WO2020027010A1 - トリコデルマ属糸状菌の変異株およびタンパク質の製造方法 - Google Patents

トリコデルマ属糸状菌の変異株およびタンパク質の製造方法 Download PDF

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WO2020027010A1
WO2020027010A1 PCT/JP2019/029563 JP2019029563W WO2020027010A1 WO 2020027010 A1 WO2020027010 A1 WO 2020027010A1 JP 2019029563 W JP2019029563 W JP 2019029563W WO 2020027010 A1 WO2020027010 A1 WO 2020027010A1
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trichoderma
amino acid
strain
culture
mutant
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French (fr)
Japanese (ja)
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雄介 加川
紳吾 平松
山田　勝成
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Toray Industries Inc
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Toray Industries Inc
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Priority to JP2019547718A priority Critical patent/JP7459509B2/ja
Priority to US17/263,685 priority patent/US12037624B2/en
Priority to AU2019313902A priority patent/AU2019313902B2/en
Priority to CN202410503273.1A priority patent/CN118240671A/zh
Priority to CA3108045A priority patent/CA3108045A1/en
Priority to BR112021001743-7A priority patent/BR112021001743A2/pt
Priority to CN201980050357.1A priority patent/CN112513249B/zh
Priority to EP19844909.2A priority patent/EP3831929A4/en
Publication of WO2020027010A1 publication Critical patent/WO2020027010A1/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungi isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/885—Trichoderma

Definitions

  • the present invention relates to a mutant strain of Trichoderma filamentous fungus capable of keeping the viscosity of a culture solution low, and a method for producing a protein using the mutant strain.
  • Trichoderma filamentous fungi are known to have a high protein-producing ability, and the production of proteins using Trichoderma filamentous fungi has been studied. Specifically, Trichoderma filamentous fungi produce cellulase, which is classified as a saccharifying enzyme among proteins, using cellulose, lactose, cellobiose and the like as inducers. In order to enhance the production of cellulase, many studies have been conducted from a long time ago, such as overexpression of factors controlling cellulase production, gene modification such as deletion, optimization of culture conditions, and the like.
  • Trichoderma filamentous fungi belong to aerobic filamentous fungi that require oxygen for growth and protein production.
  • Trichoderma filamentous fungi are characterized in that, when cultured in a liquid medium, the viscosity of the culture solution increases with growth. When the viscosity of the culture solution increases, the distribution of oxygen and nutrients becomes uneven, so when culturing Trichoderma filamentous fungi, the culture solution is agitated or the oxygen supply is increased by increasing the oxygen supply. It is necessary to prevent the decrease of the dissolved oxygen saturation and maintain it at a certain level or more. Further, as the size of the culture tank increases, the oxygen transfer capacity coefficient decreases.
  • Patent Literatures 1 to 6 disclose the dissolution during aerobic fermentation in a submerged culture compared to a parent strain by reducing the destruction or production of Sfb3, Mpg1, Gas1, Seb1, Crz1 and Tps1 proteins of Trichoderma filamentous fungi, respectively. It is disclosed that the amount of oxygen can be maintained at a low stirring rate. Patent Document 7 describes that disruption of the BXL1 gene of Trichoderma filamentous fungi can suppress a decrease in the dissolved oxygen saturation of a culture solution.
  • the present inventors have conducted intensive studies to identify the genes of Trichoderma filamentous fungi that enable the viscosity of the culture solution to be kept low. As a result, it was found that the mutation of beta-adaptin ⁇ large ⁇ subunit kept the viscosity of the culture solution low. And found that the decrease in the dissolved oxygen saturation in the culture solution could be suppressed, and completed the present invention.
  • the present invention includes the following (1) to (8).
  • a method for producing a protein comprising a step of culturing the mutant strain of the genus Trichoderma according to any one of (1) to (6).
  • a method for producing cellulase comprising a step of culturing the mutant strain of the genus Trichoderma according to any one of (1) to (6).
  • the mutant strain of the genus Trichoderma of the present invention can maintain the viscosity of the culture solution lower than that of the parent strain before the introduction of the mutation, and can also suppress a decrease in dissolved oxygen saturation in the culture solution.
  • an unexpected effect of increasing the production of proteins, particularly cellulase, is also obtained.
  • the present invention is characterized in that the viscosity of a culture solution can be kept low by introducing a mutation into a parent strain of Trichoderma filamentous fungus, which is a microorganism originally excellent in protein production ability.
  • the parent strain of Trichoderma filamentous fungus used in the present invention is not limited to a wild strain, and a mutant strain of Trichoderma filamentous fungus improved so as to enhance protein production ability can also be preferably used as a parent strain.
  • a mutant strain of Trichoderma filamentous fungus is subjected to a mutation treatment with a mutagen or ultraviolet irradiation, and a mutant strain having improved protein productivity can be used as the parent strain.
  • the parent strain include Trichoderma parareesei (ATCC MYA-4777), which is an ancestor of Trichoderma reesei, QM6a strains (NBRC31326), QM4123N, and QM9123N (QM4123N) strains which are known mutant strains belonging to Trichoderma reesei.
  • PC-3-7 strain ATCC 66589
  • QM9123 strain NBRC31327)
  • RutC-30 strain ATCC 56765
  • CL-847 strain Enzyme. Microbiol. Technol. 10, 341-346 (1988)
  • MCG77 strain Biotechnol. Bioeng. Symp. 8, # 89 (1978)
  • MCG80 strain Biotechnol.
  • Trichoderma citrinoviride (ATCC24961), Trichoderma longibrachiatum (ATCC18648), Trichoderma virens (ATCC9645), Trichoderma atroviride (ATCC20476), Trichoderma gamsii (NFCCI2177), Trichoderma asperellum (ATCC52438), Trichoderma harzianum (ATCC20846), Trichoderma @ guizhouense and the like.
  • Beta-adaptin large subunit is one of the proteins constituting an adapter protein complex composed of a tetramer. Adapter protein complexes are widely conserved in eukaryotes. It is known that the adapter protein binds to clathrin and forms vesicles involved in intracellular and extracellular transport (Proc. Nati. Acad. Sci. USA. 101, 14108-14113 (2004)). )).
  • beta-adaptin ⁇ large ⁇ subunits possessed by the filamentous fungi belonging to Trichoderma are preferably polypeptides having an amino acid sequence represented by any of SEQ ID NOs: 2 to 10.
  • the amino acid sequence represented by SEQ ID NO: 2 is derived from Trichoderma reesei
  • the amino acid sequence represented by SEQ ID NO: 3 is derived from Trichoderma citrinoviride
  • the amino acid sequence represented by SEQ ID NO: 4 is derived from Trichoderma longibrachiatum.
  • the amino acid sequence represented by SEQ ID NO: 5 is derived from Trichoderma m virens
  • the amino acid sequence represented by SEQ ID NO: 6 is derived from Trichoderma atroviride
  • the amino acid sequence represented by SEQ ID NO: 7 is derived from Trichoderma gamsii.
  • the amino acid sequence represented by SEQ ID NO: 8 is derived from Trichoderma asperellum and has the amino acid sequence represented by SEQ ID NO: 9. It is derived from Trichoderma harzianum
  • the amino acid sequence shown in SEQ ID NO: 10 is derived from a Trichoderma guizhouense.
  • the alignment results of the amino acid sequences represented by SEQ ID NOs: 2 to 10 are shown in FIGS. 1-1 and 1-2.
  • SEQ ID NOs: 2 to 10 As shown in FIGS. 1-1 and 1-2, the sequence identities of SEQ ID NOs: 2 to 10 are 90% or more, and beta-adaptin @ large ⁇ subunit has high amino acid sequence conservation in Trichoderma filamentous fungi. Understand.
  • a glutamine residue is commonly conserved at the 300th amino acid residue from the N-terminal side of the amino acid sequences represented by SEQ ID NOS: 2 to 10.
  • SEQ ID NO: 2 as an example.
  • the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 is a polypeptide belonging to Trichoderma reesei as described above, and according to National Center Center for Biotechnology Information, the adapter adapter-aprox-protein of the strain Trichoderma reesei QM6a (QM6a) is provided. It is registered as adaptin ⁇ large ⁇ subunit (EGR48910). Also, according to the National Center for Biotechnology Information, Censused Domain, Architecture, Retriever, and Tool, the 14th to 531st amino acid residues from the N-terminal side are disclosed as an Adaptin N Terminal domain.
  • having a mutation in the amino acid sequence constituting beta-adaptin large subunit may be any of amino acid deletion, substitution, or addition.
  • the glutamine residue at the 300th position from the N-terminal side of the amino acid sequence represented by any of SEQ ID NOs: 2 to 10 is mutated to an amino acid residue other than glutamine, and particularly preferably lysine. Preferably, it is mutated.
  • a specific example of a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 includes the base sequence represented by SEQ ID NO: 1.
  • a specific example of a base sequence encoding an amino acid sequence in which the glutamine residue at the 300th position from the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 is mutated to an amino acid residue other than glutamine is represented by SEQ ID NO: 1.
  • SEQ ID NO: 1 A specific example of a base sequence encoding an amino acid sequence in which the glutamine residue at the 300th position from the N-terminal side of the amino acid sequence represented by SEQ ID NO: 2 is mutated to an amino acid residue other than glutamine is represented by SEQ ID NO: 1.
  • the mutant strain of Trichoderma filamentous fungus of the present invention may be a mutant strain in which the function of beta-adaptin large subunit is deficient or reduced.
  • the loss of the function of beta-adaptin large subunit refers to the loss of all, part of the polypeptide, all of the polypeptide being changed to a different amino acid, part of the polypeptide being changed to a different amino acid, or a combination thereof. More specifically, it indicates that the amino acid sequence represented by SEQ ID NO: 2 has a sequence identity of 80% or less, and preferably 50% or less, with the amino acid sequence of beta-adaptin @ large ⁇ subunit shown above. , More preferably 20% or less, further preferably 10% or less, further preferably 5% or less, further preferably 3% or less, further preferably 1% or less, and most preferably 0% or less. %.
  • Decreased function of beta-adaptin ⁇ large ⁇ subunit refers to all or a partial deletion of beta-adaptin ⁇ large ⁇ subunit, and also introduces a mutation that reduces or reduces the expression level of beta-adaptin ⁇ large ⁇ subunit. This can also reduce the function of beta-adaptin ⁇ large ⁇ subunit.
  • the expression level of beta-adaptin ⁇ large ⁇ subunit is reduced or eliminated by mutation of the promoter or terminator region of the gene encoding beta-adaptin ⁇ large ⁇ subunit.
  • a promoter and a terminator region correspond to a region of several hundred bases before and after a gene involved in transcription.
  • Mutations in which the function of beta-adaptin @ large ⁇ subunit is deficient or reduced, or methods for introducing mutations into the amino acid sequence constituting beta-adaptin ⁇ large ⁇ subunit, can be carried out by mutation treatment using a mutagen or ultraviolet irradiation known to those skilled in the art, a selection marker.
  • Known gene mutation methods such as gene recombination such as homologous recombination using, and mutation by a transposon can be used.
  • the mutant strain of Trichoderma filamentous fungus of the present invention has a reduced viscosity of the culture solution and can also suppress a decrease in the dissolved oxygen saturation in the culture solution, as compared with the parent strain before the introduction of the mutation. Thereby, the energy required for aeration stirring and the number of rotations can be reduced. In addition, since the rotation speed of the stirring can be set low, the shear damage to the mycelium can be reduced. In particular, in the case of culturing on a large scale, it is more effective because it reduces the capacity of the blower and the stirring motor required for aeration and the stirring energy.
  • the viscosity of the culture solution is a value measured under the following conditions, and the viscosity is compared by comparing the maximum values among the values measured under the following conditions.
  • the spores of the mutant strain of the genus Trichoderma and the parent strain to be evaluated were cultured in a preculture medium such that the spores of the parent strain became 1.0 ⁇ 10 5 spores per mL of the preculture medium (an example of a specific medium composition is described in Examples. As shown in Table 1) and cultured in a shaking incubator under the conditions of 28 ° C. and 120 rpm until the amount of cells becomes about 11 g / L.
  • the preculture medium was adjusted to 10% (v / v) with respect to the main culture medium shown in Table 2 to which Arbocel B800 (manufactured by Rettenmeier) was added so as to be 100 g / L (w / v). Inoculate and submerge using a 5 L jar fermenter.
  • the culture conditions after inoculating the preculture medium into the main culture medium, deep culture is performed under the culture conditions of 28 ° C., 700 rpm, and aeration rate of 100 mL / min while controlling the pH to 5.0.
  • the viscosity of the culture solution is measured using a digital rotational viscometer.
  • the digital rotational viscometer performs zero point calibration in advance.
  • the mutant strain of Trichoderma filamentous fungus of the present invention has a lower viscosity of the culture solution than the parent strain when the parent strain before the mutagenesis is cultured under the same conditions, and the viscosity of the mutant during the culture is lower than that of the parent strain.
  • the dissolved oxygen saturation in the culture solution can be calculated by measuring the oxygen utilization rate in the culture solution.
  • the oxygen utilization rate (mM / L / hr) in the present invention refers to the oxygen consumption rate per 1 L of the culture solution per unit time 24 hours after the start of the culture.
  • the culture is performed while maintaining the culture conditions constant, the supply of oxygen is stopped at 24 hours after the start of the culture, and the dissolved oxygen (mg / L) value (DO value) is plotted every 10 seconds. Then, the slope (A) (unit: DO / sec) is obtained for plots of three or more points that logarithmically decrease in the curve.
  • the following equation 1 is used to calculate the oxygen utilization rate.
  • Oxygen utilization rate (mM / L / hr) ( ⁇ A) ⁇ (1/32) ⁇ 60 ⁇ 60 (Equation 1).
  • DO meter can be used for measuring the DO value.
  • DO meter there is no particular limitation on the DO meter used, as long as the DO value can be measured accurately. Examples include a sealed DO electrode (manufactured by Able Corporation) and a dissolved oxygen sensor (manufactured by METTLER TOLEDO).
  • the DO meter performs zero point calibration and span calibration in advance. Zero-point calibration is performed using a 2% sodium sulfite solution. In the span calibration, aeration and agitation are performed in the absence of bacterial cells under the conditions for actual culturing, wait until the dissolved oxygen becomes saturated, and then confirm that the indicated value of the instrument is stable. Calibrate according to the saturated dissolved oxygen.
  • DO measurement is performed by pressurizing the culture tank, it is necessary to perform pressure correction. Further, when the culture tank is large, it is necessary to perform hydrostatic pressure correction. When performing the correction, it is calculated using the following equation 2.
  • Dissolved oxygen saturation was measured during the culture period for saturated dissolved oxygen when the pH and temperature were set to culture conditions using a culture medium containing no bacteria and the dissolved oxygen saturation when air was aerated was 100%. Is calculated as the dissolved oxygen saturation.
  • Dissolved oxygen (mg / L) indicates the concentration of oxygen dissolved in water.
  • saturated dissolved oxygen refers to dissolved oxygen in a state in which aeration and agitation are performed in the absence of bacterial cells under culture conditions for actually culturing, and the dissolved oxygen is constant.
  • the culture conditions such as aeration conditions are not changed during the culture period. As the oxygen demand decreases, the dissolved oxygen saturation increases. The dissolved oxygen saturation is calculated according to Equation 3 below.
  • Dissolved oxygen saturation (%) (dissolved oxygen during culture) / (saturated dissolved oxygen before culture start) ⁇ 100 (formula 3).
  • the measurement results obtained by adjusting the culture conditions such as the culture medium, the oxygen supply amount, the stirring speed, the temperature, the culture volume, and the inoculated amount.
  • the amount of inoculation at the time of measurement is preferably about 10% (v / v) with respect to the main culture solution.
  • the mutant strain of Trichoderma filamentous fungus of the present invention and the dissolved oxygen of the parent strain before the introduction of the mutation are cultured under similar conditions
  • the mutant strain has a higher minimum dissolved oxygen saturation than the parent strain, and is preferably 5% or more, more preferably 6% or more, more preferably 7% or more, further preferably 8% or more, further preferably 9% or more, further preferably 10% or more, further preferably 11% or more, and still more preferably 12% or more. %, More preferably 13% or more, further preferably 14% or more, particularly preferably 15% or more.
  • the mutant strain of Trichoderma filamentous fungus of the present invention does not decrease in growth ability as compared with the parent strain before the introduction of the mutation.
  • the difference in proliferation ability can be compared by measuring the amount of cells. The cell mass is measured as the dry cell weight. 10 mL of the culture solution is subjected to suction filtration using qualitative filter paper (grade 4, GE Healthcare), and the residue is dried together with the filter paper at 100 ° C. Then, the weight is measured, and the difference between the weights of the filter paper before and after filtration is defined as the dry cell weight.
  • the mutant strain of Trichoderma filamentous fungus of the present invention may be a gene that improves or reduces protein production in addition to a mutation in which the function of beta-adaptin @ large ⁇ subunit is deficient or reduced, or a mutation in the amino acid sequence that constitutes beta-adaptin ⁇ large ⁇ subunit. It may have a mutation. Specific examples include a gene mutation that reduces the function of the polypeptide represented by SEQ ID NO: 11 and / or SEQ ID NO: 13.
  • the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11 is a polypeptide belonging to Trichoderma reesei, and is registered in National Center for Biotechnology Information as a predicated 5050 protein of Trichoderma reesei QM6a strain, which is a registered EG50 strain of QM6a.
  • the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11 is a polypeptide of unknown function, the 95th to 277th amino acids from the N-terminal side from the N-terminus according to the National Center for Biotechnology Information, Cen- sified Domain, Architecture, Retriever, Tool.
  • MIF4G domain ⁇ eukaryotic ⁇ initiation ⁇ factor ⁇ 4G domain
  • MA-3 domains are known to have the function of binding to DNA or RNA (B Ochem.44, 12265-12272 (2005), Mol.Cell.Biol.1,147-156 (2007))
  • the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 11 contains at least DNA and / or Alternatively, it is presumed to have a function of binding to RNA.
  • a specific example of the gene encoding the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 11 includes the base sequence represented by SEQ ID NO: 12.
  • the gene mutation that reduces the function of EGR50654 is defined as a total deletion of the MIF4G domain and / or the MA-3 domain of EGR50654, a partial deletion of the MIF4G domain and / or the MA-3 domain, and a difference between the MIF4G domain and the MA-3 domain.
  • Gene mutations whose steric relationship changes.
  • the function of the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 11 can also be reduced by introducing a mutation that reduces or eliminates the expression level of the polypeptide.
  • the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13 is a polypeptide belonging to Trichoderma reesei, and in National Center for Biotechnology Information, Predicated EG44 which is a registered strain of Trimoderma reesei which is a registered strain of Trimoderma reesei strain QM6a is registered in EG44.
  • the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13 is a polypeptide whose function is unknown, but according to the National Center for Biotechnology Information, Censured Domain, Architecture, Retrieval Tool, the 26th to 499th amino acids from the N-terminal to the 499th amino acid residue.
  • the group is disclosed to have a "Sugar (and other) @Transporter domain.
  • the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13 is capable of transporting sugar at least between the inside and outside of the bacterial body. It is presumed to be involved.
  • a specific example of a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13 includes the base sequence represented by SEQ ID NO: 14.
  • the gene mutation that reduces the function of EGR44419 is defined as a total deletion of the Sugar (and other) @Transporter domain, a partial deletion of the Sugar (and @ other) @Transporter domain, and a change in the configuration of the Sugar (and @ other) @Transporter domain of EGR44419.
  • Gene mutation Furthermore, the function of the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 13 can also be reduced by introducing a mutation that reduces or eliminates the expression level of the polypeptide.
  • a specific example in which the function of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13 is deficient is a mutation in which 11 bases are inserted at the 1415th position in the base sequence represented by SEQ ID NO: 14.
  • the present invention also relates to a method for producing a protein, which comprises a step of culturing a mutant strain of Trichoderma sp. Having a mutation in the amino acid sequence constituting beta-adaptin ⁇ large ⁇ subunit.
  • proteins secreted outside the cells can be efficiently produced.
  • the protein to be produced is not limited, but is preferably an enzyme, more preferably a saccharifying enzyme such as cellulase, amylase, invertase, chitinase, pectinase, and particularly preferably cellulase.
  • the cellulase produced by the present invention contains various hydrolases, including enzymes having a decomposing activity on xylan, cellulose, and hemicellulose.
  • hydrolases including enzymes having a decomposing activity on xylan, cellulose, and hemicellulose.
  • cellobiohydrolase EC 3.2.1.91
  • endoglucanase EC43.2.1.4
  • ⁇ -glucosidase which hydrolyzes cellooligosaccharides and cellobiose
  • xylanase characterized by acting on hemicellulose and particularly xylan
  • xylooligosaccharide ⁇ -xylosidase which hydrolyzes
  • the cellulase protein concentration is measured as follows. A culture solution obtained by culturing Trichoderma filamentous fungi by the method of the present invention is centrifuged at 15,000 ⁇ g for 10 minutes, and the supernatant is used as a cellulase solution. 5 ⁇ L of a cellulase solution diluted with 250 ⁇ L of Quick Start Bradford Protein Assay (manufactured by Bio-Rad) is added, and the absorbance used at 595 nm after standing at room temperature for 15 minutes is measured. Using the bovine serum albumin solution as a standard solution, the concentration of the protein contained in the saccharifying enzyme solution is calculated based on the calibration curve.
  • the culture method for culturing Trichoderma filamentous fungi in the present invention is not particularly limited, for example, a liquid culture using a centrifuge tube, a flask, a jar fermenter, a tank, or the like, or a solid culture using a plate or the like. it can.
  • the filamentous fungus of the genus Trichoderma is preferably cultured under aerobic conditions, and among these culture methods, a jar fermenter or a deep submerged culture in which aeration and stirring are performed in a tank are particularly preferable.
  • the medium composition in the culturing step is not particularly limited as long as the medium composition allows the filamentous fungus of the genus Trichoderma to produce a protein, and a well-known medium composition of the genus Trichoderma can be employed.
  • a well-known medium composition of the genus Trichoderma can be employed.
  • the nitrogen source for example, polypeptone, broth, CSL, soybean meal and the like can be used.
  • an inducer for producing a protein may be added to the medium.
  • the cellulase When producing cellulase according to the present invention, the cellulase can be cultured in a medium containing at least one or two or more inducers selected from the group consisting of lactose, cellulose and xylan.
  • cellulose or xylan may be added with biomass containing cellulose or xylan as an inducer.
  • biomass containing cellulose and xylan include seed plants, fern plants, moss plants, algae, aquatic plants, and other waste materials as well as plants. Seed plants are classified into gymnosperms and angiosperms, both of which can be preferably used.
  • Angiosperms are further classified into monocotyledons and dicotyledons, and specific examples of monocotyledons include bagasse, switchgrass, napier grass, Elianthus, corn stover, corn cob, rice straw, straw, and the like. Specific examples of plants include beet pulp, eucalyptus, oak, birch, and the like.
  • pretreated biomass containing cellulose or xylan may be used.
  • the pretreatment method is not particularly limited, and for example, a known method such as an acid treatment, a sulfuric acid treatment, a dilute sulfuric acid treatment, an alkali treatment, a hydrothermal treatment, a subcritical treatment, a fine pulverizing treatment, and a steaming treatment can be used. Pulp may be used as biomass containing pretreated cellulose and xylan.
  • the method for culturing the mutant strain of Trichoderma filamentous fungus of the present invention is not particularly limited.
  • a liquid culture using a centrifuge tube, a flask, a jar fermenter, a tank, or the like, or a solid culture using a plate or the like may be used.
  • the ventilation rate is preferably about 0.1 to 2.0 vvm, more preferably 0.3 to 1.5 vvm, and particularly preferably 0.5 to 1.0 vvm.
  • the cultivation temperature is preferably about 25 to 35 ° C, more preferably 25 to 31 ° C.
  • the pH condition in the culture is preferably pH 3.0 to 7.0, and more preferably pH 4.0 to 6.0.
  • the culturing time is not particularly limited as long as it can be carried out until a recoverable amount of the protein is accumulated under the conditions for producing the protein, but is usually 24 to 288 hours, preferably 24 to 240 hours, more preferably Is 36 to 240 hours, more preferably 36 to 192 hours.
  • the method of recovering the protein contained in the culture solution in which the mutant strain of Trichoderma filamentous fungus is cultured is not particularly limited, but the protein of Trichoderma filamentous fungus can be removed from the culture solution to recover the protein.
  • Examples of the method for removing cells include a centrifugal separation method, a membrane separation method, a filter press method and the like.
  • the cells of Trichoderma are not allowed to grow in the culture. Processing is preferred. Examples of the method for treating cells so that they cannot grow include heat treatment, chemical treatment, acid / alkali treatment, and UV treatment.
  • the culture solution treated as described above so that the cells are not removed or grown can be used as it is as the enzyme solution.
  • Protein concentration measuring method A protein concentration measuring reagent (Quick Start Bradford protein assay, manufactured by Bio-Rad) was used. 5 ⁇ L of the culture solution of the filamentous fungus diluted to 250 ⁇ L of the protein concentration measurement reagent returned to room temperature was added, and the mixture was allowed to stand at room temperature for 5 minutes, and the absorbance at 595 nm was measured with a microplate reader. BSA was used as a standard, and the protein concentration was calculated according to a calibration curve.
  • Dissolved oxygen saturation was determined by setting the pH and temperature to culture conditions using a culture medium containing no bacteria, and setting the saturated state of dissolved oxygen when air was aerated to 100%. In this case, the ratio of the dissolved oxygen to the saturated dissolved oxygen during the culture period was calculated as the dissolved oxygen saturation.
  • a closed type dissolved oxygen electrode SDOC-12F-L120 manufactured by Able Corporation was used.
  • the culture solution 39, 48, 62, 72, 86, 96, and 111 hours after the start of culture was measured using a digital rotary viscometer DV2T and a spindle.
  • the viscosity (cP) when the rotation speed was set to 0.3 rpm was determined using UL ADAPTOR (manufactured by BROOKFIELD).
  • the culture solution was subjected to suction filtration with filter paper, and the difference between the dry cell weight of the filter paper before and after suction filtration was referred to as the bacterial cell amount. did.
  • Trichoderma reesei QM9414 mutant strain I having reduced function of beta-adaptin large subunit A poly-amino acid sequence comprising amino acid sequence represented by SEQ ID NO: 2 having a mutation in the amino acid sequence constituting beta-adaptin large subunit
  • a DNA fragment comprising the gene sequence represented by SEQ ID NO: 15 was prepared as a DNA fragment containing the gene encoding the peptide, and the DNA fragment was transformed into Trichoderma reesei QM9414 strain, whereby the function of beta-adaptin large subunit was improved. Reduced Trichoderma reesei mutants were generated.
  • a mutant strain of Trichoderma reesei having a polypeptide in which the cytosine at position 1080 in SEQ ID NO: 1 has been changed to adenine and the sequence 300 in SEQ ID NO: 2 has been mutated from glutamine to lysine.
  • Acetamide and an acetamidase (AmdS) gene (amdS) capable of degrading acetamide were used as selection markers for DNA fragment introduction.
  • mutagenesis was performed so as to add a portion homologous to the gene sequence of Trichoderma reesei QM9414 strain.
  • a plasmid for use was prepared.
  • a DNA fragment obtained by treating the synthesized DNA fragment represented by SEQ ID NO: 16 with restriction enzymes KpnI and NotI was used as an upstream DNA fragment.
  • PCR was performed using genomic DNA extracted from Trichoderma reesei QM9414 strain according to a conventional method and oligo DNAs represented by SEQ ID NOs: 17 and 18, and the resulting amplified fragment was subjected to a downstream DNA fragment treated with restriction enzymes MluI and SpeI. DNA fragments were used, and the upstream and downstream DNA fragments were introduced into the plasmid into which amdS was inserted using KpnI and NotI and MluI and SpeI restriction enzymes, respectively, to construct a plasmid for mutation introduction.
  • the plasmid for mutagenesis was treated with restriction enzymes ApaI and AscI, and the obtained DNA fragment represented by SEQ ID NO: 15 was used to transform Trichoderma rereei strain QM9414 (NBRC31329).
  • the obtained mutant was used as QM9414 mutant I in the following experiments.
  • Example 2 Production test of protein using QM9414 mutant I (preculture)
  • the spores of the QM9414 mutant strain I prepared in Example 1 were diluted with physiological saline to a concentration of 1.0 ⁇ 10 7 / mL, and 2.5 mL of the diluted spore solution was placed in a 1-L baffled flask shown in Table 1.
  • the cells were inoculated into the 250 mL of the preculture medium, and cultured in a shaking incubator at 28 ° C. and 120 rpm for 72 hours.
  • Arbocel B800 (Main culture) Arbocel B800 (Rettenmeier) was added to the main culture medium shown in Table 2, and deep culture was examined using a 5 L jar fermenter (manufactured by Biot). 250 mL of a preculture of Trichoderma reesei QM9414 strain and the QM9414 mutant I prepared in Example 1 were inoculated into 2.5 L of a main culture medium supplemented with Arbocel B800. The culture conditions were as follows: after inoculation of the preculture medium into the main culture medium, deep culture was performed under the culture conditions of 28 ° C., 700 rpm, aeration rate of 100 mL / min, while controlling the pH to 5.0.
  • Example 3 Preparation of Trichoderma reesei QM9414 Mutant II with Reduced Function of beta-adaptin large Subunit Mutant of Trichoderma reesei, whose sequence number is 19 from the sequence number of the Trichoderma reesei with the reduced sequence of trichoderma reesei, has a reduced function of beta-adaptin large subunit.
  • a DNA fragment was prepared by transforming the DNA fragment into Trichoderma reesei strain QM9414. According to this method, a mutant strain of Trichoderma reesei in which amdS is inserted between positions 791 and 792 in SEQ ID NO: 1 and the function of beta-adaptin large subunit is reduced is obtained.
  • a plasmid for mutagenesis was prepared so that a portion homologous to the gene sequence of Trichoderma reesei QM9414 strain was added upstream and downstream of the DNA fragment sequence containing amdS. did.
  • PCR was performed using genomic DNA extracted from Trichoderma rereei strain QM9414 according to a standard method and oligo DNAs represented by SEQ ID NOS: 20 and 21, and the resulting amplified fragment was treated with restriction enzymes AflII and NotI. Was used as the upstream fragment.
  • PCR was performed using genomic DNA and the oligo DNAs represented by SEQ ID NOs: 22 and 23. The obtained amplified fragment was treated with restriction enzymes MluI and SpeI as a downstream fragment, and the upstream and downstream DNA fragments were designated as AflII.
  • Example 4 Production test of protein using QM9414 mutant strain II Culture was performed under the same operation and conditions as in Example 2 except that QM9414 mutant strain II was used instead of QM9414 mutant strain I prepared in Example 1. The concentration of the protein contained in the culture solution, the dissolved oxygen saturation in the culture solution, and the viscosity of the culture solution were measured.

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CN116813728B (zh) * 2023-07-07 2024-08-09 山东大学 一种真菌木质纤维素降解酶合成调控蛋白突变体及其应用

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