JP2013236641A - Pyranosone dehydratase from phanerochaete chrysosporium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide applications of sequence information relating to pyranosone dehydratase.SOLUTION: A use of pyranosone dehydratase in the conversion of AF (1,5-anhydro-D-fructose) to APP (ascopyrone P) and microthecin and the conversion of glucosone to cortalcerone is related. A polypeptide of a specific sequence is comprised, which is selected from: (i) a polynucleotide comprising the nucleotide sequence of a specific sequence or the complement thereof; (ii) a polynucleotide comprising a nucleotide sequence capable of hybridizing to the nucleotide sequence of the specific sequence, or a fragment thereof; (iii) a polynucleotide comprising a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence of the specific sequence; and (iv) a polynucleotide comprising a nucleotide sequence which is degenerated as a result of the genetic code to the polynucleotide of the specific sequence.

Description

(Field of Invention)
The present invention relates to sequences. Specifically, the present invention relates to the amino acid sequence of pyranozone dehydrase and the nucleic acid sequence encoding it.

(Technical background and prior art)
Glucose is oxidized with pyranose 2-oxidase (EC 1.1.3.10, P 2 O) to form glucosone (D-arabino-hexos-2-urose) and then converted to cortalcerone by pyranozone dehydrase (PD). It has been well reported in the literature (Non-patent document 1; Non-patent document 2). Both P2O and PD have been purified from fungi and P2O has been cloned. PD has been purified from Polyporus obtusus by Koths et al. (1992) and from Phanerochaete chrysosporium by Gabriel et al. (1993). However, to date, neither the amino acid sequence nor the nucleotide sequence of PD has been characterized.

It has been established in the art that starch can be converted to 1,5-anhydro-D-fructose (AF) (Non-patent Document 3). It was further shown that some fungal and red algae extracts could possibly convert AF to microsecins enzymatically, but the related enzymes have not been isolated, purified or characterized (Non-Patent Documents) 4; Non-patent document 5). To date, it has not been suggested that PD may play a role in the conversion of AF to microsecin.
It has also been reported that ascopyrone P (APP) can be produced from AF (Non-patent Document 6). Similarly, there was no evidence to suggest involvement of PD in this process.

Koths, K .; Halenbeck, R .; Moreland, M .; 1992, Carbohydr. Res. Vol. 232 No. 1, PP. 59-75 Gabriel, J. et al. Volc, J; Sedmera, P; Daniel, G .; Kubatova, E .; 1993, Arch. Microbi. 160: 27-34. S. Yu and J.H. Marcussen, Regent Advances in Carbohydrate Bioengineering; Gilbert, H .; J. et al. Davies, G .; J. et al. Henrissat B .; Svensson, B .; Eds. Royal Society of Chemistry (RS.C) Press, 1999, 242-250; Baute, MA. Diffieux, G .; Baute, R .; , 1986, Phytochemistry (Oxf) vol. 25: 1472-1473 Broberg, A.M. Kenne, L .; , And Pedersen, M .; (1996) Phytochemistry (Oxf), 41: 151-154. Baute, MA. Diffieux, G .; Vercauteren, J .; Baute, R .; Badoc, A .; 1993, Phytochemistry (Oxf) vol. 33 no. 1,41-45

(Summary of the Invention)
In a broad aspect, the present invention relates to the characterization of amino acid and nucleotide sequences encoding pyranozone dehydrase.

  A further aspect of the invention relates to previously undisclosed uses of pyranozone dehydrase, including the conversion of AF to microthecin and APP and the conversion of glucosone to cortalcerone.

  Aspects of the invention are set out in the claims and in the following description.

Briefly stated, some aspects of the invention relate to the following.
1. 1. New amino acid sequence 2. Novel nucleotide sequence 3. Method for preparing the amino acid sequence 4. Method for preparing the nucleotide sequence 5. Expression system comprising the nucleotide sequence 6. a method for expressing said nucleotide sequence; 7. a transformed host / host cell comprising said nucleotide sequence; 8. Use of the above amino acid sequences Use of the above nucleotide sequence.

  As used in connection with the present invention, the terms “expression”, “express”, “expressed”, and “expressible” refer to the terms “transcription”, “transcribe”, “transcribed”, and “expressed”, respectively. It is synonymous with “transferable”.

Other aspects relating to the nucleotide sequences of the present invention include: constructs comprising the sequences of the present invention; vectors comprising the sequences of the present invention; plasmids comprising the sequences of the present invention; transformed cells comprising the sequences of the present invention. A transformed tissue comprising the sequence of the invention; a transformed organ comprising the sequence of the invention; a transformed host comprising the sequence of the invention; a transformed organism comprising the sequence of the invention. The invention also includes a method of expressing a nucleotide sequence using a sequence of the invention, such as expression in a host plant cell, including a method of transferring the sequence of the invention.
For example, the present invention provides the following items.
(1) The following:

An isolated polypeptide comprising at least one amino acid sequence selected from or a variant, homolog or derivative thereof, wherein X is an unknown amino acid residue peptide.
(2) The polypeptide according to item 1, comprising at least one sequence selected from item 1.
(3) The polypeptide according to item 1 or item 2, which has pyranozone dehydrase activity.
(4) A polypeptide that is immunoreactive with an antibody raised against the purified polypeptide according to any one of items 1 to 3.
(5) An isolated polynucleotide encoding the polypeptide according to any one of items 1 to 4, or a variant, homologue, fragment or derivative thereof.
(6) Following:
(I) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or its complement;
(Ii) a polynucleotide comprising a nucleotide sequence capable of hybridizing to the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof;
(Iii) a polynucleotide comprising a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence of SEQ ID NO: 1; and (iv) a polynucleotide comprising a polynucleotide sequence that is degenerate as a result of the genetic code for the polynucleotide of SEQ ID NO: 1. ,
6. The isolated polynucleotide of item 5, selected from
(7) The nucleotide sequence according to any one of item 5, item 6, or item 7, wherein the nucleotide sequence can be obtained from Phanerochaete chrysosporium, Polyporus obtusus, or Corticium caeruleum.
(8) The nucleotide sequence according to any one of item 5, item 6, or item 7, wherein the nucleotide sequence can be obtained from the order of Pezzales, Auriculariales, Aphyllophorales, Agaricales, or Gracilariaes.
(9) The nucleotide sequence is defined as Aleuria aurantia, Pezza badia, P. et al. succosa, Sarcophaera eximia, Morchella conica, M. et al. costata, M.C. elata, M.M. esculenta, M.M. esculenta var. rotunda, M.M. hortensis, Gyromitra infula, Auricularia mesenterica, Pulcherricium caeruleum, Peniophora quercina, Phanerochaete sordida, Villeminia comendum, Sturm. sanginololentum, lopharia spadicea, sparassis laminosa, boletolpsis subsquamosa, bjerkandera adusta, trichaptum biformis, cerrenapinocrinporic, pyrenacinocor. sanguineus, junghunia nitida, ramaria flava, clauvulinopsis
helvola, C.I. helvola var. geoglossoides, V.G. pulchra, Clitocybe cyatiformis, C.I. dicolor, C.I. gibba, C.I. odora, Lepista caespitosa, L. inversa, L.A. luscina, L .; nebularis, Mycena seniii, Pleurocybella porrigens, Marasius oreales, Incybe pyriodora, Gracilaria varrucosa, Gracilaria tenurapistipitippit. Or the nucleotide sequence of any one of item 5, item 6 or item 7, which can be obtained from Gracilariopsis lemaneformis. (10) The following:
(I) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or its complement;
(Ii) a polynucleotide comprising a nucleotide sequence capable of hybridizing to the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof;
(Iii) a polynucleotide comprising a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence of SEQ ID NO: 1; and (iv) a polynucleotide comprising a polynucleotide sequence that is degenerate as a result of the genetic code for the polynucleotide of SEQ ID NO: 1. ,
An isolated polynucleotide selected from.
(11) A construct comprising the nucleotide sequence according to any one of items 5 to 10. (12) A vector comprising the nucleotide sequence according to any one of items 5 to 10.
(13) An expression vector comprising the polynucleotide sequence of any one of items 5 to 10 operably linked to a regulatory sequence capable of directing expression of the polynucleotide in a host cell.
(14) A host cell in which the nucleotide sequence according to any one of items 5 to 10 is incorporated.
(15) An isolated polypeptide encoded by the polynucleotide sequence of SEQ ID NO: 1, or a variant, homologue, fragment or derivative thereof.
(16) The isolated polypeptide according to item 15, wherein up to 7 amino acids have been removed from the N-terminus.
(17) The isolated polypeptide according to item 15 or item 16, having at least 75% identity with the polypeptide sequence encoded by SEQ ID NO: 1.
(18) An antibody capable of binding to the polypeptide according to any one of items 1 to 4 or item 15 to item 17.
(19) Item 1 to Item 4 comprising the step of expressing the nucleotide sequence according to any one of Items 5 to 10 and, if necessary, isolating and / or purifying the nucleotide sequence. Alternatively, a process for preparing the polypeptide according to any one of Items 15 to 17.
(20) A process for preparing microthecin using the polypeptide according to any one of items 1 to 4 or 15 to 17.
(21) A process for preparing ascopyrone P using the polypeptide according to any one of items 1 to 4 or items 15 to 17.
(22) The process according to item 20 or item 21, comprising the step of reacting the polypeptide with 1,5-anhydro-D-fructose.
(23) The process according to item 21, further comprising the use of APP synthase in the preparation of ascopyrone P.
(24) The process according to item 23, comprising the step of reacting APP synthase and the polypeptide with 1,5-anhydro-D-fructose.
(25) A process according to item 20, comprising a step of contacting the polypeptide with glucan lyase and dextrin starch.
(26) A process for preparing cortalcerone using the polypeptide according to any one of items 1 to 4 or 15 to 17.
(27) A process according to item 26, comprising a step of reacting the polypeptide with glucosone.
(28) A process according to item 26, comprising a step of reacting the polypeptide with glucose and pyranose 2-oxidase.
(29) A process for preparing microsesin, comprising the step of reacting pyranozone dehydrase with 1,5-anhydro-D-fructose.
(30) A process for preparing microsesin comprising reacting pyranozone dehydrase with glucose and dextrin starch.
(31) A process for preparing ascopyrone P, comprising a step of reacting pyranozone dehydrase and APP synthase with 1,5-ahydro-D-fructose.
(32) A process for preparing cortalcerone, comprising the step of reacting pyranozone dehydrase with glucosone.
(33) A process for preparing cortalcerone comprising the step of reacting pyranozone dehydrase with glucosone and pyranose 2-oxidase.
(34) Use of microsesin, cortalcerone, or a derivative or isomer thereof in the prevention and / or inhibition and / or killing of the pathogen Aphanomyces.
(35) The use according to item 34, wherein the pathogen is Aphanomyces cochlioides.
(36) The use according to item 34 or item 35, wherein the derivative of microsecin is 2-furyl-hydroxymethyl-ketone or 4-deoxy-glycero-hexo-2,3-ziruose.
(37) The use according to item 34 or item 35, wherein the derivative of cortalcerone is 2-furylglyoxal.
(38) The use according to any one of items 34 to 37 in the treatment of plants or plant seeds.
(39) Use of microsecin, cortalcerone, or a derivative or isomer thereof as a protective agent for plants or seeds.
(40) The use according to any one of items 34 to 39 in the treatment of sugar beet seeds.
(41) An enzyme having pyranozone dehydrase activity substantially as described herein and associated with the accompanying examples.
(42) A nucleotide sequence encoding an enzyme having pyranozone dehydrase activity substantially as described herein and associated with the accompanying examples.
(43) A process for preparing Ascopyrone P substantially as described herein and related to the accompanying Examples.
(44) A process for preparing microthecin substantially as described herein and related to the accompanying examples.
(45) A process for preparing cortalcelone substantially as described herein and associated with the accompanying examples.
(46) A method for preventing and / or inhibiting the growth of the pathogen Aphanomices substantially as described herein and associated with the appended examples and / or killing the pathogen Aphanomices.
For ease of reference, these and further aspects of the invention will now be discussed by the section with the appropriate headings. However, the teaching of each section is not necessarily limited to each particular section.

FIG. 1 shows electrophoresis of PD1 (pyranozone dehydrogenase isoform 1) on an 8-25% gradient gel. More particularly, FIG. 1A shows SDS-PAGE: lanes 1 and 2 (from left), Novex and Pharmacia protein markers respectively; lanes 3, 4, and 5, purified PD1. FIG. 1B shows NativePAGE: lanes 1, 2, 3, purified PD1; lane 4, Pharmacia protein marker, lane 5, partially purified Pharmacia protein marker. The gel was stained with Pharmacia's PastGel Blue R. FIG. 1 shows electrophoresis of PD1 (pyranozone dehydrogenase isoform 1) on an 8-25% gradient gel. More particularly, FIG. 1A shows SDS-PAGE: lanes 1 and 2 (from left), Novex and Pharmacia protein markers respectively; lanes 3, 4, and 5, purified PD1. FIG. 1B shows NativePAGE: lanes 1, 2, 3, purified PD1; lane 4, Pharmacia protein marker, lane 5, partially purified Pharmacia protein marker. The gel was stained with Pharmacia's PastGel Blue R. FIG. 2 shows a partial amino acid sequence of pyranozone dehydrogenase. FIG. 3A shows the use of 1,5-anhydro-D-fructose and PD for microsecin production. The reaction mixture consists of 5 μl (3.0%) 1,5-anhydro-D-fructose, 5 μl PD preparation, 65 μl sodium phosphate buffer (pH 6.0), and water to a final volume of 0.7 ml. It was. The reaction was monitored by scanning between 350-190 nm. A reaction time of 0 minutes was used as a blank. An absorption peak near 230 nm indicates the formation of microsecin. The absorption at 265 nm indicates a first formation of an AF-derived intermediate before conversion to microsecin. FIG. 3B shows the production of microsecin and its intermediates. The reaction mixture was 10 μl partially purified PD (ammonium sulfate fraction at 25-50% saturation of cell-free extract from Phanerochaete chrysosporum), 25 μl AF (3.0%, weight / volume), 100 μl sodium phosphate It consisted of a buffer (0.1 M, pH 6.5) and 0.84 ml of water. The reaction was initiated by the addition of substrate AF. The reaction was performed at 22 ° C. Microsecin and its intermediate formation were monitored at 230 nm and 260 nm, respectively. It is observed that the intermediate is first formed and stops after about 20 minutes. Microsecin formation was delayed but continued until almost all of the AF in the reaction mixture was consumed. FIG. 4 shows the pyranozone dehydrogenase (PD) from the fungus Phanerochaete chrysosporum comprising SEQ ID NO: 1 (upstream regulatory region (−1 to −288), coding region (1 to 3146), and downstream region (3147 to 3444). Encoding gene). The putative starch codon is ATG (bold) and the stop codon is TGA TAG (bold). If translation is expected to start from the bold codon ATG, the purified functional PD corresponds to the N-terminal 7-amino acid truncated PD. FIG. 4 shows the pyranozone dehydrogenase (PD) from the fungus Phanerochaete chrysosporum comprising SEQ ID NO: 1 (upstream regulatory region (−1 to −288), coding region (1 to 3146), and downstream region (3147 to 3444). Encoding gene). The putative starch codon is ATG (bold) and the stop codon is TGA TAG (bold). If translation is expected to start from the bold codon ATG, the purified functional PD corresponds to the N-terminal 7-amino acid truncated PD. FIG. 5 shows the upstream region, the coding region, and the downstream region of the pyranozone dehydrogenase (PD) gene from the fungus Phanerochaete chrysosporium. A DNA sequence can theoretically encode three proteins having different amino acid sequences. Bold amino acids were found by amino acid sequencing of purified functional PD. Underline the identified intron. FIG. 5 shows the upstream region, the coding region, and the downstream region of the pyranozone dehydrogenase (PD) gene from the fungus Phanerochaete chrysosporium. A DNA sequence can theoretically encode three proteins having different amino acid sequences. Bold amino acids were found by amino acid sequencing of purified functional PD. Underline the identified intron. FIG. 5 shows the upstream region, the coding region, and the downstream region of the pyranozone dehydrogenase (PD) gene from the fungus Phanerochaete chrysosporium. A DNA sequence can theoretically encode three proteins having different amino acid sequences. Bold amino acids were found by amino acid sequencing of purified functional PD. Underline the identified intron. FIG. 5 shows the upstream region, the coding region, and the downstream region of the pyranozone dehydrogenase (PD) gene from the fungus Phanerochaete chrysosporium. A DNA sequence can theoretically encode three proteins having different amino acid sequences. Bold amino acids were found by amino acid sequencing of purified functional PD. Underline the identified intron. FIG. 6 shows the final appearance of sugar beet seeds treated according to Example 3. FIG. 7 shows the screening of the effects of various concentrations of microsecin against the pathogen Aphanomyces cochlioides that roots sugar beet. FIG. 8 shows the screening of the effect of microsecin at various concentrations on the pathogens Phythium ultinum and Rhizotonia solani, respectively, that rot the sugar beet. FIG. 9 shows the screening of the effect of microsecin at various concentrations on each of the pathogens Phythium ultinum and Rhizotonia solani that root rot sugar beet.

(Detailed Disclosure of Invention)
One aspect of the present invention includes the following:

Relates to an isolated polypeptide comprising at least one amino acid sequence selected from wherein X is an unknown amino acid residue, or a variant, homolog or derivative thereof.

In yet a further aspect, the invention relates to a nucleotide sequence selected from:
(A) a nucleotide sequence encoding the amino acid sequence;
(B) a nucleotide sequence that is a variant, homolog, derivative or fragment of the nucleotide sequence of (a);
(C) a nucleotide sequence that is the complement of the nucleotide sequence of (a);
(D) a nucleotide sequence that is the complement of a nucleotide sequence variant, homologue, derivative, or fragment of (a);
(E) a nucleotide sequence capable of hybridizing to the nucleotide sequence of (a);
(F) a nucleotide sequence capable of hybridizing to a variant, homologue, derivative or fragment of the nucleotide sequence of (a);
(G) a nucleotide sequence that is the complement of a nucleotide sequence capable of hybridizing to the nucleotide sequence of (a);
(H) a nucleotide sequence that is the complement of a nucleotide sequence capable of hybridizing to a variant, homolog, derivative or fragment of the nucleotide sequence of (a);
(I) a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence of (a);
(J) a nucleotide sequence capable of hybridizing to the complement of a nucleotide sequence variant, homolog, derivative or fragment of (a);
(K) Any one of (a), (b), (c), (d), (e), (f), (g), (h), (i), and / or (j) A nucleotide sequence comprising

  Another aspect of the invention includes an isolated nucleotide sequence of the invention.

(Preferred aspects)
In one preferred embodiment, the present invention provides the following (i) to (xiii):

Relates to an isolated polypeptide comprising at least one amino acid sequence selected from wherein X is an unknown amino acid residue, or a variant, homolog or derivative thereof.

  In one preferred embodiment, the polypeptide comprises one sequence selected from the sequences (i) to (xiii).

  In another preferred embodiment, the polypeptide comprises two sequences selected from the sequences (i) to (xiii).

  In another preferred embodiment, the polypeptide comprises three sequences selected from sequences (i) to (xiii) above.

  In another preferred embodiment, the polypeptide comprises four sequences selected from the sequences (i) to (xiii) above.

  In another preferred embodiment, the polypeptide comprises five sequences selected from sequences (i) to (xiii) above.

  In another preferred embodiment, the polypeptide comprises six sequences selected from sequences (i) to (xiii) above.

  In another preferred embodiment, the polypeptide comprises seven sequences selected from sequences (i) to (xiii) above.

  In another preferred embodiment, the polypeptide comprises eight sequences selected from sequences (i) to (xiii) above.

  In another preferred embodiment, the polypeptide comprises nine sequences selected from sequences (i) to (xiii) above.

  In another preferred embodiment, said polypeptide comprises 10 sequences selected from sequences (i) to (xiii) above.

  In another preferred embodiment, the polypeptide comprises 11 sequences selected from sequences (i) to (xiii) above.

  In another preferred embodiment, the polypeptide comprises 12 sequences selected from sequences (i) to (xiii) above.

  In another preferred embodiment, the polypeptide comprises 13 sequences selected from sequences (i) to (xiii) above.

  Preferably, the polypeptide of the present invention has pyranozone dehydrase activity.

  One preferred embodiment relates to a polypeptide that is immune to antibodies raised against the purified amino acid sequences of the invention.

  Another aspect relates to an isolated polynucleotide sequence that encodes a polypeptide of the invention, or a variant, homolog, fragment, or derivative thereof.

Preferably, the isolated polynucleotide is:
(I) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or its complement;
(Ii) a polynucleotide comprising a nucleotide sequence capable of hybridizing to the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof;
(Iii) a polynucleotide comprising a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence of SEQ ID NO: 1; and (iv) a polynucleotide comprising a polynucleotide sequence that is degenerate as a result of the genetic code for the polynucleotide of SEQ ID NO: 1. ,
Selected from.

  Preferably, the nucleotide sequence can be obtained from Phanerochaete chrysosporium, Polyporus obtusus, or Corticium caeruleum.

  In one preferred embodiment, the nucleotide sequence is from the order of Pezzales, more preferably from Aurelia aurantia, Pezza badia, P. succosa, Sarcophaera eximia, Morchella conica, M. et al. costata, M.C. elata, M.M. esculenta, M. et al. esculenta var. rotunda, M.M. can be obtained from hortensis, or Gyromitra infula.

  In another preferred embodiment, the nucleotide sequence can be obtained from Auriculariales, more preferably Auricularia mesenterica.

  In another preferred embodiment, the nucleotide sequence is from the order of Aphyllophorales, more preferably Pulcherium caeruleum, Peniophora quercina, Phanerochaete sordida, Villeminia comedens, Sterum gausapatum, sanguinolentum, lopharia spadicea, sparitis laminosa, boletopsis subquamosa, bjerkandera adusta, trichaptum biformis, cerrenacinopor, pycnoporinopor. sanguineus, Junghunia nitida, Ramaria flava, Clavulinopsis helvola, C.I. helvola var. geoglossoides, or V. can be obtained from pulchra.

  In another preferred embodiment, the nucleotide sequence is from the order of Agalicales, more preferably Cliocyte cyatiformis, C.I. dicolor, C.I. gibba, C.I. odora, Lepista caespitosa, L. inversa, L.A. luscina, L .; can be obtained from Nebularis, Mycena seniii, Pleurocybella porrigens, Marasumius orales, or Inocypyriodora.

  In another preferred embodiment, the nucleotide sequence is from the order of Gracilariales, more preferably, Gracilaria varrucosa, Gracilaria tenuispitita, Gracilariopsis sp. Or from Gracilariopsis lemaneformis.

In another preferred embodiment, the nucleotide sequence is from the order of Melanosporaceae, more preferably Melanospora ornatha, Microthecium.
Compressum, Microthelium sobelii.

Another aspect of the present invention is the following:
(I) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or its complement;
(Ii) a polynucleotide comprising a nucleotide sequence capable of hybridizing to the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof;
(Iii) a polynucleotide comprising a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence of SEQ ID NO: 1; and (iv) a polynucleotide comprising a polynucleotide sequence that is degenerate as a result of the genetic code for the polynucleotide of SEQ ID NO: 1. ,
Relates to an isolated nucleotide selected from

  Preferably, the nucleotide sequence is operably linked to a promoter.

  Another aspect of the invention relates to a construct comprising the above polynucleotide sequence.

  Yet another aspect relates to a vector comprising the polynucleotide sequence.

  A further aspect relates to host cells into which the polynucleotide sequences of the invention have been incorporated.

  Another aspect relates to an expression vector comprising a polynucleotide sequence of the present invention operably linked to a regulatory sequence capable of directing said polynucleotide expression in a host cell.

  A further aspect relates to an isolated polypeptide encoded by the polynucleotide sequence of SEQ ID NO: 1, or a variant, homologue, fragment or derivative thereof.

  In preferred embodiments, the isolated polypeptide has up to 7 amino acids removed from the N-terminus.

  More preferably, the isolated polypeptide has at least 75% identity with the polypeptide sequence encoded by SEQ ID NO: 1.

  Yet another aspect relates to an antibody that can bind to a polypeptide of the invention.

  Another aspect relates to a method for preparing an amino acid sequence of the invention comprising expressing the nucleotide sequence of the invention and optionally isolating and / or purifying the nucleotide sequence. Preferably, the nucleotide sequence of the present invention is expressed in an environment that does not contain a substrate for the expressed enzyme (for example, in the absence of 1,5-anhydrofructose).

  A further aspect relates to the process of preparing microsesin using the amino acid sequence of the invention or the expression product of the nucleotide sequence of the invention.

  Yet another aspect relates to a process for preparing ascopyrone P using the amino acid sequence of the invention or the expression product of the nucleotide sequence of the invention.

  Preferably, the process comprises reacting said expression product of amino acid sequence or nucleotide sequence with 1,5-anhydro-D-fructose.

  Even more preferably, the process further comprises the use of APP synthase.

  In a particularly preferred aspect, the process comprises reacting APP synthase and the expression product of the amino acid or nucleotide sequence with 1,5-anhydro-D-fructose.

  In another preferred embodiment, the above process of making microsecin comprises contacting the polypeptide of the invention with glucan ligase and dextrin starch.

  A further aspect of the invention relates to a process for preparing microsesin using the expression product of the amino acid sequence of the invention or the nucleotide sequence of the invention.

  Preferably, the process comprises reacting an expression product of an amino acid sequence or nucleotide sequence with glucosone.

  In another preferred embodiment, the above process of making microsecin comprises reacting a polypeptide of the invention with glucose and pyranose 2-oxidase.

  Another aspect of the invention relates to a process for preparing microsesin comprising the step of reacting pyranozone dehydrase with 1,5-anhydro-D-fructose.

  Yet another aspect of the present invention relates to a process for preparing ascopyrone P comprising reacting pyranozone dehydrase and APP synthase with 1,5-anhydro-D-fructose.

  One aspect of the invention relates to a process for preparing microsesin comprising reacting pyranozone dehydrase with glucose and dextrin starch.

  Another aspect of the present invention relates to a process for preparing cortalcerone comprising reacting pyranozone dehydrase with glucosone.

  Yet another aspect of the invention relates to a process for preparing cortalcerone comprising the step of reacting pyranozone dehydrase with glucose and pyranose 2-oxidase.

  Another aspect of the present invention relates to the use of microsecin for preventing and / or inhibiting and / or killing the growth of microorganisms in a substance.

  Another aspect of the present invention relates to the use of cortalcerone for preventing and / or inhibiting and / or killing the growth of microorganisms in a substance.

  The invention also relates to the use of one or more microsesin, cortalcerone, or a derivative or isomer thereof for the prevention and / or inhibition and / or killing of microorganisms in a substance.

  Preferably the substance is a food product.

  Preferably, the microorganism in which cortalcerone and / or microsecin is active is a plant fungal pathogen.

  Preferably, the microorganism in which cortalcerone and / or microsecin is active is selected from microorganisms selected from the order of Rhizoctonia, Pythium, Aphanomyces, and Cercospora.

  Preferably, the microorganism in which cortalcerone and / or microsecin is active is selected from microorganisms selected from Rhizotonia solani, Pythium Ultimum, Aphanomyces cochlioides, and Cercospora beticola.

  A further aspect of the invention relates to the use of microsecin, cortalcerone, or a derivative or isomer thereof for the prevention and / or inhibition and / or killing of the pathogen Aphanomyces.

  Preferably, the pathogen is Aphanomyces cochlioides.

  In a preferred embodiment, the derivative of microsesin is 2-furyl-hydroxymethyl-ketone or 4-deoxy-glycero-hexo-2,3-ziruose.

  In a preferred embodiment, the cortalcerone derivative is 2-furylglyoxal.

  In particularly preferred embodiments, microsecin, cortalcerone, or a derivative or isomer thereof is used in the treatment of plants or plant seeds, even more preferably in the treatment of sugar beet, pea plants, or pea plant seeds.

  In another aspect, the present invention relates to the use of microsecin, cortalcerone, or a derivative or isomer thereof as a plant or seed protectant.

  Yet another aspect of the invention relates to the use of microsecin, cortalcerone, or a derivative or isomer thereof as a plant growth regulator.

(advantage)
The present invention provides amino acid and nucleotide sequence information not previously disclosed for pyranozone dehydrase.

  The present invention further relates to the use of pyranozone dehydrase in the conversion of AF to APP and microsecin and the production of cortalcerone. To date, it has not been taught or suggested in the art that PD is involved in or can cause any of these transformations.

  Thus, the present invention can facilitate large-scale production of microthecin, APP, and cortalcelone. The present invention further teaches that microthecin and cortalcerone are useful for application as antibacterial agents, and more particularly as antibacterial agents in foods.

(Assay)
The following assays can be used to characterize and identify the actual and putative amino acid sequences of the present invention.

(Isolation)
In one aspect, the sequence is preferably in an isolated form. The term “isolated” means that the sequence is not in its natural environment (ie, not found in nature). Typically, the term “isolated” means that the sequence is naturally associated and at least substantially free of at least one other component found in nature. Here, the sequence may be separated from the naturally associated at least one other component.

(Purification)
In one aspect, the sequence is preferably in purified form. The term “purified” also means that the sequence is not in its natural environment (ie not found in nature). Typically, the term “purified” means that the sequence is naturally associated and at least substantially separated from at least one other component found in nature.

(Nucleotide sequence)
The present invention includes nucleotide sequences that encode enzymes having specific properties as defined herein. As used herein, the term “nucleotide sequence” refers to oligonucleotide or polynucleotide sequences and variants, homologs, fragments, and derivatives thereof (such as portions thereof). Nucleotide sequences can be of genomic, synthetic, or recombinant origin, which can be double-stranded or single-stranded, representing either the sense strand or the antisense strand.

  The term “nucleotide sequence” in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably this means DNA, more preferably cDNA of the coding sequence of the invention.

  In a preferred embodiment, the nucleotide sequence of the invention itself does not encompass the native nucleotide sequence of the invention in its natural environment when operably linked to its naturally associated sequence that also exists in the natural environment. . For ease of reference, we refer to this preferred embodiment as a “non-natural nucleotide sequence”. In this regard, the term “native nucleotide sequence” means an entire nucleotide sequence that is operatively linked to a naturally associated promoter and, if the promoter is also present in the natural environment, in the natural environment. . However, the amino acid sequences of the invention can be isolated and / or purified after expression of the nucleotide sequence in its native organism. Preferably, however, the amino acid sequence of the invention can be expressed by a nucleotide sequence in a natural organism, provided that the nucleotide sequence is not under the control of a promoter that naturally associates in that organism. is there.

  Typically, the nucleotide sequences of the present invention are prepared using recombinant DNA techniques (ie, recombinant DNA). However, in another embodiment of the invention, nucleotide sequences can be synthesized in whole or in part using chemical methods well known in the art (Caruthers MH et al (1980) Nuc Acids Res). Symp Ser 215-233 and Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).

(Preparation of nucleotide sequence)
Identifying and / or isolating and / or isolating nucleotide sequences encoding any of the enzymes having specific properties defined herein or those suitable for modification from any cell or organism that produces the enzyme. Can be purified. Various methods for the identification and / or isolation and / or purification of nucleotide sequences are well known in the art. As an example, PCR amplification techniques can be used to prepare multiple sequences once the appropriate sequence has been identified and / or isolated and / or purified.

  As a further example, genomic DNA and / or cDNA libraries can be constructed using chromosomal or messenger DNA from an organism that produces the enzyme. If the amino acid sequence of the enzyme is known, a labeled oligonucleotide probe can be synthesized and used to identify an enzyme-coding clone from a genomic library prepared from the organism. Alternatively, a labeled oligonucleotide probe containing a sequence homologous to another known enzyme gene can be used to identify an enzyme coding clone. In the latter case, low stringency hybridization and washing conditions are used.

  Alternatively, the enzyme-coding clone is inserted into an expression vector such as a plasmid, a genomic DNA fragment is inserted, an enzyme-negative bacterium is transformed with the obtained genomic DNA library, and then the transformed bacterium is converted into an enzyme substrate (ie, maltose). Can be identified by plating to agar containing, thereby identifying enzyme-expressing clones.

  In yet another aspect, the nucleotide sequence encoding the enzyme can be obtained by an established standard method (eg, the phosphoramidide method described in Beucage SL et al (1981) Tetrahedron Letters 22, p 1859-1869). Or by the method described in Matthes et al (1984) EMBO J. 3, p 801-805). In the phosphoramidite method, oligonucleotides are synthesized, for example, on an automated DNA synthesizer, purified, annealed, ligated, and cloned into an appropriate vector.

Nucleotide sequences are prepared by ligation of fragments of synthetic DNA, genomic DNA, or cDNA origin (if necessary) by standard techniques, mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or genomic It can be a mixture of origin and cDNA origin. Each ligated fragment corresponds to a different part of the overall nucleotide sequence. DNA sequences are described, for example, in US Pat. No. 4,683,202 or Saiki RK et al (Science (1988) 239, pp.
487-491) can also be prepared by polymerase chain reaction (PCR) using the specific primers described in 487-491).

(Amino acid sequence)
The present invention also includes amino acid sequences of enzymes having specific properties as defined herein.

  As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and / or the term “protein”. In some examples, the term “amino acid sequence” is synonymous with the term “peptide”. In some examples, the term “amino acid sequence” is synonymous with the term “enzyme”.

  The amino acid sequence may be prepared / isolated from a suitable source, synthesized or may be prepared by use of recombinant DNA technology.

  The enzyme of the present invention may be used in combination with other enzymes. Thus, the present invention also encompasses enzyme combinations comprising an enzyme of the present invention and another enzyme that may be another enzyme of the present invention. This aspect is considered in the following section.

  Preferably the enzyme is not a natural enzyme. In this regard, the term “natural enzyme” refers to the entire enzyme when present in its natural environment and expressed by its natural nucleotide sequence.

(Mutant / homologue / derivative)
The invention also includes the use of any amino acid sequence variants, homologues, and derivatives of the enzymes of the invention or any nucleotide sequence encoding such an enzyme. As used herein, the term “homolog” means an entity having a certain homology with the amino acid sequences of the present invention and the nucleotide sequences of the present invention. As used herein, the term “homology” can be equated with “identity”.

  In the context of the present invention, the homologous sequence is at least 75%, 80%, 85%, or 90% identical, preferably at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of the invention. Are considered to be included. Typically, a homolog contains the same active site as the amino acid sequence of the present invention. Homology can also be regarded as similarity (ie, amino acid residues having similar chemical properties / functions), but in the context of the present invention it is preferred to express homology in terms of sequence identity.

  In the context of the present invention, homologous sequences include at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, or a nucleotide sequence encoding an enzyme of the present invention (sequence of the present invention), or Nucleotide sequences that are 90% identical, preferably at least 95%, 96%, 97%, 98%, or 99% identical are considered to be included. Typically, the homologue includes a sequence encoding the same active site as the sequence of the present invention. While homology can also be considered similarity (ie, amino acid residues having similar chemical properties / functions), in the context of the present invention, it is preferred to express homology with respect to sequence identity.

  Homology comparisons can be made visually or, more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate% homology between two or more sequences.

  % Sequence homology can be calculated (ie, one sequence is aligned with the other sequence and each amino acid in one sequence is one residue at a time with the corresponding amino acid in the other sequence) Compare directly). This is referred to as an “ungapped” alignment. Typically, such non-gap alignment is performed with only a relatively small number of residues.

  Although this is a very simple and consistent method, subsequent amino acid residues are excluded from the alignment, for example by having one insertion or deletion in an otherwise identical sequence pair Since this cannot be taken into account, the% homology can be greatly reduced when performed on the entire alignment. Thus, most sequence comparison methods are designed to provide an optimal alignment that takes into account possible insertions and deletions without penalizing excessively the overall homology score. This is done by inserting “gaps” in the sequence alignment to try to maximize local homology.

  However, in these more complex methods, for the same number of identical amino acids, a sequence alignment that contains as few gaps as possible (reflecting a higher association between the two comparison sequences) will contain a sequence alignment that contains many gaps. Assign a “gap penalty” to each gap that occurs during the alignment to get a higher score. Typically, an “affine gap cost” is used that imposes a relatively high cost on the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. Of course, a high gap penalty results in an optimized alignment that includes fewer gaps. Most alignment programs can correct gap penalties. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for gaps and -4 for each extension.

Therefore, in order to calculate the maximum homology%, it is first necessary to create an optimal alignment taking into account the gap penalty. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (Devereux et al 1984 Nuc. Acids Research 12
p387). Examples of other software than can perform sequence comparisons, BLAST package comparison tools (Ausubel et al 1999 Short Protocols in Molecular Biology, 4 th Ed - see Chapter 18), FASTA (Altschul et al 1990 J. Mol Biol. 403-410), and GENEWORKS integrated software (suite). Both BLAST and FASTA are available for offline and online searching (see Ausubel et al 1999, pages 7-58 to 7-60). However, for some applications it is preferred to use the GCG Bestfit program. A new tool called BLAST2 Sequences is also available for comparison of protein and nucleotide sequences (FEMS Microbiol Lett 1999 174 (2): 247-50, FEMS Microbiol Lett 1999 177 (1): 187-8 and tantian @@ .Nlm.nih.gov).

  Although the final% homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, we typically use a scaled similarity score matrix that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is the BLOSUM62 matrix (the default matrix for the program's BLAST integration software). The GCG Wisconsin program generally uses either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferable to use the official default values for the GCG package and for other software to use the default matrix (such as BLOSUM62).

  Alternatively,% homology was calculated using the algorithm-based DNASIS ™ (Hitachi Software) multiple alignment function, similar to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73 (1), 237-244). Can be calculated.

  Once the software has obtained an optimal alignment, it is possible to calculate% homology, preferably% sequence identity. The software typically calculates this as part of the sequence comparison and produces a numerical result.

  The sequence may also have amino acid residue deletions, insertions, or substitutions that result in silent changes resulting in functionally equivalent material. Intentional amino acid substitutions can be made based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic nature of the residue as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values include leucine, isoleucine. , Valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

  For example, conservative substitutions can be made according to the following table. Amino acids in the same block in the second column and preferably in the same row in the third column can be substituted for each other.

  The present invention also provides for possible homologous substitutions (substitution and replacement of both existing amino acid residues with another residue, ie, substitutions between analogs (basic and basic, Acidic and acidic, polar and polar, etc.) are used herein to mean). Non-homologous substitutions (ie, substitutions from one class of residues to another, or non-natural amino acids (ornithine (hereinafter Z), ornithine diaminobutyrate (hereinafter B), norleucine ornithine (hereinafter O) Substituting), including the inclusion of pyrylalanine, thienylalanine, naphthylalanine, and phenylglycine).

It is also possible to carry out this replacement by the following non-natural amino acids: alpha ^* and alpha-disubstituted ^* amino acids, N- alkyl amino acids ^*, lactic acid ^*, halide derivatives of natural amino acids (trifluoromethyl tyrosine ^*, etc.), p-Cl @ - phenylalanine ^* , P-Br-phenylalanine ^* , p-I-phenylalanine ^* , L-allyl-glycine ^* , β-alanine ^* , L-α-aminobutyric acid ^* , L-γ-aminobutyric acid ^* , L-α-aminoisobutyric acid ^* , L-ε-aminocaproic acid ^# , 7-aminoheptanoic acid ^* , L-methionine sulfone ^# , L-norleucine ^* , L-norvaline ^* , p-nitro-L-phenylalanine ^* , L-hydroxyproline ^# , L - thioproline ^*, methyl derivatives (4-methyl phenylalanine (Phe) -Phe ^*, pentamethyl -P e ^*, L-Phe (4- ^amino) #, L-Tyr ^{(methyl) *,} etc.), L-Phe (4- ^isopropyl) *, L-Tic (1,2,3,4- tetrahydroisoquinoline-3 Carboxylic acid) ^* , L-diaminopropionic acid ^# , and L-Phe (4-benzyl) ^* . The symbol ^* is used in the above description to indicate the hydrophobicity of the derivative (for homologous or non-homologous substitutions), ^# is used to indicate the hydrophilicity of the derivative, and ^** indicates amphiphilicity.

  Variant amino acid sequences can include suitable spacer groups that can be inserted between any two amino acid residues of the sequence, in addition to amino acid spacers (such as glycine or β-alanine residues). Alkyl groups (such as methyl, ethyl, or propyl groups) are included. Additional mutant forms are associated with the presence of one or more amino acid residues in the peptoid form, which is well understood by those skilled in the art. For the avoidance of doubt, the “peptoid form” is used to refer to a mutated amino acid residue in which the α carbon substituent is on the nitrogen atom of the residue and not the α carbon. Processes for preparing peptides in the peptoid form are known in the art (eg, Simon RJ et al., PNAS (1992) 89 (20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13 ( 4), 132-134).

  The nucleotide sequences used in the present invention may include synthetic or modified nucleotides within the nucleotide sequence. Several different modifications to the oligonucleotide are known in the art. These include methylphosphonate and phosphorothioate backbones and / or the addition of acridine or polylysine chains at the 3 'and / or 5' ends of the molecule. For the purposes of the present invention, it should be understood that the nucleotide sequences described herein can be modified by any method available in the art. Such modifications can be made to increase the in vivo activity or extend the lifetime of the nucleotide sequences of the present invention.

  The present invention also includes the use of nucleotide sequences that are complementary to the sequences described herein, or any derivative, fragment, or derivative thereof. If the sequence is complementary to the fragment, the sequence can be used as a probe to identify similar coding sequences in other organisms and the like.

  Polynucleotides that are not 100% homologous to the sequences of the present invention but fall within the scope of the present invention can be obtained in a number of ways. Other variants of the sequences described herein can be obtained, for example, by searching a DNA library made from a range of individuals (eg, individuals from different populations). In addition, cell homologs found in other virus / bacteria or cell homologs, particularly mammalian cells (eg, rat, mouse, bovine, and primate cells) can be obtained, such homologs and fragments thereof are In general, it can selectively hybridize with the sequences shown in the sequence listing in this specification. Such sequences are searched for cDNA libraries or genomic DNA libraries made from other animal species, and such libraries are subject to moderate to high stringency conditions in the attached sequence listing. It can be obtained by probing with a probe containing all or part of any one of the sequences. Similar considerations are applied to obtain species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.

  Variants and strain / species homologs can also be obtained using degenerate PCR using primers designed to target variants and homologues that encode conserved amino acid sequences within the sequences of the invention. . Conserved sequences can be deduced, for example, by alignment of amino acid sequences from several variants / homologs. Sequence alignment may be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used.

  Primers used in degenerate PCR contain one or more degenerate positions and are used at stringency conditions lower than those used for cloning sequences using a single sequence primer relative to a known sequence.

  Alternatively, such polynucleotides can be obtained by site-directed mutagenesis of the characterized sequence. This can be useful, for example, when silent codon sequence changes are required to optimize the codon preference of the particular host cell in which the polynucleotide sequence is expressed. Other sequence changes may be desired to introduce restriction enzyme recognition sites or to alter the nature or function of the polypeptide encoded by the polynucleotide.

  The present invention also includes polynucleotides that have undergone molecular evolution by random processes, selective mutagenesis, or in vitro recombination. As a non-limiting example, many site-specific or random mutations to the nucleotide sequence, either in vivo or in vitro, are then screened for improved functionality of the encoded polypeptide by various means. It is possible. In addition, polynucleotide sequence variations or natural variants can be recombined with either wild-type or other mutations or natural variants to produce new variants. Such novel variants can also be screened for improved functionality of the encoded polypeptide. Various methods well established in the art such as Error Threshold Mutagenesis (WO 92/18645), oligonucleotide-mediated random mutagenesis (US Pat. No. 5,723,323), DNA shuffling Rings (US Pat. No. 5,605,793), exo-mediated gene assembly (WO 00/58517)) can produce new preferred variants. Application of these and similar random directed molecular evolution methods to identify and select variants of the enzyme of the invention with favorable characteristics without any prior knowledge of protein structure or function Can produce unpredictable but advantageous mutations or variants. There are many applications of molecular evolution in the art for optimization or modification of enzyme activity, such examples include, but are not limited to, one or more of the following: in host cells or in vitro Optimized expression and / or activity, increased enzyme activity, optimized substrate specificity and / or product specificity, increased or decreased enzyme stability or structural stability, Changes in enzyme activity / specificity at temperature, pH, substrate).

  Using the polynucleotides (nucleotide sequences) of the present invention, labels that are evident by conventional means using primers (eg, PCR primers (primers for another amplification reaction)), probes (eg, radioactive or non-radioactive labels) Or the polynucleotide may be cloned into a vector. Such primers, probes, and other fragments are at least 15, preferably at least 20, such as at least 25, 30, or 40 nucleotides in length, and are also used herein as the term “ Included in “polynucleotide”.

  Polynucleotides and probes, such as DNA polynucleotides according to the present invention, can be produced by recombinant, synthetic, or any means available to those skilled in the art. These can also be cloned by standard techniques.

  In general, primers are produced by synthetic means that involve the stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for doing this using automated techniques are readily available in the art.

  In general, recombinant means are used to produce longer polynucleotides, for example using PCR (polymerase chain reaction) cloning techniques. This involves creating a primer pair (eg, about 15-30 nucleotides) adjacent to the lipid target sequence region desired to be cloned, contacting the primer with mRNA or cDNA obtained from an animal or human cell, Performing a polymerase chain reaction under conditions that amplify, isolating the amplified fragment (eg, by purification of the reaction mixture on an agarose gel), and recovering the amplified DNA. The primers may be designed to include appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into an appropriate cloning vector.

(Biologically active)
Preferably, the mutated sequence and the like are biologically active at least as much as the sequences described herein.

  As used herein, “biologically active” refers to a structural function that is similar (but not necessarily the same) as a naturally occurring sequence and / or a similar regulatory function (not necessarily the same). And / or sequences having similar biochemical functions (not necessarily to the same extent).

(Isozyme)
The polypeptides of the present invention may exist in the form of one or more different isozymes. As used herein, the term “isozyme” includes variants of polypeptides that catalyze the same reaction but differ in properties such as substrate affinity and maximum rate of enzyme-substrate reaction. Depending on the amino acid sequence difference, isozymes can be distinguished by techniques such as electrophoresis or isoelectric focusing. Different tissues often have different isozymes. In general, sequence differences provide different enzyme rate parameters that can be interpreted as fine-tuning to the specific requirements of the cell type in which the particular isozyme is found.

(Isoform)
The present invention also includes different isoforms of the polypeptides described herein. The term “isoform” refers to a protein having the same function (ie, pyranozone dehydrase activity), which is a similar gene product but a different gene product.

(Hybridization)
The present invention also includes sequences that are capable of hybridising either to a sequence that is complementary to a sequence of the invention, or to a sequence of the invention or a sequence that is complementary to a sequence of the invention.

  As used herein, the term “hybridization” includes “a process in which a strand of nucleic acid is ligated to a complementary strand by base pairing” as well as an amplification process performed in polymerase chain reaction (PCR) technology.

  The present invention also includes the use of nucleotide sequences that are capable of hybridizing to the sequences described herein, or sequences complementary to any derivative, fragment, or derivative thereof.

  The term “variant” also includes sequences that are complementary to sequences that are capable of hybridizing to the nucleotide sequences described herein.

  Preferably, the term “variant” is used under stringent conditions (eg, 50 ° C. and 0.2 × SSC {1 × SSC = 0.15 M NaCl, 0.015 M sodium citrate (pH 7.0)}). It includes sequences that are complementary to sequences that can hybridize to the nucleotide sequences described herein.

  More preferably, the term “variant” is used under highly stringent conditions (eg, 65 ° C. and 0.1 × SSC {1 × SSC = 0.15 M NaCl, 0.015 M sodium citrate (pH 7.0)}). And a sequence complementary to a sequence capable of hybridizing to the nucleotide sequence described herein.

  The present invention also relates to nucleotide sequences that are capable of hybridizing to the nucleotide sequences of the present invention (including complementary sequences of the nucleotide sequences described herein).

  The present invention also relates to nucleotide sequences that are complementary to sequences that are capable of hybridizing to the nucleotide sequences of the present invention (including complementary sequences of the nucleotide sequences described herein).

  Polynucleotide sequences that can hybridize to the nucleotide sequences described herein under moderate to maximal stringency conditions are also within the scope of the invention.

  In a preferred aspect, the present invention includes nucleotide sequences that can hybridize to the nucleotide sequence of the present invention or its complement under stringent conditions (eg, 50 ° C. and 0.2 × SSC).

  In a more preferred aspect, the invention encompasses nucleotide sequences that can hybridize to the nucleotide sequence of the invention or its complement under highly stringent conditions (eg, 65 ° C. and 0.1 × SSC).

(Site-directed mutagenesis)
Once an enzyme-encoding nucleotide sequence has been isolated or a putative enzyme-encoding nucleotide sequence has been identified, it is desirable to mutate the sequence to prepare the enzymes of the invention.

  Synthetic oligonucleotides can be used to introduce mutations. These oligonucleotides contain a nucleotide sequence that flank the desired mutation site.

  A suitable method for creating a single-stranded gap of DNA (enzyme coding sequence) in a vector carrying an enzyme gene is disclosed in Morinaga et al. (Biotechnology (1984) 2, p646-649). A synthetic nucleotide having the desired mutation is then annealed to the homologous portion of the single stranded DNA. The remaining gap is then filled with DNA polymerase I (Klenow fragment) and ligated to the construct using T4 ligase.

  US Pat. No. 4,760,025 discloses the introduction of oligonucleotides encoding multiple mutations by making minor cassette changes. However, since many oligonucleotides of various lengths can be introduced, more various mutations can be introduced at any time by the above method of Morinaga.

  Another method for introducing mutations into enzyme-encoding nucleotide sequences is described in Nelson and Long (Analytical Biochemistry (1989), 180, p 147-151). This method involves the three-step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reaction. From the PCR generated fragment, the DNA fragment carrying the mutation can be isolated by cleavage with a restriction endonuclease and reinserted into the expression plasmid.

  As an example, Sierks et al. (Protein Eng (1989) 2, 621-625 and Protein Eng (1990) 3, 193-198) describe site-directed mutagenesis in Aspergillus glucoamylase.

(Recombinant)
In one aspect of the invention, the sequence is a recombinant sequence (ie, a sequence prepared using recombinant DNA techniques).

(Synthesis)
In one aspect of the invention, the sequence is a synthetic sequence (ie, a sequence prepared by in vitro chemistry or enzymatic synthesis). This includes, but is not limited to, sequences generated using optimal codon usage for host organisms such as the methylotrophic yeasts Pichia and Hansenula.

(Enzyme expression)
The nucleotide sequence used in the present invention can be incorporated into a vector capable of recombinant replication. This vector can be used to replicate and express nucleotide sequences in enzymatic form in and / or from a compatible host cell. Both homologous and heterologous expression are included.

  For homologous expression, preferably the gene of interest or nucleotide sequence of interest is not in a naturally occurring genetic context. Where the gene of interest or nucleotide sequence of interest is in a naturally occurring genetic context, preferably expression is other than its naturally occurring expression mechanism or in addition to it (eg, by gene intervention of the gene of interest Driven by overexpression).

  Control sequences including promoter / enhancer and other expression control signals can be used to control expression. Prokaryotic promoters and promoters that are functional in eukaryotic cells can be used. Tissue specific or stimulus specific promoters can be used. Chimeric promoters comprising sequence elements derived from two or more different promoters described herein can also be used.

  Enzymes produced by the host recombinant cell by expression of the nucleotide sequence may be secreted or included within the cell depending on the sequence and / or vector used. Coding sequences can be designed using signal sequences that direct the secretion of the coding sequences of the invention through certain prokaryotic and eukaryotic membranes.

(Expression vector)
The term “expression vector” means a construct capable of in vivo or in vitro expression.

  Preferably, the expression vector is integrated into the genome of a suitable host organism. The term “integrated” preferably includes stable integration into the genome.

  The host organism may be the same as or different from the gene of the intended source organism, causing both homologous and heterologous expression.

Preferably, the vector of the present invention comprises the construct of the present invention. Alternatively, the expressed nucleotide of the present invention is preferably present in a vector, wherein the nucleotide sequence is operably linked to a regulatory sequence so that the regulatory sequence can be expressed by a suitable host organism. (Ie, the vector is an expression vector)
The vectors of the invention can be transformed into suitable host cells as described below to express the polypeptides of the invention. Accordingly, in a further aspect, the invention provides for culturing host cells transformed or transfected with an expression vector and expressing the expressed polypeptide under conditions for expression by a vector of a coding sequence encoding the polypeptide. A process for preparing a polypeptide for subsequent use according to the present invention is provided, including a step of recovering.

  The vector can be, for example, a plasmid vector, viral vector, or phage vector comprising an origin of replication, optionally a promoter for expression of the polynucleotide, and optionally a regulator of the promoter. The choice of vector often depends on the host cell into which the vector is introduced.

  The vectors of the present invention may contain one or more selectable marker genes. The most suitable selection system for industrial microorganisms is a system formed by a group of selectable markers that do not require mutation in the host organism. Suitable selection markers are B.I. subtilis or B.I. It can be a dal gene from licheniformis or a marker conferring antibiotic resistance such as ampicillin resistance, kanamycin resistance, chloramphenicol resistance, or tetracycline resistance. Another selectable marker can be an Aspergillus selectable marker such as amdS, argB, niaD, and sC or a marker that results in hygromycin resistance. Examples of other fungal selectable markers are ATP synthase, subunit 9 (oliC), orotidine-5'-phosphate decarboxylase (pvrA), phleomycin, and benomyl resistance (benA) genes. Examples of non-fungal selectable markers are bacterial G418 resistance gene (which can also be used in yeast but not in filamentous fungi), ampicillin resistance gene (E. coli), neomycin resistance gene (Bacillus) And E. coli encoding β-glucuronidase (GUS). E. coli uidA gene. Further suitable selection markers include B.I. subtilis or B.I. The dal gene from licheniformis is included. Alternatively, it can be selected by co-transformation (described in WO 91/17243).

  Vectors can be used, for example, in vitro for the production of RNA, or used to transfect or transform host cells.

  Thus, the nucleotide sequences used in the present invention can be incorporated into recombinant vectors (typically replicable vectors), such as cloning vectors or expression vectors. The vector can be used to replicate the nucleic acid in a compatible host cell. Accordingly, in a further embodiment, the invention provides for the introduction of the nucleotide sequence of the invention into a replicable vector, the introduction of the vector into a compatible host cell, and propagation of the host cell under conditions that result in replication of the vector. Provides a method for producing the nucleotide sequences of the present invention. The vector can be recovered from the host cell. Suitable host cells are described below with expression vectors.

The procedure used to ligate the DNA constructs and regulatory sequences of the present invention that encode an enzyme having the specific properties defined herein and insert them into an appropriate vector containing the information necessary for replication is as follows: Are well known to those skilled in the art (see, eg, Sambrook et al Molecular Cloning: A laboratory Manual, 2 ^nd Ed. (1989)).

  The vector may further comprise a nucleotide sequence that allows the vector to replicate in the appropriate host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1, and pIJ702.

  Expression vectors typically include cloning vector components such as, for example, elements that allow the vector to replicate autonomously in a selected host organism, and markers that can detect one or more phenotypes for selection. Expression vectors usually contain a promoter, an operator, a ribosome binding site, a translation initiation signal, and optionally a control nucleotide sequence encoding a repressor gene or one or more activator genes. In addition, the expression vector may comprise a sequence encoding an amino acid sequence capable of targeting the amino acid sequence to a host cell organelle such as peroxisome or a particular host cell compartment. In the context of the present invention, the term “expression signal” includes any of the above regulatory, repressor, or activator sequences. For expression under the direction of control sequences, the nucleotide sequence is operably linked to the control sequence in an appropriate manner for expression.

(Regulatory sequence)
In some provisions, the nucleotide sequences used in the present invention are operably linked to regulatory sequences that allow the nucleotide sequences to be expressed, such as by the selected host cell. By way of example, the present invention includes vectors comprising a nucleotide sequence of the present invention operably linked to such regulatory sequences (ie, the vector is an expression vector).

  The term “operably linked” refers to a proximal position in which the described components can function in the intended manner. A regulatory sequence “operably linked” to a coding sequence is ligated in such a way that the coding sequence is expressed under conditions compatible with the control sequences.

  The term “regulatory sequence” includes promoters and enhancers as well as other expression control signals.

  The term “promoter” is used in its ordinary sense in the art (eg, RNA polymerase binding site).

  Heterologous regulatory regions (eg, promoter regions, secretion leaders) that serve to increase expression and, if desired, the level of secretion of the protein of interest from the selected expression host and / or inductively control the expression of the enzyme of the invention. The selection of the region and the termination region) can also enhance the expression of nucleotide sequences encoding the enzyme of the present invention. In eukaryotes, the polyadenylation sequence can be operably linked to a nucleotide sequence encoding the enzyme.

  Preferably, the nucleotide sequence of the present invention can be operably linked to at least a promoter.

  In addition to the promoter native to the gene encoding the nucleotide sequence of the present invention, other promoters can be used to direct expression of the polypeptide of the present invention. A promoter can be selected for efficiency in directing expression of the nucleotide sequences of the invention in the desired expression host.

  In another embodiment, a constitutive promoter can be selected to direct expression of the desired nucleotide sequence of the invention. Such expression constructs may provide additional advantages as the need for culturing the expression host in a medium containing the induction substrate is avoided.

  Examples of suitable promoters for directing transcription of nucleotide sequences in bacterial hosts include E. coli. E. coli lac operon promoter, Streptomyces coelicolor agarase gene dagA promoter, Bacillus licheniformis α-amylase gene (amyL) promoter, Bacillus stearothermophilus maltose gene Lactococcus sp. Containing the promoter of Bacillus subtilis xylA and xylB genes, and the P170 promoter. Promoters derived from promoters are included. The nucleotide sequence is E. coli. For expression in bacterial species such as E. coli, an appropriate promoter can be selected from bacteriophage promoters including, for example, the T7 promoter and the λ phage promoter.

  Examples of useful promoters for transcription in fungal species include Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartate proteinase, Aspergillus niger neutral α-amylase, niger acid stable α-amylase, A. It is a promoter derived from a gene encoding niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, or Aspergillus nidulans acetamidase.

  Examples of strong constitutive and / or inducible promoters preferred for use in fungal expression hosts include xylanase (xlnA), phytase, ATP-synthetase, subunit 9 (oliC), triose phosphate isomerase (tpi), alcohol dehydrogenase ( AdhA), α-amylase (amy), amyloglucosidase (from the AG-glaA gene), acetamidase (amdS), and promoters obtainable from fungal genes of the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter It is. Other examples of promoters useful for transcription in fungal hosts are: oryzae TAKA amylase, S. cerevisiae-derived TPI (triosephosphate isomerase) promoter (Alber et al (1982) J. Mol. Appl. Genet. 1, p419-434), Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase; It is a promoter derived from a gene encoding nidulans acetamidase.

  Examples of suitable promoters for expression in yeast species include, but are not limited to, the Saccharomyces cerevisiae Gal1 and Gal10 promoters and the Pichia pastoris AOX1 or AOX2 promoter.

  Hybrid promoters can also be used to improve the inducible regulation of expression constructs.

  A promoter may further comprise features that ensure or increase expression in a suitable host. For example, the feature may be a storage area such as a Pribnow box or a TATA box. A promoter may further include other sequences that affect (maintain, enhance, decrease, etc.) the level of expression of the nucleotide sequences of the invention. For example, other suitable sequences include the Sh1-intron or the ADH intron. Other sequences include inductive elements (elements that can be induced by temperature, chemicals, light, stress). There may also be elements suitable for enhanced transcription or translation. An example of the latter element is the TMV 5 'signal sequence (see Sleet 1987 Gene 217, 217-225 and Dawson 1993 Plant Mol. Biol. 23:97).

(Construction)
The term “construct” (synonymous with terms such as “conjugate”, “cassette”, and “hybrid”) includes a nucleotide sequence used in the present invention that binds directly or indirectly to a promoter. An example of indirect binding is the provision of a suitable group of spacers, such as intron sequences such as Sh1 introns or ADH introns, and falls between the promoter and nucleotide sequences of the present invention. This also applies to the term “fused” with respect to the present invention, including direct or indirect binding. In some cases, the term does not cover nucleotide sequences that encode proteins normally associated with the wild-type gene promoter, and natural combinations when they are both present in the natural environment.

  The construct can include or express a marker that allows the genetic construct to be selected in, for example, an introduced bacterium, preferably a Bacillus genus (such as Bacillus subtilis) or a plant. For example, a marker encoding mannose-6-phosphate isomerase (especially for plants) or a marker indicating antibiotic resistance (eg resistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin) can be used. There are various markers.

  For some applications, preferably the construct of the present invention comprises at least a nucleotide sequence of the present invention operably linked to a promoter.

(Host cell)
The term “host cell” in relation to the present invention includes any of the nucleotide sequences or expression vectors described above and any nucleotide sequence or expression vector used in the recombinant production of an enzyme having the specific characteristics defined herein. Any cell containing is included. The nucleotide of interest may be homologous or heterologous to the host cell.

  Accordingly, a further embodiment of the present invention is to provide a host cell transformed or transfected with a nucleotide sequence that expresses an enzyme of the present invention. Preferably, the nucleotide sequence is included in a vector for replication and expression of the nucleotide sequence. The cells are selected to be compatible with the vector and can be, for example, prokaryotic cells (eg, bacteria), fungal cells, yeast cells, or plant cells.

  Examples of suitable bacterial host organisms, Bacillus subtilis, Bacilluslicheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillaceae family including Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis, such as Streptomyces murinus Lactococcus spp. Such as Streptomyces species, Lactococcus lactis. Lactobacillus spp., Including Lactobacillus reuteri. , Leuconostoc spp, Pediococcus spp. , And Streptococcus spp. Gram-positive bacterial species such as lactic acid bacteria containing Alternatively, E.I. A strain of Gram-negative bacterial species belonging to Enterobacteriaceae or Pseudomonadaceae containing E. coli can be selected as the host organism.

  Gram-negative bacteria E. coli is widely used as a host for heterologous gene expression. However, a large amount of heterologous protein tends to accumulate in the cell. A large amount of E. coli. Subsequent purification of the desired protein from E. coli intracellular proteins can often be difficult.

  E. In contrast to E. coli, Gram-positive bacteria from the genus Bacillus (B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalocilus, B. amyloliquefac. B. laurus, B. megaterium, B. thuringiensis, etc.), Streptomyces lividans, or S. Murinus may be very suitable as a heterologous host due to its ability to secrete proteins into the culture medium. Other bacteria that may be suitable as hosts are those from the genera Streptomyces and Pseudomonas.

  Depending on the nature of the nucleotide sequence encoding the enzyme of the invention and / or the desirability of further processing of the expressed protein, eukaryotic hosts such as yeast or other fungi are preferred. In general, yeast cells are preferred over fungal cells because they are easy to manipulate. However, some proteins are not very secreted from yeast cells or in some cases are not processed properly (eg hyperglycosylation in yeast). In these cases, a different fungal host organism should be selected.

  Typical fungal expression hosts are Aspergillus niger, Aspergillus niger var. tubeigenis, Aspergillus niger var. awamori, Aspergillus aculeatis, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, can be selected from Kluyveromyces lactis, and Saccharomyces cerevisiae.

Suitable filamentous fungi are, for example, strains belonging to Aspergillus species such as Aspergillus oryzae or Aspergillus niger, or Fusarium oxysporium, Fusarium gramiarum, formerly referred to as Giberberella zea,
f. sp. Cerealis synonymous), or Fusarium sulphureum (in the perfect state, called Gibberella puricaris, Fusarium trichothercioides, Fusarium bactridioides, Fusarium sambucium, Fusarium roseum, and Fusarium roseum var.graminearum synonymous), Fusarium cerealis (Fusarium crokkwellnse synonymous) Or a strain of Fusarium venenatum.

  Suitable yeast organisms can be selected from Kluyveromyces, Saccharomyces, or Schizosaccharomyces (eg, Saccharomyces cerevisiae), or Hansenulas (disclosed in UK Patent Application No. 9927801.2).

  By using appropriate host cells (such as yeast, fungi, and plant host cells), post-translational modifications (eg, myristoylation, glycosylation) that may be necessary to confer optimal biological activity to the recombinant expression product of the invention. , Truncation, lipidation, and phosphorylation of tyrosine, serine, or threonine).

  The host cell can be a protease deficient or protease deficient strain. This can be, for example, the protease deficient strain Aspergillus oryzae JaL125 lacking the alkaline protease gene called “alp”. This strain is described in WO 97/35956.

(Living)
The term “organism” in relation to the present invention may include a nucleotide sequence encoding an enzyme of the present invention and / or a product obtained therefrom and / or, if a promoter is present in an organism, the nucleotide sequence of the present invention by the promoter. Any organism capable of expressing is included.

  Suitable organisms can include prokaryotes, fungi, yeasts, or plants.

  The term “transgenic organism” in relation to the present invention includes a nucleotide sequence encoding the enzyme of the present invention and / or a product obtained therefrom, and / or allows the nucleotide sequence of the present invention to be expressed in the organism by a promoter. Any organism is included. Preferably, the nucleotide sequence is integrated into the genome of the organism.

  The term “transgenic organism” does not cover natural nucleotide coding sequences in the natural environment when present under the control of a natural promoter that also exists in the natural environment.

  Accordingly, the transgenic organism of the present invention includes a nucleotide sequence encoding the enzyme of the present invention, a construct of the present invention, a vector of the present invention, a plasmid of the present invention, a cell of the present invention, a tissue of the present invention, or a product thereof. Organisms including any one or combination are included. For example, a transgenic organism can also include a nucleotide sequence that encodes an enzyme of the invention under the control of a heterologous promoter.

(Host cell / organism transformation)
As indicated above, the host organism can be prokaryotic or eukaryotic. Examples of suitable prokaryotic hosts include E. coli. E. coli and Bacillus subtilis are included.

Teachings regarding transformation of prokaryotic hosts are well documented in the art, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and
al. , Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc. checking ... When using prokaryotic hosts, the nucleotide sequence may need to be appropriately modified, such as by removal of an intron, prior to transformation.

  Filamentous fungal cells can be transformed by processes including protoplast formation and protoplast transformation followed by cell wall regeneration in a known manner. The use of Aspergillus as a host microorganism is described in EP 0238023.

  Another host organism may be a plant. The basic principle of construction of genetically modified plants is to insert genetic information into the plant genome and maintain the inserted genetic material stably. There are several techniques for the insertion of genetic information, and two main principles are the direct introduction of genetic information and the introduction of genetic information through the use of vector systems. An overview of general techniques can be found in Potrykus (Annu Rev Plant Physiolo Plant Mol Biol [1991] 42: 205-225) and Christou (Agro-Food-Industry Hi-Tech March / April 27, published in 1994). it can. Further teachings regarding plant transformation can be found in EP-A-0449375.

  General teachings on fungal, yeast, and plant transformation are given in the following sections.

(Transformed fungus)
The host organism can be a fungus (such as mold). Examples of suitable such hosts include any member belonging to the genus Phanerochaete, Thermomyces, Acremonium, Aspergillus, Penicillium, Mucor, Neurospora, Trichoderma, and the like (Thermomyges genus). Aspergillus oryzae, Apergillus awamori, Penicillinum chrysogenem, Mucor
Javanous, Neurospora crassa, Trichoderma
viridae, Phanerochaete chrysosporium, etc.).

  In one embodiment, the host organism can be a filamentous fungus.

  For nearly a century, filamentous fungi have been widely used in many types of industries that produce organic compounds and enzymes. For example, Aspergillus sp has been used in traditional Japanese Koji and soybean fermentation. In this century, Aspergillus niger is used for the production of organic acids (particularly citric acid) used in industry and the production of various enzymes.

  There are two main reasons why filamentous fungi are so widely used in industry. First, filamentous fungi can produce large amounts of extracellular products (eg, enzymes and organic compounds such as antibiotics or organic acids). Second, filamentous fungi can grow on low cost substrates such as cereals, bran, beet pulp and the like. The same reason makes filamentous fungi an attractive organism as a heterologous expression host of the present invention.

  To prepare transgenic Aspergillus, an expression construct is prepared by insertion of a nucleotide sequence of the invention into a construct designed for expression in a filamentous fungus.

  Several types of constructs used for heterologous expression have been developed. These constructs preferably include one or more signal sequences that direct the amino acid sequence to be secreted, typically of fungal origin, and a terminator (typically active in fungi) that terminates the expression system.

  Another type of expression system has been developed in fungi that can fuse the nucleotide sequences of the present invention to a small or large portion of a fungal gene encoding a stable protein. Thereby, an amino acid sequence can be stabilized. In such a system, a cleavage site recognized by a particular protease can be introduced between the fungal protein and the amino acid sequence, thereby cleaving the produced fusion protein with a particular protease at this position. Thus, the amino acid sequence can be released. As an example, sites recognized by KEX-2-like peptidases found in at least some Aspergillus can be introduced. Such fusions cleave in vivo to produce an expression product, but not a larger fusion protein.

  Heterologous expression in Aspergillus has been reported for several genes encoding bacterial, fungal, vertebrate and plant proteins. If the nucleotide sequence of the present invention is not fused to a signal sequence, the protein can be deposited in the cell. Such proteins accumulate in the cytoplasm and are usually not glycosylated, which may be advantageous for some bacterial proteins. When the nucleotide sequence of the present invention comprises a signal sequence, the protein accumulates extracellularly.

  With respect to product stability and host strain modification, some heterologous proteins are not very stable when secreted into fungal cultures. Most fungi produce several extracellular proteases that degrade heterologous proteins. To circumvent this problem, a special novel strain with reduced protease production was used as a heterologous production host.

  The teachings on transformation of filamentous fungi are outlined in US Pat. No. 5,741,665, which describes that standard transformation techniques for filamentous fungi and fungal culture are well known in the art. N. A broad review of techniques applied to crassa can be found, for example, in Davis and de Serres, Methods Enzymol (1971) 17A: 79-143. In general, standard procedures are used for strain maintenance and conidia preparation. Lambowitz et al. J. et al. As described in Cell Biol (1979) 82: 17-31, mycelium typically grows in liquid culture for about 14 hours (25 ° C.). The host strain is generally either Vogel's minimal medium or Fries's minimal medium supplemented with appropriate nutrients such as any one or more of his, arg, phe, tyr, trp, p-aminobenzoic acid, and inositol. It can grow in.

Further teachings on the transformation of filamentous fungi are outlined in US Pat. No. 5,674,707, once the construct is obtained, DNA-mediated transformation, electroporation, gene gun bombardment, and protoplast fusion. Can be introduced into a selected filamentous fungal host in either a linear or plasmid form (eg, a pUC-based vector or other vector). Further, Ballance, 1991 (same section) describes that the transformation protocol for the preparation of transformed fungi is based on the preparation of protoplasts and the introduction of DNA into protoplasts using PEG and Ca ²⁺ ions. . The transformed protoplasts are then regenerated and transformed fungi are selected using various selectable markers.

  In order to select the resulting transformants, transformation is typically a selection in which an expression cassette has been introduced, either on the same vector or by co-transformation, into a host strain from which genetic markers can be selected. Contains possible genetic markers. A variety of marker / host systems are available, the pyrG, argB, and niaD genes for use with an auxotrophic strain of Aspergillus nidulans; the pyrG and argB genes of the Aspergillus oryzae auxotrophic strain; Penicillium chrysogenum auxotrophy Strains of pyrG, trpC, and niaD genes; and the argB gene of Trichoderma reesei auxotrophic strains. Dominant selectable markers including amdS, oliC, hyg, and phleo are also described in A.S. niger, A.M. oryzae, A.M. ficuum, P.M. Chrysogenum, Cephalosporium acremonium, Cochliobolus heterostrohus, Glosserella singulata, 95, L Available for use. Commonly used transformation markers are those that can grow high copy number fungi using acrylamide as the sole nitrogen source. Nidulans amdS gene.

  For transformation of filamentous fungi, several transformation protocols for a large number of filamentous fungi have been developed. Of the markers, many auxotrophic markers such as argB, trpC, niaD, and pyrG, antibiotic resistance markers such as benomyl resistance, hygromycin resistance, and phleomycin resistance are used for transformation.

  In one aspect, the host organism can be a host organism of the genus Aspergillus, such as Aspergillus niger.

  The transgenic Aspergillus of the present invention can also be prepared according to the following teaching: Rambosek J. et al. and Leach, J.A. 1987 (Recombinant DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6: 357-393), Davis R. et al. W. 1994 (Heterologous gene expression and protein secretion in Aspergillus In:. Martinelli S.D., Kinghorn J. R. (Editors) Aspergillus:. 50 years on Progress in industrial microbiology vol 29. Elsevier Amsterdam, 1994. pp 525-560) Ballance, D .; J. et al. 1991 (Transformation systems for Filamentous Fungi and an Overview of Fungal Gene structure In:... Leong, S.A., Berka R.M. (Editors) Molecular Industrial Mycology Systems and Applications for Filamentous Fungi Marcel Dekker Inc. New York 1991 Pp 1-29), and Turner G. et al. 1994 (Vectors for genetic manipulation. In: Martinelli S. D. Kinghorn J. R. (Editors) Aspergillus: 50 years on. Progress in industrial 29. Evidence of industriol.

(Transformed yeast)
In another embodiment, the transgenic organism can be a yeast.

  In this regard, yeast is also widely used as a medium for heterologous gene expression.

  As an example, the species Saccharomyces cerevisiae has a long history of industrial applications including use in heterologous gene expression. Heterologous gene expression in Saccharomyces cerevisiae is described by Goodey et al. (1987, Yeast Biotechnology, DR Berry et al, eds, pp 401-429, Allen and Unwin, London et al. 198 E. F. Walton and GT T. Yarronton, eds, pp 107-133, Blackie, Glasgow).

  For several reasons, Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is not pathogenic to humans and cannot produce certain endotoxins. Second, there is a history of safe use for a long time after commercial development for various purposes. This has been widely publicly approved. Thirdly, extensive commercial use and research of organisms has gained a wealth of knowledge about the genetics and physiology of Saccharomyces cerevisiae and large-scale fermentation characteristics.

A review of the principles of heterologous gene expression and gene product secretion in Saccharomyces cerevisiae can be found in E. Hinchcliffe E Kenny (1993, “Yeast as a vehicle for the expression of heterogeneous genes”, Yeasts, Vol 5,
Anthony H Rose and J Start Harrison, eds, 2nd edition, Academic Press Ltd. ).

  Several yeast vector types are available, including integrative vectors and autonomously replicating plasmid vectors that require recombination with the host genome to maintain them.

  To prepare transgenic Saccharomyces, an expression construct is prepared by inserting the nucleotide sequence of the present invention into a construct designed for expression in yeast. Several construct types have been developed for use in heterologous expression. The construct contains a promoter active in yeast, such as a promoter of yeast origin, such as the GAL1 promoter. Usually, a signal sequence of yeast origin, such as a sequence encoding a SUC2 signal peptide, is used. Terminators active in yeast stop the expression system.

  Several transformation protocols have been developed for yeast transformation. For example, the transgenic Saccharomyces of the present invention can be obtained from the following Hinnen et al. (1978, Proceedings of the National Academy of Sciences USA 75, 1929; Beggs, JD (1978, Non; , H et al. (1983, J. Bacteriology 153, 163-168).

  A variety of selectable markers can be used to select for transformed yeast cells. Of the markers, several auxotrophic markers such as LEU2, HIS4, and TRP1 and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers (eg G418) are used for transformation.

(Transformed plant / plant cell)
Preferred host organisms suitable for the present invention are plants.

  In this aspect, the basic principle in constructing genetically modified plants is to insert genetic information into the plant genome to maintain the inserted genetic material stably.

  There are several techniques for the insertion of genetic information, and two main principles are the direct introduction of genetic information and the introduction of genetic information through the use of vector systems. General techniques are reviewed in Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42: 205-225) and Christou (Agro-Food-Industry Hi-Tech March / April, 1994, 17). be able to.

  Even though the promoters of the present invention are not disclosed in EP-B-0470145 and CA-A-2006454, these two documents describe the types of techniques that can be used in the preparation of the transgenic plants of the present invention. Explains some useful backgrounds. Some of these background teachings are included in the discussion below.

  The basic principle of constructing genetically modified plants is to insert genetic information into the plant genome and maintain the inserted genetic material stably.

  Accordingly, in one aspect, the present invention relates to a vector system comprising the nucleotide sequence or construct of the present invention and capable of introducing the nucleotide sequence or construct into the genome of an organism such as a plant.

  A vector system may contain one vector but may contain two vectors. In the case of two vectors, the vector system is usually called a binary vector system. The binary vector system is described in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.

  One widely used plant cell transformation system using a given promoter or nucleotide sequence or construct is based on the use of Ti plasmids from Agrobacterium tumefaciens or Ri plasmids from Agrobacterium rhizogenes (An et al. (1986), Plant Physiol. 81, 301-305 and Butcher D. N. et al. (1980) Tissue Culture Methods for Plant Pathologies, eds .: D. S. Ingrams. .

  Several different Ti and Ri plasmids have been constructed that are suitable for the construction of the plant or plant cell constructs described above. A non-limiting example of such a Ti plasmid is pGV3850.

  In order to avoid disruption of sequences immediately surrounding the T-DNA boundary, at least one of these regions is essential for the insertion of the modified T-DNA into the plant genome, the nucleotide sequence or construct of the invention is Preferably, it should be inserted into a Ti-plasmid between the terminal sequences of the T-DNA or adjacent to the T-DNA sequence.

  As will be understood from the above description, when the organism is a plant, the vector system of the present invention is preferably a sequence necessary for plant infection (eg, vir region) and at least one border portion of the T-DNA sequence. (The boundary part is present on the same vector as the gene construct). Preferably, the vector system is an Agrobacterium tumefaciens Ti-plasmid or Agrobacterium rhizogenesRi-plasmid or derivative thereof, although it is well known that these plasmids are widely used in the construction of transgenic plants, This is because there are numerous vector systems based on plasmids or derivatives thereof.

  For construction of transgenic plants, the nucleotide sequences or constructs of the present invention can be first constructed in a microorganism capable of replicating the vector and easy to manipulate prior to insertion into the plant. Examples of useful microorganisms are E. coli. other microorganisms having the above properties can be used. The vector system defined above is E. coli. If constructed in E. coli, it is transferred to an appropriate Agrobacterium strain (eg, Agrobacterium tumefaciens) as needed. Accordingly, a Ti-plasmid having a nucleotide sequence or construct of the present invention is preferably transferred into a suitable Agrobacterium strain (eg, A. tumefaciens) to obtain Agrobacterium cells having the nucleotide sequence or construct of the present invention, The DNA is then transferred into the plant cell to be modified.

  As reported in CA-A-2006454, E.I. Large quantities of cloning vectors are available that contain a replication system in E. coli and a marker that allows selection of transformed cells. Vectors include, for example, pBR322, pUC series, M13mp series, pACYC184, and the like.

  Thus, the nucleotides or constructs of the present invention can be introduced into appropriate restriction sites in the vector. The contained plasmid was transformed into E. coli. Used for transformation in E. coli. E. E. coli cells are cultured in a suitable nutrient medium, harvested and lysed. The plasmid is then recovered. Analytical methods include commonly used sequence analysis, restriction analysis, electrophoresis, and even biochemical molecular biology methods. After each manipulation, the DNA sequence used can be digested by restriction and ligated to the next DNA sequence. Each sequence can be cloned into the same or different plasmids.

  The presence and / or insertion of additional DNA sequences may be necessary after each introduction method of the desired promoter, construct or nucleotide sequence of the present invention in the plant. For example, when using plant cell Ti-plasmid or Ri-plasmid for transformation, the right border of the T-DNA of Ti-plasmid and Ri-plasmid, but many In the case of, the left and right boundaries can be connected. The use of T-DNA for the transformation of plant cells has been extensively studied and is described in EP-A-120516; Hoekema, in: The Binary Plant Vector System-drugkerij Kants B. et al. B. Alblaserdam, 1985, Chapter V; Fraley et al. , Crit. Rev. Plant Sci. , 4: 1-46; and An et al. , EMBO J. et al. (1985) 4: 277-284.

Direct infection of plant tissue with Agrobacterium is widely used and is described in Butcher D. et al. N. et al. (1980), Tissue Culture Methods for Plant Pathologies, eds. : D.
S. Ingrams and J.M. P. A simple technique described in Helgeson, 203-208. For further teaching on this subject, see Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42: 205-225) and Christou (Agro-Food-Industry Hi-Tech March / April 1994 17). This technique can be used to infect plants to certain parts or tissues of the plant (ie leaves, roots, part of the trunk or another part of the plant).

  Typically, direct infection of plant tissue by Agrobacterium carrying a promoter and / or GOI is used, for example, by cutting the plant with a razor, puncturing the plant with a needle, or rubbing the plant with an abrasive. Hurt plants to be infected. The wound is then inoculated with Agrobacterium. The inoculated plant or plant part is then grown in a suitable culture medium and grown into mature plants.

  When plant cells are constructed, they are grown and maintained according to well-known tissue culture methods such as culturing the cells in an appropriate culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc. be able to. Using known methods of regenerating plants from cell or tissue culture (eg, by selection of transformed shoots using antibiotics and subculture of shoots in media containing appropriate nutrients, plant hormones, etc.) Transformed cells can be regenerated into genetically modified plants.

Other plant transformation techniques include ballistic transformation, silicon whisker carbide technique (Frame BR, Drayton PR, Bagnaall SV, Lewnau CJ, Bullock WP JA & Wang K (1994) Production of fertile transition genetics plants by silicon carved whiskey-mediated transformation, The Plant 9 hof I (1992) The use of cassava mosaic
see virus as a vector system for plants, Gene, 110: 213-217). Ballistic transformation techniques are shown in the following sections.

Further plant transformation techniques can be found in EP-A-0449375.
Ballistic transformation of plants and plant tissues As suggested, the techniques for producing transgenic plants are well known in the art. Typically, either whole plants, cells, or protoplasts can be transformed with an appropriate nucleic acid construct encoding a zinc finger molecule or target DNA (see, eg, the nucleic acid construct above). There are many methods for introducing a transforming DNA construct into cells, but not all are suitable for delivering DNA to plant cells. Suitable methods include Agrobacterium infection (see in particular Turpen et al., 1993, J. Virol. Methods, 42: 227-239) or, for example, PEG-mediated transformation, electroporation, or DNA-coated particles Direct DNA delivery such as acceleration of In general, the acceleration method is preferable, and includes, for example, a fine particle gun.

  Originally developed for the production of stable transformants of plant species that resist transformation by Agrobacterium tumefaciens, ballistic transformation of plant tissue that introduces DNA into cells on the surface of metal particles is a transient process. Useful for testing the performance of gene constructs in sexual expression. In this method, gene expression in a transiently transformed cell can be studied without stably incorporating the target gene, so that it is not necessary to produce a stable transformant that takes time.

  More particularly, ballistic transformation techniques (also known as gene gun techniques) were first described by Klein et al. (1987), Sanford et al. (1987), and Klein et al. (1988). Widespread due to the need for cell or tissue pretreatment.

  The principle of gene gun technology is the direct delivery of DNA-coated microprojectiles to intact plant cells by a driving force (eg, discharge or compressed air). Microparticles penetrate cell walls and cell membranes with only minor damage, after which transformed cells express the promoter construct.

  One gene gun technique that can be implemented uses the Particle Inflight Gun (PIG) developed and described by Finer et al. (1992) and Vain et al. (1993). PIG accelerates microparticles into plant cells in a helium stream with an incomplete vacuum.

  One advantage of PIG is that particulate acceleration can be controlled by adjusting a timer relay solenoid and the supplied helium pressure. The use of pressurized helium as the driving force has the advantage that it is inert, produces no residue and gives a reproducible acceleration. Vacuum reduces particle inhalation and tissue damage is reduced by diffusion of helium gas prior to impact (Finer et al. 1992).

  In some cases, the effectiveness and ease of the PIG system made a good choice for the generation of transiently transformed guar tissue and was tested for transient expression of promoter / reporter gene fusions.

  A production protocol for a typical transgenic plant (particularly monocotyledonous plant) obtained from US Pat. No. 5,874,265 is described below.

  An example of a method for delivering transforming DNA segments to plant cells is a particle gun. In this method, non-biological particles can be coated with nucleic acids and delivered to the cells by driving force. Examples of the particles include particles composed of tungsten, gold, platinum, and the like.

  A particular advantage of gene guns is that, in addition to being an effective means of stably and stably transforming both dicotyledonous and monocotyledonous plants, neither protoplast isolation nor susceptibility to Agrobacterium infection is required. is there. An exemplary embodiment of a method for delivering DNA to plant cells by acceleration is through a screen such as stainless steel or a Nytex screen on a filter surface coated with plant cells grown in suspension of DNA-coated particles. A Biolistics Particle Delivery System that can be used to propel The screen disperses the tungsten-DNA particles so that they are not delivered to the recipient cells as large aggregates. Without the use of a screen intervening between the launcher and the cells to be fired, the projectiles would aggregate and would be too large to achieve a high frequency of transformation. This can be due to damage to the recipient cell by a projectile that is too large.

  For shoot, it is preferable to concentrate the cells in suspension with a filter. Place the filter containing the cells to be shot at an appropriate distance under the particulate stopping plate. If desired, one or more screens are also placed between the gun and the cells to be shot. By using the techniques described herein, one can obtain over 1000 cell clusters that transiently express the marker gene (“focus”) on the fired filter. The number of cells in focus that express the exogenous gene product 48 hours after bombardment is often in the range of 1 to 10 and an average of 2 to 3.

  After delivery of exogenous DNA to recipient cells by any of the methods discussed above, a preferred step is the identification of transformed cells for further culture and plant regeneration. This step can include assaying the culture directly for a screenable trait or by exposing the culture bombarded with a selective agent.

An example of a marker trait that can be screened is the red pigment produced under the control of the maize R locus. This dye can be used to culture cells on a solid support containing a nutrient medium that can assist in this stage of growth, eg, incubation of cells at 18 ° C. over 180 μEm ⁻² s ⁻¹ , and colored colonies (visual Detection by selection of cells from possible cell aggregates). These cells can be further cultured in either suspension or solid media.

  Exemplary embodiments of methods for identifying transformed cells include exposure of the bombarded culture to selective agents (metabolic inhibitors, antibiotics, herbicides, etc.). Cells that have been transformed and stably incorporate a marker gene that confers resistance to the selected drug used grow and divide in the medium. Sensitive cells are not subjected to further culture.

  To use the bar-bialaphos selection system, cells shot on the filter are resuspended in non-selective liquid medium, cultured (eg, 1-2 weeks), and solid medium containing 1-3 mg / l bialaphos is obtained. Transfer to layered filter. A range of 1-3 mg / l is typically preferred, but it is proposed that a range of 0.1-50 mg / l is useful in the practice of the present invention. The type of firing filter is not particularly critical, but may include any solid, porous, inert support.

  Cells that survive the exposure to the selective agent can be cultured in a medium that aids plant regeneration. Tissues are maintained in basal medium with hormones for about 2-4 weeks and then transferred to medium without hormones. Two to four weeks later, the time for shoot growth to transfer to another medium will be signaled.

Regeneration typically requires sequential transfer to a medium whose composition has been modified so that appropriate nutrient and hormone signals are obtained during successive growth stages from transformed calli to more mature plants. It is. Growing plantlets are transferred to the soil in an environmentally controlled chamber with, for example, about 85% relative humidity, 600 ppm CO ₂ , and 250 μEm ⁻² s ⁻¹ light to harden the soil. Preferably, the plants are matured either in a growth chamber or in a greenhouse. Typically, regeneration takes about 3-12 weeks. During regeneration, cells are grown on solid media in tissue culture vessels. An exemplary embodiment of such a container is a petri dish. Regenerated plants are preferably grown at about 19 ° C to 28 ° C. After regeneration, the plants reach the stage of shoot and root development and can be transferred to the greenhouse for further growth and testing.

  Genomic DNA can be isolated from callus cell lines and plants to determine the presence of exogenous genes by use of techniques well known to those skilled in the art, such as PCR and / or Southern blotting.

There are several techniques for the insertion of genetic information, and two main principles are the direct introduction of genetic information and the introduction of genetic information through the use of vector systems. An overview of general techniques can be found in Potrykus (Annu Rev Plant Physiol Plant Mol.
Biol [1991] 42: 205-225) and Christou (Agro-Food-Industry Hi-Tech March / April 1994).
17-27).

(Culture and production)
Host cells transduced with the nucleotide sequence can be cultured under conditions that are beneficial for production of the encoded enzyme and facilitate recovery of the enzyme from the cell and / or culture medium.

  The medium used in the cell culture can be any conventional medium suitable for the growth of the relevant host cells and expression of the enzyme. Suitable media are either commercially available or can be prepared according to published recipes (eg, as described in the catalog of the American Type Culture Collection).

  Proteins produced by recombinant cells can be displayed on the cell surface. If desired, an expression vector containing a coding sequence can be designed using a signal sequence that directs secretion of the coding sequence by a particular prokaryotic or eukaryotic cell membrane, as will be appreciated by those skilled in the art. Other recombinant constructs can link the coding sequence to a nucleotide sequence encoding a polypeptide domain that facilitates purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12: 441-53).

  Enzymes can be secreted from the host cell, separated from the medium by centrifugation or filtration, and precipitation of protein components of the medium with a salt such as ammonium sulfate, followed by chromatographic procedures such as ion exchange chromatography or affinity chromatography Can be conveniently recovered from the culture medium by well-known procedures including the use of

(secretion)
Often it is desirable to have the enzyme secreted from the expression host into a culture medium where the enzyme can be more easily recovered. According to the present invention, a secretory leader sequence can be selected based on the desired expression host. Hybrid signal sequences can also be used in the context of the present invention.

  Typical examples of heterologous secretory leader sequences are the fungal amyloglucosidase (AG) gene (glaA—for example, 18 and 24 amino acid versions from Aspergillus), the factor a gene (yeast, for example, Saccharomyces, Kluyveromyces, and Hansenula) Or a sequence derived from the α-amylase gene (Bacillus).

(detection)
Various detection and measurement protocols for amino acid sequence expression are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on the POI can be used, or a competitive binding assay can be used. These and other assays are described, inter alia, in Hampton R et al. (1990, Serial Methods, A Laboratory Manual, APS Press, St Paul MN) and Madox DE et al. (1983, J Exp Med 15, 8: 121 1). Yes.

  A wide variety of labels and conjugation techniques are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybrids or PCR probes for amino acid sequence detection include oligo labeling using labeled nucleotides, nick translation, end labeling, or PCR amplification. Alternatively, NOI or any part thereof can be cloned into a vector for production of mRNA probes. Such vectors are known in the art and are commercially available and can be used for the synthesis of RNA probes in vitro by the addition of a suitable RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.

  Numerous companies (such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WI), and US Biochemical Corp (Cleveland, OH)) provide commercial kits and protocols for these procedures. Suitable reporter molecules or labels include radionuclides, enzymes, fluorescence, chemiluminescent agents, or color formers, as well as substrates, cofactors, inhibitors, magnetic particles, and the like. Patents that teach the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Alternatively, recombinant immunoglobulins can be produced as described in US Pat. No. 4,186,567.

Additional methods for quantifying the expression of amino acid sequences include radioactive labeling (Melby PC et al
1993 J Immunol Methods 159: 235-44) or biotin-treated (Dupla C et al 1993 Anal Biochem 229-36) nucleotides, simultaneous amplification of control nucleic acids, and a standard curve interpolating experimental results. Quantification of multiple samples can be accelerated by performing assays in an ELISA format that provides the oligomer of interest at various dilutions and rapidly quantifies by spectrophotometer or calorimetric response.

  The presence or absence of marker gene suggests the presence of a nucleotide sequence, but its presence and expression must be confirmed. For example, when a nucleotide sequence is inserted within a marker gene sequence, recombinant cells containing the nucleotide sequence can be identified by the absence of marker gene function. Alternatively, the marker gene can be placed in tandem with the nucleotide sequence under the control of a promoter of the invention or another promoter (preferably the same promoter of the invention). The expression of a marker gene in response to induction or selection likewise indicates normal amino acid sequence expression.

  Alternatively, host cells containing nucleotide sequences can be identified by various procedures known to those skilled in the art. These procedures include protein bioassay or immunoassay techniques, including membrane-based, solution-based, or chip-based technologies for DNA-DNA or DNA-RNA hybridization and detection and / or quantification of nucleic acids or proteins Is included, but is not limited thereto.

(Fusion protein)
The amino acid sequences of the invention can be produced as fusion proteins, for example, to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and / or transcriptional activation domain), and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to remove the fusion protein sequence. Preferably, the fusion protein does not interfere with the activity of the protein sequence.

  A fusion protein may comprise an antigen or antigenic determinant fused to a substance of the invention. In this embodiment, the fusion protein can be a non-naturally occurring fusion protein comprising a substance that can act as an adjuvant in the sense of providing systemic stimulation of the immune system. An antigen or antigenic determinant can be linked to either the amino terminus or the carboxy terminus of the substance.

  In another embodiment of the invention, the amino acid sequence can be ligated to a heterologous sequence to encode a fusion protein. For example, for screening peptide libraries for agents that can affect substance activity, it may be useful to encode a chimeric substance that expresses a heterologous epitope recognized by a commercially available antibody.

(More POI)
The sequences of the invention can be used in combination with one or more additional proteins of interest (POI) or nucleotide sequences of interest (NOI).

Non-limiting examples of POIs include proteins or enzymes involved in starch metabolism, proteins or enzymes involved in glycogen metabolism, acetylesterase, aminopeptidase, amylase, arabinase, arabinofuranosidase, carboxypeptidase, catalase, cellulase, Chitinase, chymosin, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lysase, endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β- Glucosidase, glucuronidase, hemicellulase, hexose oxidase, hydrolase, invertase, isomerase, laccase, lysase Lyase, lyase, mannosidase, oxidase, oxidoreductase, pectate lyase, pectin acetyl esterase, pectin depolymerase, pectin methyl esterase, pectin degrading enzyme, peroxidase, phenol oxidase, phytase, polygalacturonase, protease, rhamnogalacturo A nuclease, ribonuclease, thaumatin, transferase, transport protein, transglutaminase, xylanase, hexose oxidase (D-hexose: O ₂ -oxidoreductase, EC 1.1.3.5), or combinations thereof. The NOI may further be an antisense sequence of any of these sequences.

  The POI may further be a fusion protein to aid, for example, extraction and purification.

  Examples of fusion protein partners include maltose binding protein, glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and / or transcriptional activation domain), and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion component.

  The POI can also be fused to a secretory sequence. Examples of secretory leader sequences are those derived from the amyloglucosidase gene, the α factor gene, the α-amylase gene, the lipase A gene, and the xylanase A gene.

  Other sequences can also promote secretion of secreted POI or increase yield. Such a sequence can, for example, encode a chaperone protein as the Aspergillus niger cyp B gene product described in British Patent Application No. 9821198.0.

  The NOI can be engineered to alter its activity for a number of reasons, including but not limited to alterations that alter the processing and / or expression of its expression product. For example, using techniques well known in the art (e.g., site-directed mutagenesis) to insert new restriction sites, change glycosylation patterns, or change codon preference, Mutations can be introduced. As a further example, the NOI can be modified to optimize expression in a particular host cell. Other sequence changes may be desirable to introduce restriction enzyme recognition sites.

  The NOI can include synthetic or modified nucleotides therein. Many different modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3 'and / or 5' ends of the molecule. For the purposes of the present invention, it should be understood that the NOI can be modified by any method available in the art. Such modifications can be made to increase the in vivo activity of NOI or extend lifespan.

  The NOI can be modified for increased intracellular stability and half-life. Possible modifications include, but are not limited to, addition of flanking sequences at the 5 'and / or 3' end of the molecule, or the use of phosphorothioate or 2'O-methyl rather than phosphodiesterase linkages within the molecule's backbone. Not.

(antibody)
One aspect of the present invention relates to an amino acid sequence that exhibits an immune response with one or more of the amino acid sequences of claim 1.

  Antibodies can be produced by standard techniques, such as immunization with a substance of the invention or use of a phage display library.

For the purposes of the present invention, the term “antibody” includes, unless stated to the contrary, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, fragments produced by a Fab expression library, As well as mimetics thereof. Such fragments include intact antibody fragments (Fv, F (ab ′), and F (ab ′) ₂ fragments) that retain the binding activity to the target substance, as well as single chains containing the antigen binding site of the antibody. Antibodies (scFv), fusion proteins, and other synthetic proteins are included. Furthermore, the antibodies and fragments thereof can be humanized antibodies. Neutralizing antibodies (ie, antibodies that inhibit the biological activity of a substance polypeptide) are particularly preferred for diagnosis and therapy.

  If a polyclonal antibody is desired, a selected mammal (eg, mouse, rabbit, goat, horse, etc.) is immunized with a sequence of the invention (or a sequence comprising an immune epitope thereof). Depending on the host species, various adjuvants can be used to increase the immune response. Such adjuvants include Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oily emulsions, keyhole limpet hemocyanin, and dinitro Phenol is included but is not limited to these. BCG (Bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants that can be used to administer polypeptides to immunocompromised individuals for stimulation of systemic defense when the material is purified .

  Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to a sequence of the invention (or a sequence containing an immune epitope thereof) contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Polyclonal antisera production and processing techniques are known in the art. In order to be able to make such antibodies, the invention also provides fragments thereof haptenized to a polypeptide of the invention, or another polypeptide for use as an immunogen in animals or humans. .

  Those skilled in the art can also readily produce monoclonal antibodies directed against the sequences of the present invention (or sequences containing immune epitopes thereof). A general method for producing a monoclonal antibody using a hybridoma is well known. Immortal antibody-producing cell lines can also be generated by cell fusion and other techniques such as direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. A panel of monoclonal antibodies raised against an orbit epitope can be screened for a variety of properties (ie, isoforms and epitope affinity).

Monoclonal antibodies against a sequence of the invention (or a sequence comprising an immune epitope thereof) can be prepared using any technique in which antibody molecules are produced by continuous cell lines in culture. These include the hybridoma technique first described in Koehler and Milstein (1975 Nature 256: 495-497), the human B cell hybridoma technique (Kosbor et al (1983) Immunol).
Today 4:72; Cote et al (1983) Proc Natl
Acad Sci 80: 2026-2030), and EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies.
and Cancer Therapy, Alan R Lith Inc, pp
77-96), but is not limited thereto. In addition, techniques developed for the production of “chimeric antibodies” (splicing of mouse antibody genes into human antibody genes to obtain molecules with appropriate antigen specificity and biological activity) can be used (Morrison) et al (1984) Proc Natl Acad Sci 81: 6851-6855; Neuberger et al (1984) Nature.
312: 604-608; Takeda et al (1985) Nature.
314: 452-454). Alternatively, techniques described for single chain antibody production (US Pat. No. 4,946,779) can be adapted to produce substance specific single chain antibodies.

  Induction of in vivo production in lymphocyte populations or screening of a panel of recombinant immunoglobulin libraries or highly specific binding reagents as described in Orlandi et al. (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Antibodies can also be produced by Milstein C (1991; Nature 349: 293-299).

Antibody fragments that contain specific binding sites for the substance can also be generated. For example, such fragments include F (ab ′) ₂ fragments that can be produced by pepsin digestion of antibody molecules, and Fab fragments that can be generated by reduction of disulfide bonds of F (ab ′) ₂ fragments. Including, but not limited to. Alternatively, Fab expression libraries can be constructed so that monoclonal Fab fragments with the desired specificity can be quickly and easily identified (Huse WD et al (1989) Science 256: 1275-1281).

(Large scale application)
In one preferred embodiment of the invention, amino acid sequences are used for large scale applications.

  Preferably, the amino acid sequence is produced in an amount of 1 g to about 2 g per liter total cell culture volume after cultivation of the host organism.

  Preferably, the amino acid sequence is produced in an amount of 100 mg to about 900 mg per liter total cell culture volume after cultivation of the host organism.

  Preferably, the amino acid sequence is produced in an amount of from 250 mg to about 500 mg per liter of total cell culture volume after culturing of the host organism.

(Summary)
In summary, the present invention relates to amino acid and nucleotide sequences and constructs comprising the same. The invention also relates to a novel use of known enzymes.

  The invention will be further described in the following non-limiting examples with reference to the following drawings.

(Pyranozone dehydrogenase purified from the fungus Phanerochaete chrysosporium)
Phanerochaete chrysosporium (white rot fungus) is a biotechnologically important fungus due to its high optimum growth temperature (40 ° C.) and the ability to produce a range of extracellular oxidase. This fungus is therefore used in the treatment of various wastes, including explosive pollutants, insecticides, and toxic waste. In addition, Phanerochaete chrysosporium is the first basidiomycete genome that was sequenced (University of California and Department of Energy, USA).

  In the study of enzymes that metabolize anhydrofructose (AF), A thermostable pyranozone dehydrogenase (PD) purified from Chrysosporium was obtained. Studies have shown that this purified PD not only uses AF as a substrate, but also uses it more effectively than its natural substrate, glucosone. Furthermore, the product has been shown to be an antifungal agent microsesin useful in plant protection agents.

  The N-terminal sequence of PD and the endo-N-terminal sequence of PD after hydrolysis with two proteinases were elucidated. Both account for 332 amino acids, ie 37% of the total length of the PD protein based on the assumption that Mr is 97 kDa.

  A full-length PD gene was identified in Scaffold 62 by database search using the partial amino acid sequence for the fungal genome (FIG. 4). Transcription start and stop codons, both containing three introns, were identified (Figure 4). Purified PD appears to be functional, although the N-terminal 7 amino acids have been truncated. Since no enzyme PD was found in the culture medium, it could not contain a signal peptide.

(Assay method)
(Measurement of PD activity)
The reaction mixture consisted of 25 μl anhydrofructose solution (3.0%), 10 μl PD preparation, 93 μl 0.1 M sodium phosphate pH 6.5, and water to a final volume of 1 ml. The reaction was mixed and scanned from 190 to 320 nm at room temperature (22 ° C.) on a Perkin Elmer Lambda 18 uv / vis spectrophotometer every 5 minutes or after 30 minutes. The absorbance at 265 nm and 230 nm was recorded. One active unit of PD is defined as an increase of 0.01 absorbance units at 230 nm per minute at 22 ° C.

Protein assays were performed using the Bio-Rad method (Bradford method) using reagents and instructions from Bio-Rad laboratories (Peterson, GL: Determination of total protein,
Methods Enzymol. 91, 95-119 (1983)).

To separate glucose, AF, and microsecin, TLC was performed using a solvent system of ethyl acetate, acetic acid, methanol, and water (12: 3: 3: 2) as previously described (Yu S, Ahmad T, Pedersen M, Kenne L: α-1, 4-Glucan lyase, a new class of start / biocognition enzyme, III. Substrate specificity. 9 (1995)). Merck silica gel 60 (20 × 20 cm) plates with a thickness of 0.15 mm were used. 1,5-Anhydro-D-fructose was assayed by the DNS method (Yu S, Olsen CE, Marcussen J:
Methods for the assay of 1, 5-anhydro-D-Fructose and α-1, 4-glucan lyase, Carbohydr. Res. 305: 73-82 (1998)).

(PD purification)
The purification procedure used was essentially the same as that described in Gabriel et al. (1993) except that the strains used were different. Furthermore, an excess ammonium sulfate fractionation step was included. The strains used in this application were the American Type Culture Collection, Panerochaete chrysosporium (ATCC 32629) and (ATCC 24725), and the strain used by Gabriel et al. (1993) was obtained from Czechischertectorpure. .

  Cell-free extract of Phanerochaete crysosporum was subjected to up to 55% saturated ammonium sulfate. It was then gently blended for 2 hours and centrifuged at 10,000 xg for 20 minutes at 4 ° C. The precipitate with PD activity was dissolved in the same volume of extraction buffer, centrifuged again and the supernatant was used for purification of PD using the procedure described in Gabriel et al. (1993).

  After the PD purification procedure, SDS-PAGE and native-PAGE were performed using the PhastSystem (Pharmacia) using an 8-25% gradient gel according to the manufacturer's instructions. Protein bands on the gel were visualized by Coomassie brilliant blue staining (PastGel Blue R). From FIG. 1A, PD1 was estimated to have a molecular weight of 97 kDa and had a mobility similar to the protein marker phosphorylase b (97.4 kDa).

(Amino acid sequencing)
Purified PD was used for amino acid sequencing. Amino acid sequencing of PD was performed as previously described (Yu. S .; Christensen TMIE, Kragh KM, Bojsen K, Markussen J: Efficient purification, charac- terization and parti- mation and parti- mation. glucan
lyases from fungi. Biochim. Biophys Acta 1339: 311-320 (1997)). PD was first partially hydrolyzed with proteinase. The produced peptide fragments were separated by HPLC. Each polypeptide was collected, the molecular weight determined with a mass spectrometer, and sequenced with an Applied Biosystems 476A sequencer using a pulse-liquid first cycle. PD was further characterized for its optimal pH and temperature, ionic requirements for activity, stability, and other kinetic properties.

(Amino acid sequence obtained from pyranozone dehydrogenase purified from fungus Phanerochaete chrysosporium)
The following amino acid sequences are obtained by either trypsin or endoproteinase LysC digestion. Peptides are purified by reverse phase HPLC and molecular weight information is obtained by MALDI-TOF mass spectrometry. The resulting sequence is then compared to the DNA sequence found in the White Rot Genome Project launched at the University of California (Phanerochaete chrysosporium). A sequence similarity alignment is performed using the BLAST algorithm.

  All peptides with significant alignment are found at Scaffold62.

(LysC peptide)
Peptide 27.3 (N-terminal)
KPHCEPEQPPAALPLFQPQLVQGGRPDXYWVEAFFPRSDSSK (V indicates heterogeneity)
This peptide is found from base pairs 38620 to 38742. There is an initiation codon at base pair 38599, and 38317 indicates a possible signal peptide. Sequencing of the protein PD2 (isozyme) provides an independent confirmation that this is the N-terminus of the protein.

X at residue 27 is G in the database, which fits well with the MS data.
MSc + = 4669.10 MSo + = 4668.01-0.023%
N-terminus The N-terminus of pyranozone dehydrogenase isozyme I (PDI) was found to be:
KPHXEPEQPPAALPLFQPQLVV (Q) GGPPDXY
(Means X is unknown and V (Q) can be V or Q or both (due to heterogeneity))
The above N-terminal sequence (peptide 27.3) was isozyme II (PDII). The N-termini of PDI and PDII are very similar or identical.
Peptide 31.4b
SDIQMFVNPYATTNNQSSSWTPSLAKLDFPVAMHYADITK (D indicates the possibility of heterogeneity)
This peptide is found from base pairs 38788-38963. The database sequence is interspersed with introns derived from base pairs 38836 to 38889. The sequence of residues 28-41 is confirmed by trypsin peptide 8.4.

The X at residue 19 is S in the database sequence, which is consistent with the MS data.
MSc + = 4591.22 MSo + = 4591.55 + 0.007%
Trypsin peptide peptide 6a
VSWLENPGELR
This peptide is found from base pairs 39096-39128.
MSc + = 1300.44 MSo + = 1300.45 + 0.001%
Peptide 5
DGVDCLWYDGAR
This peptide is found from base pairs 39426-39461.
MSc + = 1427.48 MSo + = 1427.48
LysC peptide peptide 27.4a
PAGSPTGIVRAEWTRHVLDFGXLXXK
This peptide is found from base pair 39673-39553.
The three X's are PNGs in the database that fit well with the MS data.
MSc + = 2876.27 MSo + = 2876.80 + 0.021%
Peptide 29.4.8
HTGSIHQVVCADIDGDGEDEFFLVAMMGADPPDFQRTGVWCYK
This peptide is found from base pair 39754-39879.
MSc + = 4727.13 MSo + = 4727.70 + 0.012%
Peptide 13.11
TEMEFLDVAGK
This peptide is found from base pairs 40244-40276.
MSc + = 1240.42 MSo + = 1240.53 + 0.009%
Peptide 14.2
KLTLVVLPPFARLDVERNVSVGVK
This peptide is found from base pairs 40277-40345.
MSc + = 2552.08 MSo + = 25551.35-0.029%
Trypsin peptide peptide 10.5
SMDELVAHNLFPAYVPDSVR
This peptide is found from base pairs 40526-40585.
MSc + = 2259.55 MSo + = 2259.77 + 0.009%
LysC peptide peptide 31.4a
NDATDGTPVLALLLDGGPSPQAWNISHVPPGTDMMYEIAHAK
This peptide is found from base pair 41293-41469 and contains an intron from base pair 41362-41416.
MSc + = 4289.73 MSo + = 4289.45-0.007%
Peptide 2b
TGSLVCARCWPPVK
This peptide is found from base pairs 41470-41508.
MSc + = 1477.11 MSo + = 1472.62 + 0.062%
Peptide 2a
NQRVAGTH SPAAMGLTSRWAVTK
This peptide is found from base pairs 41509-41577.
MSc + = 2440.71 MSo + = 2441.58 + 0.036%
Peptide 11.3
GQITFRLPEAPDHGPLFLSVSAIRHQ
This peptide is found from base pairs 41641-41718.
MSc + = 2888.34 MSo + = 2888.25-0.031%
This peptide is not terminated with K, the C-terminal indicator. This sequence is also followed by a stop codon.

  The molecular weight of this protein is about 97 KD. Based on the assumption that the average molecular weight of the amino acids is 110, the expected number of residues will be 880 and the total number of base pairs will be 2640.

  The number of base pairs calculated from the database is 3100. Since the two known introns are composed of 53 and 54 base pairs, if this number is assumed normal, the database sequence is expected to contain about 8 introns.

  The total number of residues sequenced in the present invention is 332 amino acids, accounting for 37% of the protein.

Example 1: Use of 1,5-Anhydro-D-fructose and PD for microsecin production
The reaction mixture consists of 5 μl (3.0%) 1,5-anhydro-D-fructose, 5 μl PD preparation, 65 μl sodium phosphate buffer (pH 6.0), and water to a final volume of 0.7 ml. It was. The reaction was monitored by scanning from 350-190 nm. A reaction time of 0 minutes was used as a blank. An absorption peak at about 230 nm indicates the formation of microsecin. The absorption at 265 nm indicates the formation of the first intermediate from AF before conversion to microsecin.

  The microsecin formed was further confirmed by relative mobility on TLC and conversion of 2-furylhydroxymethylketone showing a typical absorption peak at 275 nm (Baute M.-A. et al., 1986).

  For larger scale microsecin production, the AF used was 0.4% to 20%. After the reaction, AF disappeared from the reaction mixture using the DNS method (Yu. S .; Christensen TMIE, Kragh KM, Bojsen K, Marcussen J, Biochim Biophys Acta 1339: 311-320 (1997)). Microsecin formation was monitored at 265 nm, which shifted to 230 nm and was further monitored by the TLC method.

  We have found that 1,5-anhydro-D-fructose is a much better substrate for pyranozone dehydrogenase (PD) than its natural substrate, glucosone. The Vmax of AF is about 4.7 times that of glucosone (Table 1).

  The reaction system consisted of AF or glucosone 1-15 μl, 25 μl sodium phosphate buffer (6.5, 0.1 M), water, 1.4 μl PD (final volume 200 μl). The reaction was carried out at 22 ° C. for 5.5 hours. The formation of microsecin from AF and cortalcerone from glucosone was monitored at 226 nm.

(Example 2: Production of cortalcelon)
Cortalcerone is obtained by incubation of starch-type substrates (starch, waxy starch, dextrin, etc.) with starch hydrolases (aminoglucosidase and debranching enzyme or cyclodextrin transferase, pyranose 2-oxidase, PD, etc.). It can be generated in a process. After incubation, the cortalcelone can be separated from the reaction mixture by ultrafiltration using a membrane with a cutoff of 300-30,000, preferably 10,000.

Example 3: Use of 1,5-anhydro-D-fructose, PD, and ascopyrone P synthase for APP production
The reaction mixture consists of 1,5-anhydro-D-fructose 50 [mu] l (3.0%), PD preparation 5 [mu] l, Ascopyrone P synthase 5 [mu] l, 0.1 ml sodium phosphate buffer (pH 6.0), and final volume. Of water to make 0.8 ml. The reaction was monitored spectroscopically by the formation of APP at 289 nm. The reaction temperature was 22 ° C. and the reaction time was 24 hours. At the end, 90% AF converted to APP. The structure of APP was confirmed using previously described NMR (WO 00/56838 filed on March 16, 2000 claiming priority from UK application No. 990647.8 filed March 19, 1999). ).

(PD gene expression)
By the techniques well known in the art and referred to herein above, Pichia pastoris, Aspergillus niger, and Hansenula
PD genes can be expressed in production organisms such as polymorph.

(Antibody production)
N Harboe and A Ingild (“Immunoglobulin Immunization, Isolation, Evaluation of Antibody Titers”, Manual of Quantitative Immunoelectrophoresis, Methods and Applications, NH Axel et al., Edited by NH Axelet et al., 73 Antibodies to the amino acids of the present invention were raised by injection of purified enzymes into rabbits and isolation of immunoglobulins from antisera according to the procedure described in "Tools", John Wiley & Sons, New York, 1977).

(Microsecin as an antifungal agent)
The growth of fungi in plants is a huge economic hit. An example is damage to sugar beet seedlings and their leaves. Sugar beet seeds are subjected to fungal attack by species such as Rhizotonia solani, Pythium ultramum, Aphanomyces cochlioides, immediately after germination in soil. In the present invention, it has been found that microsecin can inhibit the growth of these pathogens. Therefore, economical grain (sugar) seeds (such as sugar beet seeds) are coated with a paste containing 50-2000 ppm microsecin and dried before being used for planting. Alternatively, an aqueous solution of microsecin can be sprayed directly onto the plant and its leaves.

(Experiment)
Microsecin base solution: 24 mg / ml, batch number Mic2001016
Dilution used

  All solutions were filtered through a 0.22 μm filter for sterilization.

  The solution was tested against the following fungi.

  A circular plug (10 mm diameter) of fresh mycelium was placed in the center of a Petri dish (9 cm diameter) containing PDA medium. (PDA = potato dextrose agar, Difco number 213400). A 5 mm diameter well was cut out around the agar plate. Each well received 50 μl of test solution. Alternatively, 20 μl of each test solution was placed directly on the agar along the perimeter of the plate. Also, 50 μl of each test solution was placed directly on top of the fungal hyphae plug.

  The agar plate was placed under sunlight at room temperature, but protected from direct sunlight.

The fungal response (inhibition zone) to the test substance was determined as follows.
Rhizoctonia solani: After 2-3 days of growth Phythium ultimate: After 1-2 days of growth Aphanomyces cochlioides: After 3-4 days of growth Cercospora beticola: After 3-4 weeks of growth (Results)
Microsesin as a fungal growth regulator has shown inhibition to Rhizoctonia solani, Pythium ultramum, Aphanomyces cochlioides, and Cercospora beticola. The minimum inhibitory concentrations (mic) of microsecin for these fungi were 240, 480, 1200, and 2400 ppm, respectively.

(Example 4: Effect of microsecin on pelleted sugar beet seeds)
The effect of microsecin in vitro on the phytofungal pathogens Phythium ultramum, Rhizotonia solani, and Aphanomyces cochlioides was investigated by screening for pathogen growth inhibition on agar plates (FIGS. 6, 7, 8).

  FIG. 7 shows the screening effect of different concentrations of microsecin against Aphanomyces cochlioides, and FIGS. 8 and 9 show the screening effect against Pythium ultramium and Rhizoctonia solani, respectively. In each case, microsecin was dissolved in water and placed in the surrounding well. An agar block containing the pathogen was placed in the center. Pathogens were grown on PDA-agar plates for 3-5 days. These studies showed that very low concentrations of microsecin can reduce the growth of Aphanomices.

  Similar tests using other microorganisms show that microsecin is ineffective in Cercospora and slightly effective in Pseudomonads (P. fluorescens DS 96.578, P. mendocina DS 98.124), but Bacillus (B. Pumilus DS96). .734, B. megaterium DS98.124) showed no effect on growth.

  Based on these findings, the efficacy of microsecin was further investigated in a field emergence trial. The test was seeded relatively late and provided more opportunities for the presence of the pathogen Aphanomices at the test site.

  (Materials and methods)

Seeds were pelleted with standard P1 pelleting amounts with (1,2) or without (7,8) thiram.
Standard seed coating Inner coating 0.3 gai / U microsecin 14.7 gai / U himexazole 60 gai / U imidacloprid as a 0.5% aqueous solution The standard metallic green seed cover film test included the following combinations.
R FO No fungicide R FT With Tiraum (in pellets)
R FH With Himexazole R FM With Microsecin R P1 STD With Thiraum (in pellets) + With Himexazole R FTM With Thiraum (in pellets) + Microsesin test location Bukkehave, DK. (4 reps, 200 seeds / plot)
Test sowing May 21, 2002 Count May 28, 2002 (High Speed)
2. Count May 29, 2002 (high speed)
3. Count June 24, 2002 (final)
(result)
Laboratory and field appearance numbers can be found in Table 2 (Test FEHCCP034, Aphanomices). The final appearance is shown in FIG.

  Laboratory studies show that inclusion of microsecin reduces the rate of germination in the laboratory (4d), which is not reflected by the numbers 4d> 15 mm. This shows the adverse effect of hymexazole with low 4d> 15 mm germination.

  With respect to germination rate, field appearance tests show that germination is relatively slow with pellets containing hymexazole (alone or in combination with Tiraum) (expected from 4d> 15 mm laboratory germination). Pellets containing microsecin show a germination rate comparable to pellets containing only thiraum.

  In contrast to the speed of germination with the Tiraum-containing pellet, the pellet containing microsecin shows a high final germination (comparable to the Himexazole-containing pellet).

  Despite the fact that the actual attack by root rot pathogens is rather limited, the loss of plantlets in the FT-plot due to the plant attack by pathogens (most preferably Aphanomices) that can be controlled by hymexazole is 4% (approximately). The final number of plantlets in the FT-plot was less than the number of plantlets in the F0 (no fungicide) plot. This can be explained by the action of thiraum, which controls other microorganisms but not Aphanomyces, making it easier to access Aphanomyces to plantlets.

  Microsecin-containing pellets are the only pellets that exhibit both early germination and high final germination. Thus, it is believed that microthecin may be a substitute for the slightly more expensive chemical himexazole.

  All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in conjunction with specific preferred embodiments, it is to be understood that such specific embodiments do not unduly limit the claimed invention. Indeed, various described modifications of embodiments practicing the invention that are apparent to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims (1)

Invention described in this specification.