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WO2008109934A2 - Dégradation de composés à base de coumarine - Google Patents

Dégradation de composés à base de coumarine Download PDF

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Publication number
WO2008109934A2
WO2008109934A2 PCT/AU2008/000319 AU2008000319W WO2008109934A2 WO 2008109934 A2 WO2008109934 A2 WO 2008109934A2 AU 2008000319 W AU2008000319 W AU 2008000319W WO 2008109934 A2 WO2008109934 A2 WO 2008109934A2
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WO
WIPO (PCT)
Prior art keywords
seq
reductase
amino acid
acid sequence
based compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/AU2008/000319
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English (en)
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WO2008109934A3 (fr
Inventor
Matthew C Taylor
David Tattersall
Lyndall J Briggs
Nigel French
Robyn J. Russell
John G Oakeshott
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Commonwealth Scientific and Industrial Research Organization CSIRO
Grains Research and Development Corp
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Commonwealth Scientific and Industrial Research Organization CSIRO
Grains Research and Development Corp
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Publication of WO2008109934A2 publication Critical patent/WO2008109934A2/fr
Publication of WO2008109934A3 publication Critical patent/WO2008109934A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C12Y104/03005—Pyridoxal 5'-phosphate synthase (1.4.3.5), i.e. pyridoxamine 5-phosphate oxidase

Definitions

  • 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.
  • 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 B 1 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.
  • 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).
  • 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.
  • 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.
  • the present invention provides a method of degrading a coumarin based compound, the method comprising contacting the coumarin based compound with a reductase.
  • the coumarin based compound is an aflatoxin.
  • aflatoxins which can be degraded using the methods of the invention include, but are not limited to, aflatoxin Bi, aflatoxin B 2 , aflatoxin Gi, aflatoxin G 2 , aflatoxin Mi and/or aflatoxin M 2 .
  • the reductase can be purified from an Actinobacteridae, or is a fragment/mutant/variant thereof.
  • 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.
  • 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.
  • the reductase is an F 420 dependent reductase. More preferably, the
  • F 420 dependent reductase is a member of the pyridoxamine 5 '-phosphate oxidases
  • the F 420 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 F 420 dependent reductase is a member of the pyridoxamine 5 '-phosphate oxidases (PNPOx) family.
  • the PNPOx F 420 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.
  • the F 420 dependent reductase is a member of the DUF385 family.
  • the DUF385 F 420 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
  • the F 420 dependent reductase is a member of the glyoxalase/bleomycin resistant family.
  • the glyoxalase/bleomycin resistant F 420 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.
  • the F 420 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.
  • the F 420 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.
  • the F 420 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.
  • the reductase has a specific activity against aflatoxin G 1 which is at least 50, more preferably at least 250, and even more preferably at least 500 ⁇ moles/min/mg(enzyme).
  • the specific activity can be determined as outlined in Example 10.
  • the reductase has a molecular weight less than 5OkDa.
  • the reductase has a molecular weight between about 20 and about 40 kDa, more preferably between about 25 and about 35 kDa.
  • the method further comprises providing an electron donor.
  • suitable electron donors include, but are not limited to, F 420 H 2 , reduced FO, FMNH 2 , or FADH 2 .
  • the method further comprises providing an enzyme that reduces the electron donor.
  • the electron donor is F 420 H 2
  • the enzyme can be glucose-6-phosphate dehydrogenase.
  • the electron donor is FMNH 2 and the enzyme can be flavin reductase.
  • 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.
  • 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.
  • the method comprises providing a host cell producing the reductase.
  • 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
  • the present invention provides a host cell comprising an exogenous polynucleotide encoding a reductase which degrades a coumarin based compound.
  • the reductase is an F 420 dependent reductase. More preferably, the F 420 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.
  • 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.
  • 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.
  • the plant cell further comprises enzymes which convert FO to F 420 .
  • two enzymes are required to convert FO to F 420 , 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.
  • three enzymes are required to convert FO to F 420 , 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.
  • the cell further comprises an enzyme that produces reduced F 420 .
  • the enzyme that produces reduced F 420 is glucose-6-phosphate dehydrogenase.
  • 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:50
  • the present invention provides a transgenic plant comprising a host cell comprising enzymes which convert FO to F 420 .
  • the sample is selected from the group consisting of: soil, water, biological material, a feedstuff or a combination thereof.
  • the biological material is plant material.
  • the present invention provides a transgenic non-human animal comprising at least one animal cell of the invention.
  • 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.
  • a coumarin based compound such as aflatoxin
  • 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.
  • the reductase is an F 420 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
  • the method further comprises providing an electron donor.
  • suitable electron donors include, but are not limited to, F 420 H 2 , reduced FO, FMNH 2 , or FADH 2 .
  • the electron donor may be provided in the same composition as the reducatse, or administered independently.
  • 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.
  • 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).
  • the present invention provides a polypeptide produced by a method of the invention.
  • 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.
  • the present invention provides a substantially purified and/or recombinant polypeptide that degrades a coumarin based compound, wherein the polypeptide has reductase activity.
  • 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.
  • the polypeptide is a fusion protein further comprising at least one other polypeptide sequence.
  • 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).
  • 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.
  • 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.
  • the polynucleotide is operably linked to a promoter capable of directing expression of the polynucleotide in a cell.
  • the cell is a plant cell or animal cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition further comprises an electron donor.
  • suitable electron donors include, but are not limited to, F 420 H 2 , reduced FO, FMNH 2 or FADH 2 .
  • the composition further comprises an enzyme that reduces the electron donor.
  • the composition further comprises a plant or a portion thereof.
  • the composition may comprise a portion of a plant such as a peanut.
  • the composition further comprises a coumarin based compound.
  • 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.
  • the composition may further comprises a detergent.
  • the detergent is a non-ionic detergent such as Tween 80.
  • 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.
  • 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.
  • the nutritional substance is grain, hay and/or nuts.
  • 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.
  • 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.
  • 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.
  • the present invention provides a kit for degrading a coumarin based compound, the kit comprising a reductase, and/or a polynucleotide encoding the reductase.
  • the kit further comprises an electron donor.
  • the kit further comprises an enzyme that reduces the electron donor.
  • FIG. 1 AFGl was incubated with resuspended (NtLO 2 SO 4 precipitations and assayed by TLC as described in methods.
  • Aflatoxin negative control, no enzyme Lane 1; M. smegmatis control, lane 2; 60% (NH- I ) 2 SO 4 precipitation, lane 3; 50% (NtL t ) 2 SO 4 precipitation, lane 4; 40% (NR t ) 2 SO 4 precipitation, lane 5; 30% (NIL t ) 2 SO 4 precipitation, lane 6; 20% (NR I ) 2 SO 4 precipitation, lane 7; 10% (NH 4 ) I SO 4 precipitation, lane 8.
  • FIG. 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 lO ⁇ g/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 lO ⁇ g/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.
  • FIG. 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.
  • 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).
  • 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:4 - M. smegmatis reductase MSMEG0048 (Genbank ABK73917) (also known as MSMEG_0048).
  • 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: 10 M. smegmatis reductase MSMEG6537 (Genbank ABK74207) (also known as MSMEG 6576).
  • 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: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: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:34 Nucleotide sequence encoding M. smegmatis reductase MSMEG6537 (Genbank YP_890788).
  • SEQ ID NO:44 Nucleotide sequence encoding M. smegmatis reductase MSMEG3914 (Genbank YP_888200).
  • SEQ ID NO: 52 Nucleotide sequence encoding M. smegmatis flavin reductase (from
  • SEQ ID NO:55 - M. smegmatis FbiA enzyme (MSMEG1829) (Genbank ABK71517)
  • 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)
  • SEQ ID NO:102 Streptomyces coelicolor reductase (Genbank CAC14340.1).
  • 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: 120 S. coelicolor FbiC enzyme (Genbank CAB88436.1).
  • SEQ ID NO: 128 Nucleotide sequence encoding Methanococcus maripludies FbiC subunit CofH (Genbank CAF29612.1).
  • SEQ ID NO: 130 Nucleotide sequence encoding M. bovis FbiA enzyme (Genbank CAD95381.1).
  • 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: 138 Nucleotide sequence encoding S. coelicolor reductase (Genbank CAC36755.1).
  • 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 YP_001073320).
  • SEQ ID NO: 150 - M. smegmatis MSMEG2392 (Genbank ABK73289) (also known as
  • MSMEG2392 (Genbank YP_952961.1).
  • 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).
  • the term “degrades”, “degradation” and variations thereof refers to the product of reductase activity being less stable than the coumarin based compound substrate.
  • the reductase degrades AFGl to produce a compound with a molecular weigth of about 258.06 Da.
  • treating 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.
  • biological material is used herein in its broadest sense to include any product of biological origin.
  • 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.
  • 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, Sorg
  • 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.
  • the extract can be a whole portion of the organism such as the seed of a plant.
  • the term "coumarin based compound” refers to any compound that comprises, or consist of, coumarin (2-chromenone) (CAS Registry No. 91-64-5).
  • 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 B 2 (CAS Registry No. 7220-81-7), aflatoxin G 1 (CAS Registry No. 1165-39-5) and aflatoxin G 2 (CAS Registry No. 7241-98-7) are major aflatoxin derivatives produced by fungi, as well as aflatoxin M 1 (CAS Registry No. 6795-23-9) and aflatoxin M 2 (CAS Registry No. 6885-57-0), which are often detected in milk.
  • Aflatoxin B 1 is considered the most toxic and is produced by both Aspergillus flavus and Aspergillus parasiticus.
  • Aflatoxin Gi and G 2 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.
  • the coumarin based compound is an ⁇ / ⁇ unsaturated ketone or ester. Examples of such compounds include aflatoxins as well as the compounds provided in Table 1.
  • the present invention relates to the use of a reductase to degrade a coumarin based compound.
  • reductase refers to an enzyme that reduces the coumarin based compound to form a less stable product.
  • reductases 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.
  • the reductase is a member of the PNPOx protein family or DUF385 protein family.
  • PNPOx reductase 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 F 420 dependent manner (Manjunatha et al., 2006).
  • 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.
  • a reductase useful for the methods of the present invention is an F 420 dependent reductase.
  • F 420 dependent reductase refers to a reductase which can utilize F 420 H 2 , or another suitable cofactor such as FMNH 2 , or FADH 2 , as an electron donor to reduce a coumarin based compound such as aflatoxin.
  • F 420 oxidoreductase refers to a reductase which can utilize F 420 H 2 , or another suitable cofactor such as FMNH 2 , or FADH 2 , 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).
  • an F 420 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 F 420 H 2 ), 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.
  • F 420 and FO have been described by, for example, Choi et al. (2002).
  • F 420 can be produced by extraction from M. smegmatis as per the methods of Isabelle et al. (2002).
  • F 420 H 2 can be produced from F 420 using various enzymes such as an F 420 -dependent Glucose-6-Phosphate Dehydrogenase (FGD).
  • 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 F 420 H 2 is F 420 :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.
  • HPP 4-hydroxy phenylpyruvate
  • compound 6 an intermediate in riboflavin synthesis
  • 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:
  • FO can be used as a substrate for producing F 420 .
  • a phosphate group is added and a ⁇ -linked glutamate incorporated through the activity of FbiA and FbiB enzymes (see, for example, Choi et al., 2002).
  • FbiA enzymes include those which comprise an amino acid sequence provided as SEQ ID NO:55 or SEQ ID NO: 129
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a "biologically active fragment” is a portion of a polypeptide as described herein which maintains a defined activity of the full-length polypeptide.
  • a biologically active fragment of a reductase as described herein (but not a flavin reductase or a F 420 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.
  • biologically active fragments are at least 100 amino acids in length.
  • 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.
  • 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.
  • a polynucleotide described herein can be subjected to in vitro mutagenesis.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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, ⁇ -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, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ - methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogues in general.
  • 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.
  • 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.
  • 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.
  • polynucleotide is used interchangeably herein with the term “nucleic acid”.
  • 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.
  • the cell is a cell that does not naturally comprise the polynucleotide.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • Tm thermal melting point
  • 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 30 0 C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 6O 0 C 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
  • 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 65 0 C, followed by one or more washes in 0.2.xSSC, 0.01% BSA at 50 0 C.
  • 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.
  • moderate stringency hybridization conditions are hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55 0 C, followed by one or more washes in IxSSC, 0.1% SDS at 37 0 C.
  • 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.
  • 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 4O 0 C, followed by one or more washes in 2xSSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 5O 0 C.
  • Other conditions of low stringency that may be used are well known in the art, see, e.g., Ausubel et al. (supra) and Krieg
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 sarcom
  • 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.
  • 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.
  • 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.
  • transcription control signals e.g., promoters, operators, enhancers
  • translational control signals e.g., ribosome binding sites, Shine-Dalgarno sequences
  • 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,
  • 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).
  • 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,
  • plant 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ , ⁇ binding protein gene promoter.
  • CaMV cauliflower mosaic virus
  • FMV Figwort mosaic virus
  • FMV Figwort mosaic virus
  • 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.
  • source tissues of the plant such as the leaf, seed, root or stem
  • 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.
  • 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.
  • CHS glucoamylase
  • 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-H 30 symporter promoter
  • the promoter for the thylakoid membrane protein genes from spinach PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS).
  • promoters for the chlorophyll ⁇ , ⁇ -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).
  • 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 ⁇ -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.
  • 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.
  • ADPGPP ADPglucose pyrosynthase
  • 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.
  • 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.
  • 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 3 1 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.
  • Acceleration methods include, for example, microprojectile bombardment and the like.
  • microprojectile bombardment 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 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.
  • An illustrative embodiment of a method for delivering DNA into Zea mays cells by acceleration is a biolistics ⁇ -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.
  • PDS- 1000/He gun available from Bio-Rad Laboratories.
  • 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.
  • 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.
  • one or more screens are also positioned between the acceleration device and the cells to be bombarded.
  • 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).
  • 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.
  • a transgenic plant formed using Agrob ⁇ cterium 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.
  • 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 development or regeneration of plants containing the foreign, exogenous gene is well known in the art.
  • 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.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • 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.
  • 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.
  • 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.
  • Heterologous DNA can be introduced, for example, into fertilized mammalian ova.
  • 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.
  • developing embryos are infected with a retrovirus containing the desired DNA, and transgenic animals produced from the infected embryo.
  • 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 of the present invention include excipients, also referred to herein as "acceptable carriers".
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • detergents which might be used include TweenTM and sodium dodecyl sulfate (SDS).
  • 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).
  • 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.
  • a composition of the invention comprises F 420 .
  • F 420 can be extracted from Mycobaterium sp. as described by Isabelle et al. (2002).
  • F 420 can be extracted from Mycobaterium sp. as described by Isabelle et al. (2002).
  • F420 can be synthesized using the procedure as described by Choi et al. (2002).
  • Reduced F 420 (F 420 H 2 ) can be produced using a glucose-6-phosphate dehydrogenase as described by Purwantini and Daniels (1998).
  • 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).
  • FO can be synthesized using the procedure as described by Choi et al. (2002).
  • a composition of the invention comprises flavin mononucleotide (FMN). This can be obtained commercially from, for example, Sigma
  • 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.
  • a composition of the invention is a feedstuff.
  • 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
  • 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.
  • substances with nutritional value include, but are not limited to, macronutrients such as edible fats, carbohydrates and proteins.
  • 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.
  • 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.
  • vitamins and minerals 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 may also be added.
  • the components utilized in the feedstuff compositions of the present invention can be of semi-purified or purified origin.
  • semi-purified or purified is meant a material which has been prepared by purification of a natural material or by de novo synthesis.
  • the reductase is used in the production of the feedstuff.
  • 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.
  • the feedstuff comprises a plant of the invention, and/or a part of said plant, and/or an extract of said plant.
  • Example 1 Mycobacterium sme ⁇ matis degrades aflatoxin
  • Aflatoxins B 1 , B 2 , G 1 and G 2 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 4 0 C 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 Na 2 HPO 4 , 88 mM KH 2 PO 4 , 9 mM NaCl, pH 6.0) supplemented with 4 ⁇ g/mL aflatoxin G 1 or 6 ⁇ g/mL aflatoxin B 1 , and incubated for 48 h at 28 0 C on an orbital shaker (200 rpm) before 5 ⁇ L of each culture was spotted and dried onto silica gel 60 F 254 thin layer chromatography (TLC) plates (Merck).
  • TLC thin layer chromatography
  • 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).
  • 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.
  • Random insertion mutants of M. smegmatis mc 2 155 (Sutherland et al., 2002a and 2002b) were generated with the EZ::TN ⁇ R6K ⁇ ori/KAN-2> insertion kit (Epicentre).
  • the EZ::TN ⁇ R6K ⁇ ori/KAN-2> tnp transposase complex (1 ⁇ L) was electroporated into 100 ⁇ L of electrocompetent M. smegmatis me 2 155 cells.
  • Electrocompetent cells were prepared from a 100 mL culture of cells (OD 600 ⁇ 0.8) grown at 37 0 C 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 ⁇ F and 1000 ⁇ . Electrocompetent cell preparation and transformation were performed at 4 0 C.
  • the cells were resuspended in LB broth (Sambrook et al., supra) containing 0.05% (v/v) Tween-80, incubated at 37 0 C at 200 rpm, before plating on LB agar containing 20 ⁇ g/mL kanamycin. The plates were incubated at 37 0 C for 3 days to allow colony formation. Approximately 2000 mutants were obtained from one transformation event.
  • M. smegmatis me 2 155 transposon insertion mutants were individually inoculated into 2 mL square wells of 96 deep well growth blocks (Axygen). Each well contained 200 ⁇ L of PYB supplemented with 20 ⁇ g/mL kanamycin and 4 ⁇ g/mL aflatoxin G 1 . The growth blocks were sealed with silicone mats (Axygen) and incubated for 3 days (37 0 C at 200 rpm) before 5 ⁇ L 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 G 1 compared to that of wildtype cells were selected as aflatoxin G 1 - 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 ⁇ g/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).
  • FGD is the only protein that has been identified in M. smegmatis to catalyse the reduction of F 420 to F 420 H 2 (Purwantini et al, 1997).
  • F 420 was prepared from M. smegmatis me 2 155 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 F 420 containing material with 1.0 M NaCl, 20 mM Tris-HCl, pH 7.5.
  • the F 420 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 H 2 O.
  • the column was operated at approximately 5 mL/min.
  • the F 420 fraction was applied, the column washed with 20 mL of deionised H 2 O, then the bound F 420 eluted with 20% (v/v) methanol wash.
  • the F 420 was diluted 2-fold with deionised H 2 O, freeze-dried to a powder, before resuspension in deionised H 2 O and storage at -2O 0 C.
  • the cofactor F 420 was purified from M. smegmatis cultures at 0.27 ⁇ mol/g of F 420 per dry weight of M. smegmatis.
  • the resuspended solution was quantified to have a concentration of 114.2 ⁇ M and was used for subsequent experiments.
  • the M. smegmatis mc 2 155 fgd gene was cloned based on the methods of Purwantini and Daniels (1998). Briefly, the fgd gene was amplified from genomic
  • a single colony obtained from the transformation was then used to inoculate LB containing 50 ⁇ g/mL carbenicillin.
  • the culture was grown (37 0 C at 200 rpm) until an OD ⁇ oo 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 ⁇ g/mL carbenicillin.
  • the culture was grown (37 0 C at 200 rpm) until an OD 6O0 of 0.4 was reached.
  • IPTG Isopropyl- ⁇ -D-thiogalactopyranoside
  • 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 -80 0 C.
  • FGD activity was determined by a spectrophotomeric assay that measured the decrease in absorbance at 420 run due to the reduction of F 420 to F 420 H 2 (Purwantini and Daniels, 1996) and calculated using the extinction coefficient used in the quantification of purified F 420 .
  • One unit of FGD activity was defined as the amount of enzyme required to reduce one ⁇ mole OfF 420 per min. Reduction Of F 420 was performed at room temperature (22 0 C).
  • the assay mixture was buffered with 50 mM sodium phosphate, pH 7.0 and contained 25 ⁇ M F 420 , 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).
  • Soluble bacterial extracts, purified proteins, and partially purified enzymes were tested for afiatoxin G 1 -degradation activity by the following assays performed at room temperature (22 0 C) in 1.5 mL microfuge tubes in the dark.
  • the reaction mixture was typically made up to lO ⁇ l with 5 ⁇ l of sample and 5 ⁇ l of reaction buffer, containing:
  • TLC thin layer chromatography
  • 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.
  • AFG 1 is used as the rate of degradation is faster than that OfAFB 1 .
  • M. smegmatis me 2 155 was inoculated into 1.5 L of LB and grown for three days (37 0 C at 200 rpm). All the following steps were performed at 4 0 C.
  • 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 ⁇ m;
  • 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 ⁇ m filter prior to ammonium sulphate ((NHU) 2 SO 4 ) precipitation.
  • Saturated (NFLO 2 SO 4 solution (O 0 C) 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 (NH 4 ) 2 SO 4 solution (O 0 C) to give 70% saturation, the mixture was incubated overnight.
  • the pellet obtained after centrifugation (20,000 g for 30 min, 4 0 C) was resuspended in 1 M (NH 4 ) 2 SO 4 , 20 mM Tris-HCl, pH 7.5, passed through a 0.22 ⁇ m 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.
  • 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
  • the alkylated protein was then precipitated with the 2-D PAGE clean-up kit (GE Healthcare) before resuspension in 10 ⁇ L of 50 mM NH 4 HCO 3 and addition of 0.5 ⁇ g sequencing grade trypsin (Promega). Digestion was performed overnight at 28 0 C and then stopped by addition of 1 ⁇ L of 10% (v/v) formic acid.
  • Peptides (5 ⁇ L) from each digest were subjected to HPLC separation on an Agilent 1100 Series Capillary LC system by application to an Agilent Zorbax SB-C18 5 ⁇ m 150 x 0.5mm column with a flow rate of 0.1% (v/v) formic acid/5% (v/v) acetonitrile at 20 ⁇ l/min for one minute then eluted with gradients of increasing acetonitrile concentration to 0.1% formic acid/20% acetonitrile over one minute at 5 ⁇ L/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.
  • M. smegmatis soluble cell extracts were first subject to ammonium sulphate ((NILj) 2 SO 4 ) precipitation by increasing the amount of (NH t ) 2 SO 4 by 10% increments from 10% to 60% (NIL t ) 2 SO 4 ( 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) 2 SO 4 precipitates, lane 3 and 6 respectively, show the highest AFGl degradation activity of the (NILi) 2 SO 4 precipitates.
  • (NILj) 2 SO 4 ammonium sulphate
  • 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.
  • PNPOx pyridoxine 5 'phosphate oxidase family.
  • PNPOx enzymes characterised to date form dimers and bind flavin mononucleotide (FMN), which is the cofactor most similar to F 420 .
  • 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.
  • 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.
  • 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 F 420 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.
  • 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.
  • BRP glyoxalase/bleomycin resistant protein family
  • MSMEG2303 and MSMEGlOlO have identical amino acid sequence, yet are found on different regions of the M. smegmatis genome.
  • Candidate genes for aflatoxin degradation and the FbiC gene were amplified from M. smegmatis me 2 155 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 GatewayTM donor vector pDONR201 (Invitrogen), as per the manufacturers instructions.
  • Amplicons were purified away from primer dimers using either PEG 8000 following GatewayTM 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
  • GatewayTM 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-AITM 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 GatewayTM 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.
  • 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
  • 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
  • TalonTM TalonTM 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.
  • ice cold purification buffer 8M urea, 30OmM NaCl, 5OmM Sodium phosphate pH7.5
  • 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.
  • 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 ⁇ M and was used for all further experiments.
  • Example 10 - Rates of Mycobacterium sme ⁇ matis F420 dependent reductase enzymes.
  • Enzymatic assays were conducted in either lO ⁇ l or 20 ⁇ l 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 (F 420 5 ⁇ M, FGD units 0.2U/ ⁇ L, 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 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 (AFG 1 , 7.4min; AFB 1 , 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 ⁇ moles(aflatoxin)/mg(protein)/min.
  • Protein concentrations were either determined by use of the molar extinction coefficient for purified proteins as measured at A 280 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).
  • MSMEG6591 requires the reducing agent, dithiothreitol (DTT), for catalytic activity.
  • DTT dithiothreitol
  • Other enzymes closely related to MSMEG6591, namely MSMEG5954 and MSMEGlOlO showed no activity to AFG 1 .
  • 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.
  • 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.
  • FIG. 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 10 8 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 10 7 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.
  • 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.
  • 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 37 0 C, 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 -2O 0 C until further processing.
  • F 42 Q 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.
  • 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 20 ⁇ l reactions in the dark at room temperature for 16 hours.
  • the reaction mix contained:
  • 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 is reduced by incubation with M. smegmatis flavin reductase (MsFR) previously cloned in our laboratory (Sutherland et al. 2002a and 2002b).
  • MsFR M. smegmatis flavin reductase
  • Aflatoxin 30 ⁇ M 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
  • 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 F 420 , with an apparent activity of 0.0048 ⁇ mol/min/ ⁇ mol enzyme with FMN.
  • F 420 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 F 420 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 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 u n iparental 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.
  • 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 l ⁇ g/ml benzylaminopurine and 0.5 ⁇ g/ml indole acetic acid (MS9 agar) (Murashige and Skoog, 1962) and cultured for 48 hours at 25 0 C. Tobacco leaf segments were carefully transferred to fresh MS9 agar plates with antibiotics to suppress Agrobacterium growth (150 ⁇ g/ml Timentin) and to select for vector transformation (lOO ⁇ g/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.
  • MS9 agar l ⁇ g/ml benzylaminopurine and 0.5 ⁇
  • 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.
  • 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 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.
  • MSMEG2852 The activity of MSMEG2852 was confirmed using the F 42 o recycling system as in Example 10. After 48 hours incubation of AFGl with plant soluble extract and recycled F 420 , degradation of AFGl was observed by HPLC analysis. Analysis by LC- MS, (Example 10) confirmed degradation of AFGl in an F 420 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.
  • Petrikovics et al. (2000a). Toxicology Science 57: 16-21. Petrikovics et al. (2000b). Drug Delivery 7: 83-89.

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Abstract

La présente invention porte sur l'identification d'enzymes réductases qui dégradent les composés à base de coumarine tels que les aflatoxines. L'invention porte sur des procédés, comprenant ceux reposant sur des organismes transgéniques, pour la dégradation de composés à base de coumarine, tels que les aflatoxines.
PCT/AU2008/000319 2007-03-09 2008-03-07 Dégradation de composés à base de coumarine Ceased WO2008109934A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016107550A1 (fr) * 2014-12-30 2016-07-07 暨南大学 Aflatoxine-détoxifizyme ayant une résistance améliorée à la trypsine
CN108514592A (zh) * 2018-04-27 2018-09-11 山东省花生研究所 一种降解黄曲霉毒素的中药制剂及其制备方法
CN112725294A (zh) * 2021-01-29 2021-04-30 潍坊康地恩生物科技有限公司 黄曲霉毒素降解酶突变体及其高产菌株

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US5919685A (en) * 1997-08-08 1999-07-06 Incyte Pharmaceuticals, Inc. Human aflatoxin B1 aldehyde reductase

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016107550A1 (fr) * 2014-12-30 2016-07-07 暨南大学 Aflatoxine-détoxifizyme ayant une résistance améliorée à la trypsine
CN108514592A (zh) * 2018-04-27 2018-09-11 山东省花生研究所 一种降解黄曲霉毒素的中药制剂及其制备方法
CN112725294A (zh) * 2021-01-29 2021-04-30 潍坊康地恩生物科技有限公司 黄曲霉毒素降解酶突变体及其高产菌株

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