WO2015079868A1 - Lignocellulose-degradant-resistant microorganism and method for producing same, and method for producing bioethanol using said microorganism - Google Patents

Lignocellulose-degradant-resistant microorganism and method for producing same, and method for producing bioethanol using said microorganism Download PDF

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WO2015079868A1
WO2015079868A1 PCT/JP2014/079090 JP2014079090W WO2015079868A1 WO 2015079868 A1 WO2015079868 A1 WO 2015079868A1 JP 2014079090 W JP2014079090 W JP 2014079090W WO 2015079868 A1 WO2015079868 A1 WO 2015079868A1
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seq
amino acid
microorganism
yeast
protein
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French (fr)
Japanese (ja)
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浩志 北垣
林　信行
ニロシャン ジャヤコディ ラヒル
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国立大学法人佐賀大学
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    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • 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
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a microorganism having resistance to a lignocellulose degradation product and a method for producing the same.
  • the present invention also relates to a method for producing an ethanol raw material or ethanol from lignocellulosic biomass using lignocellulose degradation product-resistant microorganisms.
  • a specific gene of yeast is highly expressed.
  • lignocellulosic biomass such as wood and herbs
  • lignocellulosic biomass contains lignocellulose, which is a structure in which cellulose, hemirose, and lignin are tightly bound.
  • lignocellulose is a structure in which cellulose, hemirose, and lignin are tightly bound.
  • the degradation products of lignocellulose contain fermentation inhibitors such as furfural, HMF, glycolaldehyde, and acetic acid, and the presence of these degradation products has caused a reduction in fermentation efficiency.
  • Patent Document 1 As a method for fermenting such lignocellulose, for example, in Patent Document 1, focusing on the process of the hydrolysis process of lignocellulose, a conventionally proposed method using a chemical such as an acid, As a technique for solving problems such as mechanical processing and a method of using high-temperature and high-pressure water, a method for producing ethanol characterized by setting a vapor pressure or the like in a hydrothermal treatment step to a predetermined value is disclosed. Yes.
  • Patent Document 2 discloses a method for introducing a gene related to a mannose metabolic enzyme derived from hemicellulose into a microorganism to be transformed for efficient production of ethanol for fuel using cellulosic biomass as a raw material. Is disclosed.
  • Patent Document 3 and Non-Patent Document 1 disclose research focusing on the expression of ADH1 as a method for improving the resistance of yeast to cellulose and glycolaldehyde subjected to pressurized hot water treatment.
  • lignocellulosic biomass contains lignocellulose.
  • a degradation product of lignocellulose was produced, and this lignocellulose degradation product had a problem of inhibiting the growth of microorganisms used for fermentation. Therefore, for example, as in Patent Document 2, even if a gene related to a metabolic enzyme is introduced, growth inhibition of the microorganism itself cannot be sufficiently prevented, and sufficient fermentation may not be performed.
  • Patent Document 3 and Non-Patent Document 1 are intended to improve the tolerance of yeast by focusing on redox by ADH1 gene products, but it is possible to impart resistance to microorganisms such as yeast by a different method. If possible, it is expected that microorganisms such as yeast having higher fermentability can be obtained.
  • the present invention provides a microorganism (lignocellulose degradation product resistant microorganism) having resistance (particularly resistance to complex stress) to lignocellulose degradation products by an approach different from Patent Documents 2 and 3 and Non-Patent Document 1.
  • the task is to do.
  • Another object of the present invention is to provide a method for transforming a microorganism so as to have resistance to a lignocellulose degradation product.
  • Such a lignocellulose degradation product-resistant microorganism can make a resource recycling process such as producing ethanol from lignocellulosic biomass more efficient.
  • ⁇ 1> A lignocellulose degradation product-resistant microorganism in which a gene encoding the following protein (1) or (2) is highly expressed as compared to the wild type.
  • (1) At least one protein selected from proteins consisting of the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 (2) 1 or 2 in the amino acid sequences represented by SEQ ID NOs: 1 to 4 Several amino acids are deleted, substituted, or added, consisting of an amino acid sequence modified within the range of 80% or more identity to the corresponding amino acid sequence of SEQ ID NO., And resistance to lignocellulose degradation products to microorganisms
  • At least one or more proteins selected from the group consisting of proteins having an imparting function ⁇ 2> By including a recombinant vector containing a gene encoding the protein of (1) or (2), the gene is highly expressed The lignocellulose degradation product-resistant microorganism according to ⁇ 1>.
  • ⁇ 3> The above ⁇ 1> or ⁇ 2 wherein the microorganism is at least one microorganism selected from the genus Saccharomyces, Schizosaccharomyces, Candida, Cluyveromyces, Trichosporon, and Schwaniomyces > The microorganism described.
  • ⁇ 4> The microorganism according to ⁇ 1> or ⁇ 2>, wherein the microorganism is yeast.
  • ⁇ 5> A recombinant vector containing a gene encoding the following protein (1) or (2).
  • Several amino acids are deleted, substituted, or added, consisting of an amino acid sequence modified within the range of 80% or more identity to the corresponding amino acid sequence of SEQ ID NO., And resistance to lignocellulose degradation products to microorganisms
  • a recombinant vector containing a gene encoding the following protein (1) or (2) is introduced into a microorganism: A method for producing a lignocellulose degradation product-resistant microorganism.
  • Several amino acids are deleted, substituted, or added, consisting of an amino acid sequence modified within the range of 80% or more identity to the corresponding amino acid sequence of SEQ ID NO., And resistance to lignocellulose degradation products to microorganisms
  • the yeast is compared with the wild type as follows: A yeast characterized in that the gene encoding the protein of (1) or (2) is highly expressed.
  • the microorganisms By transforming microorganisms according to the present invention, the microorganisms can be given resistance to lignocellulose degradation products. Thereby, the transformed microorganism can maintain its own fermentation performance even in the presence of a lignocellulose degradation product. In addition, by performing fermentation using the transformed microorganism, fermentation and the like that were conventionally difficult to perform industrially can be easily performed.
  • the present invention relates to a lignocellulose degradation product-resistant microorganism in which a gene encoding the following protein (1) or (2) is highly expressed as compared to the wild type. These microorganisms have excellent resistance to lignocellulose degradation products due to high expression of these genes.
  • the present invention also relates to a method for producing a lignocellulose degradation product-resistant microorganism, characterized in that a recombinant vector containing a gene encoding the following protein (1) or (2) is introduced into the microorganism. It can also be achieved as a method for producing a lignocellulose degradation product resistant microorganism. By this method, resistance to lignocellulose degradation products can be arbitrarily imparted to the microorganism, and in particular, it can be transformed into a microorganism suitable for fermentation of lignocellulosic biomass, which is a biomass containing lignocellulose.
  • At least one or more proteins selected from the protein consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 (2) One or the number in the amino acid sequence represented by SEQ ID NOs: 1 to 4 Consisting of an amino acid sequence in which one amino acid has been deleted, substituted or added, and has been modified within a range of identity of 80% or more with respect to the amino acid sequence represented by the corresponding SEQ ID NO., And lignocellulose degradation to microorganisms At least one protein selected from the group consisting of proteins having physical resistance imparting function
  • the present invention adjusts the expression so that a gene encoding at least one protein selected from the group consisting of the proteins represented by SEQ ID NOs: 1 to 4 in yeast is highly expressed. It is characterized by that.
  • the proteins represented by SEQ ID NOs: 1 to 4 are all SUMO (Small Ubiquitin-related Modifier) proteins or proteins related to SUMOization.
  • the SUMO protein is also called a ubiquitin-like protein and is a protein having a relatively small molecular weight whose function has been elucidated in recent years.
  • One reason for the inhibition of fermentation is that proteins that contribute to the fermentation pathway are denatured and remain in the fermentation environment by fermentation inhibitors such as lignocellulose degradation products.
  • the protein represented by the SEQ ID NO of the present invention contributes to the rapid degradation of the denatured protein by enhancing the sumoylation of the protein denatured by the fermentation inhibitor.
  • the microorganism of the present invention accelerates the degradation of the denatured protein, thereby quickly removing the influence of fermentation inhibition by the fermentation inhibitor itself and the denatured protein, and shortening the growth induction period of the microorganism in the presence of the fermentation inhibitor. It is thought that there is an effect.
  • the protein represented by SEQ ID NO: 1, the protein represented by SEQ ID NO: 2, the protein represented by SEQ ID NO: 3, and the protein represented by SEQ ID NO: 4 are selected from the group consisting of four proteins.
  • a gene encoding at least one protein is used. That is, a gene encoding any one of the four proteins represented by SEQ ID NOs: 1 to 4, or any two or more proteins can be used.
  • certain lignocellulose resistance can be imparted, and by introducing these in combination, superior lignocellulose resistance can be imparted. Most preferably, four genes are used.
  • the protein represented by SEQ ID NO: 1 used in the present invention is a SUMO protein also called yeast SMT3.
  • yeast SMT3 a SUMO protein also called yeast SMT3.
  • the gene encoding this protein include the gene of yeast Saccharomyces cerevisiae shown in SEQ ID NO: 5.
  • the protein encoded by the gene of the present invention further has one or several amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NOs: 1 to 4, and an amino acid sequence represented by the corresponding SEQ ID NO: 80 % Or more, preferably 90% or more, more preferably 95% or more, particularly preferably 98% or more of a gene encoding an amino acid sequence and having a function of imparting resistance to lignocellulose degradation products to microorganisms Is included.
  • it also includes those that provide transformation of resistance to lignocellulosic degradation products of microorganisms by having similar amino acid sequences.
  • the identity with the amino acid sequence shown in the corresponding SEQ ID NO is the identity with the amino acid sequence with the closest identity among the amino acids of SEQ ID NOS: 1-4. That is, in the case of an amino acid sequence having high identity with SEQ ID NO: 1, a comparison is made with SEQ ID NO: 1. Similarly, when identity with SEQ ID NO: 2 is high, SEQ ID NO: 2 and SEQ ID NO: When the identity with 3 is high, SEQ ID NO: 3 is compared with SEQ ID NO: 4 when the identity with SEQ ID NO: 4 is high.
  • BLAST Basic Local Alignment Search Tool
  • amino acid sequences represented by SEQ ID NOs: 1 to 4 one or several amino acids are deleted, substituted or added, and the amino acid sequence represented by the corresponding SEQ ID NOs has 80% or more identity.
  • a protein having an amino acid sequence modified within the range and having a function of imparting resistance to lignocellulose degradation products to microorganisms it is preferable to use a combination of two or more of the proteins corresponding to SEQ ID NOs: 1 to 4. It is preferable to use four types in combination.
  • SEQ ID NO: 1 can be used as the protein
  • SEQ ID NO: 2 can be combined with a protein that satisfies the requirements such as identity described in (2) corresponding thereto.
  • the protein of SEQ ID NO: 2 is a protein also called yeast SLX5
  • the protein of SEQ ID NO: 3 is a protein also called yeast SLX8
  • the protein of SEQ ID NO: 4 is a protein also called UBC9.
  • genes encoding these proteins include the yeast Saccharomyces cerevisiae genes shown in SEQ ID NO: 6 (SLX5), SEQ ID NO: 7 (SLX8), and SEQ ID NO: 8 (UBC9).
  • the present invention is characterized in that it is a lignocellulose degradation product-resistant microorganism in which the gene encoding the protein (1) or (2) described above is highly expressed as compared to the wild type.
  • the method for highly expressing these genes is not particularly limited, and various methods capable of highly expressing the gene targeted by the present invention are used by a technique proposed as a so-called genome editing technique. be able to. For example, a method of additionally introducing the gene so as to highly express the gene, a method of exchanging the promoter of these genes with a promoter capable of highly expressing the gene, and the like can be mentioned.
  • a typical method for introducing a gene is to use a base sequence having a base sequence portion having a high homology with a base sequence portion of a part of a chromosome of a microorganism in which the gene is highly expressed.
  • transduces is mentioned. This is because, for example, a linear DNA fragment is prepared by PCR, and at that time, a sequence having high homology with a chromosome of a wild-type target microorganism of about 50 bp is provided at both ends and brought into contact with the target microorganism.
  • a highly homologous sequence is automatically incorporated, and the chromosomal region of the wild-type target microorganism is replaced.
  • a plurality of genes targeted by the present invention can be introduced into a linear DNA fragment so that the gene is highly expressed.
  • a method of introducing a recombinant vector can also be employed. That is, the present invention may be abbreviated as the protein represented by the above (1) or (2) (hereinafter referred to as “protein used in the present invention”) like the proteins represented by SEQ ID NOS: 1-4. And a recombinant vector containing a gene encoding), and introduced into a microorganism to be transformed.
  • this gene is not only a gene encoding the protein represented by SEQ ID NO: 1, for example, but also a gene having a certain identity and the like and having a function of imparting resistance to lignocellulose degradation products. You can also.
  • a vector used for yeast when a transformation target is yeast, a vector used for yeast can be used, and more specifically, a protein expression type vector or the like can be used.
  • a recombinant vector suitable for the microorganism to be transformed may be used as appropriate.
  • the recombinant vector can be introduced by, for example, an electroporation method, a spheroblast method, a lithium acetate method, a method using calcium ions, a protoplast method, or the like.
  • Expression vectors include plasmids, viruses, cosmids and the like.
  • the vector is a prokaryotic or eukaryotic vector, such as pMAL, pTYB vector (Daiichi Kagaku), pAUR123 (Takara Bio), pET system, pBAC system, pTri-Ex1 vector (pTri-Ex1), Examples include pCruz TM vectors (Cosmo Bio).
  • the vector used in the present invention includes, in addition to the gene sequence according to the present invention, an expression control sequence such as a promoter, enhancer, terminator, start codon, splice signal, stop codon, replication origin, selectable marker, polylin Cars can be included.
  • an expression control sequence such as a promoter, enhancer, terminator, start codon, splice signal, stop codon, replication origin, selectable marker, polylin Cars can be included.
  • Promoters include constitutive promoters or inducible promoters. When cloning in a microbial system, inducible promoters such as bacteriophage ⁇ pL and lac are exemplified.
  • Selection markers include drug resistance markers such as ampicillin, kanamycin, neomycin, teracycline, chloramphenicol, G418, hygromycin, methotrexate, and other auxotrophic markers such as URA3.
  • the present invention provides an expression vector containing the above specific gene, and a host cell (microorganism) in which the specific gene is highly expressed by the expression vector or the like as compared with the wild type.
  • the host cell (microorganism) that is the subject of the present invention is a microorganism that exhibits resistance to lignocellulose degradation products by having a gene related to SUMO and the protein used in the present invention that is involved in sumoylation.
  • Examples of the microorganism of the present invention include eukaryotic cells and yeast (for example, Saccharomyces cerevisiae).
  • this microorganism for example, yeast that is a microorganism that ferments lignocellulosic biomass is targeted, and transformation to impart lignocellulose degradation product resistance to these microorganisms is performed.
  • a fungus that performs ethanol fermentation can be selected.
  • yeast is a microorganism that performs typical ethanol fermentation.
  • Ethanol is used for various industrial uses (especially fuel as bioethanol), and further technological development is expected in the future. According to the invention, it is a preferred microorganism as a target for imparting resistance to lignocellulose degradation products.
  • examples of the genus of the microorganism that serves as a host cell include at least one microorganism selected from the genus Saccharomyces, Schizosaccharomyces, Candida, Kluyveromyces, Trichosporon, and Schwaniomyces can do.
  • a plurality of microorganisms and the like used simultaneously in fermentation can be transformed and used.
  • the microorganism that is the host cell is yeast.
  • Yeast is a general term for fungi and fungi that grow in a round shape.
  • it can be set as the yeast used as the object of this invention also including what is called incomplete yeast which has the phase which grows in a yeast type
  • yeast used for the production of transformed yeast is the genus Saccharomyces, the genus Schizosaccharomyces, the genus Candida, It can be at least one yeast selected from the genus Kluyveromyces, Trichosporon, and Schwanniomyces.
  • Saccharomyces cerevisae Saccharomyces pasteurianus
  • Saccharomyces bayanus Saccharomyces bayanus
  • Schizospomata brevices Schizospoglabrace
  • Kluyveromyces lactis Trichosporon pullulans
  • Schwanniomyces alluvius examples include Kluyveromyces lactis, Trichosporon pullulans, and Schwanniomyces alluvius.
  • the gene encoding the amino acid sequence represented by SEQ ID NO: 1 used in the present invention is known as the SMT3 gene of yeast, and yeasts originally having this gene also exist.
  • yeasts having the genes of SLX5, SLX8, and UBC9 corresponding to the proteins of SEQ ID NOs: 2 to 4.
  • the yeast is subject to growth inhibition by lignocellulose degradation products, as is the case with conventional yeasts.
  • this gene by introducing this gene into yeast using a recombinant vector, this gene can be expressed at a higher level than usual and a yeast having excellent lignocellulose degradation product resistance can be obtained.
  • the present invention provides microorganisms useful for fermentation that uses lignocellulosic biomass as resources. Moreover, fermentation resources are obtained using such microorganisms. That is, the present invention is characterized in that in the method for fermenting lignocellulosic biomass, the microorganism for performing the fermentation is a microorganism containing a recombinant vector containing a gene encoding the protein represented by SEQ ID NO: 1. And the fermentation method. In particular, in the method for producing bioethanol from lignocellulosic biomass using yeast, the yeast encoded the above-described gene encoding the protein (1) or (2) higher than the wild type. It can be set as the method of manufacturing bioethanol characterized by being yeast.
  • lignocellulosic biomass means biomass (living organic resources) that has lignocellulose mainly composed of cellulose, hemicellulose, and lignin, including woody and herbaceous plants. It is a concept to include. Specific examples include wood, rice straw, wheat straw, bagasse, bamboo, pulp, corn stover, rice husk, palm palm residue, cassava residue, hemp and the like.
  • the degradation product of lignocellulose contains furfural, HMF, glycol aldehyde, acetic acid and the like. These substances inhibit the growth of many microorganisms, and the fermentation efficiency decreases due to the inhibition of the growth of microorganisms. That is, the lignocellulose decomposition product was a fermentation inhibitor.
  • the microorganism transformed by the present invention has resistance to this lignocellulose degradation product. That is, the microorganism obtained by the present invention is a microorganism that can sufficiently grow even in the presence of the lignocellulose degradation product as described above. Thereby, the original fermentation by those microorganisms is sufficiently performed.
  • SC standard medium SC medium containing 2% glucose was used.
  • lignocellulose degradation product-containing medium As a fermentation inhibitor, glucose aldehyde, hydroxymethylfurfural (HMF), furfural, methylglyoxal, and acetic acid, which are also contained in the lignocellulose degradation product, were used. A mixture of these fermentation inhibitor substances so as to have a concentration of 10 mmol / L in an SC standard medium was used as a lignocellulose degradation medium-containing medium.
  • CSM medium CSM medium (2% (w / v) glucose (Wako, 041-00595), 790 mg / l of a complete supplement mixture (CSM complete) medium (Formedium, DCS0019), and a 0.67% (w / v) yeast nitrogen Base without amino acids and ammonium sulphate (Becton Dickinson and Company, 233520) was used as one of the media, and the CSM media was adjusted to pH 6.5 using NaOH (192-13763 made by Wako) as necessary. Adjusted as follows.
  • Yeast BY4743 Laboratory yeast BY4743 strain was used as “yeast BY4743”.
  • -Yeast BYpADH1 A strain found to show strong glycoaldehyde reduction activity by introducing the ADH1 gene derived from Saccharomyces cerevisiae into the strain BY4743 using the vector RS426 was used as "yeast BYpADH1".
  • ⁇ Preparation of standard yeast preculture solution Inoculate 1.5 ⁇ 10 6 cells of each yeast into 1.5 mL (short test tube) of the aforementioned CSM medium for culture. It was cultured at 30 ° C. for 1 day to obtain a yeast preculture solution.
  • vector (I) Preparation of a vector containing the gene represented by SEQ ID NO: 5 (hereinafter referred to as “vector (I)”)> SMT3fw (CGA GCT CTA GAG GTT AGC CAT GCT GTT TCC ATC A) and SMT3rv (GAA ATT CGC TTA GTT TGT GGC ACG TCG TGA AAG AAT).
  • the fragment containing the gene was taken out and amplified by PCR to obtain a PCR fragment.
  • PCR was performed using “PC320” manufactured by Astech Co., Ltd., and “KOD plus ver2” manufactured by Toyobo Co., Ltd. as the polymerase.
  • Vector (I) By cutting “pAUR123” used as a vector with a restriction enzyme “KpnI”, combining the vector and a PCR fragment with a Clontech Infusion kit, transforming into E. coli, and selecting the gene represented by SEQ ID NO: 5 A containing vector ("Vector (I)") was obtained. Since the gene represented by SEQ ID NO: 5 is a gene encoding the protein represented by SEQ ID NO: 1, this vector (I) is a recombinant vector containing the gene encoding the protein represented by SEQ ID NO: 1. It is. A restriction enzyme map of this vector (I) is shown in FIG.
  • vectors (II) and (III) represented by other SEQ ID NOs>
  • vectors (II) and (III) containing genes with other sequence numbers were prepared. Table 1 shows combinations of the genes included.
  • a plurality of genes were introduced by infusion cloning.
  • yeast BY4743 was transformed using the aforementioned vectors (I) to (III) according to the method of Nature Protocols 2, -31-34 (2007) High-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method.
  • yeast BY4743 + pAUR123 + SMT3 represents a yeast culture solution in which vector (I) is introduced into yeast BY4743.
  • vectors (I) to (III) were appropriately introduced into yeast BYpADH1.
  • yeast BY4743 + pAUR123 represents a yeast culture solution obtained by adding only a vector to yeast.
  • ⁇ Main culture of yeast> In the main culture of yeast, the yeast culture solution was added to the medium transferred to the test tube so that the turbidity (OD600) of the number of yeasts in the medium was 0.1. In addition, in order to adjust the addition amount of this yeast culture solution, the turbidity (OD600) when yeast culture solution was diluted 10 times was measured previously, and the addition amount was determined from this result.
  • a yeast culture solution was added to the medium to obtain a main culture solution, and the turbidity was measured to determine the turbidity immediately after the main culture. This main culture was further subjected to stationary culture at 30 ° C. for main culture.
  • B 100 ⁇ L of the above-described main culture solution was taken out every 3 hours from the start of the culture, and the turbidity was measured.
  • Example 1 Comparative Example 1, Reference Examples 1 and 2
  • a yeast growth test was performed using a combination of the medium shown in Table 2 and the yeast culture solution.
  • the measurement result of turbidity (OD600) in the main culture process of this yeast is shown in FIG.
  • Reference examples 1 and 2 were evaluated in order to evaluate the degree of yeast growth depending on the presence or absence of lignocellulose degradation products. As shown in the reference example of FIG. 2, in the medium containing no lignocellulose degradation product, all yeasts show the same growth regardless of the presence or absence of transformation.
  • Examples 2 and 3, Comparative Example 2, Reference Examples 3, 4, and 5 In accordance with the above-described method for main culture of yeast, a yeast growth test was conducted using a combination of the medium shown in Table 3 and the yeast culture solution. The measurement result of turbidity (OD600) in the main culture process of this yeast is shown in FIG. Moreover, the measurement result of the ethanol concentration in the main culture process is shown in FIG.
  • the yeast grows more easily than Comparative Example 2 using the conventional yeast culture solution. There was a significant difference at 36 hours. That is, it can be seen that the growth induction period of yeast in the presence of lignocellulose degradation product can be greatly shortened by the present invention. In addition, it is thought that the value which saturates on the measurement upper limit or main culture conditions shows the same turbidity at the time of 48-hour culture. Moreover, as shown in FIG. 4, the yeast of the comparative example 2 which introduce
  • the present invention it is possible to obtain a microorganism having characteristics suitable for fermentation, such as resistance to lignocellulose degradation products, which becomes a problem during fermentation of lignocellulosic biomass. This improves the utilization efficiency of lignocellulosic biomass and is useful.

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Abstract

　Provided is a microorganism having resistance to a lignocellulose degradant which is a factor that inhibits the fermentation of wood. One embodiment of the present invention is a lignocellulose-degradant-resistant microorganism expressing a higher level of a gene that encodes protein (1) or (2) than does the wild type. A gene that encodes a protein represented by SEQ ID NO: 1 among these genes is a SUMO gene known as an SMT3 gene in yeast. (1) At least one protein selected from the group comprising proteins that comprise amino acid sequences represented by SEQ ID NOS: 1-4; (2) at least one protein selected from the group comprising proteins that comprise amino acid sequences in which one or several amino acids in the amino acid sequences represented by SEQ ID NOS: 1-4 have been deleted, substituted, or added, the amino acid sequences being modified within the range of 80% or higher identity with the amino acid sequence of the corresponding SEQ ID NO., and that have a function for imparting lignocellulose-degradant resistance to microorganisms.

Description

リグノセルロース分解物耐性微生物およびその製造方法、ならびにその微生物を用いてバイオエタノールを製造する方法Lignocellulose degradation product resistant microorganism, method for producing the same, and method for producing bioethanol using the microorganism
　本発明は、リグノセルロース分解物に耐性を有する微生物およびその製造方法に関する。また、リグノセルロース分解物耐性微生物を用いた、リグノセルロース系バイオマスからのエタノール原料やエタノールを製造する方法に関するものである。特に、酵母の特定の遺伝子を高発現させることを特徴とする。 The present invention relates to a microorganism having resistance to a lignocellulose degradation product and a method for producing the same. The present invention also relates to a method for producing an ethanol raw material or ethanol from lignocellulosic biomass using lignocellulose degradation product-resistant microorganisms. In particular, a specific gene of yeast is highly expressed.
　環境負荷を抑えたエネルギー源や、化学品の原料として木材や草本等のリグノセルロース系バイオマスの更なる利用が求められている。リグノセルロース系バイオマスを利用するために、これらの構成成分を単糖に分解し、この単糖を原料としてバイオエタノール等の様々な物質を製造することが検討されている。より具体的に説明すると、リグノセルロース系バイオマスは、セルロースやヘミロース、リグニンが強固に結合した構造であるリグノセルロースを含んでおり、これからエタノールを製造するためには、一旦リグノセルロースを分解した後、酵母等で発酵させる必要がある。しかしながら、リグノセルロースの分解物には、フルフラールや、ＨＭＦ、グリコールアルデヒド、酢酸などの発酵阻害物質が含まれており、これらの分解物の存在は発酵効率が低下する原因となっていた。 There is a demand for further utilization of lignocellulosic biomass such as wood and herbs as an energy source with reduced environmental impact and as a raw material for chemicals. In order to use lignocellulosic biomass, it has been studied to decompose these constituents into monosaccharides and to produce various substances such as bioethanol using the monosaccharides as raw materials. More specifically, lignocellulosic biomass contains lignocellulose, which is a structure in which cellulose, hemirose, and lignin are tightly bound. To produce ethanol from this, after lignocellulose is once decomposed, It is necessary to ferment with yeast. However, the degradation products of lignocellulose contain fermentation inhibitors such as furfural, HMF, glycolaldehyde, and acetic acid, and the presence of these degradation products has caused a reduction in fermentation efficiency.
　このようなリグノセルロースを発酵させる手法として、例えば特許文献１では、特にリグノセルロースの加水分解工程のプロセスに着目し、従来提案されてきた酸などの化学薬品を使う方法や、微粉化のような機械的処理、高温高圧の水を利用する方法等の問題点を解決する手法として、水熱処理工程の蒸気圧等を所定の値とすることなどを特徴とするエタノールを製造する方法が開示されている。 As a method for fermenting such lignocellulose, for example, in Patent Document 1, focusing on the process of the hydrolysis process of lignocellulose, a conventionally proposed method using a chemical such as an acid, As a technique for solving problems such as mechanical processing and a method of using high-temperature and high-pressure water, a method for producing ethanol characterized by setting a vapor pressure or the like in a hydrothermal treatment step to a predetermined value is disclosed. Yes.
　また、特許文献２には、セルロース系バイオマスを原料とする燃料用エタノールの効率的生産に利用するために、形質転換の対象となる微生物にヘミセルロースに由来するマンノース代謝系酵素に関する遺伝子を導入する方法が開示されている。 Patent Document 2 discloses a method for introducing a gene related to a mannose metabolic enzyme derived from hemicellulose into a microorganism to be transformed for efficient production of ethanol for fuel using cellulosic biomass as a raw material. Is disclosed.
　また、特許文献３や非特許文献１には、加圧熱水処理したセルロースおよびグリコールアルデヒドに対する酵母の耐性を向上させる方法として、ＡＤＨ１の発現に着目した研究が開示されている。 Further, Patent Document 3 and Non-Patent Document 1 disclose research focusing on the expression of ADH1 as a method for improving the resistance of yeast to cellulose and glycolaldehyde subjected to pressurized hot water treatment.
特許５２７８９９１号公報Japanese Patent No. 5278991 特開２００６－２４６７８９号公報JP 2006-246789 A 特開２０１２－１５７２７０号公報JP 2012-157270 A
　前述のように、リグノセルロース系バイオマスはリグノセルロースを含有している。このリグノセルロース系バイオマスを発酵させエタノール等を得る工程において、リグノセルロースの分解物が生じ、このリグノセルロース分解物が発酵に用いられる微生物の生育を阻害する問題があった。そのため、例えば特許文献２のように、代謝系酵素に関する遺伝子を導入しても、微生物自体の生育阻害を十分に防止することができず、十分な発酵を行うことができない場合があった。この微生物の生育阻害について、従来、これらの生育阻害物質に対する個別の耐性酵母は馴養等により育種されてきたが、複合的なストレス（これらの阻害物質によるもの）耐性の微生物は育種されてこなかった。また、特許文献３や非特許文献１はＡＤＨ１遺伝子産物による酸化還元に着目することで酵母の耐性を向上させるものであるが、これとは異なる方法で酵母等の微生物に耐性を付与することができれば、より発酵能が高い酵母等の微生物を得ることができることが期待される。 As described above, lignocellulosic biomass contains lignocellulose. In the process of fermenting this lignocellulosic biomass to obtain ethanol and the like, a degradation product of lignocellulose was produced, and this lignocellulose degradation product had a problem of inhibiting the growth of microorganisms used for fermentation. Therefore, for example, as in Patent Document 2, even if a gene related to a metabolic enzyme is introduced, growth inhibition of the microorganism itself cannot be sufficiently prevented, and sufficient fermentation may not be performed. Regarding the growth inhibition of these microorganisms, individual resistant yeasts against these growth inhibitory substances have been bred by acclimatization etc., but microorganisms resistant to complex stress (due to these inhibitors) have not been bred. . Patent Document 3 and Non-Patent Document 1 are intended to improve the tolerance of yeast by focusing on redox by ADH1 gene products, but it is possible to impart resistance to microorganisms such as yeast by a different method. If possible, it is expected that microorganisms such as yeast having higher fermentability can be obtained.
　そこで、本発明は、特許文献２、３や非特許文献１とは異なるアプローチで、リグノセルロース分解物に耐性（特に複合的なストレスに対する耐性）を有する微生物（リグノセルロース分解物耐性微生物）を提供することを課題とする。また、リグノセルロース分解物に耐性を有するように微生物を形質転換させる方法を提供することを課題とする。このようなリグノセルロース分解物耐性微生物によって、リグノセルロース系バイオマスからエタノールを製造するなどの資源化工程をより効率的にすることができる。 Therefore, the present invention provides a microorganism (lignocellulose degradation product resistant microorganism) having resistance (particularly resistance to complex stress) to lignocellulose degradation products by an approach different from Patent Documents 2 and 3 and Non-Patent Document 1. The task is to do. Another object of the present invention is to provide a method for transforming a microorganism so as to have resistance to a lignocellulose degradation product. Such a lignocellulose degradation product-resistant microorganism can make a resource recycling process such as producing ethanol from lignocellulosic biomass more efficient.
　本発明者は、上記課題を解決すべく鋭意研究を重ねた結果、下記の発明が上記目的に合致することを見出し、本発明に至った。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the following inventions meet the above-mentioned object, and have reached the present invention.
　すなわち、本発明は、以下の発明に係るものである。
　＜１＞　　野生型と比較して、以下の（１）または（２）のタンパク質をコードする遺伝子が高発現されたリグノセルロース分解物耐性微生物。
　（１）配列番号１～４のいずれかの配列番号で表されるアミノ酸配列からなるタンパク質から選択される少なくとも１つ以上のタンパク質
　（２）配列番号１～４で表されるアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加され、対応する配列番号のアミノ酸配列に対して８０％以上の同一性の範囲内で修飾されたアミノ酸配列からなり、かつ微生物へのリグノセルロース分解物耐性付与機能を有するタンパク質からなる群から選択される少なくとも１つ以上のタンパク質
　＜２＞　前記（１）または（２）のタンパク質をコードする遺伝子を含有する組み換えベクターを含むことでその遺伝子が高発現された前記＜１＞記載のリグノセルロース分解物耐性微生物。
　＜３＞　前記微生物が、サッカロミセス属、シゾサッカロミセス属、キャンディダ属、クリュイベロミセス属、トリコスポロン属、シュワニオミセス属からから選択される少なくとも１以上の微生物である前記＜１＞または＜２＞記載の微生物。
　＜４＞　前記微生物が、酵母である前記＜１＞または＜２＞記載の微生物。
　＜５＞　以下の（１）または（２）のタンパク質をコードする遺伝子を含有する組み換えベクター。
　（１）配列番号１～４のいずれかの配列番号で表されるアミノ酸配列からなるタンパク質から選択される少なくとも１つ以上のタンパク質
　（２）配列番号１～４で表されるアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加され、対応する配列番号のアミノ酸配列に対して８０％以上の同一性の範囲内で修飾されたアミノ酸配列からなり、かつ微生物へのリグノセルロース分解物耐性付与機能を有するタンパク質からなる群から選択される少なくとも１つ以上のタンパク質
　＜６＞　微生物に、以下の（１）または（２）のタンパク質をコードする遺伝子を含有する組み換えベクターを導入することを特徴とするリグノセルロース分解物耐性微生物を製造する方法。
　（１）配列番号１～４のいずれかの配列番号で表されるアミノ酸配列からなるタンパク質から選択される少なくとも１つ以上のタンパク質
　（２）配列番号１～４で表されるアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加され、対応する配列番号のアミノ酸配列に対して８０％以上の同一性の範囲内で修飾されたアミノ酸配列からなり、かつ微生物へのリグノセルロース分解物耐性付与機能を有するタンパク質からなる群から選択される少なくとも１つ以上のタンパク質
　＜７＞　酵母を用いてリグノセルロース系バイオマスからバイオエタノールを製造する方法において、前記酵母が、野生型と比較して、以下の（１）または（２）のタンパク質をコードする遺伝子が高発現された酵母であることを特徴とするバイオエタノールを製造する方法。
　（１）配列番号１～４のいずれかの配列番号で表されるアミノ酸配列からなるタンパク質から選択される少なくとも１つ以上のタンパク質
　（２）配列番号１～４で表されるアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加され、対応する配列番号のアミノ酸配列に対して８０％以上の同一性の範囲内で修飾されたアミノ酸配列からなり、かつ微生物へのリグノセルロース分解物耐性付与機能を有するタンパク質からなる群から選択される少なくとも１つ以上のタンパク質 That is, the present invention relates to the following inventions.
<1> A lignocellulose degradation product-resistant microorganism in which a gene encoding the following protein (1) or (2) is highly expressed as compared to the wild type.
(1) At least one protein selected from proteins consisting of the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 (2) 1 or 2 in the amino acid sequences represented by SEQ ID NOs: 1 to 4 Several amino acids are deleted, substituted, or added, consisting of an amino acid sequence modified within the range of 80% or more identity to the corresponding amino acid sequence of SEQ ID NO., And resistance to lignocellulose degradation products to microorganisms At least one or more proteins selected from the group consisting of proteins having an imparting function <2> By including a recombinant vector containing a gene encoding the protein of (1) or (2), the gene is highly expressed The lignocellulose degradation product-resistant microorganism according to <1>.
<3> The above <1> or <2 wherein the microorganism is at least one microorganism selected from the genus Saccharomyces, Schizosaccharomyces, Candida, Cluyveromyces, Trichosporon, and Schwaniomyces > The microorganism described.
<4> The microorganism according to <1> or <2>, wherein the microorganism is yeast.
<5> A recombinant vector containing a gene encoding the following protein (1) or (2).
(1) At least one protein selected from proteins consisting of the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 (2) 1 or 2 in the amino acid sequences represented by SEQ ID NOs: 1 to 4 Several amino acids are deleted, substituted, or added, consisting of an amino acid sequence modified within the range of 80% or more identity to the corresponding amino acid sequence of SEQ ID NO., And resistance to lignocellulose degradation products to microorganisms At least one protein selected from the group consisting of proteins having an imparting function <6> A recombinant vector containing a gene encoding the following protein (1) or (2) is introduced into a microorganism: A method for producing a lignocellulose degradation product-resistant microorganism.
(1) At least one protein selected from proteins consisting of the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 (2) 1 or 2 in the amino acid sequences represented by SEQ ID NOs: 1 to 4 Several amino acids are deleted, substituted, or added, consisting of an amino acid sequence modified within the range of 80% or more identity to the corresponding amino acid sequence of SEQ ID NO., And resistance to lignocellulose degradation products to microorganisms At least one protein selected from the group consisting of proteins having an imparting function <7> In the method for producing bioethanol from lignocellulosic biomass using yeast, the yeast is compared with the wild type as follows: A yeast characterized in that the gene encoding the protein of (1) or (2) is highly expressed. A method for producing ethanol.
(1) At least one protein selected from proteins consisting of the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 (2) 1 or 2 in the amino acid sequences represented by SEQ ID NOs: 1 to 4 Several amino acids are deleted, substituted, or added, consisting of an amino acid sequence modified within the range of 80% or more identity to the corresponding amino acid sequence of SEQ ID NO., And resistance to lignocellulose degradation products to microorganisms At least one protein selected from the group consisting of proteins having an imparting function
　本発明によって微生物を形質転換させることで、微生物にリグノセルロース分解物への耐性を付与することができる。これによって、形質転換された微生物はリグノセルロース分解物存在下でも自身の発酵性能を維持することができる。また、この形質転換された微生物を用いて発酵を行うことによって、従来では工業的に行うことが困難だった発酵等を容易に行うことができる。 By transforming microorganisms according to the present invention, the microorganisms can be given resistance to lignocellulose degradation products. Thereby, the transformed microorganism can maintain its own fermentation performance even in the presence of a lignocellulose degradation product. In addition, by performing fermentation using the transformed microorganism, fermentation and the like that were conventionally difficult to perform industrially can be easily performed.
本発明の実施例において製造したベクターの制限酵素地図の一例である。It is an example of the restriction enzyme map of the vector manufactured in the Example of this invention. 本発明の実施例にかかる酵母の培養結果を示す図である。It is a figure which shows the culture result of the yeast concerning the Example of this invention. 本発明の実施例にかかる酵母の培養結果を示す図である。It is a figure which shows the culture result of the yeast concerning the Example of this invention. 本発明の実施例にかかる酵母の培養過程におけるエタノール生産量の経時変化を示す図である。It is a figure which shows the time-dependent change of the ethanol production amount in the culture | cultivation process of the yeast concerning the Example of this invention.
　以下に本発明の実施の形態を詳細に説明するが、以下に記載する構成要件の説明は、本発明の実施態様の一例（代表例）であり、本発明はその要旨を超えない限り、以下の内容に限定されない。 DESCRIPTION OF EMBODIMENTS Embodiments of the present invention will be described in detail below. However, the description of the constituent elements described below is an example (representative example) of an embodiment of the present invention. It is not limited to the contents.
　本発明は、野生型と比較して、以下の（１）または（２）のタンパク質をコードする遺伝子が高発現されたリグノセルロース分解物耐性微生物に関するものである。この微生物は、これらの遺伝子が高発現されることでリグノセルロース分解物に対して優れた耐性を有するものとなる。
　（１）配列番号１～４のいずれかの配列番号で表されるアミノ酸配列からなるタンパク質から選択される少なくとも１つ以上のタンパク質
　（２）配列番号１～４で表されるアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加され、対応する配列番号のアミノ酸配列に対して８０％以上の同一性の範囲内で修飾されたアミノ酸配列からなり、かつ微生物へのリグノセルロース分解物耐性付与機能を有するタンパク質からなる群から選択される少なくとも１つ以上のタンパク質 The present invention relates to a lignocellulose degradation product-resistant microorganism in which a gene encoding the following protein (1) or (2) is highly expressed as compared to the wild type. These microorganisms have excellent resistance to lignocellulose degradation products due to high expression of these genes.
(1) At least one protein selected from proteins consisting of the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 (2) 1 or 2 in the amino acid sequences represented by SEQ ID NOs: 1 to 4 Several amino acids are deleted, substituted, or added, consisting of an amino acid sequence modified within the range of 80% or more identity to the corresponding amino acid sequence of SEQ ID NO., And resistance to lignocellulose degradation products to microorganisms At least one protein selected from the group consisting of proteins having an imparting function
　また、本発明は、リグノセルロース分解物耐性微生物を製造する方法であって、微生物に、以下の（１）または（２）のタンパク質をコードする遺伝子を含有する組み換えベクターを導入することを特徴とするリグノセルロース分解物耐性微生物を製造する方法としても達成することができる。この方法により、微生物にリグノセルロース分解物耐性を任意で付与することができ、特にリグノセルロースを含有するバイオマスであるリグノセルロース系バイオマスの発酵に適した微生物へと形質転換することができる。
　（１）配列番号１～４のいずれかの配列番号で表されるアミノ酸配列からなるタンパク質から選択される少なくとも１以上のタンパク質
　（２）配列番号１～４で表されるアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加され、対応する配列番号で表されるアミノ酸配列に対して８０％以上の同一性の範囲内で修飾されたアミノ酸配列からなり、かつ微生物へのリグノセルロース分解物耐性付与機能を有するタンパク質からなる群から選択される少なくとも１つ以上のタンパク質 The present invention also relates to a method for producing a lignocellulose degradation product-resistant microorganism, characterized in that a recombinant vector containing a gene encoding the following protein (1) or (2) is introduced into the microorganism. It can also be achieved as a method for producing a lignocellulose degradation product resistant microorganism. By this method, resistance to lignocellulose degradation products can be arbitrarily imparted to the microorganism, and in particular, it can be transformed into a microorganism suitable for fermentation of lignocellulosic biomass, which is a biomass containing lignocellulose.
(1) At least one or more proteins selected from the protein consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 (2) One or the number in the amino acid sequence represented by SEQ ID NOs: 1 to 4 Consisting of an amino acid sequence in which one amino acid has been deleted, substituted or added, and has been modified within a range of identity of 80% or more with respect to the amino acid sequence represented by the corresponding SEQ ID NO., And lignocellulose degradation to microorganisms At least one protein selected from the group consisting of proteins having physical resistance imparting function
　本発明は、酵母における配列番号１～４のいずれかの配列番号で表されるタンパク質からなる群から選択される少なくとも１以上のタンパク質をコードする遺伝子が高発現するようにその発現性を調整することを特徴とする。この配列番号１～４で表されるタンパク質は、いずれもＳＵＭＯ（Ｓｍａｌｌ　Ｕｂｉｑｕｉｔｉｎ－ｒｅｌａｔｅｄ　Ｍｏｄｉｆｉｅｒ）タンパク質あるいは、ＳＵＭＯ化に関わるタンパク質である。ＳＵＭＯタンパク質は、ユビキチン様タンパク質ともよばれ、近年、その機能が解明されてきている比較的分子量が小さいタンパク質である。発酵阻害が生じる理由として、リグノセルロース分解物のような発酵阻害物質により、発酵経路に寄与するようなタンパク質が変性して発酵環境に残留することが、理由の一つと考えられる。ここで、本発明の配列番号で表されるタンパク質は、発酵阻害物質により変性したタンパク質のＳＵＭＯ化を亢進することによってそれら変性したタンパク質の分解を早めることに寄与していると考えられる。本発明の微生物は、この変性したタンパク質の分解を促進することにより、発酵阻害物質自体や変性タンパク質による発酵阻害の影響を速やかに取り除き、発酵阻害物質存在下における微生物の成長誘導期を短縮するといった効果を奏するものと考えられる。 The present invention adjusts the expression so that a gene encoding at least one protein selected from the group consisting of the proteins represented by SEQ ID NOs: 1 to 4 in yeast is highly expressed. It is characterized by that. The proteins represented by SEQ ID NOs: 1 to 4 are all SUMO (Small Ubiquitin-related Modifier) proteins or proteins related to SUMOization. The SUMO protein is also called a ubiquitin-like protein and is a protein having a relatively small molecular weight whose function has been elucidated in recent years. One reason for the inhibition of fermentation is that proteins that contribute to the fermentation pathway are denatured and remain in the fermentation environment by fermentation inhibitors such as lignocellulose degradation products. Here, it is considered that the protein represented by the SEQ ID NO of the present invention contributes to the rapid degradation of the denatured protein by enhancing the sumoylation of the protein denatured by the fermentation inhibitor. The microorganism of the present invention accelerates the degradation of the denatured protein, thereby quickly removing the influence of fermentation inhibition by the fermentation inhibitor itself and the denatured protein, and shortening the growth induction period of the microorganism in the presence of the fermentation inhibitor. It is thought that there is an effect.
　本発明においては、配列番号１で表されるタンパク質および配列番号２で表されるタンパク質、配列番号３で表されるタンパク質、配列番号４で表されるタンパク質の４つのタンパク質からなる群から選択される少なくとも１以上のタンパク質をコードする遺伝子を利用する。すなわち、配列番号１～４で表される４つのタンパク質のうち、いずれか１つ、或いは任意の２つ以上のタンパク質をコードする遺伝子を用いることができる。いずれか１つのタンパク質をコードする遺伝子を導入することで、一定のリグノセルロース耐性を付与することができ、これらを組み合わせて導入することでより優れたリグノセルロース耐性を付与することができる。最も好ましくは４つの遺伝子を利用することである。 In the present invention, the protein represented by SEQ ID NO: 1, the protein represented by SEQ ID NO: 2, the protein represented by SEQ ID NO: 3, and the protein represented by SEQ ID NO: 4 are selected from the group consisting of four proteins. A gene encoding at least one protein is used. That is, a gene encoding any one of the four proteins represented by SEQ ID NOs: 1 to 4, or any two or more proteins can be used. By introducing a gene encoding any one protein, certain lignocellulose resistance can be imparted, and by introducing these in combination, superior lignocellulose resistance can be imparted. Most preferably, four genes are used.
　本発明に関するタンパク質および遺伝子についてより詳しく解説する。例えば、本発明に用いる配列番号１で表されるタンパク質は、酵母のＳＭＴ３とも呼ばれるＳＵＭＯタンパク質である。このたんぱく質をコードする遺伝子として、例えば、配列番号５に示す酵母Saccharomyces cerevisiaeの遺伝子があげられる。 The protein and gene related to the present invention will be explained in more detail. For example, the protein represented by SEQ ID NO: 1 used in the present invention is a SUMO protein also called yeast SMT3. Examples of the gene encoding this protein include the gene of yeast Saccharomyces cerevisiae shown in SEQ ID NO: 5.
　本発明の遺伝子がコードするタンパク質はさらに、配列番号１～４で表されるアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加され、その対応する配列番号に示されるアミノ酸配列と８０％以上、好ましくは９０％以上、より好ましくは９５％以上、特に好ましくは９８％以上の同一性を有するアミノ酸配列をコードする遺伝子からなり、かつ、微生物にリグノセルロース分解物耐性付与機能を有するタンパク質を包含する。すわなち、アミノ酸配列が類似することで、微生物へリグノセルロース分解物耐性の形質転換を供与するものも包含する。また、ここで対応する配列番号に示されるアミノ酸配列との同一性とは、配列番号１～４のアミノ酸のうち、最も同一性が近い配列番号のアミノ酸配列との同一性である。すなわち、配列番号１との同一性が高いアミノ酸配列の場合は、配列番号１との比較を行うものであり、同様に配列番号２との同一性が高い場合は、配列番号２と、配列番号３との同一性が高い場合は配列番号３と、配列番号４との同一性が高い場合は配列番号４との比較を行う。
　配列番号１～４のアミノ酸配列と対応するアミノ酸配列の同一性を求めるアルゴリズムとしては、この分野で汎用されているアルゴリズムの一つであるＢＬＡＳＴ(Basic Local Alignment Search Tool)を用いて求めることができる。 The protein encoded by the gene of the present invention further has one or several amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NOs: 1 to 4, and an amino acid sequence represented by the corresponding SEQ ID NO: 80 % Or more, preferably 90% or more, more preferably 95% or more, particularly preferably 98% or more of a gene encoding an amino acid sequence and having a function of imparting resistance to lignocellulose degradation products to microorganisms Is included. In other words, it also includes those that provide transformation of resistance to lignocellulosic degradation products of microorganisms by having similar amino acid sequences. Here, the identity with the amino acid sequence shown in the corresponding SEQ ID NO is the identity with the amino acid sequence with the closest identity among the amino acids of SEQ ID NOS: 1-4. That is, in the case of an amino acid sequence having high identity with SEQ ID NO: 1, a comparison is made with SEQ ID NO: 1. Similarly, when identity with SEQ ID NO: 2 is high, SEQ ID NO: 2 and SEQ ID NO: When the identity with 3 is high, SEQ ID NO: 3 is compared with SEQ ID NO: 4 when the identity with SEQ ID NO: 4 is high.
As an algorithm for determining the identity of the amino acid sequence corresponding to the amino acid sequence of SEQ ID NOs: 1 to 4, it can be determined using BLAST (Basic Local Alignment Search Tool) which is one of the algorithms widely used in this field. .
　なお、この配列番号１～４で表されるアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加され、対応する配列番号で表されるアミノ酸配列に対して８０％以上の同一性の範囲内で修飾されたアミノ酸配列からなり、かつ微生物へのリグノセルロース分解物耐性付与機能を有するタンパク質を用いる場合、配列番号１～４に対応するそれぞれのタンパク質を２種以上組み合わせて用いることが好ましく、４種を組み合わせて用いることが好ましい。このとき、配列番号１については、そのタンパク質とし、配列番号２についてはそれと対応する前記（２）に記載の同一性等の要件を満足するタンパク質を組み合わせることもできる。 In the amino acid sequences represented by SEQ ID NOs: 1 to 4, one or several amino acids are deleted, substituted or added, and the amino acid sequence represented by the corresponding SEQ ID NOs has 80% or more identity. When using a protein having an amino acid sequence modified within the range and having a function of imparting resistance to lignocellulose degradation products to microorganisms, it is preferable to use a combination of two or more of the proteins corresponding to SEQ ID NOs: 1 to 4. It is preferable to use four types in combination. At this time, SEQ ID NO: 1 can be used as the protein, and SEQ ID NO: 2 can be combined with a protein that satisfies the requirements such as identity described in (2) corresponding thereto.
　ここで配列番号２のタンパク質は酵母のＳＬＸ５とも呼ばれるタンパク質であり、配列番号３のタンパク質は酵母のＳＬＸ８とも呼ばれるタンパク質であり、配列番号４のタンパク質はＵＢＣ９とも呼ばれるタンパク質である。これらのたんぱく質をコードする遺伝子として、例えば、配列番号６（ＳＬＸ５）、配列番号７（ＳＬＸ８）、配列番号８（ＵＢＣ９）に示す酵母Saccharomyces cerevisiaeの遺伝子があげられる。 Here, the protein of SEQ ID NO: 2 is a protein also called yeast SLX5, the protein of SEQ ID NO: 3 is a protein also called yeast SLX8, and the protein of SEQ ID NO: 4 is a protein also called UBC9. Examples of genes encoding these proteins include the yeast Saccharomyces cerevisiae genes shown in SEQ ID NO: 6 (SLX5), SEQ ID NO: 7 (SLX8), and SEQ ID NO: 8 (UBC9).
　本発明は、野生型と比較して、前述の（１）または（２）のタンパク質をコードする遺伝子が高発現されたリグノセルロース分解物耐性微生物とすることを特徴とする。これらの遺伝子を高発現させる方法は、特に限定されることはなく、いわゆるゲノム編集技術として提案されている手法で、本発明の対象となる遺伝子を高発現させることができる種々の方法を利用することができる。例えば、当該遺伝子を高発現するように、その遺伝子を付加的に導入する方法や、これらの遺伝子のプロモーターを当該遺伝子を高発現させるようなプロモーターと交換する方法などが挙げられる。遺伝子を導入する方法として代表的なものとしては、当該遺伝子が高発現される微生物の一部の染色体の塩基配列部分と、高い相同性を有する塩基配列部分を有する塩基配列を利用して、遺伝子を導入する相同組み換えが挙げられる。これは、例えばＰＣＲで直鎖状のＤＮＡフラグメントを作成し、そのとき、両端に約５０ｂｐ程度の野生型の対象微生物の染色体と相同性が高い配列を持たせておき対象微生物に接触させることで、当該野生型の対象微生物がＤＮＡ修復するときに自動的にその相同性が高い配列を組込み、野生型の対象微生物の染色体の領域が入れ替わるものである。この相同組み換えを利用して、直鎖状のＤＮＡフラグメントに、本発明の対象となる遺伝子を複数導入しておくなどし、その遺伝子が高発現するようにできる。 The present invention is characterized in that it is a lignocellulose degradation product-resistant microorganism in which the gene encoding the protein (1) or (2) described above is highly expressed as compared to the wild type. The method for highly expressing these genes is not particularly limited, and various methods capable of highly expressing the gene targeted by the present invention are used by a technique proposed as a so-called genome editing technique. be able to. For example, a method of additionally introducing the gene so as to highly express the gene, a method of exchanging the promoter of these genes with a promoter capable of highly expressing the gene, and the like can be mentioned. A typical method for introducing a gene is to use a base sequence having a base sequence portion having a high homology with a base sequence portion of a part of a chromosome of a microorganism in which the gene is highly expressed. The homologous recombination which introduce | transduces is mentioned. This is because, for example, a linear DNA fragment is prepared by PCR, and at that time, a sequence having high homology with a chromosome of a wild-type target microorganism of about 50 bp is provided at both ends and brought into contact with the target microorganism. When the wild-type target microorganism repairs DNA, a highly homologous sequence is automatically incorporated, and the chromosomal region of the wild-type target microorganism is replaced. By utilizing this homologous recombination, a plurality of genes targeted by the present invention can be introduced into a linear DNA fragment so that the gene is highly expressed.
　対象遺伝子を高発現させる方法として、組み換えベクターを導入する方法も採用することができる。すなわち、本発明は、配列番号１～４で表されるタンパク質のように、上記（１）、（２）で表されるタンパク質（以下、「本発明に用いるタンパク質」と省略する場合がある。）をコードする遺伝子を含有する組み換えベクターを用いて、形質転換の対象となる微生物に導入することを特徴とすることができる。また、この遺伝子は、例えば、配列番号１で表されるタンパク質をコードする遺伝子のみではなく、さらに、それと一定の同一性等を有し、微生物へリグノセルロース分解物耐性付与機能を有する遺伝子とすることもできる。この組み換えベクターとしては、例えば、形質転換の対象を酵母とする場合、酵母用に用いられるベクターを使用でき、より具体的には、タンパク発現型のベクター等を用いることができる。その他の微生物を対象とする場合も、適宜、形質転換の対象となるその微生物に適した組み換えベクターを用いればよい。宿主細胞への組み換えベクターの導入方法としては、例えば、エレクトロポレーション法、スフェロブラスト法、酢酸リチウム法、カルシウムイオンを用いる方法やプロトプラスト法等により組み換えベクターを導入することができる。 As a method for highly expressing the target gene, a method of introducing a recombinant vector can also be employed. That is, the present invention may be abbreviated as the protein represented by the above (1) or (2) (hereinafter referred to as “protein used in the present invention”) like the proteins represented by SEQ ID NOS: 1-4. And a recombinant vector containing a gene encoding), and introduced into a microorganism to be transformed. In addition, this gene is not only a gene encoding the protein represented by SEQ ID NO: 1, for example, but also a gene having a certain identity and the like and having a function of imparting resistance to lignocellulose degradation products. You can also. As this recombinant vector, for example, when a transformation target is yeast, a vector used for yeast can be used, and more specifically, a protein expression type vector or the like can be used. In the case of targeting other microorganisms, a recombinant vector suitable for the microorganism to be transformed may be used as appropriate. As a method for introducing a recombinant vector into a host cell, the recombinant vector can be introduced by, for example, an electroporation method, a spheroblast method, a lithium acetate method, a method using calcium ions, a protoplast method, or the like.
　発現ベクターには、プラスミド、ウイルス、コスミドなどが含まれる。ベクターは、原核生物用又は真核生物用ベクター、例えば、ｐＭＡＬ系、ｐＴＹＢ系ベクター（第一化学薬品）、ｐＡＵＲ１２３（タカラバイオ）、ｐＥＴ系、ｐＢＡＣ系、ｐＴｒｉ―Ｅｘ１ベクター（ｐＴｒｉ―Ｅｘ１）、ｐＣｒｕｚＴＭ系ベクター（コスモバイオ）などが挙げられる。 Expression vectors include plasmids, viruses, cosmids and the like. The vector is a prokaryotic or eukaryotic vector, such as pMAL, pTYB vector (Daiichi Kagaku), pAUR123 (Takara Bio), pET system, pBAC system, pTri-Ex1 vector (pTri-Ex1), Examples include pCruz ™ vectors (Cosmo Bio).
　ここで、本発明に用いるベクターには、本発明に係る遺伝子配列の他に、発現制御配列、例えばプロモーター、エンハンサー、ターミネーター、開始コドン、スプライスシグナル、停止コドンなど、複製開始点、選択マーカー、ポリリンカーなどを含むことができる。 Here, the vector used in the present invention includes, in addition to the gene sequence according to the present invention, an expression control sequence such as a promoter, enhancer, terminator, start codon, splice signal, stop codon, replication origin, selectable marker, polylin Cars can be included.
　プロモーターは、構成的プロモーター又は誘導性プロモーターを含む。微生物系でクローニングを行う場合、バクテリオファージλのｐＬ、ｐｌａｃなどの誘導性プロモーターが例示される。 Promoters include constitutive promoters or inducible promoters. When cloning in a microbial system, inducible promoters such as bacteriophage λ pL and lac are exemplified.
　選択マーカーは、薬剤耐性マーカー、例えばアンピシリン、カナマイシン、ネオマイシン、テロラサイクリン、クロラムフェニコール、Ｇ４１８、ハイグロマイシン、メトトレキサートなど、栄養要求性マーカー、例えばＵＲＡ３などを含む。 Selection markers include drug resistance markers such as ampicillin, kanamycin, neomycin, teracycline, chloramphenicol, G418, hygromycin, methotrexate, and other auxotrophic markers such as URA3.
　本発明は、上記の特定の遺伝子を含む発現ベクター、及び該発現ベクター等によって野生型と比較して特定の遺伝子が高発現された宿主細胞（微生物）を提供するものである。本発明の対象となる宿主細胞（微生物）は、ＳＵＭＯやＳＵＭＯ化に関与する本発明に用いるタンパク質に関連する遺伝子を有することでリグノセルロース分解物耐性を示す微生物である。このような微生物として、本発明の微生物には、真核細胞、酵母（例えばサッカロマイセス・セレビシェ）などが挙げられる。この微生物（宿主細胞）として、例えばリグノセルロース系バイオマスを発酵する微生物である酵母等を対象とすることで、これらの微生物にリグノセルロース分解物耐性を付与する形質転換を行うものである。この対象となる微生物は、エタノール発酵を行う真菌類を選択することができる。これらの中でも、酵母は代表的なエタノール発酵を行う微生物であり、エタノールは各種工業的利用（特にバイオエタノールとしての燃料）が行われ、今後、更にその技術開発が期待されるものであり、本発明によりリグノセルロース分解物耐性を付与する対象として好ましい微生物である。 The present invention provides an expression vector containing the above specific gene, and a host cell (microorganism) in which the specific gene is highly expressed by the expression vector or the like as compared with the wild type. The host cell (microorganism) that is the subject of the present invention is a microorganism that exhibits resistance to lignocellulose degradation products by having a gene related to SUMO and the protein used in the present invention that is involved in sumoylation. Examples of the microorganism of the present invention include eukaryotic cells and yeast (for example, Saccharomyces cerevisiae). As this microorganism (host cell), for example, yeast that is a microorganism that ferments lignocellulosic biomass is targeted, and transformation to impart lignocellulose degradation product resistance to these microorganisms is performed. As the target microorganism, a fungus that performs ethanol fermentation can be selected. Among these, yeast is a microorganism that performs typical ethanol fermentation. Ethanol is used for various industrial uses (especially fuel as bioethanol), and further technological development is expected in the future. According to the invention, it is a preferred microorganism as a target for imparting resistance to lignocellulose degradation products.
　本発明において、宿主細胞となる微生物のその属を例示すると、サッカロミセス属、シゾサッカロミセス属、キャンディダ属、クリュイベロミセス属、トリコスポロン属、シュワニオミセス属から選択される少なくとも１以上の微生物とすることができる。発酵に同時に用いられる微生物などを複数形質転換させておき利用することもできる。 In the present invention, examples of the genus of the microorganism that serves as a host cell include at least one microorganism selected from the genus Saccharomyces, Schizosaccharomyces, Candida, Kluyveromyces, Trichosporon, and Schwaniomyces can do. A plurality of microorganisms and the like used simultaneously in fermentation can be transformed and used.
　本発明においては宿主細胞である微生物を酵母とすることが好ましい態様の一つである。酵母とは、かび、真菌のうち、丸い形で増殖するものの総称である。また、Candidaなどのように、二形性の増殖を示し酵母型で増殖するフェーズと先端成長するフェーズを有する、いわゆる不完全酵母も含めて、本発明の対象となる酵母とすることができる。より具体的には、本発明において宿主細胞として酵母を選択するとき、形質転換酵母の作製に用いられる酵母は、サッカロミセス（Saccharomyces）属、シゾサッカロミセス（Schizosaccharomyces）属、キャンディダ（Candida）属、クリュイベロミセス（Kluyveromyces）属、トリコスポロン（Trichosporon）属、シュワニオミセス（Schwanniomyces）属から選択される少なくとも１以上の酵母とすることができる。これらの具体例としては、サッカロミセス・セレビシェ(Saccharomyces cerevisae)、サッカロミセス・パストリアヌス(Saccharomyces pasteurianus)、サッカロミセス・バイヤヌス(Saccharomyces bayanus)、シゾサッカロミセス・ボンベ(Schizosaccharomyces pombe)、キャンディダ・グラブラータ（Candida glabrata）、クリュイベロミセス・ラクチス(Kluyveromyces lactis)、トリコスポロン・プルランス(Trichosporon pullulans)、シュワニオミセス・アルビウス(Schwanniomyces alluvius)等を挙げることができる。 In the present invention, it is one of preferred embodiments that the microorganism that is the host cell is yeast. Yeast is a general term for fungi and fungi that grow in a round shape. Moreover, it can be set as the yeast used as the object of this invention also including what is called incomplete yeast which has the phase which grows in a yeast type | mold, and has the phase which grows at the tip like Candida. More specifically, when yeast is selected as a host cell in the present invention, the yeast used for the production of transformed yeast is the genus Saccharomyces, the genus Schizosaccharomyces, the genus Candida, It can be at least one yeast selected from the genus Kluyveromyces, Trichosporon, and Schwanniomyces. Specific examples of these include Saccharomyces cerevisae, Saccharomyces pasteurianus, Saccharomyces bayanus, Schizospomata brevices (Schizospoglabrace) Examples include Kluyveromyces lactis, Trichosporon pullulans, and Schwanniomyces alluvius.
　一部前述したように、本発明に用いる配列番号１で表されるアミノ酸配列をコードする遺伝子は、酵母のＳＭＴ３遺伝子として知られているものであり、この遺伝子を元来有する酵母も存在する。また、同様に、配列番号２～４のタンパク質に対応する、ＳＬＸ５、ＳＬＸ８、ＵＢＣ９の遺伝子を有する酵母も存在する。しかしながら、この遺伝子を単に有していても、従来の酵母がそうであるように、酵母はリグノセルロース分解物による生育阻害を受ける。本発明は、この遺伝子を組み換えベクターを用いて、酵母に導入することで、通常よりもこの遺伝子を高発現し、優れたリグノセルロース分解物耐性を有する酵母とすることができる。 As described above in part, the gene encoding the amino acid sequence represented by SEQ ID NO: 1 used in the present invention is known as the SMT3 gene of yeast, and yeasts originally having this gene also exist. Similarly, there are yeasts having the genes of SLX5, SLX8, and UBC9 corresponding to the proteins of SEQ ID NOs: 2 to 4. However, even with this gene simply, the yeast is subject to growth inhibition by lignocellulose degradation products, as is the case with conventional yeasts. In the present invention, by introducing this gene into yeast using a recombinant vector, this gene can be expressed at a higher level than usual and a yeast having excellent lignocellulose degradation product resistance can be obtained.
　本発明はリグノセルロース系バイオマスを資源化する発酵に有用な微生物を提供するものである。また、そのような微生物を用いて、発酵資源を得るものである。すなわち、本発明は、リグノセルロース系バイオマスを発酵する方法において、当該発酵を行うための微生物が、配列番号１で表されるタンパク質をコードする遺伝子を含有する組み換えベクターを含む微生物であることを特徴とする発酵法とすることができる。特に、酵母を用いてリグノセルロース系バイオマスからバイオエタノールを製造する方法において、前記酵母が、野生型と比較して、前述した（１）または（２）のタンパク質をコードする遺伝子が高発現された酵母であることを特徴とするバイオエタノールを製造する方法とすることができる。 The present invention provides microorganisms useful for fermentation that uses lignocellulosic biomass as resources. Moreover, fermentation resources are obtained using such microorganisms. That is, the present invention is characterized in that in the method for fermenting lignocellulosic biomass, the microorganism for performing the fermentation is a microorganism containing a recombinant vector containing a gene encoding the protein represented by SEQ ID NO: 1. And the fermentation method. In particular, in the method for producing bioethanol from lignocellulosic biomass using yeast, the yeast encoded the above-described gene encoding the protein (1) or (2) higher than the wild type. It can be set as the method of manufacturing bioethanol characterized by being yeast.
　ここで、リグノセルロース系バイオマスとは、主としてセルロース、ヘミセルロース、およびリグニンから構成されるリグノセルロースを有するバイオマス（生物由来の有機資源）のことを意味し、木質系や、草本植物系のものなどを包含する概念である。具体的には、木材、イナワラ、ムギワラ、バガス、竹、パルプ、コーンストーバー、もみがら、パーム椰子残渣、キャッサバ残渣、麻等が挙げられる。 Here, lignocellulosic biomass means biomass (living organic resources) that has lignocellulose mainly composed of cellulose, hemicellulose, and lignin, including woody and herbaceous plants. It is a concept to include. Specific examples include wood, rice straw, wheat straw, bagasse, bamboo, pulp, corn stover, rice husk, palm palm residue, cassava residue, hemp and the like.
　このリグノセルロース系バイオマスを発酵して資源化するにあたっては、特にリグノセルロースの分解物に問題があった。リグノセルロースの分解物には、フルフラールや、ＨＭＦ、グリコールアルデヒド、酢酸などが含まれている。これらの物質は、多くの微生物の生育を阻害し、微生物生育が阻害されることで発酵効率が低下する。すなわち、リグノセルロース分解物は、発酵阻害物質となっていた。 When the lignocellulosic biomass was fermented and turned into resources, there was a problem with lignocellulose degradation products. The degradation product of lignocellulose contains furfural, HMF, glycol aldehyde, acetic acid and the like. These substances inhibit the growth of many microorganisms, and the fermentation efficiency decreases due to the inhibition of the growth of microorganisms. That is, the lignocellulose decomposition product was a fermentation inhibitor.
　本発明によって形質転換された微生物は、このリグノセルロース分解物耐性を有している。すなわち、本発明により得られる微生物は、前述したようなリグノセルロース分解物の存在下においても、十分に生育することができる微生物となる。これによって、それらの微生物による本来の発酵が十分に行われる。 The microorganism transformed by the present invention has resistance to this lignocellulose degradation product. That is, the microorganism obtained by the present invention is a microorganism that can sufficiently grow even in the presence of the lignocellulose degradation product as described above. Thereby, the original fermentation by those microorganisms is sufficiently performed.
　以下、実施例により本発明を更に詳細に説明するが、本発明は、その要旨を変更しない限り以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples unless the gist thereof is changed.
［評価項目］
（濁度（ＯＤ６００））
　濁度計（株式会社島津製作所社製“ＵＶ－１８００”：試験波長６００ｎｍ、石英セル使用、光路長１ｃｍ）を用いて、発酵試験液の濁度を測定することで、菌の繁殖の程度を評価する指標とした。なお、濁度（ＯＤ６００）が０となるブランクとして、蒸留水を用いた。 [Evaluation item]
(Turbidity (OD600))
By measuring the turbidity of the fermentation test solution using a turbidimeter (Shimadzu Corporation “UV-1800”: test wavelength 600 nm, quartz cell used, optical path length 1 cm) The index to be evaluated. In addition, distilled water was used as a blank in which the turbidity (OD600) was zero.
［培地の調製］
　実施例、比較例、参考例およびそれらの実験を行うための前培養用に、以下の、ＳＣ標準培地、リグノセルロース分解物含有培地、ＣＳＭ培地を調製した。なお、いずれの培地も植菌前に、オートクレーブ（１２１℃２気圧１５分）で滅菌処理を行った。 [Preparation of medium]
The following SC standard medium, lignocellulose degradation product-containing medium, and CSM medium were prepared for the examples, comparative examples, reference examples, and pre-cultures for conducting these experiments. Each medium was sterilized by autoclaving (121 ° C., 2 atm for 15 minutes) before inoculation.
（ＳＣ標準培地）
　標準ＳＣ培地として、２％グルコースを含有するＳＣ培地を用いた。 (SC standard medium)
As the standard SC medium, SC medium containing 2% glucose was used.
（リグノセルロース分解物含有培地）
　発酵阻害物質（ｉｎｈｉｂｉｔｏｒ）として、リグノセルロースの分解物にも含まれる、グルコールアルデヒド、ヒドロキシメチルフルフラール（ＨＭＦ）、フルフラール、メチルグリオキサール、酢酸を用いた。ＳＣ標準培地に、これらの各発酵阻害物質を、各濃度が１０ｍｍｏｌ／Ｌとなるように混合したものをリグノセルロース分解物含有培地とした。 (Lignocellulose degradation product-containing medium)
As a fermentation inhibitor, glucose aldehyde, hydroxymethylfurfural (HMF), furfural, methylglyoxal, and acetic acid, which are also contained in the lignocellulose degradation product, were used. A mixture of these fermentation inhibitor substances so as to have a concentration of 10 mmol / L in an SC standard medium was used as a lignocellulose degradation medium-containing medium.
（ＣＳＭ培地）
　ＣＳＭ培地（2% (w/v) glucose　(Wako, 041-00595), 790 mg/l of a complete supplement mixture (CSM complete) medium (Formedium, DCS0019), and a 0.67% (w/v) yeast nitrogen base without amino acids and ammonium sulphate (Becton Dickinson and Company, 233520）を培地の一つとして用いた。なお、ＣＳＭ培地は、必要に応じてＮａＯＨ（Ｗａｋｏ製192-13763）を用いてｐＨ６．５となるように調整した。 (CSM medium)
CSM medium (2% (w / v) glucose (Wako, 041-00595), 790 mg / l of a complete supplement mixture (CSM complete) medium (Formedium, DCS0019), and a 0.67% (w / v) yeast nitrogen Base without amino acids and ammonium sulphate (Becton Dickinson and Company, 233520) was used as one of the media, and the CSM media was adjusted to pH 6.5 using NaOH (192-13763 made by Wako) as necessary. Adjusted as follows.
＜酵母＞
・酵母ＢＹ４７４３：実験室酵母ＢＹ４７４３株を「酵母ＢＹ４７４３」として使用した。
・酵母ＢＹｐＡＤＨ１：ＢＹ４７４３株に、Saccharomyces cerevisiae由来のＡＤＨ１遺伝子をベクターＲＳ４２６を用いて導入することで、強いグリコアルデヒドの還元活性を示すことが見出された菌株を、「酵母ＢＹｐＡＤＨ１」として使用した。（参考：非特許文献１） <Yeast>
Yeast BY4743: Laboratory yeast BY4743 strain was used as “yeast BY4743”.
-Yeast BYpADH1: A strain found to show strong glycoaldehyde reduction activity by introducing the ADH1 gene derived from Saccharomyces cerevisiae into the strain BY4743 using the vector RS426 was used as "yeast BYpADH1". (Reference: Non-patent document 1)
＜標準酵母前培養液の調製＞
　各酵母１．５×１０6ｃｅｌｌｓを、前述の培養用ＣＳＭ培地１．５ｍＬ（短い試験管）に植菌。３０℃にて、１日培養して、酵母前培養液とした。 <Preparation of standard yeast preculture solution>
Inoculate 1.5 × 10 6 cells of each yeast into 1.5 mL (short test tube) of the aforementioned CSM medium for culture. It was cultured at 30 ° C. for 1 day to obtain a yeast preculture solution.
＜配列番号５で表される遺伝子を含むベクター（以下、「ベクター（Ｉ）」）の調製＞
　ＳＭＴ３ｆｗ(CGA GCT CTA GAG GTT AGC CAT GCT GTT TCC ATC A)及びＳＭＴ３ｒｖ(GAA ATT CGC TTA GTT TGT GGC ACG TCG TGA AAG AAT)で、酵母Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ　ＢＹ４７４３株のゲノムからＳＭＴ３断片（配列番号５で表される遺伝子を含む断片）を取り出し、ＰＣＲで増幅してＰＣＲ断片を得た。なお、ＰＣＲはアステック社製“ＰＣ３２０”を使用し、ポリメラーゼとして東洋紡社製“ＫＯＤ　ｐｌｕｓ　ｖｅｒ２”を用いておこなった。ＰＣＲ時の条件として、１０倍濃縮高塩濃度緩衝液２．５μＬ、２ｍＭ　ｄＮＴＰ２．５μＬ、２５ｍＭ　ＭｇＳＯ4１．０μＬ、Ｉｎ－ｆｕｓｉｏｎ　ｐｒｉｍｅｒ（ＦＷ）０．７５μＬ、Ｉｎ－ｆｕｓｉｏｎ　ｐｒｉｍｅｒ（ＲＷ）０．７５μＬ、Ｔｅｍｐｌａｔｅ０．０１３７５μｇ、ポリメラーゼ０．５μＬを混合し、ミリＱ水を残部とし、２５μＬの反応液として用いた。このＰＣＲ反応は、変性（ｄｅｎａｔｕｒｅ）９４℃１５秒、アニール（ａｎｎｅａｌｉｎｇ）５３℃３０秒、伸長（ｅｌｏｎｇａｔｉｏｎ）６８℃２分のサイクルとし４０サイクル行った。ベクターとして用いる「ｐＡＵＲ１２３」を制限酵素「ＫｐｎＩ」で切断し、Clontech Infusion kitにて、ベクターとＰＣＲ断片を結合し、大腸菌に形質転換して選抜することで、配列番号５で表される遺伝子を含むベクター（「ベクター（Ｉ）」）を得た。配列番号５で表される遺伝子は、配列番号１で表されるタンパク質をコードする遺伝子であることから、このベクター（Ｉ）は配列番号１で表されるタンパク質をコードする遺伝子を含有する組み換えベクターである。このベクター（Ｉ）の制限酵素地図を図１に示す。 <Preparation of a vector containing the gene represented by SEQ ID NO: 5 (hereinafter referred to as “vector (I)”)>
SMT3fw (CGA GCT CTA GAG GTT AGC CAT GCT GTT TCC ATC A) and SMT3rv (GAA ATT CGC TTA GTT TGT GGC ACG TCG TGA AAG AAT). The fragment containing the gene) was taken out and amplified by PCR to obtain a PCR fragment. PCR was performed using “PC320” manufactured by Astech Co., Ltd., and “KOD plus ver2” manufactured by Toyobo Co., Ltd. as the polymerase. As conditions at the time of PCR, 10-fold concentrated high salt buffer 2.5 μL, 2 mM dNTP 2.5 μL, 25 mM MgSO 4 1.0 μL, In-fusion primer (FW) 0.75 μL, In-fusion primer (RW) 0. 75 μL, 0.01375 μg of Template and 0.5 μL of polymerase were mixed, and Milli-Q water was used as the balance, which was used as a 25 μL reaction solution. This PCR reaction was performed for 40 cycles with a denaturation of 94 ° C. for 15 seconds, an annealing of 53 ° C. for 30 seconds, and an elongation of 68 ° C. for 2 minutes. By cutting “pAUR123” used as a vector with a restriction enzyme “KpnI”, combining the vector and a PCR fragment with a Clontech Infusion kit, transforming into E. coli, and selecting the gene represented by SEQ ID NO: 5 A containing vector ("Vector (I)") was obtained. Since the gene represented by SEQ ID NO: 5 is a gene encoding the protein represented by SEQ ID NO: 1, this vector (I) is a recombinant vector containing the gene encoding the protein represented by SEQ ID NO: 1. It is. A restriction enzyme map of this vector (I) is shown in FIG.
＜他の配列番号で表されるベクター（ベクター（ＩＩ）、（ＩＩＩ））の調製＞
　前述のベクター（Ｉ）の調製に準じて、他の配列番号の遺伝子を含むベクター（ＩＩ）、（ＩＩＩ）の調製を行った。含有させた遺伝子の組み合わせを、表１に示す。なお、複数の遺伝子を導入したベクターの調製にあたっては、Infusion cloningにより複数の遺伝子を導入した。 <Preparation of vectors (vectors (II) and (III)) represented by other SEQ ID NOs>
In accordance with the preparation of the vector (I) described above, vectors (II) and (III) containing genes with other sequence numbers were prepared. Table 1 shows combinations of the genes included. In preparing a vector into which a plurality of genes were introduced, a plurality of genes were introduced by infusion cloning.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
＜ベクター（Ｉ）～（ＩＩＩ）の導入により形質転換された酵母の調製＞
　前述のベクター（Ｉ）～（ＩＩＩ）を用いて、酵母ＢＹ４７４３を、Nature Protocols 2,-31-34(2007)High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG methodの方法に従い形質転換した。なお、例えば「酵母ＢＹ４７４３＋ｐＡＵＲ１２３＋ＳＭＴ３」とは、酵母ＢＹ４７４３にベクター（Ｉ）を導入した酵母培養液を表す。
　同様の手法で、適宜、酵母ＢＹｐＡＤＨ１に、ベクター（Ｉ）～（ＩＩＩ）を導入した。 <Preparation of yeast transformed by introduction of vectors (I) to (III)>
Yeast BY4743 was transformed using the aforementioned vectors (I) to (III) according to the method of Nature Protocols 2, -31-34 (2007) High-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method. . For example, “yeast BY4743 + pAUR123 + SMT3” represents a yeast culture solution in which vector (I) is introduced into yeast BY4743.
In the same manner, vectors (I) to (III) were appropriately introduced into yeast BYpADH1.
＜ベクターｐＡＵＲ１２３のみを導入した酵母の調製＞
　配列番号５で表される遺伝子の有無による差を評価するために、前述のベクターｐＡＵＲ１２３を、配列番号５で表される遺伝子を導入する組み換えを行わないままで、酵母に導入した。なお、「酵母ＢＹ４７４３＋ｐＡＵＲ１２３」とは、酵母にベクターのみを加えた酵母培養液を表わす。 <Preparation of yeast introduced with vector pAUR123 only>
In order to evaluate the difference due to the presence or absence of the gene represented by SEQ ID NO: 5, the above-described vector pAUR123 was introduced into yeast without recombination for introducing the gene represented by SEQ ID NO: 5. “Yeast BY4743 + pAUR123” represents a yeast culture solution obtained by adding only a vector to yeast.
＜酵母の本培養＞
　酵母の本培養にあたり、試験管に移した培地に、酵母培養液を、培地中の酵母菌数の濁度（ＯＤ６００）が０．１となるように添加した。なお、この酵母培養液の添加量の調整を行うために、酵母培養液は１０倍希釈したときの濁度（ＯＤ６００）を予め測定し、この結果から添加量を決定した。
（ａ）培地に、酵母培養液を添加して本培養液とし、その濁度を測定し本培養直後の濁度を求めた。この本培養液をさらに３０℃にて、継続して静置培養し、本培養を行った。
（ｂ）前述した本培養の液を、培養開始から３時間毎に１００μＬずつ取り出し濁度を測定した。 <Main culture of yeast>
In the main culture of yeast, the yeast culture solution was added to the medium transferred to the test tube so that the turbidity (OD600) of the number of yeasts in the medium was 0.1. In addition, in order to adjust the addition amount of this yeast culture solution, the turbidity (OD600) when yeast culture solution was diluted 10 times was measured previously, and the addition amount was determined from this result.
(A) A yeast culture solution was added to the medium to obtain a main culture solution, and the turbidity was measured to determine the turbidity immediately after the main culture. This main culture was further subjected to stationary culture at 30 ° C. for main culture.
(B) 100 μL of the above-described main culture solution was taken out every 3 hours from the start of the culture, and the turbidity was measured.
［実施例１、比較例１、参考例１、２］
　前述の酵母の本培養の方法に則り、表２に示す培地と、酵母培養液の組み合わせで、酵母の生育試験を行った。この酵母の本培養経過における濁度（ＯＤ６００）の測定結果を図２に示す。 [Example 1, Comparative Example 1, Reference Examples 1 and 2]
In accordance with the above-described method for main culture of yeast, a yeast growth test was performed using a combination of the medium shown in Table 2 and the yeast culture solution. The measurement result of turbidity (OD600) in the main culture process of this yeast is shown in FIG.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　リグノセルロース分解物の有無による酵母生育の程度を評価するために、参考例１、２の評価をおこなった。図２の参考例に示すように、リグノセルロース分解物を含まない培地においては、形質転換の有無に関わらずいずれの酵母も同程度の生育を示す。
　本発明の形質転換を行った酵母培養液である「酵母ＢＹ４７４３＋ｐＡＵＲ１２３＋ＳＭＴ３」を用いた実施例１は、従来の酵母培養液に相当する「酵母ＢＹ４７４３＋ｐＡＵＲ１２３」を用いた比較例１と比し、明らかに酵母が生育しやすく、リグノセルロース分解物により初期の生育速度は低下するものの参考例の濁度まで生育することができる。
Reference examples 1 and 2 were evaluated in order to evaluate the degree of yeast growth depending on the presence or absence of lignocellulose degradation products. As shown in the reference example of FIG. 2, in the medium containing no lignocellulose degradation product, all yeasts show the same growth regardless of the presence or absence of transformation.
Example 1 using “yeast BY4743 + pAUR123 + SMT3”, which is a yeast culture solution subjected to transformation of the present invention, is clearly compared with Comparative Example 1 using “yeast BY4743 + pAUR123” corresponding to the conventional yeast culture solution. Although it is easy to grow and the initial growth rate is lowered by the lignocellulose decomposition product, it can grow to the turbidity of the reference example.
［実施例２、３、比較例２、参考例３、４、５］
　前述の酵母の本培養の方法に則り、表３に示す培地と、酵母培養液の組み合わせで、酵母の生育試験を行った。この酵母の本培養経過における濁度（ＯＤ６００）の測定結果を図３に示す。また、本培養経過におけるエタノール濃度の測定結果を図４に示す。 [Examples 2 and 3, Comparative Example 2, Reference Examples 3, 4, and 5]
In accordance with the above-described method for main culture of yeast, a yeast growth test was conducted using a combination of the medium shown in Table 3 and the yeast culture solution. The measurement result of turbidity (OD600) in the main culture process of this yeast is shown in FIG. Moreover, the measurement result of the ethanol concentration in the main culture process is shown in FIG.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　本発明の形質転換を行った酵母培養液を用いた実施例２、３は、従来の酵母培養液を用いた比較例２と比べて、酵母が生育しやすく、特に、培養開始後、２４～３６時間において有意な差を示した。すなわち、本発明により、リグノセルロース分解物存在下における酵母について、大幅にその成長誘導期を短縮できることがわかる。なお、４８時間培養時に同程度の濁度を示すのは、測定上限または本培養条件で飽和する値と考えられる。また、図４に示すように、実施例３にかかるベクター（ＩＩＩ）を導入した酵母を用いて培養を行うことで、ＡＤＨ１を高発現するようにのみ遺伝子導入した比較例２の酵母を用いた場合と比べて、エタノール生産速度が高くなることがわかった。
In Examples 2 and 3 using the yeast culture solution transformed according to the present invention, the yeast grows more easily than Comparative Example 2 using the conventional yeast culture solution. There was a significant difference at 36 hours. That is, it can be seen that the growth induction period of yeast in the presence of lignocellulose degradation product can be greatly shortened by the present invention. In addition, it is thought that the value which saturates on the measurement upper limit or main culture conditions shows the same turbidity at the time of 48-hour culture. Moreover, as shown in FIG. 4, the yeast of the comparative example 2 which introduce | transduced the gene only so that ADH1 might be highly expressed was used by culturing using the yeast which introduce | transduced the vector (III) concerning Example 3. It was found that the ethanol production rate was higher than in the case.
　本発明により、リグノセルロース系バイオマスの発酵時に問題となるリグノセルロース分解物耐性を有する等の、発酵に適した特性を有する微生物を得ることができる。これによって、リグノセルロース系バイオマスの利用効率が向上し有用である。 According to the present invention, it is possible to obtain a microorganism having characteristics suitable for fermentation, such as resistance to lignocellulose degradation products, which becomes a problem during fermentation of lignocellulosic biomass. This improves the utilization efficiency of lignocellulosic biomass and is useful.

Claims (7)

  1. 　野生型と比較して、以下の（１）または（２）のタンパク質をコードする遺伝子が高発現されたリグノセルロース分解物耐性微生物。
    　（１）配列番号１～４のいずれかの配列番号で表されるアミノ酸配列からなるタンパク質から選択される少なくとも１つ以上のタンパク質
    　（２）配列番号１～４で表されるアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加され、対応する配列番号のアミノ酸配列に対して８０％以上の同一性の範囲内で修飾されたアミノ酸配列からなり、かつ微生物へのリグノセルロース分解物耐性付与機能を有するタンパク質からなる群から選択される少なくとも１つ以上のタンパク質     A lignocellulose degradation product resistant microorganism in which a gene encoding the following protein (1) or (2) is highly expressed as compared to the wild type.
    (1) At least one protein selected from proteins consisting of the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 (2) 1 or 2 in the amino acid sequences represented by SEQ ID NOs: 1 to 4 Several amino acids are deleted, substituted, or added, consisting of an amino acid sequence modified within the range of 80% or more identity to the corresponding amino acid sequence of SEQ ID NO., And resistance to lignocellulose degradation products to microorganisms At least one protein selected from the group consisting of proteins having an imparting function
  2. 　前記（１）または（２）のタンパク質をコードする遺伝子を含有する組み換えベクターを含むことでその遺伝子が高発現された請求項１記載のリグノセルロース分解物耐性微生物。 The lignocellulose degradation product resistant microorganism according to claim 1, wherein the gene is highly expressed by including a recombinant vector containing the gene encoding the protein of (1) or (2).
  3. 　前記微生物が、サッカロミセス属、シゾサッカロミセス属、キャンディダ属、クリュイベロミセス属、トリコスポロン属、シュワニオミセス属からから選択される少なくとも１以上の微生物である請求項１または２記載の微生物。 The microorganism according to claim 1 or 2, wherein the microorganism is at least one microorganism selected from the genus Saccharomyces, Schizosaccharomyces, Candida, Kluyveromyces, Trichosporon, and Schwanniomyces.
  4. 　前記微生物が、酵母である請求項１または２記載の微生物。 The microorganism according to claim 1 or 2, wherein the microorganism is yeast.
  5. 　以下の（１）または（２）のタンパク質をコードする遺伝子を含有する組み換えベクター。
    　（１）配列番号１～４のいずれかの配列番号で表されるアミノ酸配列からなるタンパク質から選択される少なくとも１つ以上のタンパク質
    　（２）配列番号１～４で表されるアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加され、対応する配列番号のアミノ酸配列に対して８０％以上の同一性の範囲内で修飾されたアミノ酸配列からなり、かつ微生物へのリグノセルロース分解物耐性付与機能を有するタンパク質からなる群から選択される少なくとも１つ以上のタンパク質     A recombinant vector containing a gene encoding the following protein (1) or (2).
    (1) At least one protein selected from proteins consisting of the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 (2) 1 or 2 in the amino acid sequences represented by SEQ ID NOs: 1 to 4 Several amino acids are deleted, substituted, or added, consisting of an amino acid sequence modified within the range of 80% or more identity to the corresponding amino acid sequence of SEQ ID NO., And resistance to lignocellulose degradation products to microorganisms At least one protein selected from the group consisting of proteins having an imparting function
  6. 　微生物に、以下の（１）または（２）のタンパク質をコードする遺伝子を含有する組み換えベクターを導入することを特徴とするリグノセルロース分解物耐性微生物を製造する方法。
    　（１）配列番号１～４のいずれかの配列番号で表されるアミノ酸配列からなるタンパク質から選択される少なくとも１つ以上のタンパク質
    　（２）配列番号１～４で表されるアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加され、対応する配列番号のアミノ酸配列に対して８０％以上の同一性の範囲内で修飾されたアミノ酸配列からなり、かつ微生物へのリグノセルロース分解物耐性付与機能を有するタンパク質からなる群から選択される少なくとも１つ以上のタンパク質     A method for producing a lignocellulose degradation product-resistant microorganism, which comprises introducing a recombinant vector containing a gene encoding the following protein (1) or (2) into a microorganism.
    (1) At least one protein selected from proteins consisting of the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 (2) 1 or 2 in the amino acid sequences represented by SEQ ID NOs: 1 to 4 Several amino acids are deleted, substituted, or added, consisting of an amino acid sequence modified within the range of 80% or more identity to the corresponding amino acid sequence of SEQ ID NO., And resistance to lignocellulose degradation products to microorganisms At least one protein selected from the group consisting of proteins having an imparting function
  7. 　酵母を用いてリグノセルロース系バイオマスからバイオエタノールを製造する方法において、前記酵母が、野生型と比較して、以下の（１）または（２）のタンパク質をコードする遺伝子が高発現された酵母であることを特徴とするバイオエタノールを製造する方法。
    　（１）配列番号１～４のいずれかの配列番号で表されるアミノ酸配列からなるタンパク質から選択される少なくとも１つ以上のタンパク質
    　（２）配列番号１～４で表されるアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加され、対応する配列番号のアミノ酸配列に対して８０％以上の同一性の範囲内で修飾されたアミノ酸配列からなり、かつ微生物へのリグノセルロース分解物耐性付与機能を有するタンパク質からなる群から選択される少なくとも１つ以上のタンパク質     In the method for producing bioethanol from lignocellulosic biomass using yeast, the yeast is a yeast in which a gene encoding the following protein (1) or (2) is highly expressed compared to the wild type: A method for producing bioethanol, comprising:
    (1) At least one protein selected from proteins consisting of the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 (2) 1 or 2 in the amino acid sequences represented by SEQ ID NOs: 1 to 4 Several amino acids are deleted, substituted, or added, consisting of an amino acid sequence modified within the range of 80% or more identity to the corresponding amino acid sequence of SEQ ID NO., And resistance to lignocellulose degradation products to microorganisms At least one protein selected from the group consisting of proteins having an imparting function
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JP2005514025A (en) * 2002-01-07 2005-05-19 ライフセンサーズ、インク． Methods and compositions for protein expression and purification

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JP2005514025A (en) * 2002-01-07 2005-05-19 ライフセンサーズ、インク． Methods and compositions for protein expression and purification

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