KR102286791B1 - Recombinant strains expressing fusion protein which fragment of SrbA protein and golgi resident protein fused and its application - Google Patents

Abstract

The present invention relates to a recombinant vector containing a gene encoding a SrbA protein fragment and a gene encoding an early golgi resident protein, and ΔsrbA recombinant Aspergillus nidulans transformed with the vector. will be.
Therefore, the strain transformed with the recombinant vector of the present invention can overexpress higher levels of ergosterol or genes related to its synthesis compared to the wild-type strain even under normoxic conditions. can provide
Further, Ascomycetes sterol regulation of (Ascomycetes) factor binding protein (sterol regulatory element binding protein) or a homologous (homolog) activation is inhibition of the Ascomycetes is essential the protein on to growth in hypoxic condition when suppressing the DUF2014 domain of the protein Therefore, the present invention can provide a method for inhibiting hypoxic growth of Ascomycetes.

Description

Recombinant strains expressing fusion protein which fragment of SrbA protein and golgi resident protein fused and its application

The present invention relates to a method for inhibiting hypoxic growth of Ascomycetes , comprising the step of inhibiting the DUF2014 domain of a sterol regulatory element binding protein or a homolog thereof.

In general, it is known that mammalian tissue cells are significantly lower than atmospheric oxygen levels and that oxygen levels vary depending on numerous factors such as tissue type and inflammatory response. The oxygen-richest place in the mammalian body is the alveoli of the lungs, where the oxygen level is about 14%, but when oxygen diffuses from the capillaries to the tissues through the blood, the oxygen level can drop to 2-4%. there is. There is also the potential for hypoxic conditions due to fungal infections, as inflammation-induced phagocytosis and pathogen growth can disrupt blood supply.

Since most human pathogenic fungi are aerobic eukaryotic cells, overcoming the low oxygen concentration of the host cell during infection is a very important factor for the survival of the fungus. Among the human pathogenic fungi, Aspergillus fumigatus (A. fumigatus) is a fungus commonly found in soil and organic materials such as compost heaps, and is a causative agent of invasive aspergillosis (IA). . IA is a fatal disease with a fatality rate of 60 to 90%, which can be caused by several Asphagillus species, but most (>90%) are known to be due to A. fumigatus.

Aspergillus nidulans (or Emericella nidulans; A. nidulans), owing to its easy handling, rapid growth, and well-established genetic system, is a popular choice for fungal biology studies such as development (cell differentiation), cell cycle and secondary metabolite production. It is used as a fibrous model mold. Although A. nidulans rarely infects humans, it is genetically closely related to the human pathogenic fungus A. fumigatus. Indeed, as a result of cross-species complementation assay, it was confirmed that the SrbA protein essential for hypoxic adaptation shared a biological function between A. fumigatus and A. nidulans.

Sterol regulatory element binding proteins (SREBPs) of fungi are known to be important transcription factors for adaptation and survival in hypoxic conditions. In addition, SREBPs are also involved in the transcriptional activity of lanosterol 14 α-demethylase (erg11 or cyp51), which is the target protein of azole antifungal agents, very sensitive to Therefore, inhibition of fungal SREBP is expected as a new alternative to azole-based antifungals and can be used as an effective treatment through combination treatment with other antifungals. In addition, SREBPs are involved in ergosterol biosynthesis, iron acquisition, glycolysis, oxidative stress resistance, and cell wall biosynthesis.

Ergosterol (ergosterol; ergosta-5,7,22-trien-3β-ol) is a sterol found in the cell membranes of fungi and protozoa, and performs the same function as cholesterol in animal cells. Ergosterol homeostasis is important for fungal cells as ergosterol regulates membrane fluidity, plasma membrane bioformation and function, and includes transcriptional regulation of genes encoding ergosterol biosynthetic enzymes and proteins that perform sterol processing and uptake. Since many fungi and protozoa cannot survive without ergosterol under hypoxic conditions, enzymes that synthesize ergosterol have become important targets for the discovery of antifungal agents. In addition, since ergosterol can be used in various ways, such as a precursor of vitamin D2, it is also obtained by culturing various strains including A. nidulans that produce it in large quantities.

However, since it is very inefficient in terms of time and cost to create a hypoxic environment to obtain ergosterol from the strains, a countermeasure against this is required.

Republic of Korea Patent No. 10-1467448 relates to the aqpA gene of Aspergillus nidulans and a deletion mutant thereof, and by inhibiting the expression of the aqpA gene of Aspergillus nidulans, resistance to antibacterial agents and resistance to oxidative stress. An increased strain of Aspergillus nidulans is provided.

However, since the improvement method related to the oxygen concentration in the growth or culture conditions of the strain capable of producing ergosterol, particularly the Asphagillus nidulance strain, is not disclosed, a countermeasure for this is still needed.

Accordingly, the present inventors have made intensive efforts to obtain ergosterol in high yield by culturing the strain producing ergosterol, in particular, the Aspergillus nidulans strain under normoxic conditions rather than hypoxic conditions. As a result, A. nidulans When the ΔsrbA strain is expressed in a ΔsrbA strain fused with a fragment of the SrbA protein of the strain and the initial Golgi protein, ergosterol and its synthesis-related genes are expressed at a significantly increased level compared to the wild-type as well as under normoxic conditions. It was confirmed that the present invention was completed.

Accordingly, it is an object of the present invention to provide a method for inhibiting hypoxic growth of Ascomycetes , comprising the step of inhibiting the DUF2014 domain of a sterol regulatory element binding protein or a homolog thereof will be.

In order to achieve the above object, the present invention provides a method for inhibiting hypoxic growth of Ascomycetes , comprising the step of inhibiting the DUF2014 domain of a sterol regulatory element binding protein or a homolog protein thereof can provide

According to a preferred embodiment of the present invention, the homologous protein of the sterol regulator binding protein is SrbA, and the Aspergillus may be of the genus Aspergillus.

According to a preferred embodiment of the present invention, the low oxygen may be a condition in which the oxygen concentration in the atmosphere is 13% to 0.1% by volume.

The strain transformed with the recombinant vector of the present invention can overexpress a higher level of ergosterol or genes related to its synthesis compared to the wild-type strain even under normoxic conditions. can do.

In addition, when the DUF2014 domain of a sterol regulatory element binding protein or a homolog thereof of Ascomycetes is inhibited, the activation of the protein essential for the growth of Ascomycetes under hypoxic conditions is inhibited. It is possible to provide a method of inhibiting hypoxic growth of

1 shows whether SrbA cleavage is activated under normoxic or hypoxic conditions. It was confirmed that the cleavage of the SrbA precursor (SrbA-F) was significantly increased under the hypoxic condition compared to the case of the normoxic condition, and the production of mature karyotype SrbA (SrbA-N) was increased. SrbA-F1 and SrbA-N1 were speculated to be due to another cleavage occurring at a site near the N-terminus of SrbA.
2 shows 13 types of fragments (SrbA-381, -414, -440, -480, -600, -667, -758, -792, -793 , -794, -795, -823, -952). The left side shows the amino acid position of the main domain of SrbA and the length of the SrbA fragments, and the right side shows the names of the SrbA fragments and the growth degree of each strain under hypoxic conditions.
3A) shows the growth degree under normoxic or hypoxic conditions for each strain expressing the SrbA fragments prepared in Example 3 (FIG. 2), and the name of the strain is indicated by the cleaved position of SrbA. B) shows whether SrbA cleavage is activated in the strains grown for 30 minutes under hypoxic conditions. Boxes marked with gray dots represent the mature karyotype of SrbA (SrbA-N).
4A) shows the structures of YFP:SrbA and YFP:SrbA-480, which are fusion proteins in which the N-terminus of SrbA or SrbA-480 is labeled with 4×YFP. B) shows that the signal of YPF:SrbA and the signal of RFP:H2A do not overlap under normoxic conditions, but overlap under hypoxic conditions (Merge), indicating that YPF:SrbA migrated from the endoplasmic reticulum to the nucleus. C) shows that the signals of YPF:SrbA-480 and ShrA:RFP did not overlap (Merge) regardless of oxygen conditions, indicating that YPF:SrbA-480 did not migrate in the endoplasmic reticulum.
5A) is a fusion protein (SrbA-480: SedV, SrbA-480: RerA and SrbA-480) that binds a Golgi protein (early Golgi protein: SedV, RerA or late Golgi protein: TlgB) to the C terminus of SrbA-480. :TlgB). B) shows the growth degree under normoxic or hypoxic conditions of each strain expressing the fusion proteins, respectively. When fused with an early Golgi protein, growth was possible even under hypoxic conditions, but when fused with a late Golgi protein, it could not survive. C) shows whether SrbA was cleaved under normoxic or hypoxic conditions of each strain. Black arrows indicate mature karyotype SrbA (SrbA-N).
6A) shows the structure of a protein labeled with 4×YFP (SrbA-480:YFP:SedV) between SrbA-480 and SedV in SrbA-480 and SrbA-480:SedV. B) shows that the signal of SrbA-480:YFP:SedV and RFP:SedV overlapped and the fusion protein moved to the initial Golgi.
7A) shows the structures of SrbA-480 and YFP:SrbA-480:SedV. B) shows that the signals of YFP:SrbA-480:SedV and RFP:H2A overlap, indicating that the fusion protein was relocated to the nucleus regardless of the extracellular oxygen concentration.
8 shows that YFP-tagged SrbA-480:SedV functions normally. ΔsrbA and SrbA-480 did not grow under hypoxic conditions.
9A) shows whether cleavage activation of the control (SrbA ₂₆₁ :Flag) or SrbA-480:SedV under normoxic or hypoxic conditions. Compared to the control group, it was confirmed that SrbA-480:SedV was cleaved regardless of oxygen conditions. B) shows the expression levels of erg11A and erg25A genes, which encode enzymes involved in ergosterol biosynthesis of _{control (SrbA 261:Flag) or SrbA-480:SedV under normoxic or hypoxic conditions.} It was confirmed that the expression of erg11A and erg25A was significantly increased in the case of SrbA-480:SedV compared to the control group.
10 shows that SrbA-480 fused with SedV or RerA, which is an initial Golgi protein, increased resistance to itraconazole. The diagram at the top shows the strain used.
11 shows that SrbA-480 fused with SedV or RerA, which is an initial Golgi protein, increased resistance to itraconazole. In particular, it was confirmed that resistance was further increased when SrbA-480:SedV was overexpressed.
12 shows that when SrbA-480 fused with SedV, an initial Golgi protein, is expressed, ergosterol production is increased compared to that of the wild type.
13 shows the structure and amino acid sequence of SrbA protein of wild-type Asphagillus nidulance. The wild-type SrbA protein consists of a total of 1015 amino acid residues.
14 shows a sequence map of the SrbA-480:SedV vector.

Hereinafter, terms used in the present invention will be described.

In the present invention, “SrbA-F” refers to a precursor of an uncleaved SrbA protein, and “SrbA-N” refers to a cleaved SrbA protein, which refers to a mature or active form thereof. “SrbA-F1” or “SrbA-N1” refers to a protein produced by cleaving a portion other than the active site of the SrbA protein.

"ΔsrbA " of the present invention means a strain of Aspergillus nidulans in which the gene expressing the wild-type SrbA protein is deleted.

"Golgi (resident) protein" of the present invention means a protein constituting the Golgi apparatus (Golgi apparatus). ‘Early Golgi protein’ refers to the first cisternae structure (cis golgi) of the Golgi apparatus, and ‘late Golgi protein’ refers to the last cisternae structure (trans golgi) of the Golgi apparatus. “Syntaxin” refers to a protein involved in cell exocytosis.

"Vector" of the present invention means a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. A vector may be a plasmid, a phage particle, or simply a potential genomic insert. Upon transformation into an appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. In the present invention, it is used in the sense of including other forms of vectors, such as plasmids, which have an equivalent function as known or known in the art.

"Fusion protein" of the present invention means that one or more polypeptides are bound to a single-stranded polypeptide through a peptide bond, and one or more genes including a target gene are fused by a recombinant method to form one polypeptide. protein that has been translated into

"Normal oxygen" in the present invention means oxygen in general atmospheric conditions, and means an oxygen concentration of 20 to 21% by volume compared to the entire atmosphere.

"Hypoxia" of the present invention means an oxygen concentration lower than the oxygen concentration of general atmospheric conditions, and means an oxygen concentration of 13 to 0.1% by volume compared to the entire atmosphere.

Hereinafter, the present invention will be described in more detail.

As described above, in the prior art, in order to obtain a large amount of ergosterol from a strain such as Asphagillus nidulance, hypoxic conditions are essential, so there was a disadvantage that it is inefficient in terms of cost or time. In order to overcome this, a method for overcoming the hypoxic conditions and further increasing the yield of ergosterol is required, but effective strains have not been studied yet.

ΔsrbA recombinant Aspergillus nidulans strain transformed with a recombinant vector containing a gene encoding a SrbA protein fragment according to the present invention and a gene encoding an early golgi resident protein, hypoxic conditions In addition, ergosterol and genes involved in its synthesis are expressed even under normoxic conditions, and since it is expressed at a significantly increased level compared to the wild-type Asphagillus nidulance strain, it is effective as a strain for ergosterol production, and it is effective as an ergosterol production method can be used effectively.

Accordingly, the present invention relates to a gene encoding a SrbA protein fragment; And an early Golgi resident (early golgi resident) gene encoding the protein; As a recombinant vector comprising a,

The SrbA protein fragment provides a recombinant vector comprising an amino acid sequence from the first amino acid residue to the 440th amino acid to the 794th amino acid residue of the wild-type SrbA protein amino acid sequence of SEQ ID NO: 1.

The SrbA protein is a homolog of a sterol regulatory element binding protein, and is an essential protein for some fungi such as strains of Asphagillus species to survive under hypoxic conditions.

Specifically, the SrbA protein fragment is the 440th, 441th, 442th, ... from the first amino acid residue of the wild-type SrbA protein amino acid sequence of SEQ ID NO: 1. , an SrbA protein fragment selected from the group consisting of a total of 355 SrbA protein fragments consisting of up to the 792th, 793th, or 794th amino acid residue, preferably from the 1st amino acid residue to 480 of the wild-type SrbA protein amino acid sequence of SEQ ID NO: 1 Preferably, it is an SrbA protein fragment consisting of an amino acid sequence up to the first amino acid residue.

The SrbA protein fragment was cleaved from the full length of the wild-type SrbA protein to a range not including the DUF2014 domain (amino acids 491 to 754) (Example 3, FIG. 2).

The early Golgi resident protein refers to a protein belonging to the first cisternae structure (cis golgi) of the Golgi body, preferably SedV or RerA. More preferably, it is SedV.

SedV is classified as a cis (early) Golgi resident protein and is a protein involved in vesicular transport between the endoplasmic reticulum (ER) and the Golgi protein. RerA is also classified as a cis (early) Golgi resident protein and is a retrieval receptor responsible for ER recovery of ER proteins circulating in the ER and Golgi.

The recombinant vector of the present invention is preferably represented by the sequence map shown in [Fig. 14], and encodes a gene encoding a fusion protein in which an SrbA protein fragment and an initial Golgi resident protein are fused.

In addition, the present invention provides a strain ΔsrbA recombinant Aspergillus nidulans transformed with the vector.

The strain is ΔsrbA recombinant asparagyl expressing a fusion protein in which an SrbA protein fragment consisting of the amino acid sequence from the 1st amino acid residue to the 480th amino acid residue of the wild-type SrbA protein amino acid sequence of SEQ ID NO: 1 and SedV, an initial Golgi resident protein, are fused. As a sidulence ( Aspergillus nidulans ) strain, it is preferable that the strain deposited with the accession number KCTC 13598BP.

The strain of the present invention overexpresses a target gene of wild-type SrbA. Preferably, the target gene of wild-type SrbA is erg11A or erg25A. erg11A is a gene encoding 14-demethylase, and erg25A is a gene encoding 4-sterol methyl oxidase, all of which are involved in the synthesis of ergosterol. The expression level of wild-type ergosterol-producing strains such as wild-type Asphagillus nidulance significantly increased than the expression level under normoxic to hypoxic conditions, so it is suitable for mass production of ergosterol under normoxic conditions (Example 10, Fig. 9) ). Therefore, the strain can also be utilized for the production of ergosterol.

The strains having increased expression of ergosterol or genes involved in its synthesis may have increased resistance to azole antifungal agents. The azole antifungal agent usually targets 14-demethylase and acts as a non-competitive inhibitor by binding to erg11A. Types include triazoles, fluconazole, or itraconazole.

In addition, the strain can grow under normoxic conditions as well as hypoxic conditions. Specifically, it is preferable that the oxygen concentration in the atmosphere can grow under a condition of 30% to 0.1% by volume, and more preferably, it can grow under a condition of 20% to 1% by volume.

In addition, the present invention comprises the steps of i) culturing the strain; and ii) obtaining ergosterol produced from the strain of step i); It provides an ergosterol production method comprising a.

The culturing in step i) of the present invention is preferably cultured under the condition that the oxygen concentration in the atmosphere is 30% to 0.1% by volume, more preferably 25% to 0.5%, and most preferably 20% It is preferable to be able to grow under conditions of 1% to 1%.

The method for producing ergosterol of the present invention may be performed in vitro.

In addition, the present invention provides a method for inhibiting hypoxic growth of Ascomycetes , comprising the step of inhibiting the DUF2014 domain of a sterol regulatory element binding protein or a homolog thereof.

The homologous protein of the sterol regulator binding protein of the present invention is SrbA, and the ascomycetes include both SrbA and DUF2014 domains of Aspergillus genus, Schizosaccharomyces genus, Penicillium ) genus or Neurospora (Neurospora) genus, etc. may be applicable, but the genus Aspergillus is most preferred.

The low oxygen of the present invention is preferably a condition in which the oxygen concentration in the atmosphere is 13% to 0.1% by volume, more preferably 5% to 0.5%, and most preferably 3% to 1% do.

The method of inhibiting the hypoxic growth of the ascomycetes of the present invention may be performed in vitro or in mammals other than humans.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

<1-1> Preparation of strains and recombinant strains

The strains used in this study are shown in [Table 1] below.

strain name genetic information source A4 Wild-type Asphagillus nidulance strain FGSC SrbA ₂₆₁ :Flag pabaA1 ; argB2 ; pyroA4 , srbA ₂₆₁ :3×Flag; chaA1 Bat-Ochir ΔsrbA pabaA1 ; argB2 ; Δ srbA :: argB , pyroA4 ; chaA1 Bat-Ochir SrbA-(n);Δ srbA pabaA1 ; argB2 ; Δ srbA :: argB , pyroA4 ; chaA1 ;
s rbA (p): srbA 261:Flag:(1-n): qa- 4(t), pyroA
(n = amino acid position; 381, 414,
440, 480, 600, 667, 758, 792, 793, 794, 795,
823 or 952) this study YFP: SrbA-480;
Δ srbA pabaA1 ; argB2 ; Δ srbA :: argB , pyroA4 ; chaA1 ;
srbA (p):4×YFP: srbA-480 , pyroA ,
alcA (p):RFP:H2A or shrA:RFP, pabaA this study SrbA-480:SedV pabaA1 ; argB2 ; Δ srbA :: argB , pyroA4 ; chaA1 ;
srbA (p): srbA 261:Flag: 1-480 : sedV, pyroA this study SrbA-480:RerA pabaA1 ; argB2 ; Δ srbA :: argB , pyroA4 ; chaA1 ;
srbA (p): srbA 261:Flag: 1-480 : rerA, pyroA this study SrbA-480:TlgB pabaA1 ; argB2 ; Δ srbA :: argB , pyroA4 ; chaA1 ;
srbA (p): srbA 261:Flag: 1-480 : tlgB, pyroA this study YFP:SrbA-480:
SedV pabaA1 ; argB2 ; Δ srbA :: argB , pyroA4 ; chaA1 ;
srbA (p):4×YFP: srbA-480 : sedV , pyroA ,
alcA (p):RFP:H2A , pabaA this study SrbA-480:YFP:
SedV pabaA1 ; argB2 ; Δ srbA :: argB , pyroA4 ; chaA1 ;
srbA (p): srbA -480:4×YFP: sedV , pyroA ,
alcA (p):RFP: sedV, pabaA this study

SrbA-(n);Δ srbA or The vector used to construct the SrbA-480:SedV strain is pBS- pyroA - srbA (p):: qa- 4(T). This was cloned using a restriction enzyme and ligase (Engnomics) based on the pBluescriptⅡ Sk(-) vector , transformed into the E. coli strain 'XL1-blue MRF', and the recombinant plasmid was amplified. Specifically, as shown in [Fig. 14], between the Notl and BamHl positions of the pBluscript vector, the srbA promoter MCS, MCS (Xhol and Kpnl positions) qa -4 terminator (t) and sspl includes pyroA gene for the enzyme site. The pyroA gene was included for later selection of transformants. Specific sequences used in the present invention are as described in [Table 2] below. In the vector sequence of SEQ ID NO: 2, bases 780 to 1667 are srbA(p) sequences; Bases 1668 to 1724 are multiple cloning site (MCS) sequences; Bases 1725 to 2104 are qa-4(t) sequences; It is preferable that the pyroA sequence from base 2714 to base 5339 is the sequence of pBluescript II Sk(-) vector.

sequence content SEQ ID NO: Aspergillus nidulans_SrbA One Vector pBS- pyroA - srbA (p):: qa -4 (T) 2 Aspergillus nidulans_SedV 3

For transformation of the strain, conidium of A. nidulans (a total of 1 × 10 ⁸ ) was cultured in 200 ml complete medium at 37 ° C., 200 rpm for 16 hours. The mycelium was harvested by filtration with sterile miracloth, washed with 500 ml of sterile water and transferred to a 50 ml sterile tube. KCl-citric acid solution (pH 5.8; 8.2 g KCl, 2.1 g citric acid monohydrate (MW 210) in a total volume of 100 ml) 10 ml of 30 ml containing 1.28 g of Vinoflow FCE (Novozymes, Denmark) A sterile-filtered protoplasting solution was added to each tube, mixed, and incubated at 30° C. and 80 rpm for 2 hours. Protoplasts were collected by centrifugation at 3,000 rpm for 6 h and washed twice with 0.6 M KCl. The pellet was resuspended in 0.5 ml of TS-KCa buffer (0.6M KCl, 50 mM CaCl ₂ ) and 0.25 ml of PEG solution. DNA was added to a 100 μl aliquot of the protoplast suspension and the DNA-protoplast mixture was incubated on ice for 30 minutes. Then, 2x volume of PEG solution was added to the mixture and incubated at room temperature for 30 minutes, followed by incubation in selective minimal medium using 0.7% top agar.

The DNA was obtained as follows; The conidia were cultured in a flask containing 100 ml of a liquid medium, collected with 20 ml of Tween-80 solution (polysorbate 80 - 0.1 ml/L stock), and cultured at 37° C. while stirring for one day. Mycelia were harvested by filtration with Miracloth, washed with distilled water (DW), and then powdered using liquid nitrogen. 100 μg of powdered mycelium was rapidly suspended in 500 μl of Aspergillus lysis buffer (50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 3% SDS and 1% β-mercaptoethanol) and vortexed at room temperature. The lysate was heated at 65° C. for 30 minutes (inverted every 10 minutes), cooled to room temperature, and 500 μl of a phenol:chloroform:isoamyl alchohol mixture (25:24:1) was added and 2 at 4°C. were extracted twice. Finally, DNA was obtained by precipitation with 100% cold ethanol, washing once with 70% ethanol, drying, and eluting with _{50 μl H 2 O.}

<1-2> Strain culture conditions

Aspergillus nidulans strains were cultured at 37 °C for 16 hours in minimal media (MM) and complete media (CM). _{10 g of glucose, 0.5 g of magnesium sulfate (MgSO 4} ), 1 ml of Hunter's trace element solution (Table 3) and _{50 ml of 20×Salt solution-MgSO 4} (Table 4) were added per 1 L of the minimum medium. The complete medium was prepared by adding 1 g of yeast extract, 1 g of casein hydrolyzate, 2 g of Bacto-peptone and 1 ml of vitamin solution to a standard minimal medium.

In the case of normoxic culture, it was performed in an incubator under normal atmospheric conditions ( _{including 21% O 2 ).} For hypoxic culture, it was carried out in a 42" polymer hypoxic chamber (Coy Laboratory products inc.) maintaining the conditions of _{1% O 2} and 99 % N _{2 with} a gas mixer (A16 series, Coy Laboratory products inc.) at 37 ° C. For normoxic and hypoxic samples, the surface cultures were left in normoxia or transferred to a hypoxic chamber and incubated for an additional 10, 20, 30 or 60 minutes.

Determining whether SrbA cleavage is affected by extracellular oxygen concentration

To determine whether extracellular oxygen concentration affects the cleavage activation of SrbA in Aspergillus nidulans , immunoblotting analyzes were performed under normoxic or hypoxic conditions.

Specifically, for immunoblotting analysis using an anti-Flag antibody, the SrbA ₂₆₁ :Flag strain endogenously expressing 3×Flag at the 261st amino acid of SrbA was used. In order to maximize the exposure to the atmosphere, the _{conidia of the SrbA 261} :Flag strain were suspended and cultured on the surface of a liquid complete medium at 37°C for 16 hours. Then, the mycelium was further cultured for 10, 20, 30, or 60 minutes under normoxic or hypoxic conditions. The mycelium used for protein extraction was directly recovered from the surface cultures.

Immunoblotting assays were performed in which total protein was purified from frozen and powdered mycelium in TNE buffer (50 mM Tris-HCl (pH 7.4), 0.5 M NaCl, 1%) containing a protease inhibitor cocktail (Thermo Scientific, USA). Triton-X 100 and 1 mM EDTA) and 2 mM PMSF (Thermo Scientific, USA) were used for separation. Protein was quantified using a Pierce 600 nm protein assay, and 30 μg of protein was loaded onto a 10% SDS-PAGE gel. Proteins in the gel were transferred to a nitrocellulose membrane (GE Healthcare Biosciences Corp., USA) and 5% (w/v) in TTBS (20 mM Tris, 150 mM, NaCl, 0.05 % Tween-20, 0.02 % NaN _{3 ).} Blocked with skim milk. The membranes were incubated with mouse anti-Flag M2 monoclonal antibody (Sigma-Aldrich Cat. No. F3165, USA) and anti-mouse HRP-conjugated rabbit polyclonal secondary antibody (1:10,000, Abcam Cat. No. Ab6728, UK). Chemiluminescence was detected on Fuji medical X-ray film after incubation with Super Signal West Pico Chemiluminescence Substrate (Thermo Scientific, USA). SrbA-F represents the SrbA precursor, and SrbA-N represents the mature (cleaved) karyotype of SrbA. γ-Tubulin was used as a loading control for each sample.

As a result, as shown in [Fig. 1], SrbA-N was greatly increased in 10 minutes under hypoxic condition, whereas SrbA-F was mainly detected under normoxic condition. This means that cleavage of SrbA occurred rapidly under hypoxic conditions. SrbA-F1 and SrbA-N1 were assumed to be due to another cleavage occurring at a site near the N-terminus of SrbA. These data suggest that the cleavage pathway for SrbA activation may include an oxygen-sensing mechanism, as SrbA cleavage is regulated by A.nidulans extracellular oxygen concentration.

Whether the DUF2014 domain is essential for cleavage activation of SrbA

Complementation assays and immunoblotting analysis were performed to investigate whether the C-terminus of SrbA containing the DUF2014 domain (amino acids 491 to 754) is required for cleavage activation of SrbA.

Specifically, a strain expressing the full-length or truncated fragment of SrbA ectopically for complementary analysis was prepared as ΔsrbA. Deletion constructs amplified 5' and 3' DNA fragments of about 5 kb from A. nidulans wild-type (A4; FGSC) gDNA, and selectable markers A. nidulans argB or pyroA from plasmid pILJ16 or pNQ-pyroA each amplified. Three PCR amplicons (5' region, argB+ or pyroA+, 3' region) were used as templates to generate the final PCR product transformed into the A. nidulans strain. The deletion mutation was confirmed by PCR and Southern analysis analysis, and the structure is as shown in [Fig. 2]. Each strain was inoculated into a minimal medium using a toothpick and incubated at 37 °C for 3 days under _{normoxic or hypoxic (1% O 2} , 99% N _{2 ) conditions.}

As a result, as shown in A) of [Fig. 3], the expression of SrbA-381 (cleaved form by SppA) or -414 (cleaved form by RbdB), which is the expected cleavage form of SrbA, is defective hypoxic growth of Δ srbA , because it can be released from the membrane to the nucleus with the SrbA activation domain included. In contrast, expression of SrbA-440, -480, -600, -667, -758, -792, -793 or -794 lacking the C-terminal region did not compensate for the defective growth of ΔsrbA under hypoxic conditions, This means that the C-terminal domain of SrbA is important for adaptation to hypoxic conditions. However, expression of SrbA-795, -823, -952 or full-length compensated for the defective growth of ΔsrbA in hypoxia, indicating that the region after amino acid 795 of SrbA is not essential for hypoxia adaptation.

In addition, as shown in [Fig. 3] B), in the strain grown under hypoxic conditions, a band was detected at a position with a molecular weight similar to that of SrbA-N. The strain that could not grow under hypoxic conditions did not detect a band with a size similar to that of SrbA-N, which means that SrbA was not activated. However, in these strains, a band having a size similar to that of wild-type SrbA-N1 was detected. Considering that these strains did not grow under hypoxic conditions, it was assumed that the SrbA-N1 size product had no transcriptional activity. Consequently, these data suggest that the C-terminal domain of SrbA containing DUF2014 is essential for the cleavage activation of SrbA under hypoxia.

Whether the transport of SrbA-480 from the endoplasmic reticulum to the Golgi is inhibited under hypoxic conditions.

As confirmed in [Example 3], SrbA-480 did not undergo proteolytic cleavage and could not supplement growth under hypoxic conditions. Therefore, the present inventors predicted that YFP:SrbA-480 will be located in the endoplasmic reticulum because SrbA-480 has a TM (transmembrane) domain, and the transport ability of SrbA from the ER to the Golgi is defective due to the loss of the C-terminal domain of SrbA under hypoxic conditions. I wanted to check whether or not it happened.

Specifically, Δ N-terminal in srbA is a fused protein labeled with a 4 × YFP YFP: SrbA-480 , YFP: SrbA, RFP: H2A or ShrA: an RFP were respectively expressed by ISO grades. H2A was a nuclear marker, and the N-terminus was labeled with RFP. ShrA was an endoplasmic reticulum marker, and the C terminus was labeled with RFP. The strains were cultured in a liquid minimal medium at 30° C. for 14 hours and then incubated for 2 hours under normoxic conditions for 30 seconds, then transferred to a liquid minimal medium containing 1% glycerol and 1% threonine without glucose. Then, it was further cultured under hypoxic conditions for 1 hour. YFP: SrbA-480 is in ΔsrbA ShrA: RFP was expressed and at the same time. As a control, we used the YFP:SrbA;Δ srbA ;RFP:H2A strain co-expressing N-terminal YFP-labeled intact SrbA and RFP-labeled histone 2A (H2A) proteins as nuclear markers. The localization patterns of the marker-labeled fusion proteins were observed with a Zeiss Axioskop 2 Plus (Zeiss, Germany) differential interference contrast microscope equipped with a Zeiss Axiocam HRc camera. To visualize the YFP fusion protein, a GFP filter was used with an excitation wavelength of 470 nm and an emission wavelength of 509 nm. Image acquisition and processing were performed using Zeiss AxioVision Rel 4.8 software. DIC represents images observed with differential interference contrast microscopy (scale bar: 10 μm), and Merge represents YPF:SrbA and RFP:H2A images or superimposed images of YPF:SrbA-480 and ShrA:RFP images.

As a result, as shown in B) of [Fig. 4], YFP:SrbA-480 was expressed together with ShrA:RFP. In addition, in the Δ srbA strain under normoxic conditions, the signal of YFP:SrbA was localized in the endoplasmic reticulum, a region surrounding the RFP:H2A signal. Under hypoxic conditions, the YFP:SrbA signal overlapped with the RFP:H2A signal, indicating that SrbA migrates from the ER to the nucleus. In addition, as shown in [Fig. 4C), the YFP:SrbA-480 signal was overlapped with the ShrA:RFP signal, indicating that SrbA-480 is located in the endoplasmic reticulum under normoxic conditions. In contrast, the YFP:SrbA-480 signal overlapped with the ShrA:RFP signal, indicating that SrbA-480 did not migrate in the ER under hypoxic conditions. Considering that SrbA-480 is defective in release from the ER under hypoxia, these data suggest that the C-terminal domain of SrbA is important for the transport of SrbA from the ER under hypoxia.

Compensation of functional defects in the case of fusion of SrbA-480 with early Golgi protein

If the C-terminal domain of SrbA is only involved in the transport of SrbA from the endoplasmic reticulum to the Golgi, if the Golgi-resident protein is fused to SrbA lost during the C-terminal domain, whether the defective hypoxic growth of Δ srbA can be compensated wanted to check.

Specifically, the C-terminal coding region after amino acid 480 of SrbA was bound to the open reading frame (ORF) of SedV, an early Golgi syntaxin, RerA, an early Golgi protein, or TolgB, a late Golgi syntaxin, respectively ([FIG. 5] ] A)). Each strain was inoculated in a minimal medium using a toothpick, and cultured at 37 °C under _{normal oxygen (N) or hypoxic (H; 1% O 2} , 99% N _{2 ) conditions for 5 days.} Alternatively, these strains were cultured on the surface of a liquid complete medium at 37°C under normoxic conditions for 16 hours, then transferred to hypoxic conditions and incubated at 37°C for 30 minutes. As a control strain, SrbA ₂₆₁ :Flag was used.

As a result, as shown in [Fig. 5] B) and C), SrbA-480:SedV;Δ srbA and SrbA-480:RerA;Δ srbA supplemented the growth of Δ srbA under hypoxic conditions to a level similar to that of the control group. , a truncated band with a size similar to that of SrbA-N was detected. However, SrbA-480:TlgB;Δ srbA did not grow under hypoxic conditions and cleaved bands such as SrbA-N were not detected. This indicated that the functional deletion of SrbA-480 lacking the C-terminal region was restored by replacement of the early Golgi fiducial SedV and the early Golgi protein RerA. Considering the cleavage activity of SrbA-480:SedV and SrbA-480:RerA, it is suggested that the C-terminal region after amino acid 480 of SrbA may not be required for proteolytic cleavage of SrbA. It was presumed that SrbA fused with early Golgi protein bypassed the transport route from ER to Golgi by relocating SrbA-480, which was poorly released from the ER to the early Golgi.

Determining whether the position of SrbA-480:SedV in the fusion protein is in the early Golgi

The purpose of this study was to determine whether SrbA-480 located in the endoplasmic reticulum was relocated to the early Golgi due to the C-terminal fusion of SedV.

Specifically, a strain was generated expressing a 4×YFP-labeled fusion protein between SrbA-480 and SedV in Δ srbA to co-express SrbA-480:YFP:SedV and RFP:SedV in Δ srbA . The strain was cultured in a liquid minimal medium at 30 ° C. for 14 hours, transferred to a liquid minimal medium containing 1% glycerol and 1% threonine without glucose, and incubated at 30 ° C. under normoxic conditions for 2 hours, and then under hypoxic conditions 1 Additional incubation for hours. The localization pattern of the fusion protein was observed using the same microscope as in <Example 4>. DIC represents an image observed with differential interference contrast microscopy (scale bar: 10 μm), and Merge represents a superimposed image of SrbA-480:YFP:SedV and RFP:SedV.

As a result, as shown in [Fig. 6] B), the signal of SrbA-480:YFP:SedB in the Δ srbA strain overlapped the signal of RFP:SedV in normoxia and hypoxia, indicating that the fusion protein was relocated to the Golgi apparatus. it means. These results suggest that rearrangement of the C-terminal defective SrbA via fusion with the early Golgi protein bypasses the need for the C-terminal region for SrbA cleavage activation. SrbA-480 to the C-terminal labeled with YFP: SedV was filled not restore the growth defect Δ srbA in hypoxic conditions (data not shown).

SrbA-480: N-terminal localization of SedV

SrbA-480 in the steady oxygen or low-oxygen conditions: In order to determine the location of SedV, SrbA-480 Cover for the N-terminal to the 4 × YFP from natural srbA promoter in ΔsrbA:, generating the strain expressing SedV ([7 ] A). Thereafter, the localization pattern of the fusion protein was observed using the same microscope as in <Example 4>. DIC represents an image observed with differential interference contrast microscopy (scale bar: 10 μm), and Merge represents a superimposed image of YFP:SrbA-480:SedV and RFP:H2A.

As a result, as shown in [FIG. 7] B), the YFP:SrbA-480:SedV signal is different from the YFP:SrbA signal, which is a precursor inactivated in the endoplasmic reticulum under normoxic conditions, and the RFP:H2A signal under normoxic and hypoxic conditions. appeared overlapping with, which means that SrbA-480:SedV is located in the nucleus. This YFP in ΔsrbA regardless of the oxygen concentration in the extracellular: SrbA-480: indicates that the cutting of the continuous SedV activated. Therefore, it was determined that the C-terminal domain, not the N-terminal domain, of SrbA is related to the oxygen sensing device for controlling the cleavage activity of SrbA.

Verification of normal function of YFP-labeled SrbA-480:SedV

When SrbA-480:SedV was labeled with YFP, the function of the SrbA-480:SedV fusion protein was verified to determine whether or not the defective hypoxic growth complementation of ΔsrbA was affected depending on its location.

Specifically, each strain was inoculated into a minimal medium using a toothpick and incubated at 37 °C for 3 days under _{normoxic or hypoxic (1% O 2} , 99% N _{2 ) conditions.}

As a result, as shown in Fig. 8], SrbA-480 or SrbA-480 labeled with 4 × YFP: SedV is normally expressed in both the hypoxic ΔsrbA was supplemented growth defects ΔsrbA. The upper part of [Fig. 8] shows the names of the modified strains.

Verification of whether SrbA-480:SedV cleavage is active

To investigate whether the cleavage activation of SrbA-480:SedV is affected by changes in extracellular oxygen concentration, the cleavage pattern of SrbA-480:SedV under normoxic and hypoxic conditions was analyzed by immunoblot analysis using an anti-Flag antibody. tested.

Specifically, the control (SrbA ₂₆₁ :Flag) and SrbA-480:SedV strains were incubated on the surface of a liquid complete medium for 16 h at 37 °C under normoxic conditions, and then for 10, 30, 60 min under normoxic or hypoxic conditions. Further incubation.

As a result, as shown in A) of [Fig. 9], compared to the control strain, SrbA-480:SedV showed a band at a position similar to that of SrbA-N at a high level regardless of the extracellular oxygen concentration. This suggests that the C-terminal domain of SrbA is important for cleavage regulation of SrbA via oxygen sensing.

Confirmation of expression of erg11A and erg25A, which are SrbA target genes

Since the initial Golgi rearrangement of SrbA-480 via SedV fusion bypasses the cleavage regulation of SrbA, we investigated whether SrbA-480:SedV also consistently promotes the expression of SrbA target ergosterol synthesis-related genes. wanted to confirm. This was done by comparing target gene expression levels between _{control (SrbA 261} :Flag), SrbA-480:SedV and Δ srbA strains using Northern blot analysis.

Specifically, as probes used for Northern blotting analysis, cyp51A/erg11A and erg25A genes involved in ergosterol synthesis were used as SrbA target genes. In order to extract RNA, the strain was cultured for 16 hours at 37° C. under normoxic conditions, and then incubated again under normoxic conditions for 30 minutes and 60 minutes, or further cultured under hypoxic conditions for 30 minutes and 60 minutes. Equal RNA loading and RNA quality were confirmed by rRNA in RNA gel images. _{SrbA 261} :Flag was used as a control.

As a result, as shown in [Fig. 9] B), the transcription levels of cyp51A/erg11A and erg25A were significantly reduced in Δ srbA compared to the control strain, and significantly increased in the SrbA-480:SedV strain compared to the control strain. did. In the SrbA-480:SedV strain, the expression level of erg11A was particularly increased under normoxic conditions, showing results consistent with our expectations.

Relationship between relocation of SrbA-480 to Golgi and increased resistance to itraconazole

The purpose of this study was to determine whether SrbA-480 migrated to the early Golgi and increased expression of ergosterol synthesis gene increased resistance to azole antifungals, particularly itraconazole (ITZ).

^{Specifically, 2×10 5} conidia of SrbA-480:SedV and SrbA-480:RerA strains were respectively added dropwise to a minimal medium or a minimal medium containing the antifungal agent itraconazole (0.01 μg/ml or 0.02 μg/ml), and heated at 37°C. Incubated for 72 to 96 hours, and this was repeated 5 times. The radial growth of the strains in the media was measured and graphed every 24 hours. SrbA ₂₆₁ :Flag was used as a control, and C was used as a control medium as a minimal medium.

^{Alternatively, after mixing the spores (1x10 6)} of the SrbA-480:SedV and SrbA-480:RerA strains with a minimal medium (solid medium), pour the same amount into a Petri dish, and when the medium hardens, the E-test bar for itraconazole (used to measure the resistance of the strain to itraconazole) was placed on the medium as it was and incubated in an incubator at 37° C. for 48 hours. The boundary between the part where the strain was grown and the part that did not grow through the E-test in the medium (MIC: minimum inhibitory concentration - represents the minimum inhibitory concentration and the boundary between the clean zone where the strain cannot grow by the antifungal agent and the grown strain) ) was measured and indicated by arrows. SrbA ₂₆₁ :Flag was used as a control, and niiA (p): srbA-480 : sedV strain was sodium nitrate (induction of expression of niiA promoter; Induction) or sodium tartrate (inhibition of expression of niiA promoter; Non-Induction) The expression of srbA- 480 : sedV was controlled through treatment.

As a result, as shown in [Fig. 10], the growth of the strains showed a larger diameter than the control strain in the itraconazole-treated medium. This means that early Golgi relocation of the C-terminal defective SrbA increases resistance to antifungal agents, especially itraconazole. In addition, as shown in [Fig. 11], the growth of the strains increased resistance than the control strain in the itraconazole-treated medium. In particular, overexpression of SrbA-480:SedV further increased resistance. Again, this suggests that early Golgi relocation of the C-terminal defective SrbA increases resistance to antifungal agents, particularly itraconazole.

Increased ergosterol productivity due to expression and activation of SrbA-480:SedV

The purpose of this study was to determine whether the expression of SrbA-480:SedV fused with a Golgi resident protein increases ergosterol productivity.

Specifically, wild type (WT), Δ srbA , SrbA-480:SedV strains were suspended on a minimal medium and cultured for 16 hours. The cultured mycelium was crushed by freezing with liquid nitrogen after removing the medium components, and 200 mg each was recovered using a scale. The recovered samples were mixed with methanol and ethanol in a 3:2 ratio and then mixed with a solution of 25% potassium hydroxide in 10 ml each, and then left at 80° C. for 1 hour. Then, after taking 2.5 ml of the upper layer solution, it was placed in a glass bottle together with 1 ml of distilled water and 2.5 ml of a n-pentane solution, stirred for 10 minutes, and then left to stand. The separated pentane layer was placed in a new glass bottle, the pentane was evaporated, dissolved in 500 μl of methanol, and the ergosterol concentration was measured by HPLC analysis. The measured value represents the average of the values obtained by repeating the above experiment three times.

As a result, as shown in [Fig. 12], the ergosterol concentration of the SrbA-480:SedV strain was increased by about 1.5 times compared to the control (wild type). This indicates that activation of SrbA-480 by the Golgi resident protein SedV increases ergosterol productivity.

[Accession Number]

Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13598BP

Deposit date: 20180724

<110> PAICHAI UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Recombinant strains expressing fusion protein which fragment of SrbA protein and golgi resident protein fused and its application <130> 1067892 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1015 <212> PRT <213> Artificial Sequence <220> <223> Aspergillus nidulans_SrbA <400> 1 Met Ala Ala Pro Ser Met Gly Ser Asp Phe Gln Leu Phe Ser Pro Ala 1 5 10 15 Gln Ser Arg Gly Arg Lys Ser Ser Gln Gly Asp Ser Gly Ser Asp Asp 20 25 30 Thr Gly Gln Asp Trp Thr Glu Trp Met Arg Trp Asp Glu Gln Ala Phe 35 40 45 Pro Asp Asn Lys Asn Leu Pro Leu Ser Pro Ser Leu Thr Ser Pro Pro 50 55 60 Phe Ser Asp Gly Asn Lys Ser Ile Asp Leu Phe Pro Ser Gly Asp Phe 65 70 75 80 Ser Pro Ser Ile Pro Ile Asp Cys Thr Asn Ile Ser Phe Glu Pro Phe 85 90 95 Pro Ala Gln Asp Arg Ser Gly Val Leu Ser Thr Gln Ser Phe Phe Gln 100 105 110 Asp Gly Ile Asp Thr Phe Ser Ala Asn Ser Val Val Ser Gly Ser Pro 115 120 125 Leu Ser Thr Gly Ala Gly Gln Lys Arg Lys Ser Gly Ser Asp Asp Asp 130 135 140 Gly Ser Ala Gly Ser Gly Met Val Gln Glu Val Lys Lys Val Pro Ser 145 150 155 160 Lys Lys Arg Ala His Asn Val Ile Glu Lys Arg Tyr Arg Ala Asn Leu 165 170 175 Asn Glu Lys Ile Ala Glu Leu Arg Asp Ser Val Pro Ser Leu Arg Ala 180 185 190 Ser Lys Gly Asn Gly Val Leu Asp Asp Glu Asp Glu Gly Val Thr Pro 195 200 205 Ala Asn Lys Leu Asn Lys Ala Ser Ile Leu Ser Lys Ala Thr Asp Tyr 210 215 220 Ile Arg His Leu Glu Thr Arg Asn Lys Arg Leu Glu Asp Glu Asn Thr 225 230 235 240 Ala Leu Lys Val Arg Leu Arg Glu Leu Glu Lys Val Ala Asp Gln Ser 245 250 255 Leu Thr Ser Ala Ala Ser Val Ser Ser Pro Ser Asn Tyr Thr Val Ser 260 265 270 Thr Glu Ser Ala Gly Ser Ser Ser Pro Ser Ile Phe Ser Asn Pro Glu 275 280 285 Glu Ser Pro Ile Glu Pro Ser Ser Ser Ser Ser Arg Pro Ala Ala Gly 290 295 300 Met Ile Gln Leu Pro Asp Ser Phe Lys Arg Met Arg Thr Glu Gln Ser 305 310 315 320 Lys Asp Asn Leu Trp Ser Gln Ser Tyr Met Gln Cys Pro Ser Ser Asn 325 330 335 Ser Val Ser Thr Gln Ser Gly Asn Gly Arg Arg Arg Ser Tyr Tyr Pro 340 345 350 Asn Lys Tyr Val Leu Gly Thr Leu Ala Gly Leu Met Val Phe Glu Gly 355 360 365 Leu Gly Lys Glu Lys Glu Thr Asp Ser Thr Ala Lys Gly Leu Leu Ala 370 375 380 Ile Pro Tyr Asn Tyr Phe Arg Asn Val Glu Val Pro Leu Phe Tyr 385 390 395 400 Glu Ile Met Gly Arg Ser Phe Trp Ser Ser Trp His Ala Lys Ala Ile 405 410 415 Leu His Phe Leu Phe Leu Ala Val Leu Val Val Gly Ser Ala Phe Ile 420 425 430 Val Phe Val Tyr Leu Phe Asn Ser Gly Pro Gly His Gln Asn Ser Ser 435 440 445 Lys Leu Ser Thr Pro Gly Val Met Leu Ser Ser Ser Asn Phe Arg Arg 450 455 460 Gln Ala Trp Leu Thr Ser Ile Gln Arg Val Gly Val Pro Arg His Arg 465 470 475 480 Phe Phe His Glu Trp Tyr Val Val Thr Ser Arg Cys Phe Glu Tyr Val 485 490 495 Leu Arg Cys Leu Leu Gly Trp Lys Leu Tyr Ser Ser Ile Thr Gly Ile 500 505 510 Thr Glu Glu Asp Glu Lys Gly Arg Val Lys Thr Trp Asp Ile Ala Ile 515 520 525 Asp Ala Gln Leu Ser Gly Gly Asp Ala Glu Ile Ser Lys Ser Arg Leu 530 535 540 Val Leu Thr Ile Phe Ala Ala Gly Thr Leu Pro Arg Ser Pro Met Arg 545 550 555 560 Met Met Leu Lys Ala Leu His Cys Arg Ile Leu Leu Trp Arg Val Gly 565 570 575 Val Pro Gly Gln Trp Ser Tyr Arg Val Ser Asn Asp Val Ala Arg Ser 580 585 590 Leu Ala Lys Tyr Gln Trp Asp Leu Ala Arg Lys Met Asn Ala Ala Leu 595 600 605 Pro Lys Asp His Glu Asp Ala Leu Pro His Leu Glu Ala Leu Leu 610 615 620 Gln Cys Glu Ser Glu Asp Val Met Ile Asp Ser Ile Thr Gln Arg Ala 625 630 635 640 Ala Asn Leu Thr Trp Asn Asp Pro Thr Gln Glu Gly Ser Asp Gly Asp 645 650 655 Asp Ala Phe Leu Asp Val Val Glu Glu Asp Pro Ala Ile Gln Ser Ser 660 665 670 Leu Asp Ala Leu Ala Ala Trp Trp Ser His Leu Leu Gln Arg Ala 675 680 685 Leu Leu Lys Tyr Phe Glu Ala Ser Ala Arg Gly Pro Asp Ala Lys Lys 690 695 700 Ser Arg Asp Met Phe Lys Ala Lys Ile Gln Leu Ala Leu Asp Val Ala 705 710 715 720 Pro Gln Pro Ser Ala Ala His Thr Arg Ala Leu Val Met Met Ala Val 725 730 735 Phe Phe Glu Gln Asp Arg Val Lys Asn Ile Gly Ala Val Leu Ala Ala 740 745 750 Leu Pro Lys Glu Lys Ser Lys Ser Lys Gln Asn Gln Thr Phe Asn Phe 755 760 765 Leu Asp Ser Ser Leu Pro Val Ser Val Arg Glu Glu Ile Ser Ile Ala 770 775 780 Val Arg Cys Ala Met Ile Ala Ala Ile Phe Thr Ala Arg Ser Arg His 785 790 795 800 Asp Asn Ser Leu Pro Glu Ser Phe Thr Met Glu Lys Ala Val Thr Trp 805 810 815 Phe Asp Gln Leu Pro Leu Asp Pro Val Asp Leu Thr Leu Leu Gly Phe 820 825 830 Ser Ala Val Tyr His Leu Leu His Val Leu Ala Ser Asp Thr Gly Tyr 835 840 845 Leu Ser Ser Ser Asp Ser Ser Arg Pro Ser Ser Pro Met Leu Lys Asp 850 855 860 Ser Thr Leu Gly Asp Ser Ser Asp Asp Ala Glu Asp Glu Ala Glu Glu 865 870 875 880 Ser Ile Thr His Arg Lys Glu Thr Ala Pro Glu Pro Val Pro Leu Ile 885 890 895 Gly Arg Val Ala Ser Glu Leu Met Tyr Trp Ala Arg Asn Ala Tyr Asn 900 905 910 Pro Thr Phe Tyr Gly Phe Thr Ser Asp Leu Val Arg Val Ile Glu Glu 915 920 925 Glu Cys Thr Ser Leu Cys His Gly Ala Gly Val Asp Val Ala Asp Tyr 930 935 940 Ser Arg Leu His Gln Glu Arg Leu Lys Ala Ser Arg Arg Lys Asp Lys 945 950 955 960 Arg Lys Ala Lys Ser Arg Leu Ser Lys Glu Arg Ala Thr Pro Pro Ala 965 970 975 Glu Thr His Leu Ala Ala Lys Pro Ala Ala Ser Ala Ser Pro Ser Asp 980 985 990 Phe Pro Cys Pro Leu Lys Asp Gln Pro Ala Leu Val Gly Glu Ser Ile 995 1000 1005 Ala Ala Gly Arg Glu Arg Thr 1010 1015 <210> 2 <211> 6677 <212> DNA <213> Artificial Sequence <220> <223> pBS-pyroA-srbA(p)::qa-4(T) <400> 2 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 60 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 120 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 180 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 240 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 300 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 360 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 420 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 480 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 540 ggcaggacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 600 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 660 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc 720 gcgcaattaa ccctcactaa agggaacaaa agctggagct ccaccgcggt ggcggccgca 780 gtcatgtata cagtacaaat tctgccaggc ggtaaatata actggtcgct gcactgcccg 840 ctgtcaccag ccggcggttg tgccggaagg gcagattccc gcgatcatac gacgcccacc 900 accgcccttc cattggctct caggatcacc aattttcgat ggcggttggt gatgcagtgc 960 tctgccaggc acctccaagt acatggcaac tgtctctcaa gcatggagat aaccaattgt 1020 tgataactca cggacacctt tggcaactca aatcactttc atgctgtgtg aggtctcata 1080 ggattaagca gaagcaggtg ccaggaacgc aaccttgtca agtggaacaa taggctccta 1140 taattcctca tgtgaagggg aaccacggtg cctagaacca attttaccag ggcgggcggc 1200 actcaaccaa acccccaacg gtttgataag atcttcatct tcacgcaggt aaaagacgac 1260 cttctgggct tcctcagtca tccttgtctg tcattaacaa ccgcctcact agattcattc 1320 ttgatattct tcccaacgtc aaagtgactc aacttgtctc ctcaactctc ggtccgatta 1380 tcattctcca cagtgcagcg gtttagccct gttagcgcgt cggcttgggt ggctgatcgt 1440 caatcaatct tcgaacttaa ggtggcggcg ccgcggctcg aacctttgcc tttaccagtt 1500 gtcttttcac attgttgtat gttttgacgt tgagagctat catatacttc gttgctgata 1560 tctactgcca aagtgaggaa ccccgcgcgg gtgtatgata caagtcgata tacgaccacg 1620 gttaaccggg cgacggtcca tggtcacagc attctatctt gcttcaggga tccccccgggc 1680 tgcaggaatt cgatatcaag cttatcgata ccgtcgacct cgagtctctg cttcattgta 1740 aatttaacgg ttgttgttct tactacttgg tttatcgtta tagactgact ccagtccaca 1800 caaaatgctg ggtttttttt tttttgttct cttttttctt ttcgttttct cttggtacgt 1860 tggattacaa atcggagact ggcacgtgta tcaggagttg gtgacatatt ccttgagccc 1920 aggtcttttt tcttttcatt caatcatgat ttcttcatct gattcttcta gaccctgact 1980 gtagtatcat cttggacgag tggaagacaa tacacatcca ggctctacat taatagggtt 2040 ataattcctg acatcaaagc atgctttccc atgttttttg agccccctgc ttcggcgtgg 2100 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac 2160 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt 2220 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 2280 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 2340 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 2400 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 2460 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 2520 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 2580 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 2640 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 2700 tgatttaaca aaaatttaac gcgaatttta acaaaatatt agtgcgcgaa agcgtaagga 2760 gaccggaagc catttgggaa gcgcagttga gcctgagacc aatgaatact attccgtgtt 2820 tagcttcaat ttggttcgag attataaacc tatagggtgg gattcttgta gatgtcaaca 2880 agcagcatct gccctgtgcg gagtaacatg gcctttacgt catgctggaa caggaggcgg 2940 taagtagagg caatatagtc cctgcggcag tcggtgttac tttaatccgc agatcatgat 3000 ccggcaacgg caccctggct ccggcaggta aattgccatc ggaatggagg tttaactccg 3060 gtcaggtcga tcatccgtgg accgtctcat tcttaagttt cactagaata acagggcgtc 3120 aagcgctcta ttacgataca acaattggat ttttttttgt tgggccgctg gttgttgcca 3180 agcatgatcg aaaaaggcga acatgtcgct aggaagggtc cggaactcta tattgagcga 3240 tttcggagct ctatggattc gatatttgtt ccgcagatag ccacgatgtc taggcatttc 3300 gctgattgaa tcctgaagtt tatcgaataa tcaaatagtc tagaaaccac tcccgaccga 3360 gaacccagcc gagtgcgcgg cgaggcgaac gaatggtaca cggtccctgg ccaactactc 3420 cgcgggaaac gtgaaagatc tgctgcggaa tttgcacgca ccagacattg gaggggcaaa 3480 gaagctgtga ttggcgaatg gcggtttttg gcgggtaagt cagataatag acagagggg 3540 gacgttttct ggacccgaga ggcgagagct taacactgta ggctgtgagt atatagacca 3600 gtgactcacg cgcccttcac ctgacattta tttccccacg caattctcct gctttgctcc 3660 tgtccttgtc cctctctcca tcgttttctc tcgttctttt gtttctctag acgttccaca 3720 cactctctgc ctaaacttta caatggccgc caccaatgga gcctcgaacg acttcaccgt 3780 caaggccggt ctggctcaga tgctgaaggg cggtgttatt atggatgttg tcaatgcgga 3840 acaggtgtgt aatactacga ttatcaattg gcgcaatgac tgactttcaa ctctcaaggc 3900 ccgcattgcc gaagaggccg gtgccgccgc tgtcatggcc ctcgagcgtg tccccgccga 3960 tatccgcgca cagggaggcg tcgcccgcat gtccgacccc tcaatgatca aggagatcat 4020 ggaggccgtg accatccccg tcatggccaa ggcccgtatt ggacacttcg ttgagtgcca 4080 ggttcgtcgt ttgacccctc tcactctcct cgccttaacc gccatgctaa ttgacctgga 4140 tagatcctcg aagccatcgg cgtcgactat atcgatgaat ccgaagtcct cacccccgcc 4200 gacaaccttt accacgtcac aaagcacaac ttcaaagccc ccttcgtctg cggctgccgc 4260 aaccttggag aagccctccg tcgcatctcc gaaggcgcag ccatgatccg cacaaagggc 4320 gaagccggca ctggcgacgt cgtcgaagcc gtcaagcaca tgcgcaccgt caacgcggag 4380 attgcccgcg cccgcgccat ccttcagtcg tcgcctgacc ccgagcccga gctccgcgcc 4440 tacgcccgtg agctcgaggc cccctatgag cttctccgcg aagccgccga gaagggccgc 4500 cttcctgtcg tgaacttcgc ggccggtggt gtcgcgaccc ctgctgatgc tgcgcttatg 4560 atgcagcttg gctgtgatgg tgtctttgtt ggctccggca tctttaaatc tggagatgca 4620 aagaagcgcg ctaaggccat cgtccaggct gttacacact acaaggaccc taaggtgctg 4680 gctgaggtca gccagggcct cggggaggca atggttggca taaacgtttc gcatatgaag 4740 gatgaggaca agctggctaa gcgtgggtgg taataccgac acttgacttc aacttcagtt 4800 ttttgttttt ctttttgctt tgtaattttt tttttttttt tcatgagcgt tggaaaagtt 4860 agtatgtata gcacggtacg cgttcgacct gaagatttta aagctcttgc aatagatttt 4920 ctttgctgtc ttcagaggct ggcatcagac attcaattgt ttgtgtataa ccggagatgt 4980 agatattcta tctctgcaaa gttaaaacat gcccctagtg tctcttaagt ctgctctact 5040 tagctttgac ggaccttttg acatttacat ctgtggccca gtaaaactct gtttcctttc 5100 ctcaggattc gtcaatttac tggctggtga gaacacatgc acaacttgga agaagcggaa 5160 tgctaataag aacgcttcag ctactaaact gacgttaaaa aagatagata tctataaacg 5220 gaggttatta agaatacgat agagccaaga gaaagcgtca agtcagattg cgacagaaga 5280 tatgtgttat tctcaaaccc ttatttcaac accacgcgca atgcaatgct atgcgatgca 5340 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 5400 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 5460 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 5520 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 5580 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 5640 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 5700 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 5760 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 5820 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 5880 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 5940 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 6000 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 6060 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 6120 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 6180 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 6240 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 6300 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 6360 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 6420 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 6480 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 6540 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 6600 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 6660 gttggactca agacgat 6677 <210> 3 <211> 344 <212> PRT <213> Artificial Sequence <220> <223> Aspergillus nidulans_SedV <400> 3 Met Thr Gly Pro Thr Ile Gln Asp Arg Thr Ser Glu Phe Gln Ala Ile 1 5 10 15 Leu Gly Gln Ala Gln Lys Arg Ala Ala Ser Ser Lys Val Gly Ser Gln 20 25 30 Arg Gln Ala Leu Leu Thr Asp Ser Gln Arg Gln Gln Ala Asn Gly Ser 35 40 45 Pro Asn Gly Thr Ala Ala Gly Gly Lys Arg Arg Ser Glu Phe Ala Arg 50 55 60 Arg Ala Ala Glu Ile Gly Arg Gly Ile Thr Ala Thr Thr Ala Lys Leu 65 70 75 80 Arg Arg Leu Ala Glu Leu Ala Lys Arg Lys Thr Leu Phe Asp Asp Arg 85 90 95 Pro Val Glu Ile Ser Glu Leu Thr Tyr Val Ile Lys Gln Asp Leu Ala 100 105 110 Ser Leu Asn Gln Gln Ile Ala Ser Leu Gln Ala Leu Thr Leu Ser Gln 115 120 125 His Pro Lys Ser Asn Arg Ser Lys Thr Asp Gln Glu Gly Glu His Asn 130 135 140 Asp Asn Val Val Val Met Leu Gln Gly Lys Leu Ala Asp Val Gly Ala 145 150 155 160 Asn Phe Lys Asp Val Leu Glu Val Arg Thr Lys Asn Ile Gln Ala Ser 165 170 175 Arg Ser Arg Thr Glu Asn Phe Val Ser Ser Val Ser Ser Lys Ser Gln 180 185 190 Ala Ala Leu Asp Pro Gln Arg Ser Asp Ser Pro Leu Tyr Pro Ser Gly 195 200 205 Arg Arg Thr Pro Gln Pro Gly Gly Ser Ser Asp Leu Leu Thr Leu Glu 210 215 220 Pro Ser Asn Pro Ser Pro Leu Gly Arg Pro Ser Met Gln Ser Asp Gln 225 230 235 240 Gln Leu Leu Met Met Glu Glu Ala Glu Ser Ser Asn Ser Tyr Ile Gln 245 250 255 Ser Arg Gly Glu Ala Ile Asp Ala Ile Glu Arg Thr Ile Asn Glu Leu 260 265 270 Gly Gly Ile Phe Gly Gln Leu Ala Gln Met Val Ser Glu Gln Ser Glu 275 280 285 Met Ile Gln Arg Ile Asp Ala Asn Thr Glu Asp Val Val Asp Asn Val 290 295 300 Gln Gly Ala Gln Arg Glu Leu Met Lys Tyr Trp Thr Arg Val Ser Gly 305 310 315 320 Asn Arg Trp Leu Ile Ala Lys Met Phe Gly Val Leu Met Ile Phe Phe 325 330 335 Leu Leu Trp Val Leu Ile Ser Gly 340

Claims (3)

As a method of inhibiting hypoxic growth of Ascomycetes , comprising the step of inhibiting the DUF2014 domain of a sterol regulatory element binding protein or a homolog thereof,
The homologous protein of the sterol regulator binding protein is SrbA, the Aspergillus is a genus Aspergillus, and the hypoxia is a hypoxic growth inhibition method, characterized in that the oxygen concentration in the atmosphere is 4% to 0.1% by volume . delete delete

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