JP2015063522A - Ebd- and hkd-containing fusion peptide and transformant expressing peptide concerned - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microorganism which can control genetic expression by ethylene gas.SOLUTION: The invention provides a fusion peptide comprising an ethylene sensor domain derived from a plant and a filamentous fungus or a histidine kinase domain derived from a yeast; a nucleic acid molecule encoding the fusion peptide concerned; a vector comprising the nucleic acid molecule concerned; a transformant transformed with the vector concerned; and a step of culturing the transformant to produce substances after ethylene treatment; and a step of collecting the obtained substances.

Description

  The present invention relates to substance production using filamentous fungi or yeast, filamentous fungi used for production of the substance, and yeast transformants.

  Filamentous fungi are a general term for what is composed of tubular cells called mycelia, and low molecular weight compounds such as organic acids, pigments, chemicals such as agrochemicals, pharmaceuticals such as penicillins and statins; amylases, cellulases It is used for fermentation production of industrial enzymes such as protease and lipase.

  For example, in Patent Document 1, filamentous fungus culture using a step of adding a thermophilic bacterium-derived β-glucosidase to a glucose-containing solution and producing a disaccharide-containing solution by a condensation reaction, and a medium containing the disaccharide-containing solution. Describes a method for producing cellulase, which comprises the step of producing cellulase.

  Patent Document 2 includes a step of processing a fungal peptide to excise the peptide from the C-terminus and / or the peptide from the N-terminus to generate a core peptide consisting of a specific amino acid sequence having phospholipase activity. A method for producing phospholipases is described.

  Patent Documents 3 to 7 describe an expression vector constructed so as to function as a host for the purpose of efficient substance production by the filamentous fungus, and a gene encoding the same or different protein in the expression vector. A method for introducing a plasmid in which lysates are functionally linked into a filamentous fungus to produce a transformant, and that the use of the transformant contributes to an increase in the production of enzymes such as amylase and cellulase, and low molecular weight compounds such as penicillin. , Is described.

  As described above, filamentous fungi have the advantage of being able to produce a wide variety of useful substances. Yeast is also used for various fermentative productions, and bioenergy production uses such as bioethanol and biobutanol have been studied.

  Here, liquid culture is used for industrial culture of yeast, and solid culture and liquid culture are used for industrial culture of filamentous fungi. However, in order to use a solid substrate in solid industrial culture, gene expression substrate (monosaccharide and its derivatives, metal ions, etc.) and temperature (low temperature, high temperature) with high diffusibility used in liquid industrial culture are expressed. It cannot be used for induction, and gene expression induction at any time point in culture has not been possible so far.

  On the other hand, even in liquid culture, the above-described highly diffusible gene induction substrate has a problem of high cost. Therefore, in liquid culture, it is desired to induce substances and industrially produce substances using substrates other than these conventional ones.

JP 2010-227032 A JP2010-172343 JP2001-46078 JP 2005-52116 A JP 2009-118783 A Special table flat 11-506025 Special table 2007-508022

  The problem to be solved by the present invention is to provide a microorganism capable of controlling gene expression with ethylene gas. If it is ethylene gas, it can be used as an induction substrate even in solid industrial culture using a solid substrate. In liquid industrial culture, it is much cheaper than conventional gene inducers that can be used on an industrial scale. This is useful.

  Under the circumstances as described above, as a result of intensive studies, the present inventors have succeeded in producing a novel peptide in which a plant-derived ethylene sensor domain and a filamentous fungus or yeast-derived histidine kinase domain are fused. And it discovered that the said subject could be solved by using this novel fusion peptide.

Accordingly, the present invention provides the following sections:
Item 1. A fusion peptide comprising an ethylene sensor domain derived from a plant and a histidine kinase domain derived from a filamentous fungus or yeast.

  Item 2. The histidine kinase domain is derived from a filamentous fungus belonging to the genus Aspergillus, Penicillium, Trichoderma, Cephalosporium, or Acremonium or Saccharomyces, Candida, Tolropsis, Digosaccharomyces, Schizosaccharomyces, Pichia Item 5. The fusion peptide according to Item 1, which is derived from a yeast belonging to the genus Yarrowia, Hansenula, Kluyweromyces, Debariomyces, Geotrichum, Wickelhamia or Ferromies.

  Item 3. Item 3. The fusion peptide according to Item 2, wherein the histidine kinase domain is derived from Aspergillus nidulans, Aspergillus oryzae, Aspergillus soya, Aspergillus niger, Aspergillus fumigatus or Saccharomyces cerevisiae.

  Item 4. Item 4. The fusion peptide according to any one of Items 1 to 3, wherein the plant-derived ethylene sensor domain is derived from Arabidopsis thaliana, tomato, petunia, rice, tobacco, cucumber, banana, or apple.

  Item 5. Item 5. A nucleic acid molecule encoding the fusion peptide according to any one of Items 1 to 4.

  Item 6. Item 6. A vector comprising the nucleic acid molecule according to Item 5.

  Item 7. A transformant obtained by transforming a host filamentous fungus or yeast using the vector according to item 6 or a vector containing a nucleic acid molecule encoding a peptide containing a plant-derived ethylene sensor domain and a plant-derived histidine kinase domain .

Item 8. The step of culturing the filamentous fungus or yeast transformant according to Item 7,
A step of treating the transformant with ethylene to cause the transformant to produce a substance, and a step of recovering the obtained substance.
A material production method comprising:

  Microorganisms transformed with the fusion peptide of the present invention comprising a plant-derived ethylene sensor domain and a filamentous fungus or yeast-derived histidine kinase domain exhibit ethylene responsiveness. Therefore, according to the present invention, gene expression can be controlled using gaseous ethylene at low cost and at normal temperature. As a chimeric protein containing part or all of a plant-derived ethylene receptor protein, a chimeric protein of ETR1 derived from Arabidopsis thaliana and a fluorescent protein is known (The Journal of Biological Chemistry vol 285, No. 52, ppp40706). -40713, Dec, 24, 2010, etc.), this is only for examining the localization of ETR1 in cells (tissues), and for use in the presence / absence / amount / phenomenon assay. Chimeric proteins linking various Arabidopsis ethylene receptors and cyanobacterial histidine kinase have been studied (Tsukuba Journal of Biology (2013) 12, 29). Responsiveness cannot be obtained. On the other hand, in the present invention, surprisingly, ethylene-responsive transcription control is shown by combining a plant-derived ethylene sensor domain with a filamentous fungus or yeast-derived histidine kinase domain. Therefore, this effect of the present invention cannot be expected from the prior art.

The structure comparison between Arabidopsis ethylene sensor AtETR1, yeast osmotic pressure sensor ScSln1p, and filamentous fungus osmotic pressure sensor AnTcsB is shown. Each sensor consists of a transmembrane region on the N-terminal side. It has a typical two-component signal transduction system Hitidine kinase domain and Receiver domain on the C-terminal side. The outline of the fusion peptide in this invention is shown. The outline of the model of confirmation of HK function complementarity and confirmation of ethylene response in the budding yeast SLN1 gene condition expression strain in this-application test example 1 is shown. In Saccharomyces cerevisiae SLN1 gene conditional expression strain, Sln1p, which is the only HK in yeast, is inactivated at 37 ° C. and is lethal. HK functional complementation is confirmed by normal growth at 37 ° C when galactose is induced. Ethylene responsiveness is confirmed by the fact that the HK function complementary to galactose induction at 37 ° C becomes inactive by ethylene treatment and the growth deteriorates. The result of the functional complementation test of Test Example 1 is shown. The result of the ethylene responsiveness test of Test Example 1 is shown. The result of the gene expression test of Test Example 1 is shown. The outline of the Fludioxonil sensitivity model of fusion NikA in the budding yeast SLN1 gene conditional expression strain in Test Example 2 is shown. In Saccharomyces cerevisiae SLN1 gene conditional expression strain, Sln1p, which is the only HK in yeast, is inactivated at 37 ° C. and is lethal. Fusion NikA grows normally at 37 ° C upon induction of galactose. Fludioxonil is known to act on HAMPdomain and HK becomes inactive. It is confirmed that the HK function complementary to galactose induction at 37 ° C acts only on fusion NikA with HAMPdomain by Fludioxonil treatment. The result of Test Example 2 is shown. The model of confirmation of HK function complementarity and confirmation of ethylene response in filamentous fungi in Test Example 3 is shown. When Fludioxonil is treated to a filamentous fungus (wild type), it acts on the HAMP domain of AnNikA, and the HK function of NikA becomes inactive, indicating lethality. Confirm that HK functional complementation of the fused NikA grows normally during Fludioxonil treatment. Ethylene responsiveness confirms that the HK function complemented by Fludioxonil treatment becomes inactive by ethylene treatment and the growth deteriorates. The result of Test Example 3 is shown. The outline of the plasmid design in an Example is shown. 1 shows the amino acid sequence of AtETR1 (1-738) (SEQ ID NO: 1). This shows the base sequence of AtETR1 (1-2217) (SEQ ID NO: 2). This shows the amino acid sequence of AtETR1 (1-156) (SEQ ID NO: 3). This shows the base sequence of AtETR1 (1-468) (SEQ ID NO: 4). This shows the amino acid sequence of AtETR1 (1-325) (SEQ ID NO: 5). This shows the base sequence of AtETR1 (1-975) (SEQ ID NO: 6). This shows the amino acid sequence of ScSln1p (519-1220) (SEQ ID NO: 7). This shows the base sequence (SEQ ID NO: 8) of ScSLN1 (1555-3663). This shows the amino acid sequence (SEQ ID NO: 9) of AtETR1 (1-156) + ScSln1p (519-1220). This shows the base sequence (SEQ ID NO: 10) of AtETR1 (1-468) + ScSLN1 (1555-3663). This shows the amino acid sequence (SEQ ID NO: 11) of AtETR1 (1-325) + ScSln1p (519-1220). This shows the base sequence (SEQ ID NO: 12) of AtETR1 (1-975) + ScSLN1 (1555-3663). An amino acid sequence of AnTcsB (495-1070) (SEQ ID NO: 13) is shown. This shows the base sequence of AntcsB (1483-3213) (SEQ ID NO: 14). This shows the base sequence (SEQ ID NO: 16) of AtETR1 (1-156) + AntcsB (495-1070) AtETR1 (1-468) + AnTcsB (1483-3213). This shows the base sequence (SEQ ID NO: 16) of AtETR1 (1-468) + AntcsB (1483-3213). This shows the amino acid sequence of AtETR1 (1-325) + AnTcsB (495-1070) (SEQ ID NO: 17). This shows the base sequence (SEQ ID NO: 18) of AtETR1 (1-975) + AntcsB (1483-3213). An outline of plasmid design (with and without HAMP) in the examples is shown. This shows the amino acid sequence of AnNikA (708-1297) (SEQ ID NO: 19). This shows the base sequence of AnnikA (2122-3894) (SEQ ID NO: 20). This shows the amino acid sequence of AtETR1 (1-156) + AnNikA (708-1297) (SEQ ID NO: 21). This shows the base sequence (SEQ ID NO: 22) of AtETR1 (1-468) + AnnikA (2122-3894). This shows the amino acid sequence of AtETR1 (1-325) + AnNikA (708-1297) (SEQ ID NO: 23). This shows the base sequence (SEQ ID NO: 24) of AtETR1 (1-975) + AnnikA (2122-3894). This shows the amino acid sequence of AnNikA (152-1297) (SEQ ID NO: 25). This shows the base sequence of AnnikA (454-3894) (SEQ ID NO: 26). This shows the amino acid sequence of AtETR1 (1-156) + AnNikA (152-1297) (SEQ ID NO: 27). This shows the base sequence (SEQ ID NO: 28) of AtETR1 (1-468) + AnnikA (454-3894). This shows the amino acid sequence of AtETR1 (1-325) + AnNikA (152-1297) (SEQ ID NO: 29). This shows the base sequence (SEQ ID NO: 30) of AtETR1 (1-975) + AnnikA (454-3894).

Fusion peptide The present invention provides a fusion peptide comprising a plant-derived ethylene sensor domain and a filamentous fungi or yeast-derived histidine kinase domain.

  Ethylene sensor domains derived from Arabidopsis thaliana, derived from tomato (Lycopersicun esculentum), derived from Petunia hybrida, derived from rice (Oryza sativa), derived from tobacco (Nicotiana tabacum) Cucumber (Cucumis sativus), banana (Musa acuminata), apple (Malus domestica), etc., preferably Arabidopsis, tomato, petunia, etc. More preferably, those derived from Arabidopsis thaliana are mentioned. In the present invention, the ethylene sensor domain may be referred to as EBD.

  Various ethylene sensor domains can be widely used. Examples of those derived from Arabidopsis include ETR1, ETR2, ERS1, ERS2, EIN4 (Schaller (2011) Curr Biol. 21, R320- 30). In the present invention, the ethylene sensor domain ETR1 includes, for example, a peptide represented by Genbank accession No. NM_105305 and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3. As long as the effects of the present invention can be obtained, the ethylene sensor domain ETR1 has a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 3. Or a part thereof is also included.

  In the present invention, the histidine kinase domain (HKD) means a catalytic domain of a kinase reaction in a histidine kinase protein. In the present invention, as the histidine kinase domain, those derived from filamentous fungi or those derived from yeast are used.

  Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, Cephalosporium, or Acremonium. More specifically, examples of the filamentous fungi include Aspergillus nidulans, Aspergillus oryzae, Aspergillus soya, Aspergillus niger, Aspergillus fumigatus and the like.

  As the histidine kinase domain possessed by filamentous fungi, for example, histidine kinase possessed by Aspergillus nidulans, more specifically, for example, TcsB (HK1), HK2, NikA (HK3), TcsA (HK4), PhkA (HK5), PhkB (HK6), FphA (HK7), HysA (HK8-2), HK8 (HK1, HK3-7), HK9 (Kanamaru (2011) Biosci. Biotechnol. Biochem. 75, 1-6), etc. TcsB is preferable from the viewpoint of functional complementation, and NikA is preferable from the viewpoint of histidine kinase activity in filamentous fungi. Examples of the histidine kinase domain TcsB include a peptide represented by Genbank Accession No. AB — 036054, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 13, and the like. In addition, as long as the effects of the present invention can be obtained, the histidine kinase domain TcsB has a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 13. Or a part thereof is also included. In the present invention, the histidine kinase domain may contain a receiver domain in addition to the histidine kinase domain in the narrow sense as shown in FIG.

  Examples of NikA include a peptide represented by Genbank accession No. AN_4479.3, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 19, and the like. As long as the effects of the present invention can be obtained, the histidine kinase domain NikA has a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 19. Or a part thereof is also included.

  Examples of the yeast include, for example, the genus Saccharomyces, the genus Candida, the genus Torulopsis, the genus Zygosaccharomyces, the genus Schizosaccharomyces, the genus Pichia Y, Yeast belonging to the genus Hansenula, the genus Kluyveromyces, the genus Debaryomyces, the genus Geotrichum, the genus Wickerhamia, the genus Fellomyces, preferably Examples include yeasts belonging to the genus Saccharomyces, Candida, Tolropsis, Digosaccharomyces, Schizosaccharomyces, Pichia, Yarrowia, Hansenula, Kluywellomyces, Debariomyces, and the like. More specifically, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methanolica, Hansenula anomala, Kuriweromyces lactis and the like. Of these, Saccharomyces cerevisiae is preferable.

  Examples of histidine kinase domains possessed by yeast include, for example, the only histidine kinase possessed by Saccharomyces cerevisiae, and more specifically, for example, Sln1p, Mar1, Mar2, Mar3, Candida albicans possessed by Schizosaccharomyces pombe. Chk1p, Nik1p (Santos and Shinozaki (2001) Sci STKE.) And the like are preferred, and Sln1p is preferred. Examples of the histidine kinase Sln1p include a peptide represented by Genbank accession No. NM — 001179495, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, and the like. As long as the effects of the present invention can be obtained, the histidine kinase domain NikA has a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 19. Or a part thereof is also included.

Fusion peptide of the present invention, GAF between the ethylene sensor domain and a histidine kinase domain (cyclic G MP, A denylyl cyclase , F hlA) may have a domain. Examples of the GAF domain include those derived from plants, such as a peptide consisting of the 158th to 307th amino acid sequences in the amino acid sequence represented by SEQ ID NO: 1. As long as the effects of the present invention can be obtained, the GAF domain contains a peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence at positions 158 to 307 of SEQ ID NO: 1 or its Some are also included.

  The fusion peptide of the present invention has one or more (for example, 6 or less) HAMP (signal transduction domain in Histidine kinases, Adenyl cyclases, Methyl-accepting proteins and Phosphatases) between the ethylene sensor domain and the histidine kinase domain. You may have a domain. Examples of the HAMP domain include those derived from filamentous fungi or yeast. For example, in the amino acid sequence represented by SEQ ID NO: 25, a peptide consisting of the amino acid sequence at positions 193 to 708, and the like can be mentioned. As long as the effects of the present invention can be obtained, the HAMP domain contains a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence from 193 to 708 of SEQ ID NO: 25 or Some are also included. When the fusion peptide of the present invention has both a GAF domain and a HAMP domain, the ethylene sensor domain, the GAF domain, the HAMP domain, and the histidine kinase domain are preferably arranged in this order. By arranging the HAMP domain, responsiveness to fludioxonil can be imparted to the host.

  As a fusion peptide of this invention, what consists of an amino acid sequence represented by sequence number 9, 11, 15, 17, 21, 23, 27, 29, etc. are mentioned, for example. As long as the effects of the present invention are obtained, one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NOs: 9, 11, 15, 17, 21, 23, 27, 29. Also included are peptides consisting of the amino acid sequences made, or a part of the peptides.

  Since the fusion peptide of the present invention uses a filamentous fungus or yeast-derived one as the histidine kinase domain, when the gene of such fusion peptide is introduced into a microorganism, the linkage with the protein present in the host is smoother. This is preferable because it is expected to be performed.

Nucleic acid molecule The present invention provides a nucleic acid molecule encoding the fusion peptide.

  In the present specification, “nucleotide”, “oligonucleotide” and “polynucleotide” are synonymous with a nucleic acid molecule, and include both DNA and RNA. These may be double-stranded or single-stranded, and in the case of “nucleotide” having a certain sequence (or “oligonucleotide”, “polynucleotide”), it is complementary to this unless otherwise specified. “Nucleotide” (or “oligonucleotide” and “polynucleotide”) having a typical sequence is also intended to be inclusive. Furthermore, when the “nucleotide” (or “oligonucleotide” and “polynucleotide”) is RNA, the base symbol “T” shown in the sequence listing shall be read as “U”.

  In the present invention, “gene” includes double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof, unless otherwise specified. In the present invention, “gene” refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified.

  The nucleic acid molecule of the present invention has a base sequence encoding an ethylene sensor domain and a base sequence encoding a histidine kinase domain. As an example of the ethylene sensor domain, a base sequence encoding the ethylene sensor domain ETR1 is shown in SEQ ID NO: 2. The base sequence encoding histidine kinase domain TcsB is shown in SEQ ID NO: 14. The base sequence encoding histidine kinase domain NikA is shown in SEQ ID NO: 19. The base sequence encoding histidine kinase SLN1 is shown in SEQ ID NO: 8. The nucleic acid molecule of the present invention includes not only those having these sequences, but also those nucleotide sequences whose mutations do not change the encoded amino acid sequence even if they are mutations in intron sites or exons. Also included are nucleic acid molecules having silent mutations.

  Moreover, as a nucleic acid molecule of this invention, what consists of a base sequence represented by sequence number 10, 12, 16, 18, 22, 24, 28, 30 etc. is mentioned, for example. The nucleic acid molecule of the present invention includes not only those represented by the above SEQ ID NOs, but also nucleic acid molecules comprising a base sequence obtained by adding a silent mutation to these base sequences.

  In addition, the nucleic acid molecule of the present invention includes one or several nucleotide sequences represented by SEQ ID NOs: 10, 12, 16, 18, 22, 24, 28, 30 as long as the effects of the present invention are obtained. A peptide consisting of a base sequence in which a base is deleted, substituted or added, or a part thereof is also included.

Vector The present invention also provides a vector comprising a nucleic acid molecule encoding the above-described fusion peptide of the present invention.

  Various plasmid vectors, phage vectors, etc. can be used as vectors, but they can replicate in microbial cells, have appropriate drug resistance markers, specific restriction enzyme cleavage sites, and copy numbers in the microbial cells. It is desirable to use a high plasmid vector. When yeast is specifically used as a host, pYES2 (Invitrogen), pRS (Sikorski and Hieter. (1989)) Genetics 122, 19-27) and the like can be exemplified. Specifically, when a filamentous fungus is used as a host, pUC, pNGA142, pNAN8142, pNEN142 (Minetoki (2003) J. Biol Macromol. 3, 89-96) can be exemplified.

  As a method for cloning a nucleotide sequence containing a nucleic acid molecule encoding the fusion peptide of the present invention into the vector, a method known per se can be used. For example, expression control signals (transcription initiation and translation initiation signals), etc. Can be designed so that the gene can be self-expressed in microbial cells depending on the host microorganism.

Transformant The present invention provides a transformant obtained by transforming a filamentous fungus or yeast as a host using the above vector.

  The above-mentioned filamentous fungi or yeast can be used as the host microorganism.

  Many methods for transforming microorganisms have already been reported, and may be appropriately selected according to the microorganism used as a host.

  As a method for producing such a mutant strain, a method known per se (for example, the methods described in Non-Patent Documents 2 to 5) is appropriately used. For example, a gene cassette encoding a fusion peptide is constructed and the cassette into the genomic gene is used. This can be done by introducing the above.

  In another embodiment, the present invention transforms a host filamentous fungus or yeast using a vector comprising a nucleic acid molecule encoding a peptide comprising a plant-derived ethylene sensor domain and a plant-derived histidine kinase domain. Also provided is a transformant. As the plant-derived ethylene sensor domain, those described above in the description of the fusion peptide can be similarly used. Examples of the plant-derived histidine kinase domain include those derived from Arabidopsis thaliana, those derived from rice (Oryza sativa), those derived from tobacco (Nicotiana tabacum), those derived from tomato (Solanum lycopersicum), and the like. Preferably, the thing derived from Arabidopsis thaliana is mentioned. In the present invention, plant-derived histidine kinase domains include, for example, ETR1, ETR2, ERS1, ERS2, EIN4 and the like as those derived from Arabidopsis thaliana. In the present invention, the histidine kinase domain ETR1 includes, for example, the peptide represented by Genbank accession No. NM_105305, the 350th to the 585th of the amino acid sequence represented by SEQ ID NO: 1 (the 350th to the 729th when the receiver domain is included) Peptides consisting of the amino acids are included. As long as the effects of the present invention can be obtained, the histidine kinase domain ETR1 has an amino acid sequence of positions 350 to 585 (350 to 729 when the receiver domain is included) in the amino acid sequence represented by SEQ ID NO: 1. A peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, or a part thereof is also included. In the present invention, introduction of a peptide containing a plant-derived ethylene sensor domain and a plant-derived histidine kinase domain imparts ethylene responsiveness to the host filamentous fungus or yeast. In plants such as Arabidopsis thaliana, signal transduction due to ethylene stimulation is not performed by histidine kinase, and it has been reported that serine threonine kinase performs the signal transduction (Schaller (2011) Curr Biol. 21, R320-30). Therefore, the effect of the transformant of the present invention is unexpected. Examples of the peptide containing a plant-derived ethylene sensor domain and a plant-derived histidine kinase domain include a peptide comprising the amino acid sequence represented by SEQ ID NO: 1. Moreover, as long as the effect of the present invention is exhibited, the effect of the transformant of the invention is unexpected. A peptide comprising a plant-derived ethylene sensor domain and a plant-derived histidine kinase domain comprises a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1. Or a part thereof is also included.

Substance production method The present invention comprises a step of culturing a transformant of filamentous fungi or yeast,
A step of treating the transformant with ethylene to cause the transformant to produce a substance, and a step of recovering the obtained substance.
A method for producing a substance is provided.

  The useful substance that can be produced according to the present invention is not particularly limited, and examples thereof include low molecular weight compounds such as penicillin, statins, cephalosporin, succinic acid, citric acid, malic acid; amylase, cellulase, protease, and lipase. , Polymer compounds such as peptidase, esterase and oxidase. In addition to the above, useful substances include chemical substances such as organic acids, pigments, and agrochemical ingredients, and various substances used as pharmaceuticals. In the present invention, as a design for expressing a useful substance in a transformant in conjunction with the control of histidine kinase activity, a method known per se can be widely adopted. For example, phosphorylation of the histidine kinase signal transduction pathway is possible. Examples include a method of linking a transcription factor for inducing a useful substance to a promoter under the control of an acid transfer intermediate factor or an appropriate transcription factor downstream of the HOG pathway. The method of the present invention can also be applied to bioethanol production by decomposition of biomass (such as those using fungi that have been genetically modified to produce cellulase or the like at high production).

Culturing Step The method of the present invention includes a step of culturing the above-mentioned filamentous fungus or yeast transformant to cause the filamentous fungus or yeast to produce a substance. The medium used in this step is not particularly limited, and a medium that can be used for culturing filamentous fungi or yeast can be widely used. For example, CD minimum medium, YPD medium, TSB medium, malt medium, PDA medium and the like can be mentioned. In the step, a substrate used for solid culture can be used instead of the medium. Examples of such a substrate include, but are not limited to, solid cereals such as wheat bran, wheat, soybean, rice, and the like. These substrates are usually heat-treated products. These substrates are suitable for solid culture of filamentous fungi. Glucose, starch, soluble starch or the like may be added to the medium or substrate as a carbon source. Although it does not specifically limit as addition amount of this carbon source, For example, it can set suitably in 0.5 to 10%, More preferably in the range of 1 to 4%. The culture temperature is not particularly limited, and can be appropriately set in the range of 20 to 45 ° C, more preferably 25 to 37 ° C. The culture time is not particularly limited, and can be appropriately set, for example, in the range of 12 to 72 hours, more preferably 24 to 48 hours.

Recovery Step A method for recovering useful substances from the culture medium is not particularly limited, and a method known per se (centrifugation, recrystallization, distillation method, solvent extraction method, chromatography, etc.) can be appropriately used.

  EXAMPLES Hereinafter, the present invention will be specifically described using examples, but the present invention is not limited to these examples.

Example 1
AtETR1 gene
RNA was extracted from the seedlings of Arabidopsis thaliana ecotype Columbia using RNeasy Plant Mini Kit (Qiagen), and complementary DNA (cDNA) was synthesized using ReverTra Ace kit (TOYOBO). Using this cDNA as a template, the ethylene receptor gene AtETR1 (At1g66340) was amplified using a primer set to which the following restriction enzyme sites were added. AtETR1 BamHI F; 5'-CGGGATCCTAATGGAAGTCTGCAATTG-3 '(added BamHI) (SEQ ID NO: 31) and AtETR1 NotIR; 5'-TTTTCCTTTTGCGGCCGCTTACATGCCCTCGTACAGTAC-3-3 (added Not I) (SEQ ID NO: 32). The amplified fragment was subcloned into pZErO ^™ -2 vector (Invitrogen) (pZErO-AtETR1).

pYES−AtETR1
AtETR1 was excised from pZErO-AtETR1 with BamHI and NotI and introduced into a yeast expression vector pYES2 vector (Invitrogen) (pYES-AtETR1).

Ethylene bond region
Using pZErO-AtETR1 as a template, a region containing an ethylene-binding domain and a downstream GAF domain (EBD + GAF) was amplified using the following primer set. AtETR1 BamHI F1; 5′-CGGGATCCTAATGGAAGTCTGCAATTG-3 ′ (added BamHI) (SEQ ID NO: 31) and AtETR1 R2; 5′-CTTTACATGCCCTCGTACAGTACCCGG-3 ′ (SEQ ID NO: 33). The amplified fragment was subcloned into pZErO ^™ -2 vector (Invitrogen) (pYES-AtETR1 (EBD + GAF)).

To construct pYES-AtETR1 (EBD) + ScSLN1p (HKD) and pYES-AtETR1 (EBD + GAF) + ScSLN1 (HKD) EBD fusion ScSLN1p expression system, the following primers with overlapping ligation parts were designed, and the EBD fragment was Two types of AtETR1 (EBD) and AtETR1 (EBD + GAF) using pZErO-AtETR1 (EBD + GAF) as a template, ScSLN1 (HKD) fragment is pRS-ScSLN1 (Maeda et al. (1994) Nature 369, 242-245) Two types were amplified according to the connection with EBD.
AtETR1 BamHI F1; 5'-CGGGATCCTAATGGAAGTCTGCAATTG-3 '(SEQ ID NO: 31) (added BamHI) and AtETR1-ScSLN1 R1;
5'-TGTTAAATCGGAAAGTTCATCAGTGCTTCTAATCTCATGAGT-3 '(SEQ ID NO: 34) AtETR1 BamHI F1; 5'-CGGGATCCTAATGGAAGTCTGCAATTG-3' (added BamHI) (SEQ ID NO: 31) and AtETR1-ScSLN1 R2;
5′-TGTTAAATCGGAAAGTTCATCCTCCATGAGAAGGTCCCTAGC-3 ′ (SEQ ID NO: 35) AtETR1-ScSLN1 F1; 5′-ACTCATGAGATTAGAAGCACTGATGAACTTTCCGATTTAACA-3 ′ (SEQ ID NO: 36) and ScSLN1 NotI R;
5'-TTTTCCTTTTGCGGCCGCTCATTTGTTATTTTTCTTTCC-3 '(added Not I) (SEQ ID NO: 37) AtETR1-ScSLN1 F2; 5'-GCTAGGGACCTTCTCATGGAGGATGAACTTTCCGATTTAACA-3' (SEQ ID NO: 38) and ScSLN1 NotI R;
5'-TTTTCCTTTTGCGGCCGCTCATTTGTTATTTTTCTTTCC-3 '(added Not I) (SEQ ID NO: 37)
The amplified fragment was purified, and fusion PCR was performed using these as a template and primers added with the following restriction enzymes. The fused fragment was subcloned into pZErO ^™ -2 vector (Invitrogen) (pZErO-AtETR1 (EBD) + ScSLN1 (HKD)).
AtETR1 BamHI F1; 5'-CGGGATCCTAATGGAAGTCTGCAATTG-3 '(added BamHI) (SEQ ID NO: 31) and ScSLN1 NotIR;
5'-TTTTCCTTTTGCGGCCGCTCATTTGTTATTTTTCTTTCC-3 '(added Not I) (SEQ ID NO: 37)
The fusion HK fragment was excised from pZErO-AtETR1 (EBD) + ScSLN1 (HKD) and pZErO-AtETR1 (EBD + GAF) + ScSLN1 (HKD) with BamH I and Not I and introduced into the yeast expression vector pYES2 vector (Invitrogen) ( pYES−AtETR1 (EBD) + ScSLN1 (HKD) and pYES−AtETR1 (EBD + GAF) + ScSLN1 (HKD)). The base sequence of AtETR1 (EBD) + ScSLN1 (HKD) is shown in FIG. 21 (SEQ ID NO: 10), and the base sequence of AtETR1 (EBD + GAF) + ScSLN1 (HKD) is shown in FIG. 23 (SEQ ID NO: 12). It was confirmed by DNA sequencing that the fusion fragment was introduced into the vector.

Example 2
In order to construct pYES-AtETR1 (EBD) + AntcsB (HKD) and pYES-AtETR1 (EBD + GAF) + AntcsB (HKD) EBD fusion AnTcsB expression systems, the following primers with overlapping ligation parts were designed, and the EBD fragment was pZErO-AtETR1 (EBD) and pZErO-AtETR1 (EBD + GAF) were used as templates, and AntcsB (HKD) fragment was amplified using pYES-AntcsB (Furukawa et al, 2002) as a template. AtETR1 HindIII F2; 5'-CCCAAGCTTTAATGGAAGTCTGCAATTG-3 '(added Hind III) (SEQ ID NO: 39) and AtETR1-AntcsB R1;
5'-CGTCAGGTCGGTTAGTTCATCAGTGCTTCTAATCTCATGAGT-3 '(SEQ ID NO: 40) AtETR1 HindIII F2; 5'-CCCAAGCTTTAATGGAAGTCTGCAATTG-3' (SEQ ID NO: 39) and AtETR1-AntcsB R2;
5'-CGTCAGGTCGGTTAGTTCATCCTCCATGAGAAGGTCCCTAGC-3 '(SEQ ID NO: 41) AtETR1-AntcsB F1; 5'-ACTCATGAGATTAGAAGCACTGATGAACTAACCGACCTGACG-3' (SEQ ID NO: 42) and AntcsB NotI R; 5'-TTTTCCCATTTAGGG3 )
AtETR1-AntcsB F2; 5′-GCTAGGGACCTTCTCATGGAGGATGAACTAACCGACCTGACG-3 ′ (SEQ ID NO: 44) and AntcsB NotI R;
5′-TTTTCCTTTTGCGGCCGCTTACAGAGCAAAATTCACATC-3 ′ (added Not I) (SEQ ID NO: 43) The amplified fragment was purified, and fusion PCR was performed using these as a template and primers to which the following restriction enzymes were added, and the fused fragment was pZErO ^™ -2 subcloned into vector (Invitrogen) (pZErO-AtETR1 (EBD) + AntcsB (HKD) and pZErO-AtETR1 (EBD + GAF) + AntcsB (HKD)).
AtETR1 HindIII F2; 5'-CCCAAGCTTTAATGGAAGTCTGCAATTG-3 '(added Hind
III) (SEQ ID NO: 39) and AntcsB NotIR; 5'-TTTTCCTTTTGCGGCCGCTTACAGAGCAAAATTCACATC-3 '(added Not I) (SEQ ID NO: 43)
A fusion HK fragment was excised from pZErO-AtETR1 (EBD) + AntcsB (HKD) and pZErO-AtETR1 (EBD + GAF) + AntcsB (HKD) with Hind III and Not I and introduced into the yeast expression vector pYES2 vector (Invitrogen). (pYES−AtETR1 (EBD) + AntcsB (HKD) and pYES−AtETR1 (EBD + GAF) + AntcsB (HKD)). The base sequence of AtETR1 (EBD) + AntcsB is shown in FIG. 27 (SEQ ID NO: 16), and the base sequence of AtETR1 (EBD + GAF) + AntcsB (HKD) is shown in FIG. 29 (SEQ ID NO: 18). It was confirmed by DNA sequencing that the fusion fragment was introduced into the vector.

Example 3
AnnikA
Aspergills nidulans genomic DNA was extracted using DNeasy Plant Mini Kit (QIAGEN). AnnikA (AN4479.3) sequence containing HAMP domain and histidine kinase domain was amplified with the following primer set using A. nidulans genomic DNA as a template.
AnnikA F; 5′-GTATTCAACGAAACACGGCCGCCAGGGAA-3 ′ (SEQ ID NO: 45) and AnnikA NotI R1;
The 5′-TTTTCCTTTTGCGGCCGCTCATGGTCTTACCCTATCTATG-3 ′ (SEQ ID NO: 46) amplified fragment was subcloned into pZErO ^™ -2 vector (Invitrogen) (pZErO-AnnikA (HAMP + HKD)).

pYES-AtETR1 (EBD) + AnnikA (HKD) and pYES-AtETR1 (EBD + GAF) + AnnikA (HKD) To construct an EBD fusion AnNikA expression system, the following primers with overlapping ligation parts were designed, and the EBD fragment was pZErO-AtETR1 (EBD) and pZErO-AtETR1 (EBD + GAF) were used as templates, and AnnikA (HKD) fragment was amplified using pZErO-AnnikA (HAMP + HKD) (described above) as a template.
AtETR1 HindIII F2; 5'-CCCAAGCTTTAATGGAAGTCTGCAATTG-3 '(added Hind III) (SEQ ID NO: 39) and AtETR1-AnnikA R1; 5'-TTCCCTGGCGGCCGTGTTTCGAGTGCTTCTAATCTCATGAGT-3' (SEQ ID NO: 47)
AtETR1 HindIII F2; 5'-CCCAAGCTTTAATGGAAGTCTGCAATTG-3 '(added Hind III) (SEQ ID NO: 39) and AtETR1-AnnikA R2; 5'-TTCCCTGGCGGCCGTGTTTCGCTCCATGAGAAGGTCCCTAGC-3' (SEQ ID NO: 48)
AtETR1-AnnikA F1; 5'-ACTCATGAGATTAGAAGCACTCGAAACACGGCCGCCAGGGAA-3 '(SEQ ID NO: 49) and AnnikA NotI R; 5'-TTTTCCTTTTGCGGCCGCTCATGGTCTTACCCTATCTATG-3' (added Not I) (SEQ ID NO: 46)
AtETR1-AnnikA F2; 5′-GCTAGGGACCTTCTCATGGAGCGAAACACGGCCGCCAGGGAA-3 ′ (SEQ ID NO: 50) and AnnikA NotI R; 5′-TTTTCCTTTTGCGGCCGCTCATGGTCTTACCCTATCTATG-3 ′ (added Not I) (SEQ ID NO: 46)

The amplified fragments were purified, they perform Fusion PCR using primers with the addition of the following restriction enzymes as template, was subcloned fused fragment ^{pZErO TM -2 vector (Invitrogen) (} pZErO-AtETR1 (EBD) + AnnikA (HKD) and pZErO-AtETR1 (EBD + GAF) + AnnikA (HKD)).
AtETR1 HindIII F2; 5′-CCCAAGCTTTAATGGAAGTCTGCAATTG-3 ′ (added Hind III) (SEQ ID NO: 39) and AnnikA NotI R; 5′-TTTTCCTTTTGCGGCCGCTCATGGTCTTACCCTATCTATG-3 ′ (added Not I) (SEQ ID NO: 46)
The fusion HK fragment was excised from pZErO-AtETR1 (EBD) + AnnikA (HKD) and pZErO-AtETR1 (EBD + GAF) + AnnikA (HKD) with Hind III and Not I and introduced into the yeast expression vector pYES2 vector (Invitrogen). (pYES−AtETR1 (EBD) + AnnikA (HKD) and pYES−AtETR1 (EBD + GAF) + AnnikA (HKD)).
The base sequence of AtETR1 (EBD) + AnnikA (HKD) is shown in FIG. 34 (SEQ ID NO: 22), and the base sequence of AtETR1 (EBD + GAF) + AnnikA (HKD) is shown in FIG. 36 (SEQ ID NO: 24). It was confirmed by DNA sequencing that the fusion fragment was introduced into the vector.

Example 4
pYES-AtETR1 (EBD) + AnnikA (HKD) and pYES-AtETR1 (EBD + GAF) + AnnikA (HKD) (HAMP domain included)
In order to construct an EBD fusion AnNikA expression system containing a HAMP domain, the following primers with overlapping ligation parts were designed, and the EBD fragment was designed using pZErO-AtETR1 (EBD) and pZErO-AtETR1 (EBD + GAF) as templates and AnnikA ( The HKD fragment was amplified using pZErO-AnnikA (HAMP + HKD) (described above) as a template.
AtETR1 HindIII F2; 5'-CCCAAGCTTTAATGGAAGTCTGCAATTG-3 '(added Hind III) (SEQ ID NO: 39) and AtETR1-AnnikA R3;
5′-TAGCTCTTCACGGACCTGGGTAGTGCTTCTAATCTCATGAGT-3 ′ (SEQ ID NO: 51)
AtETR1 HindIII F2; 5'-CCCAAGCTTTAATGGAAGTCTGCAATTG-3 '(added Hind III) (SEQ ID NO: 39) and AtETR1-AnnikA R4;
5'-TAGCTCTTCACGGACCTGGGTCTCCATGAGAAGGTCCCTAGC-3 '(SEQ ID NO: 52)
AtETR1-AnnikA F3; 5′-ACTCATGAGATTAGAAGCACTACCCAGGTCCGTGAAGAGCTA-3 ′ (SEQ ID NO: 53) and AnnikA R2;
5′-CATTACGCACGACCAACGGTTC-3 ′ (SEQ ID NO: 54)
AtETR1-AnnikA F4; 5′-GCTAGGGACCTTCTCATGGAGACCCAGGTCCGTGAAGAGCTA-3 ′ (SEQ ID NO: 55) and AnnikA R2;
5′-CATTACGCACGACCAACGGTTC-3 ′ (SEQ ID NO: 54)

The amplified fragment was purified and subcloned into pZErO ^™ -2 vector (Invitrogen) (pZErO-AtETR1 (EBD) + AnnikA (HAMP + HKD) and pZErO-AtETR1 (EBD + GAF) + AnnikA (HAMP + HKD)).

  Cut out fusion HK fragment from pZErO-AtETR1 (EBD) + AnnikA (HAMP + HKD) and pZErO-AtETR1 (EBD + GAF) + AnnikA (HAMP + HKD) with Hind III and EcoR I, and pYES-AtETR1 (EBD) as described above + AnnikA (HKD) (Hind III and EcoR I treatment) (pYES-AtETR1 (EBD) + AnnikA (HAMP + HKD) and pYES-AtETR1 (EBD + GAF) + AnnikA (HAMP + HKD)). The base sequence of AtETR1 (EBD) + AnnikA (HAMP + HKD) is shown in FIG. 40 (SEQ ID NO: 28), and the base sequence of AtETR1 (EBD + GAF) + AnnikA (HAMP + HKD) is shown in FIG. 42 (SEQ ID NO: 30). . It was confirmed by DNA sequencing that the fusion fragment was introduced into the vector.

Test example 1
Functional complementation test of AtETR1 and fusion HK using yeast Sln1p temperature-sensitive mutant (S. cerevisiae) strain YHS-13 (MATa ura3 leu2 trp1 sln1-ts4) with a temperature-sensitive SLN1 allele (Maeda et al. (1994 ) Nature 369, 242-245) was used for functional complementation analysis. As the expression plasmid, a pYES2 vector capable of regulating expression using the galactose-inducible GAL1 promoter was used (Jonston (1987) Microbiol. Rev. 51, 458-476.).

  pRS-ScSLN1 was used as a positive control for constitutive expression of yeast SLN1 (Maeda et al. (1994) Nature 369, 242-245, Urao et al. (1999) Plant Cell 11, 1743-1754). pYES-AntcsB was used as a positive control to express A. nidulans tcsB under galactose induction at 37 ° C. (restrictive conditions) (Furukawa et al. (2002) Appl Environ Microbiol. 68, 5304-5310).

  Yeast was transformed by the lithium acetate method (Ito et al. (1983) J. Bacteriol. 153, 163-168.) Using plasmids into which various HK cDNAs were introduced, and the transformant was transformed into an YPD plate or YPG having the following composition. Functional complementation test was performed by culturing at 37 ° C (restrictive conditions) or 30 ° C for 2 days in a plate (YPD plate with glucose replaced with 2% galactose).

YPD plate composition
1% w / v Yeast extract
2% w / v Peptone
2% w / v Glucose
2% w / v Agar
The results are shown in FIG. As shown in FIG. 4, in the fused histidine kinase-introduced strain, yeast growth was observed at 37 ° C. when galactose was induced, confirming complementation of histidine kinase function.

Ethylene Treatment Ethylene treatment was set in an acrylic chamber with a capacity of 12 L so that the ethylene concentration was 50 μl / L. The yeast transformant was added to a plate in which 2% galactose was added to SD medium, and 1 × 10 ⁶ cell (left end), 1 × 10 ⁵ cell (second from the left), 1 × 10 ⁴ for each test group. cell (third from the left), 1 × 10 ³ cells (right end), spotted for 3 days at 37 ° C (restrictive conditions) with or without ethylene treatment (-) or with ethylene treatment (+) Cultured. The results are shown in FIG. Growth inhibition was observed by ethylene treatment in the fused HK-introduced strain that confirmed HK functional complementation at 37 ° C. when galactose was induced, and ethylene responsiveness was confirmed.

Analysis of HOG downstream gene ScGPD1 for confirmation of ethylene responsiveness
Ctrl. Was cultured at 30 ° C., and the complementary yeast strain was cultured for 2 days at 37 ° C. in an YP + Gal plate, followed by ethylene treatment for 10 minutes under the same conditions as described above, and quantitative PCR was performed. Specifically, yeast RNA was extracted using RNeasy Plant Mini Kit (Qiagen). The extracted RNA was subjected to DNase treatment and subjected to real-time PCR analysis using the following primer set using iScript ^™ One-Step RT-PCR Kit with SYBR Green (Bio-Rad). Used as 18S rRNA loading control. 5'-GGTTGGAAACATGTGGCTCT-3 '(SEQ ID NO: 56) and 5'-GGCAGGTTCTTCATTGGGTA-3' (ScGPD1) (SEQ ID NO: 57), 5'-TCACTACCTCCCTGAATTAGGATTG-3 '(SEQ ID NO: 58) and 5'-AGAAACGGCTACCACATCCAA-3' ( SEQ ID NO: 59) (18S rRNA).

  The results are shown in FIG. The graph shows the value obtained by normalizing the expression level for each complementary strain with 18S rRNA and dividing the gene expression level after ethylene treatment by the untreated level. As shown in FIG. 6, AtETR1 (EBD) + ScSln1p (HKD) (AtEBD-ScHKD) and AtETR1 (EBD) + AnTcsB (HKD) (AtEBD-AnHKD) are expressed by ethylene stimulation of the HOG downstream gene ScGPD1, It increased significantly compared with control, AtETR1, etc.

Test example 2
Confirmation of Fludioxonil susceptibility in Saccharomyces cerevisiae SLN1 gene conditional expression strains Nature 369, 242-245) (plasmid prepared in Example 3 (pYES-AtETR1 (EBD + GAF) + AnnikA (HKD)) and plasmid prepared in Example 4 (pYES-AtETR1 (EBD + GAF) + AnnikA (HKD) (containing HAMP domain)) and pRS-ScSLN1 as a positive control Yeast was transformed in the same manner as in Test Example 1 except that the above plasmid was used.

  Using the obtained transformant, YPD plate (+ Glc) and YGP plate (+ Gal) used in Test Example 1, and YGP plate to which Fludioxonil was added at 50 μg / ml (+ Gal + Flu), The cells were cultured at 30 ° C. or 37 ° C. for 2 days. The results are shown in FIG. In the Saccharomyces cerevisiae SLN1 gene conditional expression strain, Sln1p, which is the only HK in yeast, was inactivated at 37 ° C. and was lethal (FIG. 8). It was confirmed that the fused NikA grew normally at 37 ° C. when galactose was induced (FIG. 8). Fludioxonil is known to act on the HAMP domain and HK becomes inactive. A test was carried out to confirm that the HK function complementary to galactose induction at 37 ° C acts only on fusion NikA with HAMP domain by Fludioxonil treatment. When galactose was induced at 37 ° C., the fused NikA having a HAMP domain showed Fludioxonil sensitivity in the fused HK-introduced strain that confirmed HK functional complementation (FIG. 8).

Example 5
AnnikA (5'-UTR (promoter region) and 3'-UTR)
The upstream and downstream sequences of AnnikA were amplified with the following primer set using Aspergills nidulans genomic DNA as a template.
5'-UTR
AnnikA pro HindIII F; 5'-CCCAAGCTTTATCGTGCAGCAGCAGTCGCCCATC-3 '(added Hind III) (SEQ ID NO: 60) and AnnikA pro R; 5'-TCAATACAATTGCAGACTTCCATTTTGCCCTGGGATACTCAGTTAA-3' (SEQ ID NO: 61)
3'-UTR
AnnikA 3'UTR F; 5'-CATAGATAGGGTAAGACCATGATAC-3 '(SEQ ID NO: 62) and AnnikA 3'UTR R; 5'- ACATGCATGCCACGTACTCTTAAAGGTTGGG-3' (added Sph I site) (SEQ ID NO: 63)
The amplified fragment was subcloned into pZErO ^™ -2 vector (Invitrogen) (pZErO-AnnikApro and pZErO-AnnikA3′-UTR).

pUC−pyroA−EBD + NikA
In order to construct an EBD-fused AnNikA expression system in filamentous fungi, the following primers were designed upstream of the fusion NikA fragment used in the yeast system, overlapping the 5'-UTR junction of NikA, and pYES-AtETR1 (EBD) + AnnikA (HKD), pYES−AtETR1 (EBD + GAF) + AnnikA (HKD), pYES−AtETR1 (EBD) + AnnikA (HAMP + HKD) and pYES−AtETR1 (EBD + GAF) + AnnikA (HAMP + HKD) Amplification was performed with the following primer set as a template.
AnnikA pro-AtETR1 F; 5′-TTAACTGAGTATCCCAGGGCAAAATGGAAGTCTGCAATTGTATTGA-3 ′ (SEQ ID NO: 64) and AnnikA R2; 5′-CATCATGCACGACCAACGGTTC-3 ′ (SEQ ID NO: 54) The amplified fragments were purified, performed fusion PCR using primers with the addition of the following restriction enzymes as template, was subcloned fused fragment ^{pZErO TM -2 vector (Invitrogen) (} pZErO-nikpro + AtETR1 (EBD) + AnnikA (HKD), pZErO- nikpro + AtETR1 (EBD + GAF) + AnnikA (HKD), pZErO−nikpro + AtETR1 (EBD) + AnnikA (HAMP + HKD) and pZErO−nikpro + AtETR1 (EBD + GAF) + AnnikA (HAMP + HKD)).
AnnikA pro HindIII F; 5′-CCCAAGCTTTATCGTGCAGCAGCAGTCGCCCATC-3 ′ (added Hind III) (SEQ ID NO: 60) and AnnikA R2; 5′-CATTACGCACGACCAACGGTTC-3 ′ (SEQ ID NO: 54) Downstream of the fusion NikA fragment used in the yeast system The following primers with overlapping NikA 3′-UTR junctions were designed and amplified with the following primer set using pYES-AtETR1 (EBD) + AnnikA (HKD) as a template.

AtETR1 HindIII F2; 5′-CCCAAGCTTTAATGGAAGTCTGCAATTG-3 ′ (SEQ ID NO: 39) and AnnikA R3; 5′-TCATGGTCTTACCCTATCTATG-3 ′ (SEQ ID NO: 65) The purified fragment was purified and the AnnikA3 the '-UTR fragments using primers adding the following restriction enzyme as the template performs fusion PCR, were subcloned fused fragment ^{pZErO TM -2 vector (Invitrogen) (} pZErO-AtETR1 (EBD) + AnnikA (HKD) + nik3'UTR).
AtETR1 HindIII F2; 5'-CCCAAGCTTTAATGGAAGTCTGCAATTG-3 '(added Hind III) (SEQ ID NO: 39) and AnnikA 3'UTR R; 5'- ACATGCATGCCACGTACTCTTAAAGGTTGGG-3' (added Sph I site) (SEQ ID NO: 63)

  Furthermore, pZErO−nikpro + AtETR1 (EBD) + AnnikA (HKD), pZErO−nikpro + AtETR1 (EBD + GAF) + AnnikA (HKD), pZErO−nikpro + AtETR1 (EBD) + AnnikA (HAMP + HKD) and pZErO− Extract nikpro + fusion HK fragment from nikpro + AtETR1 (EBD + GAF) + AnnikA (HAMP + HKD) with Hind III and EcoR I, pZErO-AtETR1 (EBD) + AnnikA (HKD) + nik3'UTR (Hind III and EcoR I Process). (PZErO−nikpro + AtETR1 (EBD) + AnnikA (HKD) + nik3'UTR, pZErO−nikpro + AtETR1 (EBD + GAF) + AnnikA (HKD) + nik3'UTR, pZErO−nikpro + AtETR1 (EBD) + AnnikA ( HAMP + HKD) + nik3′UTR and pZErO−nikpro + AtETR1 (EBD + GAF) + AnnikA (HAMP + HKD) + nik3′UTR).

  pZErO−nikpro + AtETR1 (EBD) + AnnikA (HKD) + nik3'UTR, pZErO−nikpro + AtETR1 (EBD + GAF) + AnnikA (HKD) + nik3'UTR, pZErO−nikpro + AtETR1 (EBD) + AnnikA (HAMP + HKD) + nik3'UTR and pZErO-nikpro + AtETR1 (EBD + GAF) + AnnikA (HAMP + HKD) + nik3'UTR is cut out with Hind III and Sph I, pUC-pyroA2 (Hind III and Sph I Processing). (PUCpyroA−nikpro + AtETR1 (EBD) + AnnikA (HKD) + nik3'UTR, pUCpyroA−nikpro + AtETR1 (EBD + GAF) + AnnikA (HKD) + nik3′UTR, pUCpyroA−nikpro + AtETR1 (EBD) + AnnikA ( HAMP + HKD) + nik3′UTR and pUCpyroA−nikpro + AtETR1 (EBD + GAF) + AnnikA (HAMP + HKD) + nik3′UTR). It was confirmed by DNA sequencing that the fusion fragment was introduced into the vector.

Test example 3
HK functional complementation test of fusion NIkA in filamentous fungi Aspergillus nidulans ABPU1 ΔligD :: ptrA (biotin (biA1), arginine (argB2), uridine (pyrG89), pyridoxine (pyroA4) requirement strains used for functional complementation analysis The above pUCpyroA series of plasmids were linearized by restriction enzyme treatment, transformed into filamentous fungi by protoplast-PEG method, 0.02 μg / ml biotin, 0.2 mg / ml arginine, 5 mM uridine and 10 mM except pyridoxine. Czapek-Dox (CD) medium supplemented with uracil was cultured until conidia were formed, and selection of transformants with the above composition was confirmed with the following primer set.
AtETR1 HindIII F2; 5'-CCCAAGCTTTAATGGAAGTCTGCAATTG-3 '(added Hind III) (SEQ ID NO: 39) and AtETR1-AnnikA R3; 5'-TAGCTCTTCACGGACCTGGGTAGTGCTTCTAATCTCATGAGT-3' (SEQ ID NO: 51)

  The selected transformant was added to Czapek-Dox (CD) medium (control) supplemented with 0.02 μg / ml biotin, 0.2 mg / ml arginine, 0.5 μg / ml pyridoxine, 5 mM uridine and 10 mM uracil. HK functional complementation was tested by culturing at 37 ° C. in a medium supplemented with 1 μg / ml or 2 μg / ml Fludioxonil in a control medium. Wild strains, ΔnikA strains (Hagiwara et al. (2007) Biosci. Biotechnol. Biochem. 71, 844-847), and selected transformants were cultured at 37 ° C. for 2 days in a medium supplemented with 1 μg / ml Fludioxonil. It was confirmed that it grew normally. FIG. 10 shows the results of the wild-type strain, pUCpyroA-nikpro + AtETR1 (EBD) + AnnikA (HKD) + nik3′UTR transformed strain (fused NikA (without HAMP) introduced strain) and ΔNikA strain. Show. As shown in FIG. 10, growth was observed in the fused NikA-introduced strain during the treatment with Fludioxonil, and complementation of HK function was confirmed. The ΔnikA strain is resistant to Fludioxonil because it lacks NikA having a HAMP domain.

Treatment of filamentous fungi with ethylene
Response test in plate culture Ethylene treatment was set in an acrylic chamber with a volume of 12 L so that the ethylene concentration would be 100 μl / L. Incubate for days. (Ethylene concentration, 50μl ~ 100ml / L)
Analysis of HOG downstream gene for confirmation of ethylene responsiveness After culturing the above-mentioned filamentous fungal transformant on a CD plate at 37 ° C for 2 days, in an acrylic chamber with a volume of 12 L (ethylene concentration 100 μl / L The RNA of the filamentous fungus is extracted using RNeasy Plant Mini Kit (Qiagen) after 10 minutes to 24 hours. The extracted RNA is subjected to DNase treatment, and then subjected to real-time PCR analysis using the following primer set using iScript ^™ One-Step RT-PCR Kit with SYBR Green (Bio-Rad). (Ethylene concentration 50 μl to 100 ml / L) Histone is used as a loading control.
AncatA 5′-GCTACTAGGTATCACCGCCGG-3 ′ (SEQ ID NO: 66) and 5′-GCTGCTCCTGGTCTGTGTGG-3 ′ (SEQ ID NO: 67)
AngfdB 5′-CAGGTCTCCGTCATCGGCTC-3 ′ (SEQ ID NO: 68) and 5′-CTGGAGTTTCGAAGGTGTCG-3 ′ (SEQ ID NO: 69)
Anhiston 5'-CACCCGGACACTGGTATCTC-3 '(SEQ ID NO: 70) and 5'-GAATACTTCGTAACGGCCTTGG-3' (SEQ ID NO: 71)

SEQ ID NO: 9 Fusion peptide SEQ ID NO: 10 Fusion peptide SEQ ID NO: 11 Fusion peptide SEQ ID NO: 12 Fusion peptide SEQ ID NO: 15 Fusion peptide SEQ ID NO: 16 Fusion peptide SEQ ID NO: 17 Fusion peptide SEQ ID NO: 18 Fusion peptide SEQ ID NO: 21 Fusion peptide SEQ ID NO: 22 Fusion peptide SEQ ID NO: 23 Fusion peptide SEQ ID NO: 24 Fusion peptide SEQ ID NO: 27 Fusion peptide SEQ ID NO: 28 Fusion peptide SEQ ID NO: 29 Fusion peptide SEQ ID NO: 30 Fusion peptide SEQ ID NO: 31 Primer SEQ ID NO: 32 Primer SEQ ID NO: 33 Primer SEQ ID NO: 34 Primer SEQ ID NO: 35 Primer Sequence number 36 Primer sequence number 37 Primer sequence number 38 Primer sequence number 39 Primer sequence number 40 Primer sequence number 41 Primer sequence number 42 Primer arrangement Sequence number 43 Primer sequence number 44 Primer sequence number 45 Primer sequence number 46 Primer sequence number 47 Primer sequence number 48 Primer sequence number 49 Primer sequence number 50 Primer sequence number 51 Primer sequence number 52 Primer sequence number 53 Primer sequence number 54 Primer sequence number 54 Primer sequence number 55 primer sequence number 56 primer sequence number 57 primer sequence number 58 primer sequence number 59 primer sequence number 60 primer sequence number 61 primer sequence number 62 primer sequence number 63 primer sequence number 64 primer sequence number 65 primer sequence number 66 primer sequence number 67 primer SEQ ID NO: 68 Primer SEQ ID NO: 69 Primer SEQ ID NO: 70 Primer SEQ ID NO: 71 Primer

Claims (8)

A fusion peptide comprising an ethylene sensor domain derived from a plant and a histidine kinase domain derived from a filamentous fungus or yeast. The histidine kinase domain is derived from a filamentous fungus belonging to the genus Aspergillus, Penicillium, Trichoderma, Cephalosporium, or Acremonium or Saccharomyces, Candida, Tolropsis, Digosaccharomyces, Schizosaccharomyces, Pichia The fusion peptide according to claim 1, which is derived from yeast belonging to the genus Yarrowia, Hansenula, Kluyweromyces, Debariomyces, Geotrichum, Wickelhamia or Ferromies.   The fusion peptide according to claim 2, wherein the histidine kinase domain is derived from Aspergillus nidulans, Aspergillus oryzae, Aspergillus soya, Aspergillus niger, Aspergillus fumigatus or Saccharomyces cerevisiae. The fusion peptide according to any one of claims 1 to 3, wherein the plant-derived ethylene sensor domain is derived from Arabidopsis thaliana, tomato, petunia, rice, tobacco, cucumber, banana or apple. The nucleic acid molecule which codes the fusion peptide of any one of Claims 1-4. A vector comprising the nucleic acid molecule according to claim 5. A transformant obtained by transforming a host filamentous fungus or yeast using the vector according to claim 6 or a vector comprising a nucleic acid molecule encoding a peptide comprising a plant-derived ethylene sensor domain and a plant-derived histidine kinase domain. body. Culturing the transformant of the filamentous fungus or yeast according to claim 7,
A step of treating the transformant with ethylene to cause the transformant to produce a substance, and a step of recovering the obtained substance.
A material production method comprising: