WO2008109934A2

WO2008109934A2 - Degradation of coumarin based compounds

Info

Publication number: WO2008109934A2
Application number: PCT/AU2008/000319
Authority: WO
Inventors: Matthew C Taylor; David Tattersall; Lyndall J Briggs; Nigel French; Robyn J. Russell; John G Oakeshott
Original assignee: Commonwealth Scientific and Industrial Research Organization CSIRO; Grains Research and Development Corp
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO; Grains Research and Development Corp
Priority date: 2007-03-09
Filing date: 2008-03-07
Publication date: 2008-09-18
Anticipated expiration: 2009-09-09
Also published as: WO2008109934A3

Abstract

The present invention relates to the identification of reductase enzymes that degrade coumarin based compounds such as aflatoxins. Methods, including those relying on transgenic organisms, are provided for degrading coumarin based compounds such as aflatoxins.

Description

DEGRADATION OF COUMARIN BASED COMPOUNDS

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION Aflatoxins are fungal secondary metabolites that are recognised as being of economic and health importance. They are produced by at least three toxic strains of Aspergillus, namely A. flavus, A. nominus and A. parasiticus. There are various derivatives of aflatoxins, with aflatoxin B₁ being one of the most toxic.

Aflatoxins are potent carcinogens in several species of animals (Eaton and Callagher, 1994) and epidemiological studies have implicated them as acute toxicants as well as human class I hepatocarcinogens in man (IARC, 1993). Carcinogenicity is associated with renal and hepatic oxidative detoxification in contaminated foods by cytochrome P450 enzymes to yield an epoxide which is cytotoxic. Ingestion of food contaminated with fungal aflatoxins is believed to contribute to the high incidence of hepatoma and chronic liver disease in subtropical regions.

The occurrence of aflatoxin in feedstuff is quite common all over the world. Aflatoxins have been detected as contaminants of crops before harvest, between harvesting and drying, in storage, and after processing and manufacturing. Trading of aflatoxin-contaminated agricultural commodities is tightly regulated at both national and international levels. Compliance to these regulations causes the loss of millions of dollars in agricultural produce each year. Trade sanctions and health effects on aflatoxin contaminated grains add significantly to the losses (Brown et al., 1996).

Accumulation of aflatoxin B, and aflatoxin M, into eggs, and milk from food producing animals after ingestion of aflatoxin contaminated feeds has been described. Even at low concentrations aflatoxins may result in decreased milk yield and egg production in farm animals.

In experimental animals, aflatoxins have been shown to cause cancer and liver damage, have mutagenic and teratogenic activity and to be immunosuppressive (Robens and Richard, 1992; Eaton and Callagher, 1994). Thus, chronic exposure to aflatoxins may not only significantly alter food production and animal farming efficiency but direct exposure to aflatoxin-contaminated food commodities may also constitute a great risk to the consumer. It is therefore important to reduce and/or prevent human exposure by developing practical and effective methods to detoxify aflatoxin-contaminated feedstuff s.

Once food is contaminated with aflatoxin there are two options if the food is to be used: either the toxin is removed or the toxin is degraded into less toxic or non-toxic compounds. The first option is only viable when aflatoxin is present in identifiable pieces of food which can be removed from the remainder of the lot, or if a solvent system can be used to extract aflatoxin without leaving unwanted residues or markedly altering the composition of the food.

As for the second option, a variety of methods have been developed. Aflatoxin may be degraded by physical, chemical or biological methods (Park, 1993). Physical approaches to aflatoxin destruction involve treating with heat, ultraviolet light, or ionising radiation, none of which are entirely effective. Chemical degradation of aflatoxin is usually carried out by the addition of chlorinating, oxidising or hydrolytic agents. Chemical treatments require expensive equipment and may result in losses of nutritional quality of treated commodities.

It has been observed that many microorganisms, including bacteria, yeasts, moulds, actinomycetes and algae, are able to remove or degrade aflatoxin in foods and feeds (see, for example, US 5,364,788, WO 98/34503, WO 02/099142, WO 98/98450,

US 4,931,398 and US 3,428458), however biological detoxification of aflatoxin is not performed on a commercial scale.

There is a need for the identification of further enzymes and methods for degrading coumarin based compounds such as aflatoxins.

SUMMARY OF THE INVENTION The present inventors have determined that a large group of reductases are capable of degrading coumarin based compounds.

Accordingly, in a first aspect the present invention provides a method of degrading a coumarin based compound, the method comprising contacting the coumarin based compound with a reductase. In a preferred embodiment, the coumarin based compound is an aflatoxin.

Examples of aflatoxins which can be degraded using the methods of the invention include, but are not limited to, aflatoxin Bi, aflatoxin B₂, aflatoxin Gi, aflatoxin G₂, aflatoxin Mi and/or aflatoxin M₂.

In a further embodiment, the reductase can be purified from an Actinobacteridae, or is a fragment/mutant/variant thereof.

Examples of Actinobacteridae from which the reductase can be purified include, but are not limited to, Rhodococcus sp., Mycobacterium sp., Gordonia sp., Pseudonocardia sp., Streptomyces sp., Nocardia sp., Nocardiopsis sp., Nocardioides sp., Bifidobacterium sp., Actinomyces sp., Rothia sp., Saccharothrix sp., Actinoplanes sp., Frankia sp. and Clavibacter sp.

In a preferred embodiment, the Actinobacteridae is a Mycobacterium sp such as M. bovis, M. smegmatis, M. vanbaalenii, M. ulcerans. M. sp. KMS, M. sp. JLS, M. sp. MCS and M. tuberculosis.

Preferably, the reductase is an F₄₂₀ dependent reductase. More preferably, the

F₄₂₀ dependent reductase is a member of the pyridoxamine 5 '-phosphate oxidases

(PNPOx) family of reductases, the DUF385 family of reductases, or the glyoxalase/bleomycin resistant family of reductases. Even more preferably, the F₄₂₀ dependent reductase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149, and ii) an amino acid sequence which is at least 25% identical to at least one of SEQ ID NOs 1 to 24, 101 to 107, and 139 to 149. In one embodiment, the F₄₂₀ dependent reductase is a member of the pyridoxamine 5 '-phosphate oxidases (PNPOx) family. In a preferred embodiment, the PNPOx F₄₂₀ dependent reductase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 11, and ii) an amino acid sequence which is at least 25% identical to at least one of SEQ ID NOs I to 11.

In another embodiment, the F₄₂₀ dependent reductase is a member of the DUF385 family. In a preferred embodiment, the DUF385 F₄₂₀ dependent reductase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NOs 12 to 20, and ii) an amino acid sequence which is at least 25% identical to at least one of SEQ

ID NOs 12 to 20.

In yet a further embodiment, the F₄₂₀ dependent reductase is a member of the glyoxalase/bleomycin resistant family. In a preferred embodiment, the glyoxalase/bleomycin resistant F₄₂₀ dependent reductase comprises a sequence selected from: i) an amino acid sequence as provided in SEQ ID NO:21, and ii) an amino acid sequence which is at least 25% identical to SEQ ID NO:21.

In a further embodiment, the F₄₂₀ dependent reductase comprises an amino acid sequence which is at least 90% identical to at least one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149.

In a particularly preferred embodiment, the F₄₂₀ dependent reductase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NOs 1, 13, 14, 15 and 17, and ii) an amino acid sequence which is at least 25% identical to at least one of SEQ ID NOs 1, 13, 14, 15 and 17. In a further particularly preferred embodiment, the F₄₂₀ dependent reductase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NOs 14 and 139 to 149, and ii) an amino acid sequence which is at least 25% identical to at least one of SEQ ID NOs 14 and 139 to 149.

In another embodiment, the reductase has a specific activity against aflatoxin G₁ which is at least 50, more preferably at least 250, and even more preferably at least 500 μmoles/min/mg(enzyme). The specific activity can be determined as outlined in Example 10. In another embodiment, the reductase has a molecular weight less than 5OkDa.

In a further embodiment, the reductase has a molecular weight between about 20 and about 40 kDa, more preferably between about 25 and about 35 kDa.

It is also preferred that the method further comprises providing an electron donor. Examples of suitable electron donors include, but are not limited to, F₄₂₀H₂, reduced FO, FMNH₂, or FADH₂.

In a further embodiment, the method further comprises providing an enzyme that reduces the electron donor. For example, when the electron donor is F₄₂₀H₂, and the enzyme can be glucose-6-phosphate dehydrogenase. In another example, when the electron donor is FMNH₂ and the enzyme can be flavin reductase. Preferably, the glucose-6-phosphate dehydrogenase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NO:49, SEQ ID NO:115 and SEQ ID NO:116, and ii) an amino acid sequence which is at least 25% identical to SEQ ID NO:49, SEQ ID NO:115 and/or SEQ ID NO: 116.

Preferably, the flavin reductase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NO:51, SEQ ID NO: 133 and SEQ ID NO: 134, and ii) an amino acid sequence which is at least 25% identical to SEQ ID NO:51, SEQ ID NO: 133 and/or SEQ ID NO: 134.

In yet a further embodiment, the method comprises providing a host cell producing the reductase. Preferably, the host cell comprises an exogenous polynucleotide sequence selected from: i) a nucleotide sequence as provided in any one of SEQ ID NOs 25 to 48 and 108 to 114, ii) a nucleotide sequence which is at least 25% identical to at least one of SEQ ID NOs 25 to 48 and 108 to 114, iii) a nucleotide sequence which hybridizes to at least one of SEQ ID NOs 25 to

48 and 108 to 114 under stringent conditions; and iv) a nucleotide sequence encoding an F₄₂₀ dependent reductase as defined herein.

In another aspect, the present invention provides a host cell comprising an exogenous polynucleotide encoding a reductase which degrades a coumarin based compound.

Preferably, the reductase is an F₄₂₀ dependent reductase. More preferably, the F₄₂₀ dependent reductase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149, and ii) an amino acid sequence which is at least 25% identical to at least one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149.

The host cell can be any type of cell including a bacterial cell, yeast cell, plant cell or animal cell. In an embodiment, the host cell is a plant or animal cell. In a further aspect, the present invention provides a transgenic plant comprising at least one plant cell of the invention.

In an embodiment, the plant cell further comprises an enzyme which synthesizes FO using 4-hydroxy phenylpyruvate and 5-amino-6-ribitylamino-2,4 (IH, 3H)- pyrimidineione as substrates. Preferably, the enzyme comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NO:53, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122 and SEQ ID NO:123, and ii) an amino acid sequence which is at least 25% identical to SEQ ID NO:53, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122 and/or SEQ ID NO:123.

In yet a further embodiment, the plant cell further comprises enzymes which convert FO to F₄₂₀. hi an embodiment, two enzymes are required to convert FO to F₄₂₀, wherein the first enzyme comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NO:55 and SEQ ID NO: 129, and ii) an amino acid sequence which is at least 25% identical to SEQ ID NO:55 and/or SEQ ID NO: 129, and the second enzyme comprises a sequence selected from: iii) an amino acid sequence as provided in SEQ ID NO: 57 or SEQ ID NO: 131, and iv) an amino acid sequence which is at least 25% identical to SEQ ID NO:57 and/or SEQ ID NO: 131. In another embodiment, three enzymes are required to convert FO to F₄₂₀, and wherein the first enzyme comprises a sequence selected from: i) an amino acid sequence as provided in SEQ ID NO:55 or SEQ ID NO: 129, and ii) an amino acid sequence which is at least 25% identical to SEQ ID NO:55 and/or SEQ ID NO: 129, and the second enzyme comprises a sequence selected from: iii) an amino acid sequence as provided in SEQ ID NO:57 or SEQ ID NO: 131, and iv) an amino acid sequence which is at least 25% identical to SEQ ID NO:57 and/or SEQ ID NO: 131, and the third enzyme comprises a sequence selected from: v) an amino acid sequence as provided in any one of SEQ ID NOs 150 to 153, and vi) an amino acid sequence which is at least 25% identical to at least one of SEQ ID NOs 150 to 153.

In another embodiment, the cell further comprises an enzyme that produces reduced F₄₂₀. Preferably, the enzyme that produces reduced F₄₂₀ is glucose-6-phosphate dehydrogenase. Preferably, the glucose-6-phosphate dehydrogenase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NO:49, SEQ ID

NO: 115 and SEQ ID NO: 116, and ii) an amino acid sequence which is at least 25% identical to SEQ ID NO:49, SEQ ID NO:115 and/or SEQ ID NO:116.

In another aspect, the present invention provides a transgenic plant comprising a host cell comprising enzymes which convert FO to F₄₂₀.

Also provided is a method of degrading a coumarin based compound in a sample, the method comprising contacting the sample with a transgenic plant of the invention, or an extract thereof.

In a further embodiment, the sample is selected from the group consisting of: soil, water, biological material, a feedstuff or a combination thereof. In a particularly preferred embodiment, the biological material is plant material.

In yet another aspect, the present invention provides a transgenic non-human animal comprising at least one animal cell of the invention. In another embodiment, the present invention provides a method of treating toxicity caused by a coumarin based compound in a subject, the method comprising administering to the subject a composition comprising a reductase, and/or a polynucleotide encoding said reductase. Examples of the results of toxicity caused by a coumarin based compound such as aflatoxin include, but are not limited to, cancer, liver damage, mutagenic activity, teratogenic activity and immunosuppression.

Preferably, the reductase is an F₄₂₀ dependent reductase which comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 24, 101 to

107 and 139 to 149, and ii) an amino acid sequence which is at least 25% identical to at least one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149.

It is also preferred that the method further comprises providing an electron donor. Examples of suitable electron donors include, but are not limited to, F₄₂₀H₂, reduced FO, FMNH₂, or FADH₂. As the skilled addressee will appreciate, the electron donor may be provided in the same composition as the reducatse, or administered independently.

Preferably, the subject is an animal. More preferably, the animal is a mammal such as a human, cat, dog, cow, sheep, goat or horse. Even more preferably, the mammal is a human.

Also provided is the use of a reductase, or polynucleotide encoding said reductase, for the manufacture of a medicament for treating toxicity caused by a coumarin based compound in a subject. In yet a further aspect, the present invention provides a method of producing a polypeptide with enhanced ability to degrade a coumarin based compound, or altered substrate specificity for a different type of a coumarin based compound, the method comprising i) altering one or more amino acids of a reductase polypeptide, ii) determining the ability of the altered polypeptide obtained from step i) to degrade a coumarin based compound, and iii) selecting an altered polypeptide with enhanced ability to degrade the coumarin based compound, or altered substrate specificity for a different type of coumarin based compound, when compared to the polypeptide used in step i). In another aspect, the present invention provides a polypeptide produced by a method of the invention.

Whilst many of the reductases described herein have been predicted to exist through the analysis of the genome of various bacteria, no industrial use had been g determined. Hence, there was no motivation in the art to actually produce these proteins. However, as outlined herein the present inventors have surprisingly found that reductases can be used to degrade a coumarin based compound.

Thus, in a further aspect the present invention provides a substantially purified and/or recombinant polypeptide that degrades a coumarin based compound, wherein the polypeptide has reductase activity.

In yet a further aspect, the present invention provides a substantially purified and/or recombinant polypeptide comprising amino acids having a sequence as provided in any one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149, a biologically active fragment thereof, or an amino acid sequence which is at least 25% identical to at least one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149, wherein the polypeptide degrades a coumarin based compound.

In an embodiment, the polypeptide is a fusion protein further comprising at least one other polypeptide sequence. In a preferred embodiment, the at least one other polypeptide is selected from the group consisting of: a polypeptide that enhances the stability of a polypeptide of the present invention, a polypeptide that assists in the purification of the fusion protein, and a polypeptide which assists in the polypeptide of the invention being secreted from a cell (for example secreted from a plant cell).

In a further aspect, the present invention provides an isolated and/or exogenous polynucleotide comprising nucleotides having a sequence as provided in, or complementary to, any one of SEQ ID NOs 25 to 48 and 108 to 114, a sequence which is at least 25% identical to at least one of SEQ ID NOs 25 to 48 and 108 to 114, a sequence which hybridizes to one or more of SEQ ID NOs 25 to 48 and 108 to 114, or a sequence which encodes a polypeptide of the invention. Preferably, the polynucleotide comprises nucleotides having a sequence which hybridizes to one or more of SEQ ID NOs 25 to 48 and 108 to 114 under stringent conditions.

Preferably, the polynucleotide is operably linked to a promoter capable of directing expression of the polynucleotide in a cell. In an embodiment, the cell is a plant cell or animal cell.

Also provided is a vector comprising the polynucleotide of the invention.

In a further aspect, the present invention provides a method of producing the polypeptide of the invention, the method comprising expressing in a cell the polynucleotide of the invention and/or a vector of the invention. In another aspect, the present invention provides a composition for degrading a coumarin based compound, the composition comprising a polypeptide of the invention, and one or more acceptable carriers.

In a preferred embodiment, the composition is a feedstuff. The polypeptide may be provided to the composition in, for example, a purified form, or as part recombinantly produced biological material such as a transgenic plant or an extract thereof.

In yet another aspect, the present invention provides a composition for degrading a coumarin based compound, the composition comprising a host cell of the invention, or an extract thereof, and one or more acceptable carriers.

Preferably, the composition further comprises an electron donor. Examples of suitable electron donors include, but are not limited to, F₄₂₀H₂, reduced FO, FMNH₂ or FADH₂. In addition, it is preferred that the composition further comprises an enzyme that reduces the electron donor.

In yet another embodiment, the composition further comprises a plant or a portion thereof. For example, the composition may comprise a portion of a plant such as a peanut. In yet a further embodiment, the composition further comprises a coumarin based compound. Preferably, the coumarin based compound is an aflatoxin.

The composition may also comprise components for physically or chemically disrupting the cell membrane and/or cell wall of a microorganism. For example, the composition may further comprises a detergent. Preferably, the detergent is a non-ionic detergent such as Tween 80.

In yet a further aspect, the present invention provides a method for degrading a coumarin based compound, the method comprising contacting the coumarin based compound with a composition of the invention.

In a further aspect, the present invention provides a method of preparing a feedstuff, the method comprising mixing a reductase which degrades a coumarin based compound with at least one nutritional substance.

In an embodiment, the nutritional substance is grain, hay and/or nuts.

In another aspect, the present invention provides a polymeric sponge or foam for degrading a coumarin based compound, the foam or sponge comprising a polypeptide of the invention immobilized on a polymeric porous support.

In a further aspect, the present invention provides a method for degrading a coumarin based compound, the method comprising contacting compound to a sponge or foam of the invention.

In yet a further aspect, the present invention provides an extract of a host cell of the invention, a transgenic plant of the invention or a transgenic non-human animal of the invention, comprising a reductase which degrades a coumarin based compound. In another aspect, the present invention provides a kit for degrading a coumarin based compound, the kit comprising a reductase, and/or a polynucleotide encoding the reductase.

Preferably, the kit further comprises an electron donor. In addition, it is preferred that the kit further comprises an enzyme that reduces the electron donor.

As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1. AFGl was incubated with resuspended (NtLO₂SO₄ precipitations and assayed by TLC as described in methods. Aflatoxin negative control, no enzyme, Lane 1; M. smegmatis control, lane 2; 60% (NH-_I)₂SO₄ precipitation, lane 3; 50% (NtL_t)₂SO₄ precipitation, lane 4; 40% (NR_t)₂SO₄ precipitation, lane 5; 30% (NIL_t)₂SO₄ precipitation, lane 6; 20% (NR_I)₂SO₄ precipitation, lane 7; 10% (NH₄)_ISO₄ precipitation, lane 8.

Figure 2. Alignment of M. smegmatis reductases from the pyridoxamine 5' phosphate oxidase (PNPOx) subfamily which degrade aflatoxin.

Figure 3. Alignment of M. smegmatis reductases from the domain of unknown function (DUF) 385 subfamily which degrade aflatoxin.

Figure 4. Alignment of M. smegmatis reductases from the glyoxalase/bleomycin resistance protein (BRP) subfamily which degrade aflatoxin.

Figure 5. Alignment of Mycobacterium sp. reductases closely related to MSMEG5954.

Figure 6. Time course analysis of MSMEG3387 catalysed degradation of AFGl and analysed by LC-MS as described in Example 10. Panel A, 0 minute time point of lOμg/ml AFGl, stopped with formic acid and analysed by LC-MS. The trace of the total ion count is shown in the main panel, the ion species (M + H⁺) present in the peak at 7.51 minutes were extracted and are shown in the inset box on the right, with the chemical structure of AFGl shown in the inset on the left. Panel B, lOμg/ml of AFGl was degraded with MSMEG3387 for 20 minutes, stopped with acid and analysed by LC-MS. The main panel shows the trace of the total ion count, the ions that are present in the peak at 9.41 minutes were extracted and shown in the inset box on the right. The hypothesised chemical structure of the compound that represents the major ion species of this peak is shown to the left.

Figure 7. DNA PCR amplification of genes transformed into N. tabacum, using gene specific primers. Representative amplification products for MSMEG0772 (lanes 1-3), MSMEG1828 (lanes 4-6), MSMEG1829 (lanes 7-9), MSMEG 2852 (lanes 10-12), MSMEG5113 (lanes 13-15) are shown. For each amplification a negative control (lanes 1, 4, 7, 10, 13) and a plasmid positive control (lanes 3, 6, 9, 12, 15) are shown. N. tabacum transformed with MSMEG0772 clone 1 (lane 2), MSMEGl 828 clone 2 (lane 5), MSMEG1829 clone 1 (lane 8), MSMEG2852 clone 2 (lane 11) and MSMEG5113 clone 1 (lane 14).

KEY TO THE SEQUENCE LISTING

SEQ ID NO:1 - M. smegmatis reductase MSMEG3387 (Genbank ABK72884) (also known as MSMEG_3380). SEQ ID NO:2 - M. smegmatis reductase MSMEG5692 (Genbank ABK72164) (also known as MSMEG_5717).

SEQ ID NO:3 - M. smegmatis reductase MSMEG5784 (Genbank ABK70272) (also known as MSMEG_5819).

SEQ ID NO:4 - M. smegmatis reductase MSMEG0048 (Genbank ABK73917) (also known as MSMEG_0048).

SEQ ID NO: 5 - M. smegmatis reductase MSMEG2792 (Genbank ABK71846) (also known as MSMEG_2791).

SEQ ID NO:6 - M. smegmatis reductase MSMEG6811 (Genbank ABK75254) (also known as MSMEG_6848). SEQ ID NO:7 - M. smegmatis reductase MSMEG5653 (Genbank ABK69700) (also known as MSMEG_5675).

SEQ ID NO:8 - M. smegmatis reductase MSMEG3882.2 (Genbank ABK75472) (also known as MSMEG 3880).

SEQ ID NO:9 - M. smegmatis reductase MSMEG5154 (Genbank ABK72943) (also known as MSMEG_5170).

SEQ ID NO: 10 - M. smegmatis reductase MSMEG6537 (Genbank ABK74207) (also known as MSMEG 6576). SEQ ID NO: 11 - M smegmatis reductase MSMEG6445 (Genbank ABK70022) (also known as MSMEG_6485).

SEQ ID NO: 12 - M. smegmatis reductase MSMEG2029 (Genbank ABK75334) (also known as MSMEG_2027). SEQ ID NO: 13 - M. smegmatis reductase MSMEG3018 (Genbank ABK74167) (also known as MSMEG_3004).

SEQ ID NO: 14 - M. smegmatis reductase MSMEG5954 (Genbank ABK71916) (also known as MSMEG 5998).

SEQ ID NO: 15 - M. smegmatis reductase MSMEG3364 (Genbank ABK75759) (also known as MSMEG_3356).

SEQ ID NO:16 - M. smegmatis reductase MSMEG5014 (Genbank ABK74375) (also known as MSMEG_5030).

SEQ ID NO: 17 - M. smegmatis reductase MSMEG2852 (Genbank ABK73624) (also known as MSMEG_2850). SEQ ID NO: 18 - M. smegmatis reductase MSMEG5199 (Genbank ABK72597) (also known as MSMEG_5215).

SEQ ID NO: 19 - M. smegmatis reductase MSMEG6285 (Genbank ABK73368) (also known as MSMEG_6325).

SEQ ID NO:20 - M. smegmatis reductase MSMEG3914 (Genbank ABK74557) (also known as MSMEG_3909).

SEQ ID NO:21 - M. smegmatis reductase MSMEG6591 (Genbank ABK74785) (also known as MSMEG_6630).

SEQ ID NO:22 - M. smegmatis reductase MSMEGlOlO (Genbank ABK70690) (also known as MSMEGJ 021 ). SEQ ID NO:23 - M. smegmatis reductase MSMEG5870 (Genbank ABK75944) (also known as MSMEG_5910).

SEQ ID NO:24 - M. smegmatis reductase MSMEG2006 (Genbank ABK71883) (also known as MSMEG_2006).

SEQ ID NO:25 - Nucleotide sequence encoding M. smegmatis reductase MSMEG3387 (Genbank YP_887686).

SEQ ID NO:26 - Nucleotide sequence encoding M. smegmatis reductase MSMEG5692

(Genbank YP_889950).

SEQ ID NO:27 - Nucleotide sequence encoding M. smegmatis reductase MSMEG5784

(Genbank YP_890047). SEQ ID NO:28 - Nucleotide sequence encoding M. smegmatis reductase MSMEG0048

(Genbank YP 884466).

SEQ ID NO:29 - Nucleotide sequence encoding M. smegmatis reductase MSMEG2792

(Genbank YP 887122). SEQ ID NO:30 - Nucleotide sequence encoding M. smegmatis reductase MSMEG6811

(Genbank YP_891052).

SEQ ID NO:31 - Nucleotide sequence encoding M. smegmatis reductase MSMEG5653

(Genbank YP_889908). SEQ ID NO:32 - Nucleotide sequence encoding M. smegmatis reductase

MSMEG3882.2 (Genbank YP_888171).

SEQ ID NO:33 - Nucleotide sequence encoding M. smegmatis reductase MSMEG5154

(Genbank YP_889416).

SEQ ID NO:34 - Nucleotide sequence encoding M. smegmatis reductase MSMEG6537 (Genbank YP_890788).

SEQ ID NO:35 - Nucleotide sequence encoding M. smegmatis reductase MSMEG6445

(Genbank YP_890698.

SEQ ID NO:36 - Nucleotide sequence encoding M. smegmatis reductase MSMEG2029

(Genbank YP_886389). SEQ ID NO:37 - Nucleotide sequence encoding M. smegmatis reductase MSMEG3018

(Genbank YP_887322).

SEQ ID NO:38 - Nucleotide sequence encoding M. smegmatis reductase MSMEG5954

(Genbank YP_890224).

SEQ ID NO:39 - Nucleotide sequence encoding M. smegmatis reductase MSMEG3364 (Genbank YP_887663).

SEQ ID NO: 40 - Nucleotide sequence encoding M. smegmatis reductase MSMEG5014

(Genbank YP_889280).

SEQ ID NO:41 - Nucleotide sequence encoding M. smegmatis reductase MSMEG2852

(Genbank YP 887170). SEQ ID NO:42 - Nucleotide sequence encoding M. smegmatis reductase MSMEG5199

(Genbank YP_889461).

SEQ ID NO:43 - Nucleotide sequence encoding M. smegmatis reductase MSMEG6285

(Genbank YP_890543).

SEQ ID NO:44 - Nucleotide sequence encoding M. smegmatis reductase MSMEG3914 (Genbank YP_888200).

SEQ ID NO:45 - Nucleotide sequence encoding M. smegmatis reductase MSMEG6591

(Genbank YP_890838).

SEQ ID NO:46 - Nucleotide sequence encoding M. smegmatis reductase MSMEGlOlO

(Genbank YP_885420). SEQ ID NO:47 - Nucleotide sequence encoding M. smegmatis reductase MSMEG5870

(Genbank YP_890137).

SEQ ID NO:48 - Nucleotide sequence encoding M. smegmatis reductase MSMEG2006

(Genbank YP_886370). SEQ ID NO:49 - M. smegmatis FGD (MSMEG0772) (Genbank ABK75077) (also known as MSMEG_0777).

SEQ ID NO:50 - Nucleotide sequence encoding M. smegmatis FGD (MSMEG0772)

(Genbank YP_885182). SEQ ID NO:51 - M. smegmatis flavin reductase (from WO 03/104271).

SEQ ID NO: 52 - Nucleotide sequence encoding M. smegmatis flavin reductase (from

WO 03/104271).

SEQ ID NO:53 - M. smegmatis FbiC enzyme (MSMEG5113) (Genbank ABK71669)

(also known as MSMEG_5126). SEQ ID NO:54 - Nucleotide sequence encoding M, smegmatis FbiC enzyme

(MSMEG5113) (Genbank YP_889372).

SEQ ID NO:55 - M. smegmatis FbiA enzyme (MSMEG1829) (Genbank ABK71517)

(also known as MSMEGJ 830).

SEQ ID NO: 56 - Nucleotide sequence encoding M. smegmatis FbiA enzyme (MSMEGl 829) (Genbank YP_886200).

SEQ ID NO:57 - M. smegmatis FbiB enzyme (MSMEGl 828) (Genbank ABK69662)

(also known as MSMEG l 829).

SEQ ID NO:58 - Nucleotide sequence encoding M. smegmatis FbiB enzyme

(MSMEG1828) (Genbank YP_886199). SEQ ID NOs 59 to 100 - Oligonucleotide primers.

SEQ ID NO: 101 - M. tuberculosis reductase (Genbank CAB05059.1).

SEQ ID NO:102 - Streptomyces coelicolor reductase (Genbank CAC14340.1).

SEQ ID NO: 103 - Nocardiafarcinica reductase (Genbank BAD58191.1).

SEQ ID NO:104 - Rhodococcus sp. RHAl reductase (Genbank YP 704621). SEQ ID NO: 105 - Frankia sp. reductase (Genbank ABD 11484.1 ).

SEQ ID NO:106 - M. tuberculosis reductase (Genbank CAA16076.1).

SEQ ID NO: 107 - Rhodococcus sp. RHAl reductase (Genbank YP 704038).

SEQ ID NO: 108 - Nucleotide sequence encoding M. tuberculosis reductase (Genbank

CAB05059.1). SEQ ID NO: 109 - Nucleotide sequence encoding Streptomyces coelicolor reductase

(Genbank CAC 14340.1).

SEQ ID NO:110 - Nucleotide sequence encoding Nocardia farcinica reductase

(Genbank BAD58191.1).

SEQ ID NO:111 - Nucleotide sequence encoding Rhodococcus sp. RHAl reductase (Genbank YP_704621).

SEQ ID NO: 112 - Nucleotide sequence encoding Frankia sp. reductase (Genbank

ABDl 1484.1). SEQ ID NO: 113 - Nucleotide sequence encoding M. tuberculosis reductase (Genbank

CAAl 6076.1).

SEQ ID NO: 114 - Nucleotide sequence encoding Rhodococcus sp. RHAl reductase

(Genbank YP_704038). SEQ ID NO:115 - M. bovis FGD (Genbank CAD93278.1).

SEQ ID NO:116 - M. bovis FGD2 (Genbank CAD92998.1).

SEQ ID NO:117 - Nucleotide sequence encoding M. bovis FGD (Genbank

CAD93278.1).

SEQ ID NO:118 - Nucleotide sequence encoding M. bovis FGD2 (Genbank CAD92998.1).

SEQ ID NO: 119 - M bovis FbiC enzyme (Genbank CAD94067.1).

SEQ ID NO: 120 - S. coelicolor FbiC enzyme (Genbank CAB88436.1).

SEQ ID NO: 121 - Rhodococcus sp. FbiC enzyme (Genbank ABG97721).

SEQ ID NO: 122 - Methanococcus maripludies FbiC subunit CofG (Genbank CAF30432.1).

SEQ ID NO: 123 - Methanococcus maripludies FbiC subunit CofH (Genbank

CAF29612.1).

SEQ ID NO: 124 - Nucleotide sequence encoding M. bovis FbiC enzyme (Genbank

CAD94067.1). SEQ ID NO: 125 - Nucleotide sequence encoding S. coelicolor FbiC enzyme (Genbank

CAB88436.1).

SEQ ID NO: 126 - Nucleotide sequence encoding Rhodococcus sp. FbiC enzyme

(Genbank ABG97721).

SEQ ID NO: 127 - Nucleotide sequence encoding Methanococcus maripludies FbiC subunit CofG (Genbank CAF30432.1).

SEQ ID NO: 128 - Nucleotide sequence encoding Methanococcus maripludies FbiC subunit CofH (Genbank CAF29612.1).

SEQ ID NO:129 - M. bovis FbiA enzyme (Genbank CAD95381.1).

SEQ ID NO: 130 - Nucleotide sequence encoding M. bovis FbiA enzyme (Genbank CAD95381.1).

SEQ ID NO:131 - M. bovis FbiB enzyme (Genbank CAD95382.1).

SEQ ID NO: 132 - Nucleotide sequence encoding M. bovis FbiB enzyme (Genbank

CAD95382.1).

SEQ ID NO:133 - S. coelicolor flavin reductase (Genbank CAB95302.1). SEQ ID NO: 134 - Rhodococcus erythropolis flavin reductase (Genbank BAB 18470).

SEQ ID NO: 135 - Nucleotide sequence encoding S. coelicolor flavin reductase

(Genbank CAB95302.1). SEQ ID NO: 136 - Nucleotide sequence encoding Rhodococcus erythropolis flavin reductase (Genbank AB051429).

SEQ ID NO: 137 - S. coelicolor reductase (Genbank CAC36755.1).

SEQ ID NO: 138 - Nucleotide sequence encoding S. coelicolor reductase (Genbank CAC36755.1).

SEQ ID NO: 139 - M. vanbaalenii reductase closely related to M. smegmatis

MSMEG5954 (Genbank YP_956038).

SEQ ID NO: 140 - Mycobacterium sp. JLS reductase closely related to M. smegmatis

MSMEG5954 (Genbank YP_001068346). SEQ ID NO: 141 - Mycobacterium sp. KMS reductase closely related to M. smegmatis

MSMEG5954 (Genbank ABL89280).

SEQ ID NO: 142 - Mycobacterium sp. JLS reductase closely related to M. smegmatis

MSMEG5954 (Genbank YP_001073320).

SEQ ID NO: 143 - Mycobacterium sp. KMS reductase closely related to M. smegmatis MSMEG5954 (Genbank ABL93956).

SEQ ID NO: 144 - M. avium reductase closely related to M. smegmatis MSMEG5954

(Genbank AAS02836).

SEQ ID NO: 145 - M. avium reductase closely related to M. smegmatis MSMEG5954

(Genbank ABK69399). SEQ ID NO: 146 - M. ulcerans reductase closely related to M. smegmatis MSMEG5954

(Genbank YP 907625).

SEQ ID NO: 147 - M. tuberculosis FIl reductase closely related to M. smegmatis

MSMEG5954 (Genbank ZP_01686464).

SEQ ID NO: 148 - M. tuberculosis C reductase closely related to M. smegmatis MSMEG5954 (Genbank ZP 00876350).

SEQ ID NO: 149 - M. bovis reductase closely related to M. smegmatis MSMEG5954

(Genbank CAD95763).

SEQ ID NO: 150 - M. smegmatis MSMEG2392 (Genbank ABK73289) (also known as

MSMEG_2391). SEQ ID NO: 151 - M. vanbaalenii enzyme closely related to M. smegmatis

MSMEG2392 (Genbank YP_952961.1).

SEQ ID NO: 152 - M. ulcerans enzyme closely related to M. smegmatis MSMEG2392

(Genbank YP_905891.1).

SEQ ID NO: 153 - M. tuberculosis enzyme closely related to M. smegmatis MSMEG2392 (Genbank ZP_02248312.1).

SEQ ID NO: 154 - Nucleotide sequence encoding M. smegmatis MSMEG2392

(Genbank CP000480 - reverse complement). SEQ ID NO: 155 - Nucleotide sequence encoding M. vanbaalenii enzyme closely related to M. smegmatis MSMEG2392 (Genbank NC_008726 - reverse complement). SEQ ID NO: 156 - Nucleotide sequence encoding M. ulcer am enzyme closely related to M. smegmatis MSMEG2392 (Genbank NC_008611 - reverse complement). SEQ ID NO: 157 - Nucleotide sequence encoding M. tuberculosis enzyme closely related to M. smegmatis MSMEG2392 (Genbank NZ_AASN01000046).

DETAILED DESCRIPTION OF THE INVENTION

General Techniques and Selected Definitions Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, bioremediation, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, molecular biology and biochemical techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present). As used herein, the term "degrades", "degradation" and variations thereof refers to the product of reductase activity being less stable than the coumarin based compound substrate. In an embodiment, the reductase degrades AFGl to produce a compound with a molecular weigth of about 258.06 Da.

As used herein the terms "treating", "treat" or "treatment" include administering a therapeutically effective amount of a reductase, or a polynucleotide encoding therefor, sufficient to reduce or eliminate at least one symptom of toxicity caused by a coumarin based compound such as afiatoxin.

The term "biological material" is used herein in its broadest sense to include any product of biological origin. Such products include, but are not restricted to, food products for humans and animal feeds. The products include liquid media including water and liquid foodstuffs such as milk, as well as semi-solid foodstuffs such as yoghurt and the like. The present invention also extends to solid foodstuffs, particularly animal feeds. In an embodiment, it is preferred that the biological material is plant material. Examples include plant material from the families Gramineae, Composite, or Leguminosae, more preferably plant material from the genera: Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, Malus, Apium, Agrostis, Phleum, Dactylis, Sorgum, Setaria, Zea, Oryza, Triticum, Secale, Avena, Hordeum, Saccharum, Poa, Festuca, Stenotaphrum, Cynodon, Coix, Olyreae, Phareae, Glycine, Pisum, Cicer, Phaseolus, Lens, or Arachis, and even more preferably plant material from corn, rice, triticale, rye, cotton, soybean, sorghum, wheat, oats, barley, millet, sunflower, canola, peas, beans, lentils, peanuts, yam beans, cowpeas, velvet beans, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria, sweetpea or a nut plant. In a further embodiment, the biological material is distillers grain.

As used herein, an "extract" relates to any portion obtained from the ogransim which comprises the reductase. The extract may be a partially purified portion obtained following an homogenisation step. Alternatively, the extract can be a whole portion of the organism such as the seed of a plant.

Coumarin Based Compounds

As used herein, the term "coumarin based compound" refers to any compound that comprises, or consist of, coumarin (2-chromenone) (CAS Registry No. 91-64-5). In a preferred embodiment, the coumarin based compound is an aflatoxin. At least 13 different types of aflatoxin are produced in nature. These include, for example, aflatoxin Bi (CAS Registry No. 1162-65-8) and its derivatives, as well as aflatoxin precursors. Aflatoxin B₂ (CAS Registry No. 7220-81-7), aflatoxin G₁ (CAS Registry No. 1165-39-5) and aflatoxin G₂ (CAS Registry No. 7241-98-7) are major aflatoxin derivatives produced by fungi, as well as aflatoxin M₁ (CAS Registry No. 6795-23-9) and aflatoxin M₂ (CAS Registry No. 6885-57-0), which are often detected in milk.

Aflatoxin B₁ is considered the most toxic and is produced by both Aspergillus flavus and Aspergillus parasiticus. Aflatoxin Gi and G₂ are produced exclusively by A. parasiticus. While the presence of Aspergillus in food products does not always indicate harmful levels of aflatoxin are also present, it does imply a significant risk in consumption of that product. In a further embodiment, the coumarin based compound is an α/β unsaturated ketone or ester. Examples of such compounds include aflatoxins as well as the compounds provided in Table 1.

Table 1 - Coumarin based com ounds that have an α/β unsaturated ketone or ester.

Reductases and Co-Factors Thereof

The present invention relates to the use of a reductase to degrade a coumarin based compound. As used herein, the term "reductase" refers to an enzyme that reduces the coumarin based compound to form a less stable product. There are numerous families of related reductases which can be used for the methods of the invention. These include members of the pyridoxamine 5 '-phosphate oxidases (PNPOx) family of reductases, members of the DUF385 family of reductases, and members of the glyoxalase/bleomycin resistant family of reductases. In a preferred embodiment, the reductase is a member of the PNPOx protein family or DUF385 protein family.

Members of the "PNPOx reductase" family typically have the conserved domain (L/M)ATVxPDGxP, with the G and P residues being most highly conserved. Figure 2 provides an alignment of some PNPOx reductases. Other PNPOx family members not shown in Figure 2 include human pyridoxamine 5'-phosphate oxidase (Musayev et al., 2003), RvI 155 (Biswal et al., 2005; Canaan et al., 2005) and Rv2074 (Biswal et al., 2006).

"DUF385" refers to domain of unknown function (DUF) 385 protein family, and are shown herein to have reductase activity. Figure 3 provides an alignment of some proteins from this family. DUF385 protein family has two highly conserved regions, the first domain is defined by the sequence GAKSGKxRxTPLMY, with the G and P residues being most highly conserved. The second domain comprising the sequence SxGGAPKxPxWYHN has four highly conserved residues SxxxxxxxPxWxxN. This protein family includes M. smegmatis MSMEG5954 which is the closest M. smegmatis homologue to the M. tuberculosis lab strain rv3547 enzyme, sharing 46.5% amino acid identity. Rv3547 was recently shown to protonate the anti-tubercolis drug, PA-824, in an F₄₂₀ dependent manner (Manjunatha et al., 2006).

The MSMEG6591 reductase which degrades aflatoxin is detected by NCBI

BLAST as having an Glyoxalase BRP putative conserved domain. Members of the glyoxalase/bleomycin resistant protein (BRP) family typically share a conversed glyoxalase/BRP conserved domain comprising the sequence: FYxxxLG. Examples of this protein family are provided in Figure 4.

In a particularly preferred embodiment, a reductase useful for the methods of the present invention is an F₄₂₀ dependent reductase. As used herein, the terms "F₄₂₀ dependent reductase", "F₄₂₀ oxidoreductase", and "F₄₂₀ reductase" refers to a reductase which can utilize F₄₂₀H₂, or another suitable cofactor such as FMNH₂, or FADH₂, as an electron donor to reduce a coumarin based compound such as aflatoxin. Examples of such reductases include those with a sequence provided as any one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149 (see also Manjuntha et al., 2006). As indicated above, an F₄₂₀ dependent reductase used in the methods of the invention require an electron donor to degrade a coumarin based compound. Some cell expression systems, including transgenic organisms, will inherently produce a suitable electron donor (such as F₄₂₀H₂), however, in other instances it may be necessary to provide a gene(s) encoding an enzyme(s) which can be used to synthesize the desired electron donor. Compositions for degrading a coumarin based compound preferably comprise an electron donor, and possibly an enzyme capable of reducing the corresponding oxidized form of the electron donor to ensure that the supply of electron donor does not limit the activity of the reductase.

The structures of F₄₂₀ and FO have been described by, for example, Choi et al. (2002). F₄₂₀ can be produced by extraction from M. smegmatis as per the methods of Isabelle et al. (2002). F₄₂₀H₂ can be produced from F₄₂₀ using various enzymes such as an F₄₂₀-dependent Glucose-6-Phosphate Dehydrogenase (FGD). Examples of FGD enzymes include those with a sequence provided as any one of SEQ ID NO:49, SEQ ID NO:115 and SEQ ID NO:116 (see also Purwantini and Daniels (1996 and 1998)). An alternate type of enzyme that can be used to produce F₄₂₀H₂ is F₄₂₀ :N ADP+ dependent reductase, an example of which is provided as SEQ ID NO: 137.

FO (7,8-didemethyl-8-hydroxy-5-deazaribofiavin) can be made by cloning of an FbiC gene into E. coli and extracted in a similar method to that of Isabelle et al. (2002). FbiC uses 4-hydroxy phenylpyruvate (HPP) (which is the precursor of tyrosine) and 5- amino-6-ribitylamino-2,4(l/f,3//)-pyrimidinedione (compound 6) (an intermediate in riboflavin synthesis) to produce FO. Both bacteria and plants produce compound 6 and HPP. In some bacterial species such as Methanococcus jannaschii and M. maripludies FO is synthesized by the action of two genes encoding CofG (example provided as SEQ ID NO:122) and CofH (example provided as SEQ ID NO:123) (Graham et al., 2003).

FO can be used as a substrate for producing F₄₂₀. A phosphate group is added and a γ-linked glutamate incorporated through the activity of FbiA and FbiB enzymes (see, for example, Choi et al., 2002). Examples of FbiA enzymes include those which comprise an amino acid sequence provided as SEQ ID NO:55 or SEQ ID NO: 129, whereas examples of FbiB enzymes include those which comprise an amino acid sequence provided as SEQ ID NO:57 or SEQ ID NO: 131. Proteins related to MSMEG 2392 (SEQ ID NO: 150) have also been shown to be involved in the synthesis of F_42O (Guerra-Lopez et al., 2007).

Flavin mononucleotide (FMN) Riboflavin 5 '-phosphate can be purchased commercially from Sigma, catalogue numbers (F8399, F2253, F6750, F 1392, 83810). FMN can be reduced using, for example, an M. smegmatis flavin reductase MSMEG3271 cloned into pET14b and expressed in E. coli (Sutherland et al., 2002a and b). Examples of other flavin reductases include, but are not limited to, those comprising an amino acid sequence provided in SEQ ID NO: 133 and SEQ ID NO: 134.

Polypeptides

By "substantially purified" or "purified" we mean a polypeptide that has been separated from one or more lipids, nucleic acids, other polypeptides, or other contaminating molecules with which it is associated in its native state. It is preferred that the polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. The term "recombinant" in the context of a polypeptide refers to the polypeptide when produced by a cell, or in a cell-free expression system, in an altered amount or at an altered rate compared to its native state. In one embodiment the cell is a cell that does not naturally produce the polypeptide. However, the cell may be a cell which comprises a non-endogenous gene that causes an altered, preferably increased, amount of the polypeptide to be produced. A recombinant polypeptide of the invention includes polypeptides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is produced, and polypeptides produced in such cells or cell-free systems which are subsequently purified away from at least some other components. The terms "polypeptide" and "protein" are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors. The terms "proteins" and "polypeptides" as used herein also include variants, mutants, biologically active fragments, modifications, analogous and/or derivatives of the polypeptides such as those described herein.

The % identity of a polypeptide is determined by the AlignX application of the Vector NTI Advance 10.1.1 program (Invitrogen), which is based on Clustal X algorithim (Thompson et al., 1994), with a gap creation penalty of 10 and a gap extension penalty of 0.03 for multiple alignment. Where relevant, the query sequence is at least 25 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 25 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.

As used herein a "biologically active fragment" is a portion of a polypeptide as described herein which maintains a defined activity of the full-length polypeptide. For example, a biologically active fragment of a reductase as described herein (but not a flavin reductase or a F₄₂₀ glucose-6-phosphate dehydrogenase (FGD)) is able to degrade a coumarin based compound. Biologically active fragments can be any size as long as they maintain the defined activity. Preferably, biologically active fragments are at least 100 amino acids in length.

With regard to a defined polypeptide, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide comprises an amino acid sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO. In an embodiment, a polypeptide of the invention that degrades a coumarin based compound is not M. tuberculosis RvI 155 (Biswal et al., 2005; Canaan et al., 2005) or Rv2074 (Biswal et al., 2006).

Amino acid sequence mutants of a polypeptide described herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid defined herein, or by in vitro synthesis of the desired polypeptide. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics.

Mutant (altered) polypeptides can be prepared using any technique known in the art. For example, a polynucleotide described herein can be subjected to in vitro mutagenesis. Such in vitro mutagenesis techniques may include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL-I red (Stratagene) and propagating the transformed bacteria for a suitable number of generations. In another example, the polynucleotides of the invention are subjected to DNA shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they are able to confer the desired phenotype such as enhanced activity and/or altered substrate specificity.

In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues. Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites identified as important for function. Other sites of interest are those in which particular residues obtained from various strains or species are identical (see, for example, Figures 2 to 5). These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 2. able 2 - Exemplary substitutions.

In addition to the enzymes provided in the Sequence Lisiting, further examples of other enzymes useful for the methods of the invention, and/or for designing suitable mutants/variants thereof are provided below.

FbiA orthologues of M. smegmatis MSMEGl 829 include but are not limited to: M. sp MCS YP638492; M. sp. KMS YP_937343; M. sp. JLS, YP_001069653; M. vanbaalenii pyr-1, YP_952562; M. tuberculosis, NP_217778; Rhodococcus sp RHAl, YP_706247; Nocardia farcinica, YPJ20836; Salinispora arenicola, YP_001535795; and Frankia sp. EANl, YP_001510120.

FbiB orthologues of M. smegmatis MSMEGl 828 include but are not limited to: M. vanbaalenii pyr-1, YP_952561; M. sp. KMS, YP_937342; M. sp MCS, YP_638491; M. sp. JLS, YPJ)01069652; M. ulcerans Agy99, YP_906409; M. tuberculosis, NP_217779; Rhodococcus sp RHAl, YP_706246; Nocardia farcinica, YPJ20835; and Frankia alni, YP_873720.

FbiC orthologues of M. smegmatis MSMEG5113 include but are not limited to:

M sp. JLS, YP_001072524; M. sp MCS, YP_641193; M. sp. KMS, YP940089; M. vanbaalenii pyr-1 YP 955314; M. tuberculosis, NP_215689; M. ulcerans Agy99,

YP_905099; Rhodococcus sp RHAl, YP 705879; Nocardia farcinica, YP 120968; and

Frankia alni, YP_711516.

FGD orthologues of M. smegmatis include, but are not limited to: M. vanbaalenii pyr-1 YP_951542; M. sp MCS, YP_637709; M. sp KMS, YP_936550; M. tuberculosis, NP_214921; M. ulcerans Agy99, YP_906579; Rhodococcus sp RHAl, YP_702169; and Nocardia farcinica, YP_121571.

Orthologues of MSMEG2852 and MSMEG3364 include but are not limited to: Rhodococcus sp RHAl, ABG92320; M. vanbaalenii pyr-1 YP_951851, YP_953430, YP_952028; Frankia alni, YP_714223, YP_712425; M. sp. KMS, ABL91560, ABL91870; M. sp. JLS, YP_001070630, YPJ)01070930; Salinispora arenicola, EAX28712; Nocardia farcinica, YP_119109, YP_121046; and M. avium paratuberculosis, AAS03382.

MSMEG3387 orthologues in other bacteria include, but are not limited to: Janibacter sp. HTCC2649, EAP98198; Frankia alni ACNHa, CAJ60274, CAJ61661; Frankia Ss. CcB ABDl 1484; Rubrobacter xylanophilus DSM994; Saccharopolyspora erythraea CAL99708; Thermobifidia fusca YX AAZ56351; Frankia alni 14a CAJ61126; Frankia sp. EANlpec EAN13955, EANl 1738; Streptomyces avermitilis SAV6262; Roseiflexus castenholzzii EAV26042; and Roseiflexus EAT25916.

Orthologues of MsFR FMN oxidorecductase include but are not limited to: M. vanbaalenii pyr-1 YP 951204; Rhodococcus erythropolis CAJ00429; Nocardia facinica, YP l 16588; Frankia alni ACN14a, YP_714037; Streptomyces coelicolor, NP 625377; and Arthrobacter aurescens YP_947482.

MSMEG 2392 orthologues in other species include, but are not limited to: M. vanbaalenii pyr-1 YP_952961.1; M. ulcerans Agy99, YP_905891.1; M. tuberculosis ZP_02248312.1, NP_217499.1, NP 337576.1, YP_02552273.1; M. sp JLS, YP_001070185.1; M. sp KMS, YP_937961.1; M. sp MCS, YP_639091.1; Rhodococcus sp RHAl, YP 706435.1; Nocardia farcinica, YP 120420.1; and Streptomyces coelicolor A3, NP_629692.1.

Furthermore, if desired, unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into a polypeptides described herein.

Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, 2- aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β- methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogues in general.

Also included within the scope of the invention are polypeptides of the present invention which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the polypeptide.

Polypeptides described herein can be produced in a variety of ways, including production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides. In one embodiment, an isolated polypeptide of the present invention is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide. A preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit polypeptide production. An effective medium refers to any medium in which a cell is cultured to produce a polypeptide of the present invention. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.

Polynucleotides By an "isolated polynucleotide", including DNA, RNA, or a combination of these, single or double stranded, in the sense or antisense orientation or a combination of both, dsRNA or otherwise, we mean a polynucleotide which is at least partially separated from the polynucleotide sequences with which it is associated or linked in its native state. Preferably, the isolated polynucleotide is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated. Furthermore, the term "polynucleotide" is used interchangeably herein with the term "nucleic acid". The term "exogenous" in the context of a polynucleotide refers to the polynucleotide when present in a cell, or in a cell-free expression system, in an altered amount compared to its native state. In one embodiment, the cell is a cell that does not naturally comprise the polynucleotide. However, the cell may be a cell which comprises a non-endogenous polynucleotide resulting in an altered, preferably increased, amount of production of the encoded polypeptide. An exogenous polynucleotide of the invention includes polynucleotides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is present, and polynucleotides produced in such cells or cell-free systems which are subsequently purified away from at least some other components.

The % identity of a polynucleotide is determined by the AlignX application of the Vector NTI Advance 10.1.1 program (Invitrogen), which is based on Clustal X algorithim (Thompson et al., 1994), with a gap creation penalty of 10 and a gap extension penalty of 0.03 for multiple alignment. Where relevant, the query sequence is at least 45 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences over their entire length.

With regard to the defined polynucleotides, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that a polynucleotide of the invention comprises a sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO. As used herein, the term "hybridizes" refers to the ability of two single stranded nucleic acid molecules being able to form at least a partially double stranded nucleic acid through hydrogen bonding.

As used herein, the phrase "stringent conditions" refers to conditions under which a polynucleotide, probe, primer and/or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30⁰C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 6O⁰C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide. Stringent conditions are known to those skilled in the art and can be found in

Ausubel et al. (supra), Current Protocols In Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, as well as the Examples described herein. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6xSSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65⁰C, followed by one or more washes in 0.2.xSSC, 0.01% BSA at 50⁰C. In another embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs 25 to 48 and 108 to 114, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55⁰C, followed by one or more washes in IxSSC, 0.1% SDS at 37⁰C. Other conditions of moderate stringency that may be used are well-known within the art, see, e.g., Ausubel et al. (supra), and Kriegler, 1990; Gene Transfer And Expression, A Laboratory Manual, Stockton Press, NY. In yet another embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising any one of the nucleotide sequences SEQ ID NOs 25 to 48 and 108 to 114, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5xSSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 4O⁰C, followed by one or more washes in 2xSSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 5O⁰C. Other conditions of low stringency that may be used are well known in the art, see, e.g., Ausubel et al. (supra) and Kriegler, 1990, Gene Transfer And Expression, A Laboratory Manual, Stockton Press, NY.

Polynucleotides of the present invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site- directed mutagenesis on the nucleic acid).

Some polynucleotides disclosed herein which encode enzymes useful for the methods of the invention have GTG or TTG as the first codon. As is known in the art, these codons can encode a methionine in bacteria (Suzek et al., 2001). As the skilled addressee will appreciate, to express these genes so they produce the desired protein in other organisms, such as eukaryotes, it will be necessary to replace the GTG or TTG start codon with ATG. Usually, monomers of a polynucleotide or oligonucleotide are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a relatively short monomelic units, e.g., 12-18, to several hundreds of monomelic units. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate and phosphoramidate.

Recombinant Vectors

One embodiment of the present invention includes a recombinant vector, which comprises at least one isolated polynucleotide molecule described herein, and/or a polynucleotide encoding a polypeptide as described herein, inserted into any vector capable of delivering the polynucleotide molecule into a host cell. Such a vector contains heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to polynucleotide molecules of the present invention and that preferably are derived from a species other than the species from which the polynucleotide molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a transposon (such as described in US 5,792,294), a virus or a plasmid. One type of recombinant vector comprises the polynucleotide(s) operably linked to an expression vector. The phrase operably linked refers to insertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors include any vectors that function (i.e., direct gene expression) in recombinant cells, including in bacterial, fungal, endoparasite, arthropod, animal, and plant cells. Vectors of the invention can also be used to produce the polypeptide in a cell-free expression system, such systems are well known in the art.

"Operably linked" as used herein refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element to a transcribed sequence. For example, a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell and/or in a cell-free expression system. Generally, promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cz^'s-acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

In particular, expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotide molecules of the present invention. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, arthropod, nematode, plant or animal cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SPOl, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells.

Host Cells

Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules described herein or progeny cells thereof. Transformation of a polynucleotide molecule into a cell can be accomplished by any method by which a polynucleotide molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed polynucleotide molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.

Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the present invention. Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing polypeptides described herein or can be capable of producing such polypeptides after being transformed with at least one polynucleotide molecule as described herein. Host cells of the present invention can be any cell capable of producing at least one protein defined herein, and include bacterial, fungal (including yeast), parasite, nematode, arthropod, animal and plant cells. Examples of host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells, CRFK cells, CV-I cells, COS (e.g., COS-7) cells, and Vero cells. Further examples of host cells are E. coli, including E. coli K- 12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Particularly preferred host cells are plant cells.

Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.

Transgenic Plants

Plants contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. Target plants include, but are not limited to, the following: cereals (for example, wheat, barley, rye, oats, rice, maize, sorghum and related crops); beet (sugar beet and fodder beet); pomes, stone fruit and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and blackberries); leguminous plants (beans, lentils, peas, soybeans); oil plants (peanut, rape, mustard, poppy, olives, sunflowers, coconut, castor oil plants, cocoa beans, groundnuts); cucumber plants (marrows, cucumbers, melons); fibre plants (cotton, flax, hemp, jute); citrus fruit (oranges, lemons, grapefruit, mandarins); vegetables (spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, paprika); lauraceae (avocados, cinnamon, camphor); or plants such as tobacco, nuts, coffee, sugar cane, tea, vines, hops, turf, bananas and natural rubber plants, as well as ornamentals (flowers, shrubs, broad-leaved trees and evergreens, such as conifers). Crops frequently effected by Aspergillus sp. infection which are target plants of the invention include, but are not limited to, cereals (maize, sorghum, pearl millet, rice, wheat), oilseeds (peanut, soybean, sunflower, cotton), spices (chile peppers, black pepper, coriander, turmeric, ginger), and tree nuts (almond, pistachio, walnut, coconut). In a preferred embodiment, the plant is from the families Gramineae, Composite, or Leguminosae, more preferably from the genera: Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, Malus, Apium, Agrostis, Phleum, Dactylis, Sorgum, Setaria, Zea, Oryza, Triticum, Secale, Avena, Hordeum, Saccharum, Poa, Festuca, Stenotaphrum, Cynodon, Coix, Olyreae, Phareae, Glycine, Pisum, Cicer, Phaseolus, Lens, or Arachis, and even more preferably from corn, rice, triticale, rye, cotton, soybean, sorghum, wheat, oats, barley, millet, sunflower, canola, peas, beans, lentils, peanuts, yam beans, cowpeas, velvet beans, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria, sweetpea or a nut plant.

The term "plant" as used herein as a noun refers to a whole plants such as, for example, a plant growing in a field for commercial wheat production. A "plant part" or "plant portion" refers to vegetative structures (for example, leaves, stems), roots, floral organs/structures, seed (including embryo, endosperm, and seed coat), plant tissue (for example, vascular tissue, ground tissue, and the like), cells and progeny of the same.

Transgenic plants, as defined in the context of the present invention include plants (as well as parts and cells of said plants) and their progeny which have been genetically modified using recombinant techniques to cause production of at least one polypeptide of the present invention in the desired plant or plant organ. Transgenic plants can be produced using techniques known in the art, such as those generally described in A. Slater et al., Plant Biotechnology - The Genetic Manipulation of Plants, Oxford University Press (2003), and P. Christou and H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).

A "transgenic plant" refers to a plant that contains a gene construct ("transgene") not found in a wild-type plant of the same species, variety or cultivar. A "transgene" as referred to herein has the normal meaning in the art of biotechnology and includes a genetic sequence which has been produced or altered by recombinant DNA or RNA technology and which has been introduced into the plant cell. The transgene may include genetic sequences derived from a plant cell. Typically, the transgene has been introduced into the plant by human manipulation such as, for example, by transformation but any method can be used as one of skill in the art recognizes.

In a preferred embodiment, the transgenic plants are homozygous for each and every gene that has been introduced (transgene) so that their progeny do not segregate for the desired phenotype. The transgenic plants may also be heterozygous for the introduced transgene(s), such as, for example, in Fl progeny which have been grown from hybrid seed. Such plants may provide advantages such as hybrid vigour, well known in the art.

A polynucleotide of the present invention may be expressed constitutively in the transgenic plants during all stages of development. Depending on the use of the plant or plant organs, the polypeptides may be expressed in a stage-specific manner. Furthermore, the polynucleotides may be expressed tissue-specifically.

Regulatory sequences which are known or are found to cause expression of a gene encoding a polypeptide of interest in plants may be used in the present invention. The choice of the regulatory sequences used depends on the target plant and/or target organ of interest. Such regulatory sequences may be obtained from plants or plant viruses, or may be chemically synthesized. Such regulatory sequences are well known to those skilled in the art. A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al, Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally- regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

A number of constitutive promoters that are active in plant cells have been described. Suitable promoters for constitutive expression in plants include, but are not limited to, the cauliflower mosaic virus (CaMV) 35 S promoter, the Figwort mosaic virus (FMV) 35 S, the sugarcane bacilliform virus promoter, the commelina yellow mottle virus promoter, the light-inducible promoter from the small subunit of the ribulose-l,5-bis-phosphate carboxylase, the rice cytosolic triosephosphate isomerase promoter, the adenine phosphoribosyltransferase promoter of Arabidopsis, the rice actin 1 gene promoter, the mannopine synthase and octopine synthase promoters, the Adh promoter, the sucrose synthase promoter, the R gene complex promoter, and the chlorophyll α,β binding protein gene promoter. These promoters have been used to create DNA vectors that have been expressed in plants; see, e.g., PCT publication WO 8402913. All of these promoters have been used to create various types of plant- expressible recombinant DNA vectors. For the purpose of expression in source tissues of the plant, such as the leaf, seed, root or stem, it is preferred that the promoters utilized in the present invention have relatively high expression in these specific tissues. For this purpose, one may choose from a number of promoters for genes with tissue- or cell-specific or -enhanced expression. Examples of such promoters reported in the literature include the chloroplast glutamine synthetase GS2 promoter from pea, the chloroplast fructose- 1,6- biphosphatase promoter from wheat, the nuclear photosynthetic ST-LS 1 promoter from potato, the serine/threonine kinase promoter and the glucoamylase (CHS) promoter from Arabidopsis thaliana. Also reported to be active in photosynthetically active tissues are the ribulose-l,5-bisphosphate carboxylase promoter from eastern larch {Larix laricina), the promoter for the Cab gene, Cab6, from pine, the promoter for the Cab-1 gene from wheat, the promoter for the Cab-1 gene from spinach, the promoter for the Cab IR gene from rice, the pyruvate, orthophosphate dikinase (PPDK) promoter from Zea mays, the promoter for the tobacco Lhcbl*2 gene, the Arabidopsis thaliana Suc2 sucrose-H³⁰ symporter promoter, and the promoter for the thylakoid membrane protein genes from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS).

Other promoters for the chlorophyll α,β -binding proteins may also be utilized in the present invention, such as the promoters for LhcB gene and PsbP gene from white mustard (Sinapis alba). A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals, also can be used for expression of RNA-binding protein genes in plant cells, including promoters regulated by (1) heat, (2) light (e.g., pea RbcS-3A promoter, maize RbcS promoter); (3) hormones, such as abscisic acid, (4) wounding (e.g., Wunl); or (5) chemicals, such as methyl jasminate, salicylic acid, steroid hormones, alcohol, Safeners (WO 9706269), or it may also be advantageous to employ (6) organ-specific promoters.

For the purpose of expression in sink tissues of the plant, such as the tuber of the potato plant, the fruit of tomato, or the seed of soybean, canola, cotton, Zea mays, wheat, rice, and barley, it is preferred that the promoters utilized in the present invention have relatively high expression in these specific tissues. A number of promoters for genes with tuber-specific or -enhanced expression are known, including the class I patatin promoter, the promoter for the potato tuber ADPGPP genes, both the large and small subunits, the sucrose synthase promoter, the promoter for the major tuber proteins including the 22 kD protein complexes and proteinase inhibitors, the promoter for the granule bound starch synthase gene (GBSS), and other class I and II patatins promoters. Other promoters can also be used to express a protein in specific tissues, such as seeds or fruits. The promoter for β-conglycinin or other seed-specific promoters such as the napin and phaseolin promoters, can be used. A particularly preferred promoter for Zea mays endosperm expression is the promoter for the glutelin gene from rice, more particularly the Osgt-1 promoter. Examples of promoters suitable for expression in wheat include those promoters for the ADPglucose pyrosynthase (ADPGPP) subunits, the granule bound and other starch synthase, the branching and debranching enzymes, the embryogenesis-abundant proteins, the gliadins, and the glutenins. Examples of such promoters in rice include those promoters for the ADPGPP subunits, the granule bound and other starch synthase, the branching enzymes, the debranching enzymes, sucrose synthases, and the glutelins. A particularly preferred promoter is the promoter for rice glutelin, Osgt-1 gene. Examples of such promoters for barley include those for the ADPGPP subunits, the granule bound and other starch synthase, the branching enzymes, the debranching enzymes, sucrose synthases, the hordeins, the embryo globulins, and the aleurone specific proteins.

Root specific promoters may also be used. An example of such a promoter is the promoter for the acid chitinase gene. Expression in root tissue could also be accomplished by utilizing the root specific subdomains of the CaMV 35S promoter that have been identified.

The 5' non-translated leader sequence can be derived from the promoter selected to express the heterologous gene sequence of the polynucleotide of the present invention, and can be specifically modified if desired so as to increase translation of mRNA. For a review of optimizing expression of transgenes, see Koziel et al. (1996). The 5' non-translated regions can also be obtained from plant viral RNAs (Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus, Alfalfa mosaic virus, among others) from suitable eukaryotic genes, plant genes (wheat and maize chlorophyll a/b binding protein gene leader), or from a synthetic gene sequence. The present invention is not limited to constructs wherein the non-translated region is derived from the 5' non-translated sequence that accompanies the promoter sequence. The leader sequence could also be derived from an unrelated promoter or coding sequence. Leader sequences useful in context of the present invention comprise the maize Hsp70 leader (U.S. 5,362,865 and U.S. 5,859,347), and the TMV omega element. The termination of transcription is accomplished by a 3¹ non-translated DNA sequence operably linked in the chimeric vector to the polynucleotide of interest. The 3' non-translated region of a recombinant DNA molecule contains a polyadenylation signal that functions in plants to cause the addition of adenylate nucleotides to the 3' end of the RNA. The 3' non-translated region can be obtained from various genes that are expressed in plant cells. The nopaline synthase 3' untranslated region, the 3' untranslated region from pea small subunit Rubisco gene, the 3' untranslated region from soybean 7S seed storage protein gene are commonly used in this capacity. The 3' transcribed, non- translated regions containing the polyadenylate signal of Agrobacterium tumor-inducing (Ti) plasmid genes are also suitable. Four general methods for direct delivery of a gene into cells have been described: (1) chemical methods (Graham et al., 1973); (2) physical methods such as microinjection (Capecchi, 1980); electroporation (see, for example, WO 87/06614, US 5,472,869, 5,384,253, WO 92/09696 and WO 93/21335); and the gene gun (see, for example, US 4,945,050 and US 5,141,131); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis et al., 1988); and (4) receptor-mediated mechanisms (Curiel et al., 1992; Wagner et al., 1992).

Acceleration methods that may be used include, for example, microprojectile bombardment and the like. One example of a method for delivering transforming nucleic acid molecules to plant cells is microprojectile bombardment. This method has been reviewed by Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microprojectiles) are coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like. A particular advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly transforming monocots, is that neither the isolation of protoplasts, nor the susceptibility of Agrobacterium infection are required. An illustrative embodiment of a method for delivering DNA into Zea mays cells by acceleration is a biolistics α-particle delivery system, that can be used to propel particles coated with DNA through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with corn cells cultured in suspension. A particle delivery system suitable for use with the present invention is the helium acceleration PDS- 1000/He gun, available from Bio-Rad Laboratories. For the bombardment, cells in suspension may be concentrated on filters. Filters containing the cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate. If desired, one or more screens are also positioned between the gun and the cells to be bombarded.

Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth herein one may obtain up to 1000 or more foci of cells transiently expressing a marker gene. The number of cells in a focus that express the exogenous gene product 48 hours post-bombardment often range from one to ten and average one to three.

In bombardment transformation, one may optimize the pre-bombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro- or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. It is believed that pre-bombardment manipulations are especially important for successful transformation of immature embryos. In another alternative embodiment, plastids can be stably transformed. Method disclosed for plastid transformation in higher plants include particle gun delivery of

DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination (U.S. 5, 451,513, U.S. 5,545,818, U.S. 5,877,402, U.S. 5,932479, and WO 99/05265).

Accordingly, it is contemplated that one may wish to adjust various aspects of the bombardment parameters in small scale studies to fully optimize the conditions. One may particularly wish to adjust physical parameters such as gap distance, flight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors by modifying conditions that influence the physiological state of the recipient cells and that may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. The execution of other routine adjustments will be known to those of skill in the art in light of the present disclosure.

Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (see, for example, US 5,177,010, US 5,104,310, US 5,004,863, US 5,159,135). Further, the integration of the T-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome.

Modern Agrobαcterium transformation vectors are capable of replication in E. coli as well as Agrobαcterium, allowing for convenient manipulations as described (Klee et al., In: Plant DNA Infectious Agents, Hohn and Schell, eds., Springer- Verlag, New York, pp. 179-203 (1985). Moreover, technological advances in vectors for Agrobacterium-mcdiated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes. The vectors described have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobαcterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant varieties where Agrobαcterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.

A transgenic plant formed using Agrobαcterium transformation methods typically contains a single genetic locus on one chromosome. Such transgenic plants can be referred to as being hemizygous for the added gene. More preferred is a transgenic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selling) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants for the gene of interest.

It is also to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating exogenous genes. Selling of appropriate progeny can produce plants that are homozygous for both exogenous genes. Back-crossing to a parental plant and out-crossing with a non- transgenic plant are also contemplated, as is vegetative propagation. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in Fehr, In: Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments. Application of these systems to different plant varieties depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described (Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).

Other methods of cell transformation can also be used and include but are not limited to introduction of DNA into plants by direct DNA transfer into pollen, by direct injection of DNA into reproductive organs of a plant, or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach et al., In: Methods for Plant Molecular Biology, Academic Press, San Diego, Calif, (1988). This regeneration and growth process typically includes the steps of selection of transformed cells; culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign, exogenous gene is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired exogenous nucleic acid is cultivated using methods well known to one skilled in the art.

Methods for transforming dicots, primarily by use of Agrobacterium tumefaciens, and obtaining transgenic plants have been published for cotton (U.S. 5,004,863, U.S. 5,159,135, U.S. 5,518,908); soybean (U.S. 5,569,834, U.S. 5,416,011); Brassica (U.S. 5,463,174); peanut (Cheng et al., 1996); and pea (Grant et al., 1995).

Methods for transformation of cereal plants such as wheat and barley for introducing genetic variation into the plant by introduction of an exogenous nucleic acid and for regeneration of plants from protoplasts or immature plant embryos are well known in the art, see for example, Canadian Patent Application No. 2,092,588, Australian Patent Application No 61781/94, Australian Patent No 667939, US Patent No. 6,100,447, International Patent Application PCT/US97/10621, U.S. Patent No. 5,589,617, U.S. Patent No. 6,541,257, and other methods are set out in Patent specification WO99/14314. Preferably, transgenic wheat or barley plants are produced by Agrobacterium tumefaciens mediated transformation procedures. Vectors carrying the desired nucleic acid construct may be introduced into regenerable wheat cells of tissue cultured plants or explants, or suitable plant systems such as protoplasts.

With specific regard to peanuts, transgenic Arachis hypogaea can be produced generally using the methods described by Chu et al. (2008). The regenerable wheat cells are preferably from the scutellum of immature embryos, mature embryos, callus derived from these, or the meristematic tissue.

To confirm the presence of the transgenes in transgenic cells and plants, a polymerase chain reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. Expression products of the transgenes can be detected in any of a variety of ways, depending upon the nature of the product, and include Western blot and enzyme assay. One particularly useful way to quantitate protein expression and to detect replication in different plant tissues is to use a reporter gene, such as GUS. Once transgenic plants have been obtained, they may be grown to produce plant tissues or parts having the desired phenotype. The plant tissue or plant parts, may be harvested, and/or the seed collected. The seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics.

Transgenic Non-Human Animals A "transgenic non-human animal" refers to an animal, other than a human, that contains a gene construct ("transgene") not found in a wild-type animal of the same species or breed. A "transgene" as referred to herein has the normal meaning in the art of biotechnology and includes a genetic sequence which has been produced or altered by recombinant DNA or RNA technology and which has been introduced into an animal cell. The transgene may include genetic sequences derived from an animal cell. Typically, the transgene has been introduced into the animal by human manipulation such as, for example, by transformation but any method can be used as one of skill in the art recognizes.

Techniques for producing transgenic animals are well known in the art. A useful general textbook on this subject is Houdebine, Transgenic animals - Generation and Use (Harwood Academic, 1997).

Heterologous DNA can be introduced, for example, into fertilized mammalian ova. For instance, totipotent or pluripotent stem cells can be transformed by microinjection, calcium phosphate mediated precipitation, liposome fusion, retroviral infection or other means, the transformed cells are then introduced into the embryo, and the embryo then develops into a transgenic animal. In a highly preferred method, developing embryos are infected with a retrovirus containing the desired DNA, and transgenic animals produced from the infected embryo. In a most preferred method, however, the appropriate DNAs are coinjected into the pronucleus or cytoplasm of embryos, preferably at the single cell stage, and the embryos allowed to develop into mature transgenic animals.

Another method used to produce a transgenic animal involves microinjecting a nucleic acid into pro-nuclear stage eggs by standard methods. Injected eggs are then cultured before transfer into the oviducts of pseudopregnant recipients.

Transgenic animals may also be produced by nuclear transfer technology. Using this method, fibroblasts from donor animals are stably transfected with a plasmid incorporating the coding sequences for a binding domain or binding partner of interest under the control of regulatory sequences. Stable transfectants are then fused to enucleated oocytes, cultured and transferred into female recipients.

Compositions

Compositions of the present invention include excipients, also referred to herein as "acceptable carriers". An excipient can be any material that the animal, plant, plant or animal material, or environment (including soil and water samples) to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal or o-cresol, formalin and benzyl alcohol. Excipients can also be used to increase the half- life of a composition, for example, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.

Furthermore, a polypeptide described herein can be provided in a composition which enhances the rate and/or degree of degradation of a coumarin based compound, or increases the stability of the polypeptide. For example, the polypeptide can be immobilized on a polyurethane matrix (Gordon et al., 1999), or encapsulated in appropriate liposomes (Petrikovics et al., 2000a and b). The polypeptide can also be incorporated into a composition comprising a foam such as those used routinely in fire- fighting (LeJeune et al., 1998). As would be appreciated by the skilled addressee, the polypeptide of the present invention could readily be used in a sponge or foam as disclosed in WO 00/64539. A composition and/or method of the invention may also comprise means for disrupting the cell membrane and/or cell wall of a microorganism such as Aspergillus sp. The means for disrupting a cell membrane and/or cell wall can be chemical or mechanical. For example, cells can be lysed by using means such as, but not limited to: sonication, osmotic disruption, grinding/beading, French press, homogenization, explosive decompression, treatment with a detergent, critical point extraction and freeze/thaw cycles. Examples of detergents which might be used include Tween™ and sodium dodecyl sulfate (SDS).

One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal, plant, animal or plant material, or the environment (including soil and water samples). As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Preferred controlled release formulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention into soil or water which is in an area comprising a coumarin based compound. The formulation is preferably released over a period of time ranging from about 1 to about 12 months. A preferred controlled release formulation of the present invention is capable of effecting a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.

The concentration of the polypeptide, vector, or host cell of the present invention that will be required to produce effective compositions for degrading a coumarin based compound, will depend on the nature of the sample to be decontaminated, the concentration of the coumarin based compound, in the sample, and the formulation of the composition. The effective concentration of the polypeptide, vector, or host cell within the composition can readily be determined experimentally, as will be understood by the skilled artisan. In one embodiment, a composition of the invention comprises F₄₂₀. F₄₂₀ can be extracted from Mycobaterium sp. as described by Isabelle et al. (2002). Alternatively,

F420 can be synthesized using the procedure as described by Choi et al. (2002).

Reduced F₄₂₀ (F₄₂₀H₂) can be produced using a glucose-6-phosphate dehydrogenase as described by Purwantini and Daniels (1998). In a further embodiment, a composition of the invention comprises FO (7,8- didemethyl-8-hydroxy-5-deazariboflavin). FO can be extracted from Mycobaterium sp. as described by Isabelle et al. (2002). Alternatively, FO can be synthesized using the procedure as described by Choi et al. (2002).

In another embodiment, a composition of the invention comprises flavin mononucleotide (FMN). This can be obtained commercially from, for example, Sigma

(Catalogue No. F8399, F2253, F6750, F1392 and 83810). FMN can be reduced as described by Sutherland et al. (2002a and b).

Enzymes of the invention, and/or host cells encoding therefor, can be used in coating compositions as generally described in WO 2004/112482 and WO 2005/26269.

Feedstuffs

In a particularly preferred embodiment, a composition of the invention is a feedstuff. For purposes of the present invention, "feedstuffs" include any food or preparation for human or animal consumption (such as cattle, horses, goats and sheep) (including for enteral and/or parenteral consumption) which when taken into the body

(a) serve to nourish or build up tissues or supply energy; and/or (b) maintain, restore or support adequate nutritional status or metabolic function. Feedstuffs of the invention include nutritional compositions for babies and/or young children.

The feedstuffs include nutritional substances such as edible macronutrients, vitamins, and/or minerals in amounts desired for a particular use. The amounts of these ingredients will vary depending on whether the composition is intended for use with normal individuals or for use with individuals having specialized needs, such as individuals suffering from metabolic disorders and the like. Examples of substances with nutritional value include, but are not limited to, macronutrients such as edible fats, carbohydrates and proteins. Examples of such edible fats include, but are not limited to, coconut oil, borage oil, fungal oil, black current oil, soy oil, and mono- and diglycerides. Examples of such carbohydrates include (but are not limited to): glucose, edible lactose, and hydrolyzed starch. Additionally, examples of proteins which may be utilized in the nutritional composition of the invention include (but are not limited to) soy proteins, electrodialysed whey, electrodialysed skim milk, milk whey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to the feedstuff compositions of the present invention: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and

Vitamins A, E, D, C, and the B complex. Other such vitamins and minerals may also be added.

The components utilized in the feedstuff compositions of the present invention can be of semi-purified or purified origin. By semi-purified or purified is meant a material which has been prepared by purification of a natural material or by de novo synthesis.

In an embodiment, the reductase is used in the production of the feedstuff. For example, the reductase can be used in the production of biofuels from plant material such as corn, where distiller grain by-products obtained therefrom are used in, or are used for the preparation of, a feedstuff.

In a further preferred embodiment, the feedstuff comprises a plant of the invention, and/or a part of said plant, and/or an extract of said plant.

EXAMPLES

Example 1 - Mycobacterium smeεmatis degrades aflatoxin

Materials and Methods Aflatoxin stocks

Aflatoxins B₁, B₂, G₁ and G₂ were obtained from Sigma- Aldrich and Fermentek (Israel). Stocks were dissolved in HPLC grade acetonitrile (Sigma-Aldrich) at approximately 1 mg/mL and stored at 4⁰C in the dark. Actual concentrations were determined using the method of Nessheim et al. (1999).

Bacterial screening assays for aflatoxin degradation Bacterial strains were first grown on Luria-Bertani (LB) agar (Sambrook et al. supra) before inoculation into PYB (9 g/L peptone, 4.5 g/L yeast extract, 23 mM Na₂HPO₄, 88 mM KH₂PO₄, 9 mM NaCl, pH 6.0) supplemented with 4 μg/mL aflatoxin G₁ or 6 μg/mL aflatoxin B₁, and incubated for 48 h at 28⁰C on an orbital shaker (200 rpm) before 5 μL of each culture was spotted and dried onto silica gel 60 F₂₅₄ thin layer chromatography (TLC) plates (Merck). Chloroform/acetone/acetic acid (40:10:1 by volume) was used as the developing solvent and aflatoxin fluorescence was detected by viewing under ultraviolet light (365 nm). Images of TLC plates were recorded by an Alphalmager 2200 Imaging System (Alpha Innotech) fitted with an ethidium bromide bandpass filter (Alpha Innotech).

Results

Over twenty different bacterial strains previously isolated from various environmental samples in our laboratory for the purposes of pesticide degradation were tested for their ability to degrade aflatoxin in broth culture. Screening of these lab strains found that mycobacterium ESD (Sutherland et al. 2002a and 2002b) species was able to degrade aflatoxin. Simultaneous screening of soil samples identified M. smegmatis as having aflatoxin degrading activity. Subsequently the lab strain of M. smegmatis (me²155) was assayed and shown to have aflatoxin degradation activity.

These results are in accordance with those of other researchers that have shown that other species of mycobacteria, and actinomycetes, have the ability to degrade aflatoxin (Hormisch et al., 2004; Teniola et al., 2005; Alberts et al., 2006).

M. smegmatis soluble extracts were able to degrade AFGl, AFBl, AFG2 and AFB2 as measured by TLC. Since this activity was inactivated by heating the soluble extracts, degradation is probably enzymatic.

Example 2 - Identification of cofactor Εan requirements for aflatoxin degradation

Materials and Methods Transposon mutagenesis of M. smegmatis

Random insertion mutants of M. smegmatis mc²155 (Sutherland et al., 2002a and 2002b) were generated with the EZ::TN <R6Kγori/KAN-2> insertion kit (Epicentre). The EZ::TN <R6Kγori/KAN-2> tnp transposase complex (1 μL) was electroporated into 100 μL of electrocompetent M. smegmatis me²155 cells. Electrocompetent cells were prepared from a 100 mL culture of cells (OD₆₀₀ ~ 0.8) grown at 37⁰C at 200 rpm. Cells were harvested by centrifugation (2500 g, 10 min.), resuspended in 40 mL of 0.05% (v/v) Tween-80 and centrifuged as previously. The cells were pelleted and resuspended twice more in 0.05% (v/v) Tween-80 before final resupension in 0.5 mL of 0.05% (v/v) Tween-80. Electroporation was performed using 2mm gap cuvettes and an electroporator (BioRad) set at 2.5 kV, 25 μF and 1000Ω. Electrocompetent cell preparation and transformation were performed at 4⁰C.

After electroporation the cells were resuspended in LB broth (Sambrook et al., supra) containing 0.05% (v/v) Tween-80, incubated at 37⁰C at 200 rpm, before plating on LB agar containing 20 μg/mL kanamycin. The plates were incubated at 37⁰C for 3 days to allow colony formation. Approximately 2000 mutants were obtained from one transformation event.

Isolation and characterisation of aflatoxin Gj-degradation defective M. smegmatis mutants

M. smegmatis me²155 transposon insertion mutants were individually inoculated into 2 mL square wells of 96 deep well growth blocks (Axygen). Each well contained 200 μL of PYB supplemented with 20 μg/mL kanamycin and 4 μg/mL aflatoxin G₁. The growth blocks were sealed with silicone mats (Axygen) and incubated for 3 days (37⁰C at 200 rpm) before 5 μL of each culture was examined for aflatoxin degradation by TLC as described previously.

Mutants that exhibited detectable growth but had a decreased ability to degrade aflatoxin G₁ compared to that of wildtype cells were selected as aflatoxin G₁- degradation defective mutants. The genomic regions of selected mutants containing the EZ::TN <R6Kgori/KAN-2> transposon were isolated by plasmid rescue. Genomic DNA was isolated using the Bactozol DNA isolation kit (Molecular Research Center), digested with EcoKL, self-ligated and electroporated into E. coli TransforMax EClOOD pir-116 (Epicentre). Transformants containing the M. smegmatis transposon-interrupted genomic DNA were selected by plating transformants onto LB agar containing 40 μg/mL kanamycin. The resulting plasmids were isolated and the genomic DNA regions flanking the transposon were sequenced using primers supplied with the EZ::TN <R6Kgori/KAN-2> insertion kit (Epicentre).

Results Five mutants from the M. smegmatis random insert transposon library were identified that were not capable of degrading AFGi. Sequencing of the gene region in which the transposon was inserted showed that all mutations were associated with the F₄₂₀ biosynthetic pathway.

One of the mutants identified in the screen was due to disruption of the Fgd gene, MSMEG0772, encoding the F₄₂₀-dependent glucose-6-phosphate dehydrogenase, which is responsible for the reduction of F₄₂₀ to F₄₂₀H₂. FGD is the only protein that has been identified in M. smegmatis to catalyse the reduction of F₄₂₀ to F₄₂₀H₂ (Purwantini et al, 1997).

The other four mutants were found to be due to the disruption of the FbiC gene, MSMEG5113, which catalyses the last step in FO biosynthesis from intermediates from the riboflavin synthesis pathway and tyrosine pathway (Graham et al, 2003). FO is phosphorylated and stabilised by the addition of a chain of up to six γ-linked glutamate residues by FbiA and FbiB in mycobacterium (Choi et al., 2001) to form F₄₂₀. A similar study on the hydrolysis of the nitroimidazopyran anti-tuberculosis drug, PA-824, has also shown that FbiC and FGD are important in providing reduced F₄₂₀ in M. tuberculosis for the reduction and activation of PA-824 by rv3547 (Manjunatha et al., 2006).

Example 3 - Coenzyme F.nn purification Materials and Methods

F₄₂₀ was prepared from M. smegmatis me²155 cell free extracts based on the methods of Isabelle et al. (2002). The extract was loaded at a flow rate of 2 mL/min onto a 1.6 x 10 cm Macro-Prep High Q anion exchange column (BioRad) equilibrated in 20 mM Tris-HCl, pH 7.5 (buffer A). Thereafter, the column was operated at a flow rate of 5 mL/min. The column was washed with buffer A for 10 min before proteins were eluted with a linear gradient starting with buffer A and finishing in 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5 developed over 60 min. The column was then washed with 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5 for 10 min, before elution of highly fluorescent F₄₂₀ containing material with 1.0 M NaCl, 20 mM Tris-HCl, pH 7.5. The F₄₂₀ fraction was boiled for two minutes before the soluble material was further purified over a High Capacity Cl 8 Extract-Clean (1O g bed volume) solid phase extraction column (Alltech) pre-wetted in methanol and equilibrated in deionised H₂O. The column was operated at approximately 5 mL/min. The F₄₂₀ fraction was applied, the column washed with 20 mL of deionised H₂O, then the bound F₄₂₀ eluted with 20% (v/v) methanol wash.

The F₄₂₀ was diluted 2-fold with deionised H₂O, freeze-dried to a powder, before resuspension in deionised H₂O and storage at -2O⁰C. The concentration of F₄₂₀ was quantified by absorbance at 420 nm using the extinction coefficient of ε₄₂₀ at pH 7 = 38.5 mM-W¹ (Bair et al., 2001).

Results

The cofactor F₄₂₀ was purified from M. smegmatis cultures at 0.27μmol/g of F₄₂₀ per dry weight of M. smegmatis. The resuspended solution was quantified to have a concentration of 114.2μM and was used for subsequent experiments.

Example 4 - Recombinant His-tagged FGD production and purification

Materials and Methods

The M. smegmatis mc²155 fgd gene was cloned based on the methods of Purwantini and Daniels (1998). Briefly, the fgd gene was amplified from genomic

DNA using Pfu turbo polymerase (Stratagene) and the primer pair: 5'-

CGCATATGGCTGAATTGAAGCTAGGTTAC-3' (SEQ ID NO:97) and 5'-

CGGGATCCTCAGGCCAGCTTGCGCAACCG-3' (SEQ ID NO:98). The amplicon was digested with BamHl and cloned into pBluescript KS II (Stratagene) digested with BarnRl and EcoKV. The resultant construct was then digested with Ndel and BamHl and the fgd gene cloned into pET14b (Novagen), similarly digested, to produce pET14b-FGD. E. coli BL21 (DE3) Tuner cells (Novagen) were transformed with pET14b-FGD and grown overnight at 37⁰C on LB agar containing 100 μg/mL ampicillin. A single colony obtained from the transformation was then used to inoculate LB containing 50 μg/mL carbenicillin. The culture was grown (37⁰C at 200 rpm) until an ODβoo of 0.6-0.8 was reached and a glycerol stock prepared by diluting cultures 1:1 with sterile 50% glycerol. A few microlitres of the glycerol stock was used to inoculate 200 mL of LB containing 50 μg/mL carbenicillin. The culture was grown (37⁰C at 200 rpm) until an OD_6O0 of 0.4 was reached. Isopropyl-β-D-thiogalactopyranoside (IPTG) was then added to a final concentration of 0.4 mM and the culture incubated (2O⁰C at 200 rpm) for a further 16h. The cells were harvested and the pellet resuspended and lysed according to the QisLβxpressionist handbook (June 2003; Qiagen) under native conditions (Protocol

9).

The His-tagged FGD was purified from the resulting soluble protein fraction by batch purification over a ImL Ni-NTA superflow column (Qiagen) according to Protocol 12 of the Qiaexpressionist handbook (June 2003; Qiagen) through the use of standard buffers. FGD was stored by precipitation with ice cold saturated ammonium sulphate and stored at -80⁰C.

FGD activity was determined by a spectrophotomeric assay that measured the decrease in absorbance at 420 run due to the reduction of F₄₂₀ to F₄₂₀H₂ (Purwantini and Daniels, 1996) and calculated using the extinction coefficient used in the quantification of purified F₄₂₀. One unit of FGD activity was defined as the amount of enzyme required to reduce one μmole OfF₄₂₀ per min. Reduction Of F₄₂₀ was performed at room temperature (22⁰C). The assay mixture was buffered with 50 mM sodium phosphate, pH 7.0 and contained 25 μM F₄₂₀, 5 mM glucose-6-phosphate (G6P) (Sigma-Aldrich) and recombinant FGD. The assay mixture was assembled in a quartz cuvette, sparged with high purity nitrogen and stoppered with a Subaseal (Sigma-Aldrich).

Results

Recombinant FGD was expressed and purified and shown to decrease the absorbance of F₄₂₀ at 420nm, thus reducing F₄₂₀ to F₄₂₀H₂ in the presence of G6P. The activity was calculated to be 6.5U/μl. Example 5 - High through-put aflatoxin-degradation assays

Materials and Methods

Soluble bacterial extracts, purified proteins, and partially purified enzymes were tested for afiatoxin G₁ -degradation activity by the following assays performed at room temperature (22⁰C) in 1.5 mL microfuge tubes in the dark. The reaction mixture was typically made up to lOμl with 5μl of sample and 5μl of reaction buffer, containing:

F420 units 5 μM

FGD units 0.2U/μL

G6P 2.5 mM

Tris-HCl, pH 7.5 2O mM

Afiatoxin 62.5 μM

5 μL of each reaction mixture was spotted and dried onto silica gel 60 F₂₅₄ thin layer chromatography (TLC) plates (Merck). Chloroform/acetone/acetic acid (40:10:1 by volume) was used as the developing solvent and afiatoxin fluorescence was detected by viewing under ultraviolet light (365 run). Images of TLC plates were recorded by an Alphalmager 2200 Imaging System (Alpha Innotech) fitted with an ethidium bromide bandpass filter (Alpha Innotech).

Results

This method provided a quick and simple analytical technique that was used for subsequent assays in determining the proteins involved in afiatoxin degradation in M. smegmatis. AFG₁ is used as the rate of degradation is faster than that OfAFB₁.

Example 6 - Identification of AFG^-degrading proteins in M. smeεmatis

Materials and Methods

M. smegmatis me²155 was inoculated into 1.5 L of LB and grown for three days (37⁰C at 200 rpm). All the following steps were performed at 4⁰C. The cells were harvested (10,000 g for 30 min) and the 11 g pellet washed, and resuspended in 50 mL of 20 mM Tris-HCl pH 7.5, 1 mg/mL lysozyme (Sigma), 5 mM DTT and 1 mM phenylmethylsulphonylfluoride (PMSF). Acid- washed glass beads (150-212 μm;

Sigma) were added (0.1 v/v) and the cells disrupted by sonication using a Branson Sonifier 250 on maximal output control and constant duty cycle using ten 10 s bursts with a minute cooling period between each burst.

The extract was centrifuged (30 min at 20,000 g) and the pellet discarded. The supernatant (cell free extract) was passed through a 0.22 μm filter prior to ammonium sulphate ((NHU)₂SO₄) precipitation. Saturated (NFLO₂SO₄ solution (O⁰C) was added to the supernatant to give 40% saturation, the solution was mixed and incubated for 30 min, followed by centrifugation (15min at 20,00Og). The resulting supernatant was re- precipitated by the addition of saturated (NH₄)₂SO₄ solution (O⁰C) to give 70% saturation, the mixture was incubated overnight.

The pellet obtained after centrifugation (20,000 g for 30 min, 4⁰C) was resuspended in 1 M (NH₄)₂SO₄, 20 mM Tris-HCl, pH 7.5, passed through a 0.22 μm filter then loaded onto a 1.6 x 25 cm phenyl sepharose high performance hydrophobic interaction column (GE Healthcare; HIC) equilibrated with the same buffer and operated at a flow rate of 2 mL/min. After loading the column was washed for 30 min with equilibration buffer before proteins were eluted with a linear gradient starting with equilibration buffer and finishing in 20 mM Tris-HCl, pH 7.5 (buffer A) developed over 100 min. The column was washed with buffer A for a further 30 min.

Fractions of 6 mL were collected and those in the three activity peaks of interest containing AFd-degrading activity were individually pooled (designated activity peaks 1, 2 and 3), buffer exchanged into 150 mM NaCl, 20 mM Tris-HCl, pH 7.5 (buffer B) and concentrated to ~1 mL using an Amicon-Ultra centrifugal filter device with a molecular weight cut-off of 10,000 (Millipore). Each of the activities were chromatographed individually on a Superdex 200 Hi Load 26/60 gel filtration column (Pharmacia) equilibrated and run with buffer B at a flow rate of 4 mL/min. Fractions of 6 mL were collected and those of interest containing AFG₁ -degrading activity were pooled, buffer exchanged into buffer A and concentrated to ~1 mL as above.

Each of the activities were chromatographed individually on a MonoQ HR 5/5 anion exchange column (Pharmacia) equilibrated in buffer A and operated at a flow rate of 1 mL/min. After loading the column was washed with buffer A for 5 min before proteins were eluted with a linear gradient starting with buffer A and finishing in 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5 developed over 30 min. The column was finally washed with 1.0 M NaCl, 20 mM Tris-HCl, pH 7.5 for 10 min. Fractions of 1 mL were collected and those of interest containing AFGj-degrading activity were concentrated to -0.1 mL as above. The proteins derived from HIC activity peak 3 were pooled, buffer exchanged into buffer A and concentrated to ~1 mL as above, and re-chromatographed over the MonoQ HR 5/5 column as above, except 0.5 mL fractions were collected. Those of interest containing AFGi-degrading activity were concentrated to ~0.1 mL as above. Protein quantification was performed by the Bradford assay (Biorad) according to manufacturer's instructions. Gel filtration chromatography

Gel filtration chromatography was used to calculate the molecular weight of the active proteins from the HIC separated peaks. Each activity peak from HIC was separated by gel filtration chromatography on a Superdex 200 HR 26/60 column (GE

Healthcare) at 4⁰C in 2OmM Tris-HCl pH7.5, 15OmM NaCl at 4ml/min. ImI of sample was loaded onto the column and 6ml fractions were collected every 1.5min. Gel filtration chromatography standards (Amersham/GE Healthcare) were separated on identical runs as a molecular weight standard. A standard curve (elution by log MW) was drawn. The approximate elution time of each activity peak was determined by measuring the activity of each fraction, and the apparent molecular weight of each activity peak was extrapolated.

Identification of enzyme candidates for the degradation ofaflatoxin Gi Approximately 2 to 5 μg of protein present in the partially purified aflatoxin G₁- degrading activity fractions were digested with trypsin. The proteins were first reductively alkylated by incubation at 5O⁰C for 30 min in 50 mM NH₄HCO₃, 10 mM DTT, 0.3% (v/v) SDS followed by addition of iodoacetamide to a final molar ratio 2.4 times that of the DTT. The reaction was then incubated at 37⁰C in the dark for 1 h. The alkylated protein was then precipitated with the 2-D PAGE clean-up kit (GE Healthcare) before resuspension in 10 μL of 50 mM NH₄HCO₃ and addition of 0.5 μg sequencing grade trypsin (Promega). Digestion was performed overnight at 28⁰C and then stopped by addition of 1 μL of 10% (v/v) formic acid. Peptides (5 μL) from each digest were subjected to HPLC separation on an Agilent 1100 Series Capillary LC system by application to an Agilent Zorbax SB-C18 5μm 150 x 0.5mm column with a flow rate of 0.1% (v/v) formic acid/5% (v/v) acetonitrile at 20μl/min for one minute then eluted with gradients of increasing acetonitrile concentration to 0.1% formic acid/20% acetonitrile over one minute at 5μL/min, then to 0.1% (v/v) formic acid/50% (v/v) acetonitrile over 28 minutes, then to 0.1% (v/v) formic acid/95% (v/v) acetonitrile over one minute.

Eluate from the column was introduced to an Agilent XCT ion trap mass spectrometer through the instrument's micronebuliser electrospray ion source. As peptides were eluting from the column, the ion trap collected full spectrum positive ion scans (100 to 2200m/z) followed by four MS/MS scans of ions observed in the full spectrum avoiding the selection of ions that carried only a single charge. When an ion was selected for MS/MS analysis all others were excluded from the ion trap and the selected ion was fragmented according to the instrument's recommended "SmartFrag" and "Peptide Scan" settings. Once two fragmentation spectra were collected for any particular m/z value it was excluded from selection for analysis for a further 30 seconds to avoid collecting redundant data. Mass spectral data sets were analysed using Agilent's Spectrum Mill software to match the data with annotated protein sequences present in the M. smegmatis MC² CMR database (Jeremy et al., 2001). The software generated scores for the quality of each match between experimentally observed sets of masses of fragments of peptides and predictions from sequences in the database. The sequence matches reported here generally received scores greater than 20, the default setting for automatic, confident acceptance of valid matches.

Results

M. smegmatis soluble cell extracts were first subject to ammonium sulphate ((NILj)₂SO₄) precipitation by increasing the amount of (NH_t)₂SO₄ by 10% increments from 10% to 60% (NIL_t)₂SO₄ (Figure 1). AFGl was not degraded and fluoresced in the negative control, lane 1, and was completely degraded by M. smegmatis soluble cell extract in lane 2. The 60% and 30% (NILi)₂SO₄ precipitates, lane 3 and 6 respectively, show the highest AFGl degradation activity of the (NILi)₂SO₄ precipitates.

When the (NIL_t)₂SO₄ precipitates were incubated with AFGl overnight, AFGl was degraded in all fractions from 20 to 80% (NILi)₂SO₄, but no degradation was observed in the 10% precipitate. These results suggest that aflatoxin degrading proteins precipitate over a range of concentrations of (NILi)₂SO₄ and that there was increased aflatoxin degrading protein in the 30% and 60% precipitates.

Further separation of enzymatic activity was achieved by applying a 50%-80% (NILi)₂SO₄ precipitate onto a phenyl sepharose HIC column. This gave three activity peaks eluting in the following fractions: peak 1, fraction #37/38; peak 2, fraction #49; and peak 3, fraction #59. These fractions were concentrated, buffer exchanged into 2OmM Tris-HCl pH 7.5 and then separated by anion exchange chromatography on a mono Q HR 5/5 column; the active fractions are shown in Table 3 below. HIC fractions were also separated by gel filtration on a Superdex 200HR column to determine the molecular weight of the active proteins, the calculated MW is shown in Table 4.

Table 3 - Molecular wei ht of the active roteins.

Mono Q fractions were analysed by SDS-PAGE to determine the purity of samples and for identification of bands by time of flight mass spectrometry (MS-TOF). Sixteen bands in total were carefully excised from the mono Q fractions. These were digested and analysed by MS-TOF. Five candidate enzymes, based on their pi and the fact that four proteins belonging to two protein families were identified, were suggested as candidate aflatoxin degrading proteins (Table 4). Candidates appear to be dimers as determined by gel filtration studies as all the candidate enzymes are between 12 and 18 KDa, but elute off the gel filtration column between 25 and 32kDa.

Table 4 - Candidate aflatoxin de radin enz mes b MS

Example 7 - Identification of candidate enzymes by database homology

Materials and Methods

Further candidate enzymes were identified by database homology searches on both the National Institute of Health http://www.ncbi.nlm.nih.gov/BLAST/ and The Institute of Genomic Research (TIGR) comprehensive microbial resource (CMR) http://cmr.tigr.org/. The complete M. smegmatis genome has been sequence by TIGR (released October 2004). At the time of discovery many of the genes did not have SWISS-PROT/TrEMBL accession numbers or a GenBank ID. Accordingly the TIGR CMR annotation has been used to identify all genes (in the format e.g. MSMEG3387). Annotation can be searched on the web page http://cmr.tigr.org/tigr- scripts/CMR/shared/MakeFrontPages.cgi?page=searches&crumbs=searches.

Subsequently the entire genome sequence for M. smegmatis has been submitted to Genbank and the new locus tags and Genbank accession numbers are listed in the "Key to the Sequence Listing".

Results

Protein BLAST analysis using NCBI BLAST tool identified both MSMEG3387 and MSMEG5692 as belonging to a large family of enzymes that is conserved from bacteria to higher order mammals and is called the pyridoxine 5 'phosphate oxidase (PNPOx) family. PNPOx enzymes characterised to date form dimers and bind flavin mononucleotide (FMN), which is the cofactor most similar to F₄₂₀.

PNPOxfamily

Protein BLAST analysis of MSMEG3387 and MSMEG5692 on the TIGR CMR database identified the following M. smegmatis proteins: MSMEG0048, MSMEG2792, MSMEG5653, MSMEG5784, MSMEG6811, MSMEG5154, MSMEG6537 and MSMEG6445. Proteins that were visually identified for PNPOx conserved domain (L/MATVxPDGxP) were subject to NCBI BLAST analysis to confirm the presence of the PNPOx domain.

The sequence identities between these pyridoxamine 5 '-phosphate oxidases are provided in Table 5, whereas Figure 2 provides an alignment of the proteins.

Other PNPOx family members include human pyridoxamine 5 '-phosphate oxidase (Musayev et al., 2003), RvI 155 (Biswal et al., 2005; Canaan et al, 2005), Rv2074 (Biswal et al., 2006) and Rv2991 (Structure unpublished, available on NCBI Molecular Modelling DataBase, http://www.ncbi.nlm.nih.gov/Stmcture/rnmdb/mmdbsrv.cgi?form=6&db^;=t&Dopt=s&ui d=30793).

Table 5 - Amino acid sequence identities in the F₄₂₀ dependent reductase subfamily: pyridoxamine 5 '-phosphate oxidase.

DUF385 family

MSMEG2029 and MSMEG3018 share conserved sequence domains with a class of enzymes found only in bacteria and known as domain of unknown function (DUF) 385, which includes MSMEG5954, MSMEG5014, MSMEG2852, MSMEG6285, MSMEG3364, MSMEG5199 and MSMEG3914. The sequence identities between these domain of unknown function (DUF) 385 reductases are provided in Table 6, whereas Figure 3 provides an alignment of the proteins.

MSMEG5954 is the only enzyme in the M. smegmatis genome that has been classified as a member of the DUF385 superfamily on the TIGR database. MSMEG5954 is the closest M. smegmatis homologue to the M. tuberculosis lab strain rv3547 enzyme, sharing 46.5% amino acid identity. Rv3547 was recently shown to protonate the anti-tubercolis drug, PA-824, in an F₄₂₀ dependent manner (Manjunatha et al., 2006). By use of the PHYRE protein fold recognition server www.sbg.bio.ic.ac.uk/phyre/ Manjuantha and co-workers (2006) also showed that the DUF385 subfamily proteins have similar structures to those of the PNPOx family.

Table 6 - Sequence identities among domain of unknown function (DUF) 385 reductases.

Bleomycin resistant protein family

MSMEG6591 identified in this study is related to the glyoxalase/bleomycin resistant protein family (BRP). NCBI BLAST searching shows that it has the glyoxalase/BRP conserved domain; it is also predicted to have similar protein folds to crystallised glyoxalase/BRP proteins as predicted by PHYRE.

The M. smegmatis proteins most similar to MSMEG6591 are MSMEG2303 and MSMEGlOlO, as well as MSMEG5870. MSMEG2303 and MSMEGlOlO have identical amino acid sequence, yet are found on different regions of the M. smegmatis genome.

The sequence identities between these glyoxalase/BRPs are provided in Table 7, whereas Figure 4 provides an alignment of the proteins.

Table 7 - Sequence identities of the glyoxalase/bleomycin resistance protein (BRP) family, MSMEG2303 has been omitted as it has 100% identity to MSMEGlOlO.

Example 8 - Cloning of recombinant proteins

Candidate genes for aflatoxin degradation and the FbiC gene were amplified from M. smegmatis me²155 genomic DNA using Platinum high fidelity Taq (Invitrogen) using the primer pairs in Table 8. Primers were designed to incorporate the AttB recombination sites for recombination into the Gateway™ donor vector pDONR201 (Invitrogen), as per the manufacturers instructions.

Table 8 - Primers for ene am lification into Gatewa ™ vectors (Invitrogen).

Amplicons were purified away from primer dimers using either PEG 8000 following Gateway™ instructions, or by PCR purification spin columns (Zymo Research), and recombined into pDONR201 using BP clonase (Invitrogen), as per the recombination protocol (Invitrogen). Entry vectors were transformed into one shot TOPlO chemically competent cells (Invitrogen) on kanamycin LB plates. Colonies were screened by carefully picking half a colony for PCR, whilst making sure the remaining half colony was not contaminated, and clearly numbered on the underside of the plate to enable the same colony to be picked for inoculation into an overnight culture. The plasmid was amplified by PCR using the gene specific primers (Table 8) and Taq polymerase (Invitrogen), with an initial denaturation at 95°C for 3 minutes to crack open bacterial cell walls. PCR products were screened on a 1% agarose gel. Colonies with the correct insert size were inoculated and grown overnight and plasmid DNA was extracted using QIAGEN plasmid miniprep kit. Sequence identity was confirmed by sequencing with Big Dye terminator 3.1 and run on an Applied Biosystems 3730S Genetic Analyser, at Micromon DNA sequencing facility (Monash, Vic). The sequencing primers used were: pDONR201 forward TCGCGTTAACGCTAGC ATGGATCTC (SEQ ID NO:99) pDONR201 reverse GTAACATCAGAGATTTTGAGACAC (SEQ ID NO: 100). Plasmids with the correct size insert were subject to LR recombination with the

Gateway™ destination pDEST17, which contains an N-terminal His tag, and transformed into one shot TOPlO chemically competent cells on LB ampicillin plates. Colonies were confirmed by colony cracking PCR with gene specific primers and 2 colonies of each gene were grown in 5ml LB cultures for glycerol stock and plasmid purification for transformation into BL21-AI™ Arabinose Inducible (Invitrogen) cells for protein expression. Results

Candidate proteins were amplified from M. smegmatis genomic DNA with AttB recombination sites for cloning into the 6XHis tagged Gateway™ expression vector, pDEST17. All nucleotide sequences of clones were confirmed for errors or sequence variation from the published sequences on TIGR CMR database, and all sequences were identical to those published. Glycerol stocks were made of each construct and the expression constructs were subsequently transformed into arabinose inducible BL21-AI (invitrogen) cells for protein expression.

Example 9 - Expression of recombinant proteins

Materials and Methods

Optimisation of the expression of proteins from the pDEST17 vector was determined by small scale expression in 10ml cultures. An overnight culture of pDEST17 in BL21-AI cells was subcultured 1 :20 and grown for 2 hours before induction by the addition of 0.2% arabinose. Samples were taken at 0 hrs, 1.5 and 4 hours from both induced and un-induced cultures. Centrifuged cell pellets were resuspend in 5OmM Tris-HCl pH 7.5 and sonicated using two 5 second bursts, at setting

7 on a Branson sonicator fitted with a microprobe. Expression of recombinant protein was confirmed by separation of both whole cell and soluble fractions by either 10% or 15% polyacrylamide gel electrophoresis (PAGE). Large scale induction experiments were based upon initial optimisation experiments.

For medium scale expression of recombinant proteins, cells grown in 200ml cultures were centrifuged at 5000g and resuspended in 20ml of 5OmM Tris-HCl pH7.5 ImM phenylmethylsulphonyl fluoride (PMSF). Cultures were lysed using either a pre- cooled French press (Thermo electron Incorporated) at 1800 Mpa pressure or by sonication, using three 10 second bursts on a Branson sonicator at setting 7, with a microprobe. Cell debris and insoluble material from the lysed cells were sedimented by centrifugation at 2000Og in a Beckman centrifuge for 15 minutes. Soluble fractions were frozen at -20⁰C for further processing. Protein was quantified using Biorad DC assay in a microtitre plate format following the manufacturer's instructions. Quantification of recombinantly expressed protein was determined by separation by 15% PAGE and the separated bands recorded and analysed by Alphalmager 2200 Imaging System (Alpha Innotech), using Alphalmager software to quantify band intensity. Soluble bacterial extracts were purified by cobalt agarose metal affinity

(Talon™) chromatography at 4°C and in the presence of PMSF to prevent protein degradation. Talon resin was equilibrated with 5OmM Tris-HCl pH7.5, 30OmM NaCl (Buffer A). Resin was poured into glass chromatography columns (Biorad) and soluble extracts in Buffer A were passed over the column twice with fresh PMSF. Up to 1OmM imidazole was added to the soluble bacterial extracts to prevent non specific binding. The Talon columns were washed with Buffer A containing fresh PMSF and 0-2OmM imidazole, depending on the enzyme being purified. Proteins were eluted off the Talon column using Buffer A containing 40-25OmM imidazole, but no PMSF. The fractions eluting off the Talon column were analysed by SDS-PAGE. The fractions containing protein were pooled and either dialysed or concentrated and buffer exchanged using an Amicon MWClO filter to remove imidazole. Protein concentrations were determined by measuring the absorbance at 280nm using a NanoDrop Spectrophotometer NDlOOO and calculated based on the extinction co-efficient for each protein as determined using Vector NTI software (Invitrogen).

MSMEG5954, which was mostly insoluble under normal purification conditions, was refolded following the methods of Whitbread et. al. (2005). Briefly the cells were resuspend in ice cold purification buffer (8M urea, 30OmM NaCl, 5OmM Sodium phosphate pH7.5), lysed by French Press and the cell debris was removed by centrifugation at 10 00Og for 15 minutes. MSMEG5954 was bound to a NiAg column which had been equilibrated with purification buffer, the bound extract was washed with 500ml of purification buffer to remove non bound proteins before refolding. The refolding protocol cyclically lowers the concentration of Urea from 8M over thirteen 30 minute steps at 0.5ml/min on a Biorad FPLC according to the protocol of Whitbread (2005). MSMEG5954 was eluted over a gradient of 0-50OmM imidazole over 20 minutes and 1 minute fractions were collected. This method can be followed for any other enzymes which are insoluble when prepared by another procedure.

Results

All 15 proteins were expressed, and MSMEG2029, MSMEG3018, MSMEG3387, MSMEG5692 and MSMEG6591 were purified by cobalt agarose affinity chromatography. The other 10 proteins were analysed as soluble fractions rather than purified protein. Optimum expression for all proteins was at 1.5 hours, except for MSMEG3018, MSMEG3387 and MSMEG6591, which was at 4 hours. MSMEG5954 was largely insoluble, and no protein could be detected by SDS PAGE in the soluble fraction. However, this soluble fraction had aflatoxin degrading activity, as determined by high throughput aflatoxin-degradation assays.

MSMEG5954 was subsequently repurified under denaturing conditions and refolded. The refolded protein maintained activity, had a concentration of 1.09 μM and was used for all further experiments. Example 10 - Rates of Mycobacterium smeεmatis F420 dependent reductase enzymes.

Materials and Methods

Enzymatic assays were conducted in either lOμl or 20μl reactions in the dark at room temperature. Aflatoxins were purchased from Sigma and dissolved in acetonitril to lmg/ml, which was diluted to 10mg/ml in reaction buffer (F₄₂₀ 5 μM, FGD units 0.2U/μL, G6P 2.5 mM, Tris-HCl, pH 7.5 20 mM) and enzyme or E. coli soluble extract. Reactions were incubated for 0.5 to 24 hours depending on the enzyme and type of aflatoxin used. The reaction was stopped by the addition of formic acid to a final concentration of 2%, and incubated on ice for 15 minutes. Protein was pelleted by centrifugation for 5 to 10 minutes at 14 000g.

Samples were carefully transferred into glass vials with inserts (low volume, Agilent) and 5μl was injected onto an Agilent zobax XDB Cl 8 column (3.5micron, 2.1x30mm) on an Agilent 1100 Series Binary LC with diode array detector and in-line Time of Flight Mass Spectrometer (MSD TOF). Aflatoxin and its detoxification products were separated over a gradient of 5% acetonitrile (v/v) and 0.1% formic acid (v/v) from 0.5 minutes to 20% acetonitrile at 2 minutes, then a less steep gradient to 50% acetonitrile at 10 minutes, at a flow rate of 0.3ml/min. AFGl elutes at 7.4 minutes. Following each run the gradient was increased to 100% acetonitrile, then brought back to 5% and allowed to equilibrate for 4 minutes before the next injection.

Samples were quantified using Analyst QS software calculating the peak area of ions extracted (AFGl, 329-330; AFG2, 331-332; AFBl, 313-314; AFB2, 315-316) at the time when the aflatoxin elutes (AFG₁, 7.4min; AFB₁, 8.0min), and concentration determined by a standard curve from 0.25mg/ml to lOmg/ml). The specific activity of reaction of the enzymes were determined as μmoles(aflatoxin)/mg(protein)/min. Protein concentrations were either determined by use of the molar extinction coefficient for purified proteins as measured at A₂₈₀ or in soluble extracts by measuring the total protein by Biorad DC assay and determining the percentage of expressed protein by gel densitometry (Alpha imager).

Results

All enzymes were shown to degrade AFG₁, AFG₂, AFB₁ and AFB₂ at varying rates, except for the protein expressed from the MSMEG2029 construct for which no activity was detected. The specific activities for AFGl, based on loss of substrate, are shown in Tables 9 and 10. Table 9 - Specific activities of various MSMEG proteins for AFGl (based on loss of substrate .

¹ * not yet tested

² ND not detected i.f. activity only in insoluble fraction 4 requires DTT for activity

Specific activities for AFG₂ were similar to those for AFG₁, while those for AFB₁ and AFB₂ were at least an order of magnitude less. MSMEG6591 requires the reducing agent, dithiothreitol (DTT), for catalytic activity. Other enzymes closely related to MSMEG6591, namely MSMEG5954 and MSMEGlOlO showed no activity to AFG₁. For MSMEG2792, no activity was detected in the soluble bacterial extract, although on re-testing with the whole cell fraction activity was detected. No activity was detected for the soluble fraction of MSMEG2029 even though the expressed protein was mainly in the soluble fraction and purified by Cobalt agarose. This apparent lack of activity is surprising given the degree of identity and similarity to two other DUF385 family members which have high activity for aflatoxin, MSMEG5954 (32% identity, 45% similarity) and MSMEG2852 (36%, 50%). The loss of activity may be due to altered post-translational modification in E. coli as compared to M. smegmatis. The loss of activity of MSMEG2029 was found to be due to an incorrectly predicted start codon of the protein sequence, the correct start codon is the methionine at residue 20 (SEQ ID No 12). Re-expression of the truncated sequence produced an enzyme with activities as shown in Table 10.

Table 10 - Specific activities of various MSMEG proteins for aflatoxins (based on loss of substrate).

Activity (μmol/min/μmol enzyme)

Protein Protein family (MSMEG) AFGl AFG2 AFBl AFB2

DUF385 5954 83.391 61.411 9.122 7.650

3364 8.079 3.148 1.168 1.332

2852 11.982 7.976 1.259 0.408

3018 1.632 1.256 0.307 0.619

2029 0.661 0.308 0.212 0.161

PNPOx 3387 1.556 0.322 0.129 0.033

6811 0.927 0.433 0.095 0.086

5154 0.238 0.144 0.328 0.091

5692 0.003 0.003 0.003 0.002

5653 0.576 0.292 - -

BRP 6591 0.013 0.008 0.003 0.002

The most active enzyme was MSMEG5954. Accordingly, enzymes closely related to this reductase from other Mycobacterium sp. will be particularly useful degrading coumarin based compounds. Figure 5 provides an alignment of reductases closely related to MSMEG5954.

Example 11 - Mechanism of degradation of aflatoxin by M. smeεmatis F420 dependent reductases

Materials and Methods

Many of the Materials and Methods are as described in Example 10. Enzymes in both the PNPOx family and DUF385 family degrade all types of aflatoxin by the transfer of electrons from F420 and the reaction products are the same for each enzyme. The detailed results of the analysis of reaction products from MSMEG3387 is described.

The results were analysed using Agilent Analyst software. The loss of AFG₁ was monitored in a BMG FLUOstar galaxy fluorescent plate reader using 355 excitation filter and 460 emission filter, monitored over a 60 minute period taking a reading every minute.

Results

To study the mechanism of action of the M. smegmatis F₄₂o dependent reducatases, the reaction products were analysed by LC-MS. AFGl was degraded at a much faster rate than AFBl for all enzymes, suggesting that the difference in structure between AFGl and AFBl is important for either enzyme recognition or for catalytic activity. Given the difference in catalytic activity, AFGl was primarily used to determine the mechanism of action of the aflatoxin degrading enzymes.

Analysis of AFGl degradation by MSMEG3387 over a time period of 1 hour, with time points taken at 0, 10, 20, 40 and 60 minutes, revealed that AFGl was degraded to an unstable intermediate with a molecular weight of 258.06. Figure 6A shows the total ion count trace of the 0 minute time point, AFGl eluted at 7.5 minutes with a peak area of 1.1 x 10⁸ counts. The MS profile of this peak is shown in the inset, with the main ion species of 329 corresponding to the MW of AFGl (328) plus H⁺ ion. Figure 6B shows the total ion count trace of AFGl after a 20 minute reaction with MSMEG3387; the trace shows a decrease in AFGl peak area at 7.5 minutes to 5.8 x 10⁷ counts. The MS profile of the major peak formed, which eluted at 9.4 minutes, after a 20 minute incubation with MSMEG3387 has a MW of 258.06 + 1H+ ion, which corresponds to the MW of the predicted degradation product shown in the inset of Figure 6B. On reanalysis of these reactions 24 hours later, the reaction product at 9.4 minutes was not present, there was no change in the peak area of the AFGl peak, thus suggesting that the loss of reaction product was not enzymatic. When monitoring the MSMEG3387 catalysed degradation of AFGl, by fluorescence spectroscopy, the decrease in fluorescence due to the loss of AFGl appeared much slower than that predicted by LC-MS. The same sample was removed from the fluorescent spectrometer and assayed by LC-MS within 10 minutes. AFGl was completely degraded and the main peak present eluted at 9.4 minutes with a MW of 258.06. These results suggest that the unstable intermediate (MW 258.06) is fluorescent and that the coumarin ring structure is intact.

Further degradation products of AFGl are also detected by LC-MS; however, these elute in multiple peaks between 4 and 8 minutes, and limited information can be extracted regarding their structure. The most common ion species are 262.09, 244.08, and 218.07.

Analysis of AFBl degradation by LC-MS showed a new peak being resolved at 10, minutes with the major ion species having a molecular weight of 314.09 corresponding to the addition of two hydrogen ions to AFBl (MW 312.07). These results indicate that the enzymes are transferring two H⁺ ions from F₄₂₀H₂ to AFBl. Further evidence on the mechanism of degradation of aflatoxin was achieved by assaying the degradation of other similar structures. Both coumarin and 4- methylumbelliferone were incubated with MSMEG3387. MSMEG3387 was able to degrade coumarin at rates similar to the degradation of AFBl, but was unable to degrade 4-methylumbelliferone.

Example 12 - Cofactor analysis Coenzyme FO production and purification

Coenzyme FO was produced in E. coli by cloning the M. smegmatis FbiC gene, MSMEG 5113, into the Gateway pDEST17 vector, using the methods of Graham et al. (2003). FbiC was expressed in the E. coli strain BL21-AI, which was grown in LB broth overnight at 37⁰C, reinoculated 1:20, and grown for a further 2 hours. Expression of FbiC was induced by transferring culture to M9 media (without amino acids) containing 6mM tyrosine and 0.2% arabinose. Cells were induced for 2 hours, then centrifuged and both supernatants and cell pellet stored at -2O⁰C until further processing.

The presence of FO in the media was detected using a fluorescent plate reader (Molecular Devices) with an excitation wavelength at 420nm and emission wavelength of 480nm (Graham et al., 2003). Large scale purification, adapted from the methods of Isabelle et al. (2002) was performed as described above for coenzyme F₄₂₀ production (Example 3). However, for coenzyme FO purification, the column was washed with buffer containing 100mm NaCl and the FO eluted in buffer containing 25OmM NaCl. The eluted FO was further purified on a Cl 8 column and eluted in 20% methanol.

FO analysis by fluorescent HPLC

The presence of FO, FMN and F420 of samples was determined by analytical HPLC using tetrabutylammonium phosphate as an ion pairing agent and detection by fluorescence at pH 7.0 (excitation 420nm and emission 470nm). An Agilent 1200 HPLC with solvent degasser, binary pumps, autosampler, diodode array detector, and multiwavelength fluorescent detector was used for the separation of cofactors.

Bacterial or plant material was heated at 90⁰C for 15 minutes in an equal volume of sodium phosphate buffer, pH 7.0. After centrifugation, for 10 minutes at 14 000 g, FO and F₄₂₀ are separated on a Zorbax Eclipse XDB-C 18 3.5μm (1.5 x 30mm) column equilibrated in 95% 5mM tetrabutylammonium phosphate 1OmM ammonium phosphate pH 7.0 (solution A) and 5% Acetonitril (solution B). Separation of FO was achieved by holding the percent of acetonitril for 1 minute, at which time the percent acetonitril was increased to 10% at 2 minutes to allow for the elution of FMN. F₄₂Q species were eluted by increasing the percent acetonitril to 30% from 2 to 30 minutes and holding the concentration of acetonitril at 30% until 11 minutes. The column was flushed with a 60% acetonitril before being re-equilibrated to 5% solution B. FO elutes at 1.2 minutes, FMN elutes at 3.5 minutes, F₄₂₀ peaks elute from around 3 minutes, the F₄₂₀ species with the least amount of glutamates eluting first.

FO aflatoxin degrading assays

FO was substituted into the standard F₄₂₀ dependent assay, replacing F₄₂₀.

Enzyme activity is dependent on reduced FO and this was recycled using FGD and G6P as previously described in Examples 5 and 10. Enzymatic degradation of AFGl was conducted in 20μl reactions in the dark at room temperature for 16 hours. The reaction mix contained:

FO 5 μM, FGD 0.2U/μL,

G6P 2.5 mM,

Tris-HCl, pH 7.5 20 mM

AFGl 62.5μM

Aflatoxin was added to start the reaction. Upon completion the reaction was stopped with 2% formic acid, left on ice followed by centrifugation for 5 to 10 minutes to pellet protein. The degradation of aflatoxin was monitored by LC-MS as previously described in Example 10.

FMN aflatoxin degrading assays

FMN is reduced by incubation with M. smegmatis flavin reductase (MsFR) previously cloned in our laboratory (Sutherland et al. 2002a and 2002b). The assay typically contains:

FMN 500μM MsFR 12μg/ml

NADH 5mM

Sodium formate 5OmM

Sodium dehydrogenase 0.05 - 0.1 units

Tris-HCl, pH 7.5 2OmM Protein lOμM

Aflatoxin 30μM Degradation of aflatoxin will be monitored by LC-MS, as described in Example 10.

Results FO was isolated from boiled M. smegmatis cell extracts and E. coli expressing

MSMEG5113, FO was present in low concentrations and eluted with FMN upon purification. FO was not reduced by FGD in the presence of glucose-6-phosphate, indicating that a different enzyme such as a flavin reductase may be required to produce reduced FO. AFGl was shown to be degraded by MSMEG3387 in the presence of reduced

FMN. The enzymatic rate of activity with FMN is slower than it is with F₄₂₀, with an apparent activity of 0.0048 μmol/min/μmol enzyme with FMN. These results demonstrate that aflatoxin degrading enzymes can use FMN as a cofactor in the absence OfF₄₂₀ to degrade aflatoxin.

Example 13 - Transformation of F^n dependent reductase enzymes and cofactor Ψiia synthesis enzymes in Nicotiana tabacum.

The genes encoding F₄₂₀ reductases that are required for aflatoxin degradation were expressed in Tobacco leaf explants from Nicotiana tabacum and transformed using Agrobacterium tumefaciens based on the methods of Horsch (Horsch et al., 1985). Similarly, each gene that is required for F₄₂₀ biosynthesis (FbiC, FbiA, FbiB and FGD) was cloned into Tobacco leaf explants. Each gene was cloned into a suitable expression vector and subsequently transformed into A. tumefaciens which was then used to transform N. tabacum. Transformation of each plant was confirmed by PCR, RT-PCR and the expression of the protein was determined by biochemical assays.

The DNA encoding all of the F₄₂₀ reductases and the F₄₂₀ biosynthesis genes (FbiA, FbiB, FbiC and FGD) were cloned into the Agrobacterium transfer vector, p277rfC. (a generous gift from Dr C. Helliwell, CSIRO plant industry, Canberra, Australia). Genes were recombined using Gateway™ recombination protocols as described in Example 8. The vector p277 was derived from the plant expression plasmids pART7 and pART27 (Gleave, 1992) and incorporates the cauliflower mosaic virus 35S promoter, octopine synthase gene (OCS) 3 '-untranslated region with polyadenylation signal, and the nptll gene for kanamycin resistance in plants. The Gateway modified version of this vector, p277rfC has the Gateway cloning cassette inserted into the multiple cloning site between the promoter and terminator, allowing for simple recombination of the gene desired to be expressed.

Transformation of the Agrobacterium tumefaciens strain GV3101 was achieved using the uⁿiparental mating method. This involves co-streaking cultures of A. tumefaciens GV3101, E. coli carrying a helper plasmid, RK2013, and E. coli carrying the desired recombinant p277rfC plasmid onto a non-selective LB plate. Overnight incubation at 28°C results in a mixed culture which was collected and dilution streaked onto LB plates which selected for A. tumefaciens GV3101 carrying the p277rfC recombinant plasmid.

For each gene construct and control, tobacco leaf strips 5- 10mm wide from surface-sterilised leaves were placed in the Agrobacterium suspension for 10-20 minutes. After gentle shaking the strips were blotted dry and incubated upper surface down on Murashige and Skoog agar with lμg/ml benzylaminopurine and 0.5μg/ml indole acetic acid (MS9 agar) (Murashige and Skoog, 1962) and cultured for 48 hours at 25⁰C. Tobacco leaf segments were carefully transferred to fresh MS9 agar plates with antibiotics to suppress Agrobacterium growth (150μg/ml Timentin) and to select for vector transformation (lOOμg/ml kanamycin). Shoots formed in 2 to 3 weeks and were excised and plated on Murashige and Skoog agar (MSO) with antibiotics. Transformed shoots grew expanded green leaves and began to take root.

Transformation was confirmed by PCR analysis, RT-PCR analysis, followed by in vitro biochemical analysis. Incorporation of plasmid DNA into transformed N. tabacum was confirmed by isolation of DNA using a Qiagen DNeasy plant mini kit, following the manufacturers instructions, and amplification using gene specific primers as in Table 8. Transcription into mRNA was confirmed by extraction of RNA from leaves ground on dry ice using the Qiagen RNeasy plant miniprep kit, following the manufacturers instructions with the addition of DNase.

Expression of each of the F₄₂₀ dependent reductases was confirmed by biochemical assays as described in Example 10, substituting E. coli extract for plant extract. Plant extracts were prepared by grinding approximately 1Og of tobacco leaves in liquid nitrogen to a fine powder. The ground leaf extract was resuspended in 2ml of buffer per gram of leaf tissue and centrifuged at 20 00Og for 30 minutes at 4°C. Both soluble extract and resuspended insoluble material was tested for activity by biochemical methods. FO and F₄₂₀ produced by expression of F₄₂₀ synthesis genes were assayed by the biochemical methods in Example 12. It is expected that expression of FbiC will produce FO in plants and analysis will be directly from plant material. FbiA and B will require a source of FO to convert to F₄₂₀, and analysis of FGD will be able to convert oxidised F₄₂₀ to F₄₂₀H₂ as measured in Example 4.

Results

Constructs encoding aflatoxin degrading genes (MSMEG2852, 3364, 5954, 3387) and F₄₂₀ synthesis genes (MSMEG5113-FbiC, MSMEGl 828, MSMEGl 829 and MSMEG0772-FGD) were recombined into p277rfc and transformed into A. tumefaciens by the triparental mating method. The genes encoding MSMEG2852 and the F₄₂₀ synthesis genes were transformed into N. tabacum, leaf segments. Approximately 30 leaf segments of each construct were plated onto MS9 plates with Kanamycin to select for transformed shoots. Approximately six individual shoots for each transformation were carefully excised and replated onto MSO agar with Kanamycin.

Transformation was confirmed by extracting DNA and RNA from two plants of each gene that was transformed into N. tabacum and analysed by amplification using gene specific primers. PCR analysis of DNA isolated from plants transformed with MSMEG2852, MSMEG5113, MSEG1828, MSMEG1829 and MSMEG0772 (Figure 7) demonstrated that both transformed plants contained the insert except for clone 1 of MSMEG2852 and clone 1 of MSMEGl 828. RNA analysis confirmed that DNA is being transcribed into RNA in all of the plants that had been confirmed to be transformed by DNA analysis. DNase was used to ensure that no DNA contamination was present in the reverse transcribed cDNA, and PCR amplification from RNA was used to confirm that no DNA was present in the RNA sample before reverse transcription.

The activity of MSMEG2852 was confirmed using the F₄₂o recycling system as in Example 10. After 48 hours incubation of AFGl with plant soluble extract and recycled F₄₂₀, degradation of AFGl was observed by HPLC analysis. Analysis by LC- MS, (Example 10) confirmed degradation of AFGl in an F₄₂₀ dependent fashion, characterised by the appearance of the 245 m/z and 263 m/z ion species. These ion species correspond to the ion species that are found when AFGl is degraded by bacterially expressed and purified MSMEG2852. Furthermore, these ion species were not identified in plant extracts transformed with GUS or in MSMEG2852 plant 1 which was not confirmed to have MSMEG2852 expressed by PCR analysis. Tobacco plants show some innate capacity to modify AFGl by the loss of 2 H⁺ ions as shown by a decrease in size from 329 to 327 m/z.

Synthesis of FO by plants transformed with MSMEG5113 was confirmed by qualitative HPLC as described in Example 12. FO elutes at 1.2 minutes for FO purified from M. smegmatis, E. coli expressing MSMEG5113 and tobacco plants confirmed to express MSMEG5113, FbiC, DNA. No fluorescent peak was observed at this time in the negative controls. FO expressed in plants had the same fluorescent profile as FO from M. smegmatis, using excitation wavelength of 420nm.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All publications discussed and/or referenced herein are incorporated herein in their entirety. The present application is a divisional of US 60/893,950 filed 9 March 2007, the entire contents of which are incorporated herein by reference.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form 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.

REFERENCES

Abdullah et al. (1986) Biotechnology 4:1087.

Alberts et al. (2006) Int J Food Microbiol 109:121-126. Bair et al. (2001) Arch Microbiol 176: 37-43.

Biswal et al. (2005) Acta Crystallogr D Biol Crystallogr 61 : 1492-1499.

Biswal et al. (2006) Acta Crystallogr Sect F Struct Biol Cryst Commun 62:735-742.

Brown et al. (1996) PNAS 93:14873-14877.

Canaan et al. (2005) FEBS Lett 579: 215-221. Capecchi (1980) Cell 22:479-488.

Cheng et al. (1996) Plant Cell Rep. 15:653-657.

Choi et al. (2001) J Bacterid 183:7058-7066.

Choi et al. (2002) J Bacteriol 184:2420-2428.

Chu et al. (2008) Plant Cell Rep 27:85-92. Clapp (1993) Clin. Perinatol. 20:155-168.

Curiel et al. (1992) Hum. Gen. Ther. 3:147-154.

Eaton and Callagher (1994) Ann Rev Pharmacol Toxicol 34:135-172.

Eglitis et al. (1988) Biotechniques 6:608-614.

Fujimura et al. (1985) Plant Tissue Culture Letters 2:74. Gleave (1992) Plant MoI Biol 20: 1203-1207.

Gordon et al. (1999). Chemical-Biological Interactions 14:463-470.

Graham et al. (1973) Virology 54:536-539.

Graham et al. (2003) Arch Microbiol 180:455-464.

Grant et al. (1995) Plant Cell Rep. 15:254-258. Guerra-Lopez et al. (2007) Microbiol Sgm 153:2724.

Harayama (1998) Trends Biotechnol. 16:76-82.

Hormisch et al. (2004) Syst Appl Microbiol 27:653-660.

Horsch et al. (1985) Science 227: 1229-1231.

International Agency for Research on Cancer (1993). Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins.

LARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 56

(Lyon,IARC).

Isabelle et al. (2002) Appl Environ Microbiol 68:5750-5755.

Jeremy et al. (2001) Nucleic Acids Research 29:123-125. Koziel et al. (1996) Plant MoI Biol 32:393-405.

LeJuene et al. (1998). Nature 395:27-28.

Lu et al. (1993) J. Exp. Med. 178:2089-2096.

Manjunatha et al. (2006) PNAS 103:431-436. Murashige and Skoog (1962) Physiologia Plantarum 15: 473-497.

Musayev et al. (2003) Protein Sci 12:1455-1463.

Park (1993) Food Tech October:92-96.

Petrikovics et al. (2000a). Toxicology Science 57: 16-21. Petrikovics et al. (2000b). Drug Delivery 7: 83-89.

Purwantini and Daniels (1996) J Bacteriol 178:2861-2868.

Purwantini and Daniels (1998) J Bacteriol 180:2212-2129.

Purwantini et al. (1997) FEMS Microbiol Lett 146:129-134.

Robens and Richard (1992) Rev Environ Contain Toxicol 127:69-74. Sutherland et al. (2002a) J Appl Microbiol 93:380-389.

Sutherland et al. (2002b) Appl Environ Microbiol 68:6237-6245.

Suzek et al. (2001) Bioinformatics 17:1123-1130.

Teniola et al. (2005) Int J Food Microbiol 105:111-117.

Toriyama et al. (1986) Theor. Appl. Genet. 205:34. Wagner et al. (1992) Proc. Natl. Acad. Sci. USA 89:6099-6103.

Whitbread et al. (2005) Methods Enzymol 401:78-99.

Claims

1. A method of degrading a coumarin based compound, the method comprising contacting the coumarin based compound with a reductase.

2. The method of claim 1, wherein the coumarin based compound is an aflatoxin.

3. The method of claim 2, wherein the aflatoxin is aflatoxin B₁, aflatoxin B₂, aflatoxin G₁, aflatoxin G₂, aflatoxin M₁ or aflatoxin M₂.

4. The method according to any one of claims 1 to 3, wherein the reductase can be purified from an Actinobacteridae.

5. The method of claim 4, wherein the Actinobacteridae is a Mycobacterium sp.

6. The method according to any one of claims 1 to 5, wherein the reductase is an F₄₂₀ dependent reductase.

7. The method of claim 6, wherein the F₄₂₀ dependent reductase is a member of the pyridoxamine 5 '-phosphate oxidases (PNPOx) family of reductases, the DUF385 family of reductases, or the glyoxalase/bleomycin resistant family of reductases.

8. The method of claim 6 or claim 7, wherein the F₄₂₀ dependent reductase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 24, 101 to

9. The method of claim 8, wherein the F₄₂₀ dependent reductase comprises an amino acid sequence which is at least 90% identical to at least one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149.

10. The method according to any one of 1 to 9 which further comprises providing an electron donor.

11. The method of claim 10, wherein the electron donor is F₄₂₀H₂, reduced FO, FMNH₂ Or FADH₂.

12. The method of claim 10 or claim 11 which further comprises providing an enzyme that reduces the electron donor.

13. The method of claim 12, wherein the electron donor is F₄₂₀H₂, and the enzyme is glucose-6-phosphate dehydrogenase.

14. The method of claim 13, wherein the glucose-6-phosphate dehydrogenase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NO:49, SEQ ID

NO:115 and SEQ ID NO:116 , and ii) an amino acid sequence which is at least 25% identical to SEQ ID NO:49, SEQ ID NO:115 and/or SEQ ID NO:116.

15. The method of claim 12, wherein the electron donor is FMNH₂ and the enzyme is flavin reductase.

16. The method of claim 15, wherein the flavin reductase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NO:51, SEQ ID

NO: 133 and SEQ ID NO: 134, and ii) an amino acid sequence which is at least 25% identical to SEQ ID NO:51, SEQ ID NO: 133 and/or SEQ ID NO: 134.

17. The method according to any one of claims 1 to 16, wherein the sample is selected from the group consisting of: soil, water, biological material, a feedstuff or a combination thereof.

18. The method according to any one of claims 1 to 17 which comprises providing a host cell producing the reductase.

19. The method of claim 18, wherein the host cell comprises an exogenous polynucleotide sequence selected from: i) a nucleotide sequence as provided in any one of SEQ ID NOs 25 to 48 and 108 to 114, ii) a nucleotide sequence which is at least 25% identical to at least one of SEQ ID NOs 25 to 48 and 108 to 114, iii) a nucleotide sequence which hybridizes to at least one of SEQ ID NOs 25 to 48 and 108 to 114 under stringent conditions; and iv) a nucleotide sequence encoding an F₄₂₀ dependent reductase as defined in claim 8.

20. A host cell comprising an exogenous polynucleotide encoding a reductase which degrades a coumarin based compound.

21. The host cell of claim 20, wherein the reductase is an F₄₂₀ dependent reductase.

22. The host cell of claim 21, wherein the F₄₂₀ dependent reductase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149, and ii) an amino acid sequence which is at least 25% identical to at least one of SEQ

ID NOs 1 to 24, 101 to 107 and 139 to 149.

23. The host cell according to any one of claims 20 to 22 which is a plant or animal cell.

24. A transgenic plant comprising at least one plant cell of claim 23.

25. The transgenic plant of claim 24, wherein the cell further comprises an enzyme which synthesizes FO using 4-hydroxy phenylpyruvate and 5-amino-6-ribitylamino-2,4 (7H,3H)-pyrimidineione as substrates.

26. The transgenic plant of claim 25, wherein the enzyme comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NO:53, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122 and SEQ ID NO:123, and ii) an amino acid sequence which is at least 25% identical to SEQ ID NO:53, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122 and/or SEQ ID NO: 123.

27. The transgenic plant of claim 25 or claim 26, wherein the cell further comprises enzymes which convert FO to F₄₂₀.

28. The transgenic plant of claim 27, wherein two enzymes are required to convert FO to F₄₂₀, and wherein the first enzyme comprises a sequence selected from: i) an amino acid sequence as provided in SEQ ID NO:55 or SEQ ID NO: 129, and ii) an amino acid sequence which is at least 25% identical to SEQ ID NO:55 and/or SEQ ID NO: 129, and the second enzyme comprises a sequence selected from: iii) an amino acid sequence as provided in SEQ ID NO:57 or SEQ ID NO: 131, and iv) an amino acid sequence which is at least 25% identical to SEQ ID NO:57 and/or SEQ ID NO: 131.

29. The transgenic plant of claim 27, wherein three enzymes are required to convert FO to F₄₂₀, and wherein the first enzyme comprises a sequence selected from: i) an amino acid sequence as provided in SEQ ID NO:55 or SEQ ID NO: 129, and ii) an amino acid sequence which is at least 25% identical to SEQ ID NO:55 and/or SEQ ID NO: 129, and the second enzyme comprises a sequence selected from: iii) an amino acid sequence as provided in SEQ ID NO:57 or SEQ ID NO: 131, and iv) an amino acid sequence which is at least 25% identical to SEQ ID NO:57 and/or SEQ ID NO: 131, and the third enzyme comprises a sequence selected from: v) an amino acid sequence as provided in any one of SEQ ID NOs 150 to 153, and vi) an amino acid sequence which is at least 25% identical to at least one of SEQ ID NOs 150 to 153.

30. The transgenic plant according to any one of claims 24 to 29, wherein the cell further comprises an enzyme that produces reduced F₄₂₀.

31. The transgenic plant of claim 30, wherein the enzyme that produces reduced F₄₂₀ is glucose-6-phosphate dehydrogenase.

32. The transgenic plant of claim 31, wherein the glucose-6-phosphate dehydrogenase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NO:49, SEQ ID NO:115 and SEQ ID NO:116, and ii) an amino acid sequence which is at least 25% identical to SEQ ID NO:49, SEQ ID NO:115 and/or SEQ ID NO:116.

33. A method of degrading a coumarin based compound in a sample, the method comprising contacting the sample with a transgenic plant according to any one of claims 24 to 32, or an extract thereof.

34. A transgenic non-human animal comprising at least one animal cell of claim 23.

35. A method of treating toxicity caused by a coumarin based compound in a subject, the method comprising administering to the subject a composition comprising a reductase, and/or a polynucleotide encoding said reductase.

36. The method of claim 35, wherein the reductase is an F₄₂₀ dependent reductase which comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149, and ii) an amino acid sequence which is at least 25% identical to at least one of SEQ

ID NOs 1 to 24, 101 to 107 and 139 to 149.

37. The method of claim 35 or claim 36, wherein the composition further comprises an electron donor.

38. The method of claim 37, wherein the electron donor is F₄₂₀H₂, reduced FO, FMNH₂, or FADH₂.

39. Use of a reductase, or polynucleotide encoding said reductase, for the manufacture of a medicament for treating toxicity caused by a coumarin based compound in a subject.

40. A method of producing a polypeptide with enhanced ability to degrade a coumarin based compound, or altered substrate specificity for a different type of coumarin based compound, the method comprising i) altering one or more amino acids of a reductase polypeptide, ii) determining the ability of the altered polypeptide obtained from step (i) to degrade a coumarin based compound, and iii) selecting an altered polypeptide with enhanced ability to degrade the coumarin based compound, or altered substrate specificity for a different type of coumarin based compound, when compared to the polypeptide used in step (i).

41. The method of claim 40, wherein the reductase is an F₄₂₀ dependent reductase.

42. The method of claim 41, wherein the F₄₂₀ dependent reductase comprises a sequence selected from: i) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149, and ii) an amino acid sequence which is at least 25% identical to at least one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149.

43. A polypeptide produced by the method according to any one of claims 40 to 42.

44. A substantially purified and/or recombinant polypeptide that degrades a coumarin based compound, wherein the polypeptide has reductase activity.

45. A substantially purified and/or recombinant polypeptide comprising amino acids having a sequence as provided in any one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149, a biologically active fragment thereof, or an amino acid sequence which is at least 25% identical to at least one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149, wherein the polypeptide degrades a coumarin based compound.

46. The polypeptide of claim 45 which is at least 90% identical to at least one of SEQ ID NOs 1 to 24, 101 to 107 and 139 to 149.

47. The polypeptide of claim 45 or claim 46, which is a fusion protein further comprising at least one other polypeptide sequence.

48. An isolated and/or exogenous polynucleotide comprising nucleotides having a sequence as provided in, or complementary to, any one of SEQ ID NOs 25 to 48 and 108 to 114, a sequence which is at least 25% identical to at least one of SEQ ID NOs 25 to 48 and 108 to 114, a sequence which hybridizes to one or more of SEQ ID NOs 25 to 48 and 108 to 114, or a sequence which encodes a polypeptide according to any one of claims 43 to 47.

49. The polynucleotide of claim 48 which comprises nucleotides having a sequence which hybridizes to one or more of SEQ ID NOs 25 to 48 and 108 to 114 under stringent conditions.

50. The polynucleotide of claim 48 or claim 49 which is operably linked to a promoter capable of directing expression of the polynucleotide in a cell.

51. The polynucleotide of claim 50, wherein the cell is a plant cell or animal cell.

52. A vector comprising the polynucleotide according to any one of claims 48 to 51.

53. A method of producing the polypeptide according to any one of claims 44 to 47, the method comprising expressing in a cell the polynucleotide according to any one of claims 48 to 51 and/or a vector of claim 52.

54. A composition for degrading a coumarin based compound, the composition comprising a polypeptide according to any one of claims 44 to 47, and one or more acceptable carriers.

55. A composition for degrading a coumarin based compound, the composition comprising a host cell according to any one of claims 20 to 23, or an extract thereof, and one or more acceptable carriers.

56. The composition of claim 54 or claim 55 which further comprises an electron donor.

57. The composition of claim 56, wherein the electron donor is F₄₂₀H₂, reduced FO, FMNH₂ or FADH₂.

58. The composition of claim 56 or claim 57 which further comprises an enzyme that reduces the electron donor.

59. The composition of claim 58, wherein the electron donor is F₄₂₀H₂ and the enzyme is glucose-6-phosphate dehydrogenase.

60. The composition of claim 58, wherein the electron donor is FMNH₂ and the enzyme is flavin reductase.

61. The composition according to any one of claims 54 to 60, wherein the composition further comprises a plant or a portion thereof.

62. The composition according to any one of claims 54 to 61, wherein the composition further comprises a coumarin based compound.

63. The composition according to any one of claims 54 to 62, wherein the composition further comprises a detergent.

64. The composition according to any one of claims 54 to 63 which is a feedstuff.

65. A method for degrading a coumarin based compound, the method comprising contacting a coumarin based compound with a composition according to any one of claims 54 to 64.

66. A method of preparing a feedstuff, the method comprising mixing a reductase which degrades a coumarin based compound with at least one nutritional substance.

67. A polymeric sponge or foam for degrading a coumarin based compound, the foam or sponge comprising a polypeptide according to any one of claims 43 to 47 immobilized on a polymeric porous support.

68. A method for degrading a coumarin based compound, the method comprising contacting the coumarin based compound to a sponge or foam of claim 67.

69. An extract of a host cell according to any one of claims 20 to 23, a transgenic plant according to any one of claims 24 to 32 or a transgenic non-human animal of claim 34 comprising a reductase which degrades a coumarin based compound.

70. A transgenic plant comprising a host cell comprising enzymes which convert FO to F₄₂₀.

71. A kit for degrading a coumarin based compound, the kit comprising a reductase, and/or a polynucleotide encoding the reductase.

72. The kit of claim 71 which further comprises an electron donor.

73. The kit of claim 72 which further comprises an enzyme that reduces the electron donor.