Citric acid (monohydrate)

Added separately after autoclaving/sterilisation:

Glucose added to a final concentration of 25 g/L (stock solution = 660 g/L).

2.5 mL/L 1 M Magnesium sulphate heptahydrate

(MgS0 ₄ .7H ₂ 0)

5 mL/L Trace metal solution (see below for formulation)

0.13 mL/L 0.1 M Thiamine hydrochloride

Antibiotics as required

Antifoam as required

The pH of the medium was adjusted to pH 7.0 using 10% (v/v) ammonia solution and the bioreactor pH controller.

Composition of Trace metal solution (per litre):

CuS0 ₄ .5H ₂ 0 2.00 g

Nal 0.08 g

MnS0 ₄ .H ₂ 0 3.00 g

NaMo0 ₄ .2H ₂ 0 0.20 g

C0CI2.6H2O 0.50 g

ZnCI ₂ 7.00 g

FeS0 ₄ .7H ₂ 0 22.0 g

CaS0 ₄ .2H ₂ 0 0.50 g

Sulphuric acid 1 mL Trace metal solution was filtered sterilised (0.2 pm) and stored at 4°C. E. coli minimal medium:

EM medium; K ₂ HP0 ₄ 7 g L-i , KH ₂ P0 ₄ 3 g L ^~1 , sodium citrate tribasic dihydrate 0.57 g L ^"1 , MgS0 ₄ .7H ₂ 0 0.2 g L ^"1 , (NH ₄ ) ₂ S0 ₄ 1 g L ^"1 , glucose 2 g L ^"1 , 50 mL L ^"1 1 M TRIS pH 7.4. Medium was filtered sterilised (0.2 pm) and stored at room temperature.

Example 1 : Isolation of a Cineole-Metabolising Microorganism

[0218] Microorganisms capable of metabolising 1 ,8-cineole were isolated from activated sludge (obtained from Lilydale Sewage Treatment Plant, Victoria, Australia) using a modified continuous culture method in combination with selection based on growth with 1 ,8-cineole as the sole source of carbon and energy. The medium used for the enrichment process was a fully defined mineral salt medium (DM2). One isolate was chosen because of its ability to grow in relatively high concentrations of 1 ,8-cineole (Dumsday et al., 2007). Once established as a pure culture the strain was shown to be able to utilise 1 ,8-cineole as a sole carbon source and could grow in the presence of 1 ,8-cineole concentrations up to 2 g L ¹ .

[0219] A blastn search using the "16S ribosomal sequences (Bacteria and Archae)" database from NCBI (accessed on 15/01/2015) using the 1492 bp 16S rRNA gene sequence from the selected isolate resulted in a number of hits with bacteria belonging to the genus Sphingobium. All characteristic 16S rRNA signatures that distinguish this genus (Takeuchi et al., 2001 ) were present in the sequence of the isolate. The top three hits are listed in Table 1 . The isolate best matched members of the species Sphingobium yanoikuyae and hence the new isolate was designated Sphingobium yanoikuyae. This identification matches the previously described colony morphology (yellow on NA) and microscopic appearance (gram-negative rod-shaped bacterium) (Dumsday et al., 2007).

[0220] Table 1 : 16S rRNA identification of the new isolate from activated sludge using the blastn algorithm. The best 3 hits are shown when the 1492 bp 16S rRNA gene sequence from the isolate was blasted against the " 16S ribosomal sequences (Bacteria and Archae)" database from NCBI. The new isolate was identified as Sphingobium yanoikuyae.

¹ Type strain

[0221 ] Based on the 16S rRNA sequence comparison the new isolate was designated a Sphingobium yanoikuyae.

Example 2: Production, Purification and Structural Analysis of 1,8-cineole Metabolites Produced by S. yanoikuyae

[0222] In an effort to identify metabolites derived from 1 ,8-cineole, S. yanoikuyae was grown in DM2 with 1 ,8-cineole as the sole carbon source and metabolites were extracted using dichloromethane and/or purified and analysed from the aqueous phase of culture supernatants. In total, five different metabolites were identified in cultures growing on 1 ,8-cineole which were produced at different growth stages; T series compounds were produced during exponential growth and declined as the culture moved into stationary phase, while the 'P' series compounds were only produced in significant quantities during stationary phase. Gas Chromatography (GC) analysis of shake flask cultures of S. yanoikuyae in exponential phase showed the presence of intermediate compounds 11 -13 as well as significant levels of 1 ,8-cineole. In stationary phase cultures little or no 1 ,8-cineole remained and two other intermediates were observed; P1 and P2. Exponentially growing cultures produced a total 11 -13 concentration of approximately 40 mg L ^~1 . Stationary phase cultures produced approximately 100 mg L ^"1 of P1 and P2. Each of the compounds was isolated and purified from culture supernatants (typically S. yanoikuyae was grown in 500 mL of DM medium in a 2 L Erlenmeyer flask) and once purified the structure of each compound was partially elucidated using Nuclear Magnetic Resonance (NMR) and Gas Chromatography Mass Spectrometry (GC MS). The putative identification of each metabolic product is shown in Table 2.

[0223] Table 2: Putative identification of metabolic intermediates produced by S. yanoikuyae when growing on 1 ,8-cineole as a sole carbon source. The intermediates were purified from solvent extracts of culture supernatants and partially characterised using NMR and GC MS. P1 was observed in stationary phase culture supernatant analysed using GC, but could not be recovered by solvent extraction. P1 was suspected to be the intermediate Baeyer-Vi Niger oxidation product of the oxo- and hydroxycineole and could rearrange to the lactone (P2) in organic solvents. Therefore in order to structurally elucidate P2, P1 and P2 were purified from the aqueous culture supernatant. The P1 NMR spectrum was determined after subtraction of the known spectrum for P2, with confirmation by Electrospray Mass Spectroscopy (ES MS). Optical rotation data was obtained the oxo-cineole metabolite (I2) which indicated that the oxygen was located in the "two position" and it was assumed that this compound was derived from the hydroxylated metabolites (11 and I3). It was therefore concluded that 11 and I3 are hydroxylated in the "two position" as shown in the table.

Chemical name

Compound structure

(according to Azerad et al., 2014)

1 ,8-cineole 1 ,8-cineole

[0224] The five compounds isolated from culture supernatants of S. yanoikuyae growing on 1 ,8-cineole were confirmed as those that were previously reported in the literature (e.g. Carman and Fletcher, 1984; MacRae et al., 1979). Based on the identities of the five metabolites a possible pathway for the conversion of 1 ,8-cineole by S. yanoikuyae was proposed. First, 1 ,8-cineole is transformed into hydroxycineole (11 and/or I3) and then further oxidised to oxo-cineole (I2). (It is possible that both hydroxycineoles are made by different enzymes, or that oxo-cineole is further converted to one hydroxycineole and then converted back to oxo-cineole). The oxo- cineole then undergoes a Baeyer-Vi Niger oxidation where an additional oxygen atom is inserted into the ring structure to form a lactone which is in turn opened, supposedly through a spontaneous process to give the ring-opened hydroxy acetate. A spontaneous oxidation then occurs to give the keto-acid, which lactonises to give a five-member lactone ring. These findings demonstrated that S. yanoikuyae has the ability to produce a range of compounds from 1 ,8-cineole and at least one of these compounds is an hydroxylated intermediate.

Example 3: S. yanoikuyae Whole Genome Sequence

[0225] The genomic DNA of the S. yanoikuyae isolate was extracted using a standard DNA extraction technique. Illumina® HiSeq sequencing of the isolated DNA and assembly using Velvet delivered a draft genome sequence of approximately 5.9 Mbp containing 263 contigs. The Sphingobium yanoikuyae strain genome was automatically annotated using the PROKKA software (http://www.vicbioinformatics.com) and 9 putative P450 genes were identified in the draft genome.

Example 4: Production of 1 ,8-cineole-hydroxylating enzymes in Sphingobium yanoikuyae

[0226] To enable the production of a large amount of cells expressing enzymes involved in cineole-oxidation, a (fed-) batch bioprocess was developed. A single S. yanoikuyae colony grown on NA supplemented with ca. 1 mL L ^"1 1 ,8-cineole was used to inoculate a primary seed culture (10 mL of DM3 in a 50 mL tube) and grown for ca. 3 days at 30°C shaking at 200 rpm. A secondary seed culture (500 mL of DM3 in a 2 L baffled Erlenmeyer flask) was inoculated with 9 mL primary seed culture and grown for 24 h at 30°C shaking at 200 rpm. The main culture (1 .6 L of DM3 in a 3 L glass stirred tank bioreactor connected to a Sartorius Biostat B controller (Sartorius, Germany)) was inoculated with 101 mL secondary seed to obtain an initial calculated OD ₆ oo of 0.25. At the time of inoculation, 0.8 ml_ 1 ,8-cineole was added to the medium. S. yanoikuyae cells were initially cultivated in batch mode. The temperature was maintained at 30 °C and the pH at 7.0 via automatic addition of either 10% (v/v) H ₃ P0 ₄ and 10% (v/v) ammonia solution. Dissolved oxygen control was via a cascade control system (stirrer followed by airflow) with the dissolved oxygen maintained at 30% of saturation. OD ₆₀ o and the level of glucose (YSI 2700 SELECT Glucose analyser) were measured regularly. When the OD ₆ oo had reached about 28 and the level of glucose had decreased to approximately 2 g Γ ¹ , glucose and cineole feeds were started at rates of 3.5 g h ^_1 and 0.32 ml h ^_1 , respectively. (Cineole was fed to the culture to "induce" proteins involved in cineole metabolism.) When an OD ₆ oo of ca. 34 was attained, 4 ml_ of 1 M magnesium sulphate heptahydrate was added aseptically. At an OD ₆ oo of about 63, the glucose feed was turned off and the cineole feed increased to 3.2 ml I ¹ for a further 5 h. The final OD ₆ oo was 66 and at the end of the process a concentrated buffer stock was added to the culture and aliquots of 150 ml_ were stored at -80°C.

Example 5: Isolation and Identification of Cineole-binding P450s from S. yanoikuyae Cell Extracts

[0227] To enable identification of proteins involved in the early stages of cineole metabolism in S. yanoikuyae, proteins that could bind 1 ,8-cineole (as indicated by an increased absorbance at 417 nm) were purified from cell lysates. S. yanoikuyae cells expressing enzymes involved in cineole metabolism (Example 4) were purified from a clarified cell lysate using a multistep purification process. Cells resuspended in buffer were cooled to less than 10°C and lysed by three passages through a high pressure homogeniser at 700 bar. The cell lysate was then clarified by centrifugation (12,000 x g, 30 minutes, 4°C). Ammonium sulphate precipitation was used as the first purification step and the lysate was partitioned into three different fractions (0-20%, 20-40% and 40-60% ammonium sulphate cuts). These fractions were loaded onto an ion-exchange column (IEX) and upon addition of 1 ,8-cineole, a transition from the low-spin (LS) to the high-spin (HS) state (Luthra et al., 201 1 ) was observed. Two distinct peak fractions, PF1 and PF2 were further purified using gel filtration. After the 3-step purification procedure, PF1 was purified to apparent homogeneity on SDS- PAGE. Throughout the purification peak fractions were tested for absorbance shift from 417 nm to 390 nm after addition of 1 ,8-cineole (this was done to confirm the presence of cineole-binding proteins). The purity was also reflected by the Reinheitszahl (RZ) which is defined as the ratio between P450 absorbance at its typical Soret absorbance for the LS state (in this case 417 nm) and the total protein absorbance at 280 nm (A417 nm/A280 nm). A higher RZ is indicative of higher protein purity (Hawkes et al. , 2002). PF1 had an RZ of 1 .4 which correlates with the purity shown by SDS-PAGE analysis. See Figure 1 .

[0228] After concentration, reduction and alkylation PF1 was further purified using reverse-phase chromatography. N-terminal sequencing of the fraction that contained a protein with a molecular mass of approximately 46 kDa (typical size for P450s) produced a readable N-terminal sequence of 22 amino acids.

Example 6: Identification of gene encoding P450 Monooxygenases from S. yanoikuyae

[0229] An in silico protein translation was performed based on the genomic sequence and the N-terminal amino acid sequence (Example 5) was used to interrogate the translated draft genome sequence to enable identification of the full length gene encoding P450. Three P450 monooxygenases with lengths of 410 (P450BUI ), 406 (P450 _B u2) and 410 (P450 _BU 3) amino acids were found. Protein identities were confirmed by a tryptic digest of the reduced and alkylated proteins (excised from an SDS-PAGE gel; see Figure 1 ) followed by MALDI-MS fingerprint mapping.

[0230] This predicted protein sequences were compared to the Non-Redundant protein database using blastp. The alignment results indicated that P450 _B ui and P450 _B u2 shared a limited amount of amino acid similarity to a number of proteins in the database. P450 _B u3 appeared to share significant sequence identity to a hypothetical protein of unknown function. The following table summarises the three proteins with the highest amino acid similarities to each of P450 _B ui , P450 _BU 2 and P450 _B u3. The blastp search was performed on 20 July 2015. [0231 ] Table 3: Sequence comparison between the P450 protein of the present invention and known P450 proteins. The three proteins with the highest amino acid similarities are shown. All three are uncharacterised proteins but have features typical of P450 proteins.

[0232] These P450 proteins were designated as P450 _B ui , P450 _B u2 and P450 _B u3- As P450 _B ui shares more than 40% sequence identity with P450s that are member of the CYP101 family, pairwise amino acid alignment was performed using the amino acid sequence of P450 _B ui and other members of the CYP101 protein family as well as CYP176A1 (P450 _cin from C. braakii, the only bacterial P450 known to hydroxylate 1 ,8-cineole). The sequence similarities were calculated from the alignment and are summarised Table 4.

[0233] Table 4: Amino acid sequences similarities between P450 _B ui , P450 _B u2 and P450BU3 and other members of the CYP101 family and CYP176A1 (P450 _cin from C. braakii). The values in the table are expressed as "alignment length (sequence identity) (sequence similarity)".

[0234] P450 _B ui shares the highest amino acid similarity to CYP101 B1 which has been shown to oxidise β-ionone (Bell and Wong, 2007). The sequence identities of the P450 proteins of the present invention with the P450 from C. braakii (CYP176A1 ) is very low indicating that the P450 proteins of the present invention are is very different to this protein. Example 7: Comparative analysis of the S. yanoikuyae isolate of the Present Invention and S. yanoikuyae DSM 7462

[0235] To determine whether the ability to grow on 1 ,8-cineole is ubiquitous in S. yanoikuyae strains, the S. yanoikuyae isolate of the present invention and S. yanoikuyae DSM 7462 (available from DSMZ) were tested for their ability to grow in DM2 medium supplemented with 0.25 to 1 mL L ^"1 of 1 ,8-cineole or a-terpineol or 0.25 to 1 g L ^"1 (1 R)-(+)-camphor or (1 S)-(-)-camphor as the sole source of carbon and energy. After 7 days of incubation at 30 °C shaking at 200 rpm, visual examination showed that S. yanoikuyae DSM 7462 did not grow on any of the substrates tested whilst the new S. yanoikuyae isolate grew on both 1 ,8-cineole and α-terpineol, but not on either of the camphor enantiomers (Table 5).

[0236] Table 5: Growth of S. yanoikuyae strains on a range of terpenes. Cultures were grown in 50 mL screw-capped tubes at 30°C. Growth was scored by visual examination of the cultures: +, growth; -, no growth.

[0237] Pairwise genomic comparison between the translated S. yanoikuyae genome of DSM7462 with the P450 _B ui _, P450 _B u2 or P450 _B u3 amino acid sequence yielded no proteins with significant identity to P450 _B ui , P450 _BU 2 or P450 _BU 3. These findings suggest that genes encoding 1 ,8-cineole-hydroxylating P450s are not present in all S. yanoikuyae strains. Example 8: Recombinant Expression of P450 _B ui, P450 _B u2 and P450 _B u3

[0238] The gene encoding the P450 monooxygenases of the present invention were amplified using polymerase chain reaction (PCR) using S. yanoikuyae genomic DNA as the template and the oligonucleotide primers used for the amplification are set out in Table 6. Restriction sites for Ndel and Xhol were introduced into the PCR product by the two primers (sequences for the restriction enzymes sites are underlined) for cloning into the multiple cloning site of the vector pET28a(+). The pET28a(+) vector introduced an in-frame N-terminal 6x histidine tag to P450 _B ui , this 6xHis tag allows the purification of the expressed the P450 monooxygenases of the present invention using immobilised metal affinity chromatography (IMAC).

[0239] Table 6: DNA sequences of the oligonucleotide primers used to amplify the P450 monooxygenases of the present invention from DNA. Underlined nucleotides are for introducing Ndel and Xhol restriction enzyme sites into the final PCR product.

The PCR cycling conditions were as follows (30 cycles of steps b and c): a. Initial denaturation at 98 °C for 1 min; b. Denaturation at 98 °C for 10 seconds; c. Annealing and extension at 72 °C for 40 seconds; and d. Final extension step at 72 °C for 10 minutes.

[0241 ] The endogenous STOP codon (TGA) was substituted to the alternative STOP codon TAA. After ligation into the vector, DNA sequencing reactions were performed using T7 Promoter Primer and T7 Terminator Primer (Table 7) using the BigDye Terminator, version 3.1 (Applied Biosystems) kit.

[0242] Table 7: DNA sequences of the oligonucleotide primers used to confirm the correct insertion of the P450 genes of the present invention into pET28a(+).

[0243] DNA sequencing and base calling was performed by MICROMON, Monash University, Victoria, Australia. Sequencing was used to confirm that the insert was in- frame and had the correct sequence.

[0244] Chemically competent Escherichia coli BL21 (DE3) (Life Technologies, USA) bacteria were separately transformed with pET28a(+) vectors, each encoding one of P450 _B ui , P450 _B U2 or P450 _B u3 and plated onto LB agar containing 50 g mL ^"1 kanamycin sulphate (Kan) as the selection antibiotic and incubated for 18 hours at 37 °C. A single colony from each transformation was used to inoculate a 50 mL TB (in a 250 mL non-baffled flask) containing 50 g mL ^"1 Kan. The seed cultures were incubated for 18 h at 30°C and 180 rpm and then used to inoculate (to an initial OD ₆ oo of approximately 0.1 ) 500 mL of TB supplemented with 50 g mL ^"1 Kan in a 2 L baffled flask. The cultures were incubated under the same conditions until the OD ₆ oo reached 0.6-0.8. Protein expression was induced with 1 mM isopropyl β-D-l - thiogalactopyranoside (IPTG) and incubated for a further 18 h and then harvested by centrifugation at 6000 x g for 20 minutes at 4°C. The cell pellets were washed in 50 mM Tris, 150 mM NaCI buffer, pH 7.4 and then stored at -80 °C. The cell pellets were resuspended in the same buffer and then disrupted by two passages through an Avestin cell disruptor. After centrifugation, the clarified cell lysates were each loaded onto a separate column with cobalt-charged resin (TALON® Superflow metal affinity resin; Clontech) equilibrated with 50 mM Tris, 150 mM NaCI and 5 mM imidazole, pH 7.4. After a wash step the His-tagged P450 _B ui , P450 _B u2 or P450 _B u3 were eluted by a stepwise increase (20, 60, 100 and 200 mM) in imidazole concentration. Fractions with similar purities as judged by SDS-PAGE were pooled and buffer-exchanged into 50 mM Tris, pH 7.4 by repeated cycles of concentration and dilution using Amicon Ultra-15 Centrifugal Filter Units with a molecular weight cut of 10 kDa.

[0245] P450 _B ui , P450 _B U2 or P450 _B u3's estimated molecular weights inclusive of the 6xHis tag were approximately 48.7 kDa, 47.8 kDa and 48.4 kDa, respectively. The purified proteins were visualised on a Coomassie-stained SDS-PAGE gel (4-12% Bis-Tris, MOPS running buffer) and the size of the main expression product corresponded with the estimated size (see Figure 2).

[0246] The purity of the P450 _BU i , P450 _BU 2 or P450 _BU 3 preparations were reflected by the RZ values of 1 .6, 1 .0 and 1 .5, respectively. The yield of active protein was estimated in the purified pool by performing carbon monoxide difference spectroscopy in the presence of 1 ,8-cineole (see Figure 3 and Example 7). P450 _BU i and P450 _BU 2 were both recovered in good yields with more than 30 mg purified protein per litre original culture, while P450 _BU 3 was expressed in lower quantities with approximately 1 .5 mg of purified protein per litre of original culture. The concentration of functional P450's were estimated using CO difference spectroscopy (Omura and Sato, 1964). This value is based on the extinction coefficient of another P450 and is used extensively by P450 researchers to estimate P450 concentration.

Example 7: Characterisation of P450 _B ui, P450 _B u2 and P450 _B u3

[0247] To confirm that P450 _BU i , P450 _BU 2 and P450 _BU 3 from S. yanoikuyae had properties of a P450 enzyme, P450 _BU i , P450 _BU 2 and P450 _BU 3 protein solutions were prepared in 50 mM Tris, pH 7.4. Substrate-induced absorbance shifts were demonstrated by adding 1 μί of undiluted 1 ,8-cineole to 1 mL of protein solution. Each of the P450 proteins were then reduced by the addition of a small amount (a few milligrams) of solid sodium dithionite (Sigma Aldrich, USA) and incubation for 1 minute. Subsequently, carbon monoxide (CO) was bubbled through the cuvette for 30 seconds. Spectra were recorded from 350 to 700 nm. The purified P450's showed the characteristic spectroscopic properties as shown in Figure 3.

[0248] The absorbance maxima for the oxidised and substrate-free, oxidised in the presence of 1 ,8-cineole, reduced with sodium dithionite in the presence of 1 ,8- cineole and reduced and in complex with CO in the presence of 1 ,8-cineole forms are summarised in Table 8.

[0249] Table 8: Characteristic absorbance maxima of the four different forms of P450 _B ui , P450 _B U2 and P450 _B u3- All three P450 proteins showed the typical Soret absorbance maximum (~41 7 nm) which shifts to a lower wavelength (~392 nm) upon addition of the substrate, 1 ,8-cineole. The addition of 1 , 8-cineole results in a very large shift indicating that this compound may be the preferred substrate for these P450 proteins.

[0250] Purified P450 _BU 1 , P450 _BU 2 and P450 _BU 3 were diluted in 50 mM Tris, pH 7.4, or 50 mM Tris, 200 mM KCI, pH 7.4 buffer to approximately 2 μΜ. 1 ,8-cineole stock solutions in ethanol (EtOH) in the concentration range of 1 - 600 mM were prepared by serial dilution. To 1 ml_ of each protein solution, 1 μΙ_ aliquots of undiluted 1 ,8- cineole stock solutions of varying concentrations were added sequentially. After mixing, an absorbance spectrum between 350 and 450 nm was measured. Reference spectra of protein solutions containing EtOH instead of substrate solutions were subtracted from the sample absorbance spectra to obtain the 1 ,8-cineole induced difference spectra. Further 1 μΙ_ aliquots of undiluted 1 ,8-cineole stock solutions were added until the absorbance difference between peak and trough (ΔΑ) reached its maximum, the maximum total amount of added substrate solution was 1 % (v/v) of the sample. K _D values were calculated by fitting ΔΑ against 1 ,8-cineole concentration ([S]) to the hyberbolic function (Bell et al. , 2010):

₌ AA _max x [S]

D + [S]

[0251 ] The dissociation constants (K _D ) were determined with 1 ,8-cineole as the substrate and are summarised in Table 9. The dissociation constant was decreased by a factor of approximately 2 when 0.2 M KCI was present.

[0252] Table 8: The dissociation constant of P450 _B ui , P450 _B u2 and P450 _B u3 for 1 ,8-cineole in the presence or absence of 0.2 M KCI.

[0253] Several alternative substrates were also screened for the induction of absorbance shifts using the same procedure as above albeit with higher substrate concentrations. Solutions of 2-adamantanone, β-ionone, (1 R)-(+)-camphor and (1 S)- (-)-camphor, in EtOH were added to the protein solutions until the ΔΑ did not increase any further. Under the tested conditions, AA _max was largest using 1 ,8-cineole in the presence of KCI. None of the other tested substrates triggered a larger shift than 1 ,8- cineole. [0254] Table 9: The dissociated constant of P450 _B ui and P450 _B u ₂ proteins for 1 ,8- cineole, 2-adamantanone, β-ionone, (1 S)-(-)-camphor and (1 R)-(+)-camphor in the present and absence of 0.2 M KCI.

Example 9: Whole Cell Biotransformation of 1 ,8-cineole Using Recombinant

[0255] E. coli BL21 (DE3) was transformed with the P450 _B ui -expressing vector (pET28a(+)) to demonstrate whole-cell biotransformation of 1 ,8-cineole and gas chromatography-mass spectrometry (GC-MS) analysis was used to demonstrate hydroxylation of 1 ,8-cineole. E. coli BL21 (DE3) transformed with an empty pET28a(+) vector was used as the negative control.

[0256] Transformed E. coli was cultured in a 500 ml_ of TB (30°C and shaking at 180 rpm) until the OD ₆ oo reached 0.6 to 0.8. The culture was then induced with 1 mM IPTG and at the same time 250 μΙ_ of undiluted 1 ,8-cineole were added to the culture. The culture was incubated for a further 45 hours and then the cells were removed by centrifugation at 6000 x g for 20 minutes at 4°C. The culture supernatant was extracted with ethyl acetate (EtOAc) and the organic phase was dried over Na ₂ S0 ₄ and concentrated under reduced pressure. The crude solvent extract was dissolved in 3 ml_ of EtOAc, and then diluted 1000-fold in EtOAc and analysed using GC-MS. GC-mass spectra were obtained with a ThermoQuest MAT95XL GC mass spectrometer using electron impact ionisation in the positive ion mode with ionisation energy of 70 eV. The gas chromatography was performed with a SGE SOLGEL-1 MS column (30 m x 0.25 mm ID, 0.25 pm film thickness), with a temperature program of 50°C for 2 minutes, then heating at 23°C min ^"1 to 300°C where the temperature was held for 7 minutes with a splitless injection, an injector temperature of 300°C and the transfer line was set to 300°C. High-purity helium was used as carrier gas with a flow rate of 0.8 ml_ min ^"1 .

[0257] After 45 hours of biotransformation a single peak corresponding to hydroxycineole was detected and no residual 1 ,8-cineole was detected in the supernatant extract. Analysis of the supernatant of the negative control showed presence of 1 ,8-cineole but hydroxycineole was not detected. We therefore concluded that E. coli expressing P450 _B ui can transform 1 ,8-cineole to hydroxycineole. However, GC analysis does not necessarily distinguish between certain hydroxylated intermediates as the boiling temperatures of these molecules are very similar if not identical.

[0258] Accordingly, we spiked the hydroxycineole produced by P450 _B ui with (1 R)- ββ-hydroxy-l ,8-cineole (produced from the bicistronic plasmid pCW-P450cin/Cdx (Slessor et al., 2012) which expressed P450 _C i _n ) before analysing the sample using GC MS. Two separate gas chromatography peaks with identical mass spectra were observed. Since the hydroxycineole from this study can be distinguished from (1 R)- ββ-hydroxy-l ,8-cineole using non-chiral gas chromatography the product of the biotransformation of 1 ,8-cineole by P450 _B ui could be either (1 S)-2a-hydroxy-1 ,8- cineole or (1 R)-6a-hydroxy-1 ,8-cineole.

[0259] In order to distinguish the biotransformation product of P450 _B ui from P450 _cin the optical rotation of purified hydroxycineole produced using P450 _BU i was determined. The optical rotation was +28.2 degrees (c = 0.3, EtOH) with the biotransformation product dissolved in ethanol. According to Carman and Fletcher (1984) and Carmen et al. (1986), (1 S)-2a-hydroxy-1 ,8-cineole should have an optical rotation of +30.2 degrees (c=0.36, EtOH) or 31 .9 degrees (c=1 .3, EtOH), respectively. When this data is combined with the results from NMR and GC-MS, it was concluded that the biotransformation product of P450 _BU i was mainly (1 S)-2a-hydroxy-1 ,8-cineole and not (1 R)-6p-hydroxy-1 ,8-cineole which is produced by P450 _cin . [0260] Whole-cell biotransformation using E. coli BL21 (DE3) followed by GC-MS analysis demonstrated that all three P450s (P450 _B ui , P450 _B u2, P450 _B u3) hydroxylate 1 ,8-cineole. After 45 h incubation a peak with a retention time (RT) of 4.29 min was detected in supernatant solvent extracts from cultures expressing (separately) each of the three P450s. This peak had a mass increase of 16 units with a molecular peak at m/z = 170 and the characteristic fragmentation pattern for monohydroxylated 1 ,8- cineole. It was identified with a 91 -94% probability match to (1 S)-2-hydroxy-1 ,8- cineole in the National Institutes Standards Technology (NIST) 14 (MS Database and MS Search Program v.2.2) library containing spectra for 242466 chemical compounds. This peak was not detected in the negative control (E. coli BL21 (DE3) harbouring an empty pET28a(+) vector). Analysis of a mixture of chemically synthesised a and β forms of (1 S)-2-hydroxy-1 ,8-cineole showed that the two stereoisomers can be separated using non-chiral GC-MS (RT 4.19 and 4.29 min). Analysis of mixtures of (1 R)-6p-hydroxy-1 ,8-cineole (RT of 4.19 min) and the solvent extracts from cultures expressing P450 _B ui , P450 _BU 2 and P450 _BU 3, resulted in two distinct GC peaks (RTs of 4.29 min and 4.19 min) with highly similar MS data. Since monohydroxylated 1 ,8-cineole from this study separates from the beta-form ((1 R)-6p- hydroxy-1 ,8-cineole) using non-chiral GC, we putatively identified the products of P450 _B ui , P450 _B u2 and P450 _BU 3 as (1 S)-2a-hydroxy-1 ,8-cineole or its enantiomer (1 R)- 6a-hydroxy-1 ,8-cineole. See Figure 13.

Example 10: S. yanoikuyae Ferredoxins and Ferredoxin Reductases

[0261 ] Putative ferredoxins and ferredoxin reductases were identified from the genome of S. yanoikuyae on the basis of sequence homology to known ferredoxin and ferredoxin reductases.

[0262] The identified S. yanoikuyae ferredoxins and ferredoxin reductases were cloned from genomic DNA. In order to optimise the translation of these ferredoxins and ferredoxin reductases in E. coli, where the original START codon was not ATG and/or where the original STOP codon was not TAA, the START/STOP codons were substituted with ATG or TAA, respectively. The codon changes for each ferredoxin and ferredoxin reductases are summarised in Table 10. Table 10: Summary of codon changes for each ferredoxin and ferredoxin

[0264] The final sequences of the ferredoxins and ferredoxin reductases are as summarised in Figure 9.

[0265] The polynucleotide and polypeptide sequences of each ferredoxin and ferredoxin reductases were compared to sequences within the Non-redundant nucleotides and proteins database of NCBI using the blastn and blastp programs, respectively. The top three hits for each are shown below. No results are shown for alignments that did not identify any significant similarity. The blast comparisons were performed on 20 July 2015.

FdX1 polynucleotide - SEQ ID NO: 4 Query Identity

Coverage (%) (%)

No significant similarity found FdX1 polypeptide - SEQ ID NO: 3 Query Identity

Coverage (%) (%) ferredoxin [Sphingobium czechense LL01 ] - 100 70 KMS54512.1

ferredoxin [Sphingomonas sp. PR0901 1 1 -T3T-6A] - 100 70 WP 019830942.1

ferredoxin [Sphingobium baderi] - WP_021244124.1 100 70

FdX2 polynucleotide - SEQ ID NO: 6 Query Identity

Coverage (%) (%)

Altererythrobacter atlanticus strain 26DY36, complete 61 78 genome - CP01 1452.1

Sphingomonas sanxanigenens NX02, complete genome 77 76 - CP006644.1

Variovorax paradoxus B4 chromosome 1 , complete 16 94 sequence - CP00391 1 .1

FdX2 polypeptide - SEQ ID NO: 5 Query Identity

Coverage (%) (%) ferredoxin [Sphingobium yanoikuyae] - 100 100 WP 037522960.1

ferredoxin [Sphingobium yanoikuyae] - 100 99 WP 03751 1909.1

ferredoxin [Sphingobium yanoikuyae] - 100 99 WP 010339184.1

FdX3 polynucleotide - SEQ ID NO: 8 Query Identity

Coverage (%) (%)

No significant similarity found

FdX4 polynucleotide - SEQ ID NO: 10 Query Identity

Coverage (%) (%)

No significant similarity found FdX4 polypeptide - SEQ ID NO: 9 Query Identity

Coverage (%) (%) hypothetical protein [Sphingobium yanoikuyae] - 100 98 WP_004209122.1 reductase [Sphingobium yanoikuyae] - 100 97 WP_010336187.1 reductase [Sphingobium yanoikuyae] - 100 96 WP 037522017.1

FdX5 polynucleotide - SEQ ID NO: 12 Query Identity

Coverage (%) (%)

Sphingobium chiorophenoiicum L-1 chromosome 2, 61 75 complete sequence - CP002799.1

Rhizobium etli bv. mimosae str. IE4771 , complete 16 89 genome - CP006986.1

Rhizobium etli CIAT 652, complete genome - 16 89 CP001074.1

FdX5 polypeptide - SEQ ID NO: 11 Query Identity

Coverage (%) (%) ferredoxin [Sphingobium czechense LL01 ] - 100 67 KMS53059.1

ferredoxin [Sphingomonas sp. DC-6] - WP_030090025.1 100 67 ferredoxin [Sphingomonas sp. YL-JM2C] - 100 67 WP 029992007.1

FdX6 polynucleotide - SEQ ID NO: 14 Query Identity

Coverage (%) (%)

Sphingobium japonicum UT26S DNA, chromosome 1 , 99 84 complete genome - AP010803.1

Sphingobium quisquiliarum strain DC-2 FdX1 gene, 99 83 complete cds - KJ186091 .1

Sphingobium chiorophenoiicum L-1 chromosome 1 , 99 82 complete sequence - CP002798.1

FdX6 polypeptide - SEQ ID NO: 13 Query Identity

Coverage (%) (%)

WP_004210272.11 ferredoxin [Sphingobium 100 100 yanoikuyae] - WP_004210272.1

ferredoxin [Sphingobium sp. AP49] - WP_007712740.1 100 99 ferredoxin [Sphingobium czechense LL01 ] - 100 91 KMS54512.1

FdX7 polynucleotide - SEQ ID NO: 16 Query Identity

Coverage (%) (%)

PREDICTED: Pantholops hodgsonii adrenodoxin 98 99 homolog, mitochondrial-like (LOC102341306), mRNA - XM 005954782.1

Sphingobium sp. YBL2, complete genome - 99 82 CP010954.1

FdX7 polypeptide - SEQ ID NO: 15 Query Identity

Coverage (%) (%)

(2Fe-2S) ferredoxin [Sphingobium yanoikuyae] - 100 100 WP 010337134.1

hypothetical protein [Sphingobium yanoikuyae] - 100 99 WP 004207705.1

PREDICTED: adrenodoxin homolog, mitochondrial-like 98 100 [Pantholops hodgsonii] - XP_005954844.1

FdR1 polynucleotide - SEQ ID NO: 18 Query Identity

Coverage (%) (%)

Sphingobium quisquiliarum strain DC-2 hypothetical 100 85 protein, Red1 , and potassium transporter Kup genes,

complete cds - KJ020538.1

Sphingobium baderi strain DE-13 99 82 aminodeoxychorismate lyase gene, partial cds; and

hypothetical protein, ferredoxin reductase 2, and

hypothetical protein genes, complete cds

KJ020539.1

Sphingobium chlorophenolicum L-1 chromosome 1 , 99 86 complete sequence - CP002798.1

FdR1 polypeptide - SEQ ID NO: 17 Query Identity

Coverage (%) (%) pyridine nucleotide-disulfide oxidoreductase [alpha 100 99 proteobacterium LLX12A] - WP_017501 171 .1

pyridine nucleotide-disulfide oxidoreductase 100 99

[Sphingobium yanoikuyae] - WP_037508006.1

pyridine nucleotide-disulfide oxidoreductase 100 99

[Sphingobium yanoikuyae] - WP_037516721 .1

FdR2 polynucleotide - SEQ ID NO: 20 Query Identity

Coverage (%) (%)

Sphingomonas sp. XLDN2-5 insertion sequence IS6100, 100 99 complete sequence; and CarR (carR), CarAa (carAa),

CarBb (carBa), CarBb (carBb), CarC (carC), and CarAc

(carAc) genes, complete cds - GU123624.1

FdR2 polypeptide - SEQ ID NO: 19 Query Identity

Coverage (%) (%)

MULTISPECIES: pyridine nucleotide-disulfide 100 99 oxidoreductase [Sphingobium] - WP_010338887.1

putative ferredoxin reductase [Sphingomonas 100 63 parapaucimobilis NBRC 15100] - GAL99746.1

pyridine nucleotide-disulfide oxidoreductase 100 63

[Sphingomonas parapaucimobilis] - WP_042483557.1

FdR3 polynucleotide - SEQ ID NO: 22 Query Identity

Coverage (%) (%)

Stenotrophomonas maltophilia strain DI-6 DdmA1 90 78 (ddmA1 ) gene, complete cds - AY786444.1

Stenotrophomonas maltophilia strain DI-6 DdmA2 90 78 (ddmA2) gene, complete cds - AY786445.1

Sphingomonas sp. KSM1 kdR gene for FAD-dependent 81 71 ferredoxin reductase, complete cds - AB744217.1

FdR3 polypeptide - SEQ ID NO: 21 Query Identity

Coverage (%) (%) pyridine nucleotide-disulfide oxidoreductase [alpha 99 99 proteobacterium LLX12A] - WP_017499284.1

pyridine nucleotide-disulfide oxidoreductase 99 66

[Sphingomonas sp. MM-1 ] - WP_015456934.1

pyridine nucleotide-disulfide oxidoreductase 99 63

[Sphingomonas sp. PR0901 1 1 -T3T-6A] - WP 026359323.1

FdR4 polynucleotide - SEQ ID NO: 24 Query Identity

Coverage (%) (%)

Altererythrobacter marensis strain KCTC 22370, 83 72 complete genome - CP01 1805.1

Sphingobium jiangsuense reductase gene, complete cds 80 73 - KJ009326.1

Sphingopyxis alaskensis RB2256, complete genome - 80 72 CP000356.1

FdR4 polypeptide - SEQ ID NO: 23 Query Identity

Coverage (%) (%)

NAD(P)H-nitrite reductase [Sphingobium yanoikuyae] - 100 97 KEZ21015.1

NAD(P)H-nitrite reductase [Sphingobium yanoikuyae] - 88 94 KEZ12866.1

pyridine nucleotide-disulfide oxidoreductase 88 68

[Novosphingobium aromaticivorans] - WP_01 1443878.1

FdR4s polynucleotide - SEQ ID NO: 26 Query Identity

Coverage (%) (%)

Altererythrobacter marensis strain KCTC 22370, 93 72 complete genome - CP01 1805.1

Sphingobium jiangsuense reductase gene, complete cds 91 73 - KJ009326.1

Sphingopyxis alaskensis RB2256, complete genome - 90 72 CP000356.1

Example 11 : Biotransformation of 1 ,8-cineole with P450 _B ui in Combination with Recombinant Ferredoxins from S. yanoikuyae

[0266] The biotransformation of 1 ,8-cineole in the previous example utilised E. coli that recombinantly expressed P450 _B ui , which interacted with native E. coli electron transporters in order to mediate hydroxylation of 1 ,8-cineole.

[0267] The aim of this experiment was to determine whether the efficiency of the whole-cell hydroxylation of 1 ,8-cineole can be improved by recombinantly co- expressing S. yanoikuyae ferredoxins with P450 _B ui -

[0268] The S. yanoikuyae ferredoxins are as described in Example 10 and their polynucleotide sequences are as follows: FdX1 (SEQ ID NO: 4), FdX2 (SEQ ID NO: 6), FdX3 (SEQ ID NO: 8), FdX4 (SEQ ID NO: 10), FdX5 (SEQ ID NO: 12), FdX6 (SEQ ID NO: 14) and FdX7 (SEQ ID NO: 16). The genes were amplified by PCR from S. yanoikuyae genomic DNA and cloned into pRSFDuet expression vector. The experimental groups are summarised in Table 1 1 . [0269] Table 11 : Summary of the expression vectors and the genes they express for each experimental group.

[0270] Chemically competent E. coli BL21 (DE3) were transformed according to the experimental groups listed in Table 1 1 . See Figure 4A for the schematics.

[0271 ] Accordingly, the transformed E. coli of some of the experimental groups were co-expressing P450 _B ui with specific S. yanoikuyae ferredoxins. E. coli was transformed with three empty vectors was used as a negative control and did not hydroxylate 1 ,8-cineole. A 10 mL TB seed culture in a 50 mL Falcon tube containing 30 g mL ^"1 Kan, 50 g mL ^"1 Ampicillin sodium salt (Amp) and 50 g mL ^"1 streptomycin sulphate (Sm) was inoculated with a single transformant and incubated at 180 rpm and 30°C for 18-20 hours. 1 mL of the seed culture was then used to inoculate 50 mL TB containing 30 g mL ^"1 Kan, 50 g mL ^"1 Amp and 50 g mL ^"1 Sm in a 250 mL non- baffled shake flask. The culture was grown for 3 hours and then induced with 1 mM IPTG. 18 hours post induction, the cells were harvested by centrifugation at 6000 x g for 10 min and the supernatant discarded. The pellet was washed with 5 mL of EM medium and centrifuged again. EM medium was discarded and the pellet centrifuged again for 2 mins to remove any residual EM medium. The pellet was then weighed and resuspended in EM medium containing 30 g mL ^"1 Kan, 50 g mL ^"1 Amp and 50 g mL ^"1 Sm (1 g pellet was resuspended in 1 mL EM medium). 500 μί of the suspension were then transferred into a fresh 15 ml_ tube and 2 μΙ_ of undiluted 1 ,8- cineole were added (final concentration of 4 mM). The reaction was then incubated for 3 hours at 180 rpm and 30°C. After 3 hours, an internal standard was added and the suspension mixed thoroughly. Then 990 μΙ_ EtOAc were added and the suspension was mixed thoroughly. The suspension was then centrifuged at 2000 xg, 2 min, 4 °C to separate phases, the organic phase was removed and analysed using GC-MS split 10. The GC-MS peak areas were standardised using an internal standard (alpha-Terpineol).

[0272] The results of the effect of partnering P450 _B ui with the native E. coli electron transport partners or with recombinant S. yanoikuyae ferredoxins are shown in Figure 4B. The results demonstrated that P450 _B ui was more efficient at hydroxylating 1 ,8-cineole when it was partnered with an S. yanoikuyae ferredoxin with the exception of ferredoxin 7 (FdX7).

Example 12: Biotransformation of 1 ,8-cineole with P450 _B ui in Combination with Recombinant Ferredoxin Reductases from S. yanoikuyae

[0273] The biotransformation of 1 ,8-cineole in the Examples 9 and 10 utilised E. coli that recombinantly expressed P450 _B ui , which interacted with native E. coli electron transporters in order to mediate hydroxylation of 1 ,8-cineole.

[0274] The aim of this experiment was to determine whether the efficiency of the whole-cell hydroxylation of 1 ,8-cineole can be improved by recombinantly co- expressing S. yanoikuyae ferredoxin reductases with P450 _BU i .

[0275] The S. yanoikuyae ferredoxin reductases are as described in Example 10 and their polynucleotides sequences are as follows: FdR1 (SEQ ID NO: 18), FdR2 (SEQ ID NO: 20), FdR3 (SEQ ID NO: 22), FdR4 (SEQ ID NO: 24) and FdR4s (SEQ ID NO: 26) were amplified from S. yanoikuyae genomic DNA by PCR and cloned into pETDuet expression vectors. The experimental groups were as summarised in Table 12. [0276] Table 12: Summary of the expression vectors and the genes they express for each experimental group.

[0277] Chemically competent E. coli BL21 (DE3) were transformed according to the experimental groups listed in Table 12. See Figure 5A for the schematics.

[0278] Accordingly, the transformed E. coli of some of the experimental groups were co-expressing P450 _B ui with specific S. yanoikuyae ferredoxin reductases. The method used was the same as that described in Example 1 1 .

[0279] The results of the effect of partnering P450 _B ui with E. coli ferredoxin electron transport partners or with recombinant S. yanoikuyae ferredoxin reductases are shown in Figure 5B. There was no statistically significant difference between P450 _B ui partnered with the E. coli electron transport partners and with S. yanoikuyae ferredoxin reductases. The only exception was when P450 _B ui was co-expressed with ferredoxin reductase 1 (FdR1 ) as this combination resulted in significantly less hydroxycineole. Example 13: Biotransformation of 1,8-cineole with P450 _B ui in Combination with Recombinant Ferredoxin and Ferredoxin Reductases from S. yanoi ^' kuyae

[0280] The aim of this experiment was to investigate whether the efficiency of hydroxylating 1 ,8-cineole can be further improved by recombinantly co-expressing S. yanoi ^' kuyae ferredoxin reductases and ferredoxins with P450 _B ui The method used was the same as that described in Example 12.

[0281 ] Table 13: Summary of the expression vectors and the genes they express for each experimental group.

Group Name pETDuet pRSFDuet pCDFDuet

Negative Control Empty vector Empty vector Empty vector

P450BU1 only Empty vector Empty vector P450E3UI

P450BU1 -FdR1 -FdX1 FdR1 FdX1 P450BUI

P450BU1 -FdR1 -FdX2 FdR1 FdX2 P450BUI

P450BU1 -FdR1 -FdX3 FdR1 FdX3 P450BUI

P450BU1 -FdR1 -FdX4 FdR1 FdX4 P450BUI

P450BU1 -FdR1 -FdX5 FdR1 FdX5 P450BUI

P450BU1 -FdR1 -FdX6 FdR1 FdX6 P450BUI

P450BU1 -FdR1 -FdX7 FdR1 FdX7 P450BUI

P450BU1 -FdR2-FdX1 FdR2 FdX1 P450BUI

P450BU1 -FdR2-FdX2 FdR2 FdX2 P450BUI

P450BU1 -FdR2-FdX3 FdR2 FdX3 P450BUI

P450BU1 -FdR2-FdX4 FdR2 FdX4 P450BUI

P450BU1 -FdR2-FdX5 FdR2 FdX5 P450BUI

P450BU1 -FdR2-FdX6 FdR2 FdX6 P450BUI

P450BU1 -FdR2-FdX7 FdR2 FdX7 P450BUI

P450BU1 -FdR3-FdX1 FdR3 FdX1 P450BUI

P450BU1 -FdR3-FdX2 FdR3 FdX2 P450BUI

P450BU1 -FdR3-FdX3 FdR3 FdX3 P450BUI

P450BU1 -FdR3-FdX4 FdR3 FdX4 P450BUI

P450BU1 -FdR3-FdX5 FdR3 FdX5 P450BUI Group Name pETDuet pRSFDuet pCDFDuet

P450BU1 -FdR3-FdX6 FdR3 FdX6 P450BUI

P450BU1 -FdR3-FdX7 FdR3 FdX7 P450BUI

P450BU1 -FdR4-FdX1 FdR4 FdX1 P450BUI

P450BU1 -FdR4-FdX2 FdR4 FdX2 P450BUI

P450BU1 -FdR4-FdX3 FdR4 FdX3 P450BUI

P450BU1 -FdR4-FdX4 FdR4 FdX4 P450BUI

P450BU1 -FdR4-FdX5 FdR4 FdX5 P450BUI

P450BU1 -FdR4-FdX6 FdR4 FdX6 P450BUI

P450BU1 -FdR4-FdX7 FdR4 FdX7 P450BUI

P450BU1 -FdR4s-FdX1 FdR4s FdX1 P450BUI

P450BU1 -FdR4s-FdX2 FdR4s FdX2 P450BUI

P450BU1 -FdR4s-FdX3 FdR4s FdX3 P450BUI

P450BU1 -FdR4s-FdX4 FdR4s FdX4 P450BUI

P450BU1 -FdR4s-FdX5 FdR4s FdX5 P450BUI

P450BU1 -FdR4s-FdX6 FdR4s FdX6 P450BUI

P450BU1 -FdR4s-FdX7 FdR4s FdX7 P450BUI

[0282] The results of the effect of partnering P450 _B ui with E. coli electron transporters or recombinant S. yanoikuyae ferredoxins and ferredoxin reductases are shown in Figure 6.

[0283] This experiment demonstrated that P450 _B ui was significantly more efficient at hydroxylating 1 ,8-cineole when partnered with specific S. yanoikuyae ferredoxins and ferredoxin reductases. Specifically, P450 _B ui in any combination with FdX7 (i.e. with FdR1 , FdR2, FdR3, FdR4 and FdR4s) showed little to no improvement in the efficiency of converting 1 ,8-cineole to hydroxycineole as compared to E. coli transformed with P450 _B ui only. P450 _B ui when combined with any one of FdRs and FdXs (except FdX7) demonstrated the efficient hydroxylation of 1 ,8-cineole. Example 14: Evaluating the Biotransformation of 1 ,8-cineole with P450 _B ui in Combination with Selected Ferredoxin Reductases and Ferredoxins from S. yanoikuyae

[0284] The aim of this experiment was to establish the kinetics of the biotransformation of 1 ,8-cineole to hydroxycineole by P450 _B ui when recombinantly expressed in combination with any one of FdR2-FdX5, FdR3-FdX2 or FdR4s-FdX6.

[0285] Chemically competent E. coli BL21 (DE3), were transformed with plasmids according to Table 14.

[0286] Table 14: Summary of the expression vectors and the genes they express for each experimental group.

[0287] Accordingly, the transformed E. coli of each experimental group were co- expressing P450 _B ui with a specific combination of S. yanoikuyae ferredoxin and a specific S. yanoikuyae ferredoxin reductase. A 50 ml_ TB seed culture in 250 ml_ non-baffled shake flask containing 30 g ml_ ^"1 Kan, 50 g ml_ ^"1 Amp and 50 g ml_ ^"1 Sm was inoculated with a single colony of transformant and incubated at 180 rpm and 30°C for 18 hours. The seed culture was used to inoculate 500 ml_ TB containing 30 g ml_ ^"1 Kan, 50 g ml_ ^"1 Amp and 50 g ml_ ^"1 Sm in a 2 L baffled shake flask. The culture was grown for 3 hours and then induced with 1 mM IPTG. 18 hours post induction, the cells were harvested by centrifugation at 6000 x g for 20 min and the supernatant removed The pellet was washed with 20 ml_ EM medium and centrifuged again for 10 min. EM medium was removed and the pellet centrifuged again for 2 mins to remove any residual EM medium. The pellet was then weighed and resuspended in EM medium containing 30 g mL ^"1 Kan, 50 g mL ^"1 Amp and 50 g mL ^"1 Sm (1 g pellet was resuspended in 1 mL EM medium). 1 mL aliquots of the suspension were then transferred to fresh 15 mL tubes and 4 μί of 1 ,8-cineole were added (final concentration of 4 mM). Aliquots were extracted at certain times between 0 and 3 hours. To aliquots that had not been extracted after 3 hours, an additional 4 μί 1 ,8-cineole was added. To aliquots that had not been extracted after 6 hours, an additional 12 μί 1 ,8-cineole was added. An internal standard, which was terpineol, was added and the suspension was mixed thoroughly. 3 mL of EtOAc was added and the suspension was mixed thoroughly. The suspension was centrifuged at 2000 x g, 2 min, 4°C to separate the phases, the organic phase was removed and analysed using GC-MS split 10 (as discussed before). The GC-MS peak areas were standardised using the internal standard.

[0288] Furthermore, the ferredoxin-expressing pRSFDuet vectors were the same as those in Example 1 1 and the ferredoxin reductase-expressing pETDuet vectors were the same as those in Example 12. The rates in 2-hydroxycineole production by each of the tested enzyme combinations are shown in Figure 7.

[0289] This experiment demonstrated that P450 _B ui in combination with FdR3 and FdX2 provided the highest rate of hydroxylation of 1 ,8-cineole out of the different combinations tested.

Example 15: Biotransformation of 1,8-cineole with P450 _B ui in combination with FdR3 and FdX2 in a bioreactor #1

[0290] The following settings were used for the bioreactor

[0291 ] Temp Setp 37/30 °C (min = 0%, max = 62%, XP = 20%, Tl = 800 sec, TD = 200 sec, DEADB = 0.0 °C)

• Stirrer Setp 200 rpm (min = 1%, max = 100%)

• Air Setp 0.1 Ipm (min = 0%, max = 100%)

• 0 ₂ Setp 0 Ipm (min = 0%, max = 100%) • pH Setp 7.0 (min = -100 %, max = 100 %, XP = 30%, Tl = 30 sec, TD = 0 sec, DEAD = 0.02)

• Foam (Cycle = 10 sec, Pulse = 10 sec, sensitivity low)

• p02 Setp 30 % (cascade = stirrer-airsparger-0 ₂ sparger; min = 0%, max = 100%, XP = 90%, Tl = 50 sec, TD = 0 sec, DEADB = 0.0% Sat)

• Cascade (Start cascade: 0%)

Stirrer:

OUT Setp

0 500

20 1250

40 2000

60 2000

80 2000

100 2000

Airsparger:

OUT Setp

0 0.3

20 0.3

40 0.3

60 1 .5

80 1 .5

100 1 .5 0 ₂ sparger:

[0292] Chemically competent E. coli BL21 (DE3) was transformed with P450 _BU i - FdR3-FdX2 (according to Table 14). A primary seed culture (10 mL of TB in a 50 mL Falcon tube containing 30 g mL ^"1 Kan, 50 g mL ^"1 Amp and 50 g mL ^"1 Sm) was inoculated with a single transformant and incubated at 180 rpm and 37°C for ca. 24 hours. 500 μί of the primary seed culture were then used to inoculate a secondary seed culture (500 mL TB containing 30 g mL ^"1 Kan, 50 g mL ^"1 Amp and 50 g mL ^"1 Sm in a 2L baffled shake flask) and incubated at 180 rpm and 37°C for 16 hours. The main culture (1 .4 L of DM3) was inoculated with the secondary seed to a OD ₆ oo of 0.25 in a 2 L glasses bioreactor controlled by a Biostat B (Sartorius, Germany) control unit. The temperature was maintained at 37 °C and the pH of 7.0 was automatically controlled using 10% (v/v) H ₃ PO ₄ and 10% (v/v) ammonia solution. Foaming was controlled using 10% (v/v) Sigma Antifoam 204. The bioreactor was operated in cascade mode maintaining a dissolved oxygen of at least 30% by increasing stirrer speed, airflow and oxygen flow as required. When the OD ₆ oo had reached about 30 and the level the glucose was depleted (after ca. 8 hours), a Glucose/Mg feed (800 mL 660 g L ^"1 Glucose supplemented with 80 mL 1 M MgSO ₄ .7H ₂ O) was started at a flow rate of 36 mL h ^"1 which was decreased to 18 mL h ^"1 after a further 2 h when the temperature was decreased to 30°C. After another 15 min the culture was induced with 2 mmol IPTG and simultaneously a 1 ,8-cineole feed was started at a flow rate of 0.9 mL h ^"1 . 14 h post induction, when the OD ₆ oo reached ca. 67 the 1 ,8-cineole feed was increased to 1 .8 ml_ h ^"1 and 16 h post induction further increased to 2.7 ml_ h ^"1 before the cineole feed was stopped 18 h post induction and then restarted at 1 .8 ml_ h ^"1 18.5 h post induction. 20 h post induction, glucose started to accumulate in the culture and the Glucose/Mg feed was decreased to 9 ml_ h ^~1 . 23 h post induction, the 1 ,8-cineole feed was decreased to 0.6 ml_ h ^~1 . 38 h post induction, when the OD ₆ oo had decreased to ca. 28 and glucose had accumulated, both, the glucose/Mg and 1 ,8-cineole feed were turned off. The production rate of 2-hydroxycineole over the complete process is shown in Figure 8.

[0293] A total of 38.7 ml_ 1 ,8-cineole was fed into the culture. 40 h post induction, the culture was harvested and the cells were removed by centrifugation at 38,400 xg for 30 min at 4°C. The supernatant was then acidified with cone. HCI to precipitate extracellular proteins/proteins released during cell lysis and centrifuged again. The resulting supernatant was neutralised with 4 M NaOH and sterile filtered through a Sartopore 2 300 filter capsule (Sartorius, Germany) and stored at 4°C. Products of 1 ,8-cineole conversion were then extracted using dichloromethane using a continuous extraction process. The combined organic phase was then filtered through a phase separation filter and concentrated under reduced pressure. This procedure yielded 23 g of crude 2-hydroxycineole (confirmed by 1 H-NMR and GC-MS analysis) and small amounts of impurities (e.g. residual 1 ,8-cineole and oxocineole).

Example 15: Biotransformation of 1,8-cineole with P450 _B ui in combination with FdR3 and FdX2 in a bioreactor #2

[0294] This experiment was performed using the same method used in Example 14 except for slight changes in temperature and feed rates of 1 ,8-cineole and Glucose/Mg. The glucose/Mg feed (800 ml_ 660 g L ^"1 Glucose supplemented with 80 ml_ 1 M MgS0 ₄ .7H ₂ 0) was started 8 hours post inoculation at a flow rate of 36 ml_ h ^"1 which was decreased to 18 ml_ h ^"1 after further 4 h when the temperature was decreased to 30°C. After another 15 min, at an OD ₆ oo of ca 104 the culture was induced with 2 mmol IPTG and simultaneously a 1 ,8-cineole feed was started at 1 ml_ h ^"1 and increased by 1 ml_ h ^"1 every hour up to 8 mL h ^~1 . The 1 ,8-cineole feed of 8 ml_ h ^"1 was maintained until 10.08 hours post induction and then turned off, when the accumulation of glucose became apparent and the OD ₆ oo had decreased to ca. 50. A total of 52 ml_ of 1 ,8-cineole was fed to the culture. 10.4 h post induction the culture was harvested and the cells removed as per Example 14 which resulted in 1 .65 L of culture supernatant. All further downstream processing was done as per Example 15. This process yielded ca. 39 g of crude 2-hydroxycineole.

Example 16: Biotransformation of 1,8-cineole with P450 _B u2 in combination with FdR3 and FdX2 in a bioreactor #2

[0295] The following settings were used for the bioreactor

[0296] Temp Setp 37/30 °C (min = 0%, max = 62%, XP = 20%, Tl = 800 sec, TD = 200 sec, DEADB = 0.0 °C)

• Stirrer Setp 200 rpm (min = 1 %, max = 100%)

• Air Setp 0.1 Ipm (min = 0%, max = 100%)

• 0 ₂ Setp 0 Ipm (min = 0%, max = 100%)

• pH Setp 7.0 (min = -100 %, max = 100 %, XP = 30%, Tl = 30 sec, TD = 0 sec, DEAD = 0.02)

• Foam (Cycle = 10 sec, Pulse = 10 sec, sensitivity low)

• p02 Setp 30 % (cascade = stirrer-airsparger-0 ₂ sparger; min = 0%, max = 100%, XP = 90%, Tl = 50 sec, TD = 0 sec, DEADB = 0.0% Sat)

• Cascade (Start cascade: 0%)

Stirrer:

OUT Setp

0 500

20 1250

40 2000

60 2000

80 2000

100 2000 Airsparger:

0 ₂ sparger:

[0297] Chemically competent E. coli BL21 (DE3) was transformed with P450 _B u2- FdR3-FdX2. A primary seed culture (10 mL of TB in a 50 mL Falcon tube containing 30 Mg mL ^"1 Kan, 50 \ g mL ^"1 Amp and 50 \ g mL ^"1 Sm) was inoculated with a single transformant and incubated at 180 rpm and 37°C for ca. 24 hours. 500 μί of the primary seed culture were then used to inoculate a secondary seed culture (500 mL TB containing 30 g mL ^"1 Kan, 50 g mL ^"1 Amp and 50 g mL ^"1 Sm in a 2L baffled shake flask) and incubated at 180 rpm and 37°C for 16 hours. The main culture (1 .4 L of DM3) was inoculated with the secondary seed to a OD ₆ oo of 0.25 in a 2 L glasses bioreactor controlled by a Biostat B (Sartorius, Germany) control unit. The temperature was maintained at 37 °C and the pH of 7.0 was automatically controlled using 10% (v/v) H ₃ P0 ₄ and 10% (v/v) ammonia solution. Foaming was controlled using 10% (v/v) Sigma Antifoam 204. The bioreactor was operated in cascade mode maintaining a dissolved oxygen of at least 30% by increasing stirrer speed, airflow and oxygen flow as required. The glucose/Mg feed (800 mL 660 g L ^"1 Glucose supplemented with 80 mL 1 M MgS0 ₄ .7H ₂ 0) was started 8 hours post inoculation at a flow rate of 36 mL h ^"1 which was decreased to 18 mL h ^"1 after further 4 h when the temperature was decreased to 30°C. After another 15 min, at an OD ₆ oo of ca 97, the culture was induced with 2 mmol IPTG and simultaneously a 1 ,8-cineole feed was started at 1 mL h ^"1 and increased by 1 mL h ^"1 every hour up to 8 mL h ^~1 . The 1 ,8- cineole feed of 8 mL h ^"1 was maintained until 10 hours post induction and then turned off, when the accumulation of glucose became apparent and the OD ₆ oo had decreased to ca. 68. During feeding of 1 ,8-cineole, samples were taken to determine production of hydroxylated 1 ,8-cineole. A total of 52 mL of 1 ,8-cineole was fed to the culture.

[0298] 10.4 hours after induction the culture was harvested and the cells were removed by centrifugation at 38,400 xg for 30 min at 4°C. The supernatant was then acidified with cone. HCI to precipitate extracellular proteins/proteins released during cell lysis and centrifuged again. The resulting supernatant was neutralised with 4 M NaOH and sterile filtered through a Sartopore 2 300 filter capsule (Sartorius, Germany) and stored at 4°C. Products of 1 ,8-cineole conversion were then extracted using dichloromethane using a continuous extraction process. The combined organic phase was filtered through a phase separation filter and concentrated under reduced pressure. This procedure yielded 22.8 g of crude 2-hydroxycineole (confirmed by 1 H- NMR and GC-MS analysis) and small amounts of impurities (e.g. residual 1 ,8-cineole and oxo-cineole). A sample was placed in a polarimeter tube using the D line of a sodium lamp (589nm), the optical rotation at 25°C of BU2 was 23.3° (c = 0.3, ethanol). See Figure 9. Example 17: Biotransformation of 1 ,8-cineole with P450 _B u3 in combination with FdR3 and FdX2 in a bioreactor

[0299] This experiment was completed using the same method described in Example 16 except chemically competent E. coli BL21 (DE3) was transformed with P450 _BU 3-FdR3-FdX2. Glucose supplemented with 80 ml_ 1 M MgS0 ₄ .7H ₂ 0) was started 8 hours post inoculation at a flow rate of 36 ml_ h ^"1 which was decreased to 18 ml_ h ^"1 after further 4 h when the temperature was decreased to 30°C. After another 15 min, at an OD ₆ oo of ca 94, the culture was induced with 2 mmol IPTG and simultaneously a 1 ,8-cineole feed was started at 1 ml_ h ^"1 and increased by 1 ml_ h ^"1 every hour up to 8 ml_ h ^~1 . The 1 ,8-cineole feed of 8 ml_ h ^"1 was maintained until 10 hours post induction and then turned off, when the accumulation of glucose became apparent and the OD ₆ oo had decreased to ca. 108. During feeding of 1 ,8-cineole, samples were taken to determine production of hydroxylated 1 ,8-cineole. A total of 52 mL of 1 ,8-cineole was fed to the culture.

[0300] 10.4 h post induction the culture was harvested and the cells removed as per Example 1 . This procedure yielded 39.9 g of crude 2-hydroxycineole (confirmed by 1 H-NMR and GC-MS analysis) and small amounts of impurities (e.g. residual 1 ,8- cineole and oxocineole). See Figure 10.

Example 18: Biotransformation of 1,8-cineole with P450 _B u3 in combination with FdR3 and FdX2 in a bioreactor (reduced temperature and later addition of 1,8- cineole)

[0301 ] This experiment was completed using the same method described in Example 17 except the temperature was reduced to 20°C before induction and the 1 ,8-cineole feed was started 5 hours after induction. Glucose supplemented with 80 mL 1 M MgS0 ₄ .7H ₂ 0) was started 8 hours post inoculation at a flow rate of 36 mL h ^"1 which was decreased to 18 mL h ^"1 after further 4 h when the temperature was decreased to 20°C. After another 15 min, at an OD ₆ oo of ca 80, the culture was induced with 2 mmol IPTG and 5 hours later a 1 ,8-cineole feed was started at 1 mL h ^{~ 1} and increased by 2 mL h ^"1 every hour up to 8 mL h ^~1 . The 1 ,8-cineole feed of 8 mL h ^"1 was maintained until 13 hours post induction and then turned off, 1 1 hours after induction was 120 and had decreased to 105 after another 15 hours. During feeding of 1 ,8-cineole, samples were taken to determine production of hydroxylated 1 ,8- cineole. A total of 52 mL of 1 ,8-cineole was fed to the culture. 26 h post induction the culture was harvested and the cells removed as per Example 1 . Optical Activity of samples was calculated using polar 3005 polarimeter. A sample was placed in a polarimeter tube using the D line of a sodium lamp (589nm), the optical rotation at 25°C of BU3 was 23.8° (c = 0.3, ethanol). See Figure 1 1 .

Experiment 19: Biotransformation of (1 R)-6B-hydroxy-1,8-cineole with P450 _B ui in combination with FdR3 and FdX2 in a bioreactor

[0302] This experiment was completed using the same method described in Example 16 except the temperature was maintained at 30°C for the entire process and Terrific broth was used as the growth medium. Terrific broth contained (per litre): Tryptone 12 g, Yeast Extract 24 g, glycerol 4 mL, K ₂ HPO ₄ 9.4 g, KH ₂ PO ₄ 2.2 g. Protein expression was induced 3 hours after inoculation with 1 mM IPTG. Three hours after induction 1 .2 grams of (1 R)-6B-hydroxy-1 ,8-cineole was added to the culture and another 1 .0 grams was added 3 hours later. Samples were taken periodically and after solvent extraction were analysed using GC MS to estimate levels of cineole derivatives. See Figure 12.

[0303] The relative concentrations of each derivative are shown in the Figure 12. As expected no cineole-derived compounds were detected before addition of the (1 R)-6B-hydroxy-1 ,8-cineole. Immediately after addition both (1 R)-6B-hydroxy-1 ,8- cineole and (1 R)-2-oxo-1 ,8-cineole were detected. (1 R)-2-oxo-1 ,8-cineole was detected because the (1 R)-6B-hydroxy-1 ,8-cineole contained a small amount of this compound. After 3 hours (and before the next addition of (1 R)-6B-hydroxy-1 ,8- cineole) a majority of the (1 R)-6B-hydroxy-1 ,8-cineole had been converted to oxo- hydroxy-1 ,8-cineole. At the end of the process all the added substrate (a total of 2.3 grams and including (1 R)-2-oxo-1 ,8-cineole) had been converted to oxo-hydroxy-1 ,8- cineole

[0304] While the foregoing written description of the invention enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The invention should therefore not be limited by the above described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention as broadly described herein.

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