WO2023149583A1 - セルロース合成微生物の形質転換体およびその使用 - Google Patents
セルロース合成微生物の形質転換体およびその使用 Download PDFInfo
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- WO2023149583A1 WO2023149583A1 PCT/JP2023/003994 JP2023003994W WO2023149583A1 WO 2023149583 A1 WO2023149583 A1 WO 2023149583A1 JP 2023003994 W JP2023003994 W JP 2023003994W WO 2023149583 A1 WO2023149583 A1 WO 2023149583A1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Definitions
- the present invention relates to transformants of cellulose-synthesizing microorganisms and uses thereof.
- Glucose is an important energy source for living organisms, and animals mainly ingest glucose produced from CO 2 through photosynthesis in plants and the like. Glucose is useful as a food, a feed, a nutrient source for microorganisms, and as a raw material for chemical products, and various methods have been investigated as more efficient methods for producing glucose.
- Patent Document 1 describes a yeast capable of producing glucose from cellulose and a method for producing glucose from cellulose using the yeast.
- Non-Patent Document 1 describes a technique of producing glucose inside cells using cyanobacteria and excreting the produced glucose outside the cells using a transporter.
- the present invention has been made in view of the above problems, and aims to realize a transformant of a cellulose-synthesizing microorganism capable of producing glucose or cellobiose from a carbon source such as methanol.
- the present inventors conducted intensive studies and found that a transformant in which a cellulase gene and a gene that expresses the cellulase is introduced into the cell surface layer, or cellulase is introduced into the cell surface layer by introducing the oprI gene.
- the inventors have found that glucose or cellobiose can be produced from a carbon source such as methanol by using a transformant of a bacterium of the family Methylobacteriaceae that has become capable of expression, and have completed the present invention.
- the present invention includes the following configurations.
- a transformant in which a cellulase gene and a cell surface protein gene are introduced into a cellulose-synthesizing microorganism is introduced into a cellulose-synthesizing microorganism.
- a transformant of a cellulose-synthesizing microorganism capable of producing glucose or cellobiose from a carbon source such as methanol can be provided.
- FIG. 1 is a schematic diagram of a transformant according to an example of the present invention.
- FIG. 4 is a graph showing fluorescence intensity of cellulose-synthesizing bacteria according to Examples of the present invention.
- 1 is a schematic diagram of a transformant according to an example of the present invention.
- FIG. 1 is a graph showing methanol consumption, dry cell weight, and glucose production of wild strains of cellulose-synthesizing bacteria according to Examples of the present invention.
- 1 is a graph showing methanol consumption, dry cell weight, and glucose production of cellulose-synthesizing bacterial transformants according to Examples of the present invention.
- 1 is a graph showing cellulase activity of cellulose-synthesizing bacteria according to Examples of the present invention.
- 1 is a graph showing changes over time in cellulase activity of cellulose-synthesizing bacteria according to Examples of the present invention.
- 2 is a graph showing the turbidity of the culture solution and the amount of glucose produced by changing the substrate of the cellulose-synthesizing bacterium wild strain according to the examples of the present invention.
- 4 is a graph showing the turbidity of the culture solution and the amount of glucose produced by the cellulose-synthesizing bacterial transformant according to the example of the present invention when the substrate is changed.
- 1 is a schematic diagram of a transformant according to an example of the present invention.
- FIG. 4 is a graph showing cellobiose production of cellulose-synthesizing bacterial transformants according to Examples of the present invention.
- FIG. 2 is an HPLC chromatogram of a cellulose-synthesizing bacterial transformant according to an example of the present invention;
- a transformant according to one embodiment of the present invention (hereinafter also referred to as the present transformant) is obtained by introducing a cellulase gene and a cell surface protein gene into a cellulose-synthesizing microorganism.
- cellulase By containing a cellulase gene and a cell surface protein gene in this transformant, cellulase can be expressed on the cell surface. Conventionally, there has been no technique for expressing cellulase on the cell surface of cellulose-synthesizing microorganisms. Furthermore, according to this transformant, it becomes possible to produce glucose or cellobiose from cellulose without adding cellulase from the outside. In addition, this transformant has a high productivity (specific productivity) per cell, and can produce glucose or cellobiose at a higher yield.
- Cellulose-synthesizing microorganisms can produce cellulose from carbon sources via the serine cycle and the like. Therefore, according to the present invention, it is possible to produce glucose or cellobiose from CO 2 without relying on photosynthesis by using, for example, commercially produced methanol derived from CO 2 as the carbon source.
- glucose or cellobiose can be obtained even in non-cultivated land.
- the resulting glucose or cellobiose can be used as food, feed, nutrient source for microbial culture, and raw material for chemical products, thus enabling biochemical synthesis of various substances.
- this can contribute to the achievement and realization of Goal 2 of the Sustainable Development Goals (SDGs), such as ensuring sustainable food production systems.
- SDGs Sustainable Development Goals
- the introduction of the cellulase gene and the cell surface protein gene into the cellulose-synthesizing microorganism can be performed according to a known method. Specifically, for example, a plasmid vector having a cellulase gene sequence and a sequence encoding a protein that causes the cellulase gene to be expressed on the cell surface is introduced into the cellulose-synthesizing microorganism using an electroporation method or the like. obtained by
- the term "cellulose-synthesizing microorganisms” means bacteria, archaea, and fungi capable of synthesizing cellulose.
- the cellulose-synthesizing microorganism is preferably a bacterium, that is, a cellulose-synthesizing bacterium. Therefore, the "productivity per cell" can also be said to be more specifically the productivity per cell.
- the cellulose-synthesizing bacterium is not particularly limited as long as it is a bacterium capable of synthesizing cellulose from a carbon source. good.
- the bacteria of the family Methylobacteriaceae may be bacteria of the genus Methylorubrum capable of synthesizing cellulose.
- bacteria of the genus Methylorubrum capable of synthesizing cellulose include Methylorubrum extorquens, Methylorubrum podarium, Methylorubrum populi, Methylorubrum rhodesianum, Methylorubrum salsugnis, and Methylorubrum zatmani.
- Methylorubrum extorquens is preferred, and Methylorubrum extorquens AM1 is more preferred.
- Methylorubrum extorquens are bacteria that do not assimilate glucose, the produced glucose is less likely to be consumed by the Methylorubrum extorquens. Therefore, glucose production is improved.
- Bacteria capable of synthesizing cellulose from carbon sources other than Methylobacteriaceae bacteria are preferably one or more selected from Agrobacterium tumefaciens, Rhizobium leguminosarum, Salmonella enterica, Escherichia coli, Komagataeibacter medellinensis, and Komagataeibacter hansenii. These cellulose-synthesizing bacteria have well-established genetic recombination methods, so that transformants can be easily produced.
- the cell surface protein gene in this transformant means a gene that expresses cellulase on the cell surface.
- the cell surface protein may be a bacterial surface protein.
- Examples of the cell surface protein gene include lipoprotein gene, S-layer protein gene, pilin gene, flagellin gene, and outer membrane protein gene.
- Examples of the outer membrane protein gene include the oprI gene, lamB gene, ompT gene, and lpp-ompA gene, among which the oprI gene is preferred.
- the oprI gene is a gene encoding Major outer membrane lipoprotein (oprI), which is an extracellular membrane protein derived from Pseudomonas aeruginosa.
- the oprI gene is preferably any DNA selected from the group consisting of (1) to (6) below.
- Said SEQ ID NO: 1 corresponds to the full-length nucleotide sequence of said oprI gene.
- SEQ ID NO: 2 corresponds to the full-length amino acid sequence encoded by the oprI gene.
- the homology of the base sequences is preferably 92% or more, more preferably 95% or more, and still more preferably 99% or more.
- the homology of the nucleotide sequences of genes can be determined using a gene analysis program, BLAST (http://blast.genome.ad.jp) or FASTA (http://fasta.genome.ad.jp/SIT/FASTA.html). and so on.
- stringent conditions refer to conditions under which double-stranded polynucleotides specific to a so-called base sequence are formed and non-specific double-stranded polynucleotides are not formed.
- conditions for hybridization between highly homologous nucleic acids for example, a temperature in the range from the melting temperature (Tm value) of perfectly matched hybrids to a temperature 15°C, preferably 10°C, more preferably 5°C lower. It can also be said.
- the temperature is 60-68° C., preferably 65° C., more preferably 68° C. C. for 16 to 24 hours, and then in a buffer containing 20 mM Na.sub.2HPO.sub.4 , pH 7.2, 1% SDS, and 1 mM EDTA at a temperature of 60 to 68.degree. C., preferably 65.degree. can be exemplified by washing twice for 15 minutes at 68°C.
- Other examples include 25% formamide, more stringent 50% formamide, 4x SSC (sodium chloride/sodium citrate), 50 mM Hepes pH 7.0, 10x Denhardt's solution, 20 ⁇ g/mL denatured salmon sperm DNA. After prehybridization is performed overnight at 42°C in a hybridization solution, a labeled probe is added and hybridization is performed by incubating at 42°C overnight.
- the washing solution and temperature conditions for subsequent washing are about "1 x SSC, 0.1% SDS, 37°C", and more severe conditions are about "0.5 x SSC, 0.1% SDS, 42°C". As a more severe condition, it can be carried out at about "0.2 ⁇ SSC, 0.1% SDS, 65° C.”.
- the stricter the washing conditions for hybridization the higher the specificity of hybridization.
- the combination of conditions of SSC, SDS and temperature described above is exemplary, and a person skilled in the art will appreciate the above or other factors that determine the stringency of hybridization (e.g., probe concentration, probe length, hybridization reaction, etc.). time, etc.), it is possible to achieve the same stringency as above. This is described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001).
- the identity of the amino acid sequences is preferably 92% or more, more preferably 95% or more, and still more preferably 99% or more.
- the identity of the amino acid sequences can be determined, for example, using the BLASTX program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993).
- one or more amino acids have been deleted, substituted, or added means substitution, deletion, insertion, Or, as many amino acids as can be added are intended to be substituted, deleted, inserted or added.
- the number of the one or more amino acids is preferably 10 or less, more preferably 5 or less, still more preferably 1.
- the position where one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence is not limited.
- whether or not the polypeptide is expressed on the cell surface is determined according to a known method (for example, the method described in Example 1 below). be able to. Specifically, for example, a fusion protein of a polypeptide and green fluorescent protein (GFP) is expressed in cellulose-synthesizing bacteria or the like. Next, after confirming whether fluorescence is observed, an enzyme capable of degrading proteins such as proteinase K is added, and changes in fluorescence intensity are observed. When the fluorescence intensity is reduced by the enzyme, it can be determined that the polypeptide was expressed on the cell surface.
- GFP green fluorescent protein
- a transformant according to another embodiment of the present invention is obtained by introducing the oprI gene into a bacterium of the family Methylobacteriaceae. Containing the oprI gene in this transformant enables the desired protein to be expressed on the cell surface.
- the Methylobacteriaceae family which is a cellulose-synthesizing bacterium, there has been no technology to express proteins on the cell surface.
- cellulase which will be described later, as the protein, glucose or cellobiose can be produced from cellulose without adding cellulase from the outside.
- productivity per cell is high, making it possible to produce glucose or cellobiose at a higher yield.
- the oprI gene is as described above.
- a transformant according to another embodiment of the present invention is preferably further introduced with a cellulase gene.
- a cellulase gene When adding cellulase to cellulose-synthesizing bacteria to produce glucose, it is necessary to continuously add cellulase from the outside. However, if a cellulase gene is introduced into the present transformant, cellulase is expressed in the cell surface layer, and cellulase activity can be maintained without the addition of cellulase.
- the cellulase is not particularly limited as long as it can decompose cellulose, and examples thereof include endoglucanase, exoglucanase, and ⁇ -glucosidase.
- endoglucanase endoglucanase
- exoglucanase exoglucanase
- ⁇ -glucosidase ⁇ -glucosidase
- the transformant preferably contains at least one selected from endoglucanase genes and exoglucanase genes as the cellulase gene. More preferably, it contains both an endoglucanase gene and an exoglucanase gene.
- the exoglucanase gene is preferably one or more DNAs selected from the group consisting of (1) to (6) below.
- SEQ ID NO: 5 corresponds to the full-length base sequence of the cex gene used in the Examples.
- SEQ ID NO: 11 corresponds to the full-length amino acid sequence encoded by the cex gene.
- the endoglucanase gene is preferably one or more DNAs selected from the group consisting of (1) to (6) below.
- SEQ ID NO: 6 corresponds to the full-length nucleotide sequence of the cenA gene used in the Examples.
- SEQ ID NO: 12 corresponds to the full-length amino acid sequence encoded by the cenA gene.
- This transformant preferably further contains a ⁇ -glucosidase gene as the cellulase gene.
- the ⁇ -glucosidase gene is preferably one or more DNAs selected from the group consisting of (1) to (6) below.
- SEQ ID NO: 4 corresponds to the full-length nucleotide sequence of the bglC gene used in the Examples.
- SEQ ID NO: 10 corresponds to the full-length amino acid sequence encoded by the bglC gene.
- a method for producing glucose or cellobiose according to an embodiment of the present invention includes a culturing step of culturing the present transformant using a carbon source that can be assimilated by the transformant. According to the production method, addition of cellulase or the like is unnecessary, so glucose or cellobiose can be produced more efficiently than conventionally. In addition, this transformant has a higher productivity (specific productivity) per cell (bacteria) than conventional transformants, and can produce glucose or cellobiose at a higher yield.
- Examples of the assimilable carbon source include methanol, ethanol, L-malic acid, L-lactic acid, succinic acid, methylamine, fumaric acid, formic acid, acetic acid, betaine, and salts thereof.
- L-malic acid, L-lactic acid, succinic acid, methylamine, fumaric acid, and salts thereof, which are carbon sources suitable for culture, as well as methanol and ethanol are preferred.
- Methanol is more preferable from the viewpoint of commercial production of the product.
- the type of salt is not particularly limited, it may be, for example, sodium salt or hydrochloride.
- the conditions for culturing the present transformant can be appropriately set according to the type of the present transformant.
- Such culture conditions are known to those skilled in the art.
- the medium may optionally contain a nitrogen source, inorganic salts and the like, and may be either a natural medium or a synthetic medium.
- the medium can be, for example, MP medium as described in PLoS ONE 8(4):e62957 (2013).
- the culture step is preferably carried out under aerobic conditions such as shaking culture or aeration stirring culture.
- the culture temperature may be, for example, 20 to 40° C.
- the medium pH may be, for example, 5 to 9
- the culture time may be, for example, 0 to 13 days.
- the sugar produced by the production method is glucose or cellobiose can be determined by the combination of cellulase genes introduced into the present transformant described above.
- the ⁇ -glucosidase gene may be introduced in addition to the endoglucanase gene or exoglucanase gene, and for cellobiose production, the ⁇ -glucosidase gene may not be introduced.
- a method for producing a composition according to one embodiment of the present invention includes a step of producing glucose or cellobiose by the above production method. That is, the composition contains glucose or cellobiose obtained by the production method.
- Such compositions include, for example, a culture containing glucose or cellobiose and the present transformant, a bacterium other than the present transformant that uses glucose or cellobiose as a nutrient source, or a transformant that is the present transformant. Examples thereof include cultures obtained by co-culturing.
- composition containing glucose or cellobiose further contains assimilable carbon sources such as methanol, ethanol, L-malic acid, L-lactic acid, succinic acid, methylamine, fumaric acid, formic acid, acetic acid, betaine and salts thereof.
- assimilable carbon sources such as methanol, ethanol, L-malic acid, L-lactic acid, succinic acid, methylamine, fumaric acid, formic acid, acetic acid, betaine and salts thereof.
- the concentration of glucose or cellobiose in the composition containing glucose or cellobiose is preferably 0.05 g/L to 0.5 g/L, more preferably 0.1 g/L to 0.4 g/L.
- a method for manufacturing an article according to one embodiment of the present invention includes a step of manufacturing glucose or cellobiose by the manufacturing method.
- the article is not particularly limited as long as it is an article containing glucose or cellobiose, or an article manufactured using glucose or cellobiose.
- Examples of the articles include foods, feeds, nutrient sources for microorganisms, raw materials for chemical products, and the like.
- the method for producing an article may further include a step of producing an article (e.g., food, feed, nutrient source for microorganisms, raw material for chemicals, etc.) using the glucose or cellobiose produced by the production method. good.
- the composition containing glucose or cellobiose produced by the production method may be used in the production method of the article.
- Food, feed, or nutrient source for microorganisms for example, glucose or cellobiose, and a protein source and sugar source obtained by drying a culture containing a transformant, or excluding impurities such as a transformant from the culture and refined sugar sources.
- Such foods, feeds, or nutrient sources for microorganisms may further include any product produced by bacteria other than the present transformant, or by transformants using glucose or cellobiose as a nutrient source.
- the above-mentioned arbitrary product can be appropriately changed according to food, feed, or nutrient source of microorganisms.
- raw materials for the chemicals include plastic raw materials obtained by chemically converting glucose or cellobiose purified from cultures. It is preferable that the raw materials of the chemical product do not contain impurities (this transformant, etc.) other than glucose or cellobiose.
- ⁇ 3> The transformant according to ⁇ 2>, wherein the cellulose-synthesizing bacterium belongs to the family Methylobacteriaceae.
- ⁇ 4> The transformant according to ⁇ 3>, wherein the Methylobacteriaceae bacterium belongs to the genus Methylorubrum.
- ⁇ 5> The transformant according to ⁇ 4>, wherein the Methylorubrum bacterium is Methylorubrum extorquens.
- ⁇ 6> The transformant according to any one of ⁇ 1> to ⁇ 5>, wherein the cell surface protein gene is the oprI gene.
- ⁇ 7> A transformant obtained by introducing an oprI gene into a bacterium of the family Methylobacteriaceae.
- ⁇ 8> The transformant according to ⁇ 7>, wherein the Methylobacteriaceae bacterium belongs to the genus Methylorubrum.
- ⁇ 9> The transformant according to ⁇ 8>, wherein the bacterium belonging to the genus Methylorubrum is Methylorubrum extorquens.
- ⁇ 10> The transformant according to any one of ⁇ 6> to ⁇ 9>, wherein the oprI gene is any DNA selected from the group consisting of (1) to (6) below.
- ⁇ 11> The transformant according to any one of ⁇ 7> to ⁇ 10>, further comprising a cellulase gene.
- ⁇ 12> The transformant according to any one of ⁇ 1> to ⁇ 6> and ⁇ 11>, wherein the cellulase gene comprises at least one selected from an endoglucanase gene and an exoglucanase gene.
- the exoglucanase gene is one or more DNAs selected from the group consisting of (1) to (6) below.
- the endoglucanase gene is one or more DNAs selected from the group consisting of (1) to (6) below.
- ⁇ 15> The transformant according to any one of ⁇ 12> to ⁇ 14>, further comprising a ⁇ -glucosidase gene as the cellulase gene.
- ⁇ 16> The transformant according to ⁇ 15>, wherein the ⁇ -glucosidase gene is one or more DNAs selected from the group consisting of (1) to (6) below.
- ⁇ 17> A method for producing glucose or cellobiose, comprising a culturing step of culturing the transformant according to any one of ⁇ 1> to ⁇ 16> using a carbon source that can be assimilated by the transformant.
- ⁇ 18> A method for producing an article, comprising a step of producing glucose or cellobiose by the production method according to ⁇ 17>.
- ⁇ 19> A composition containing glucose or cellobiose obtained by the production method according to ⁇ 17>.
- FIG. 1 is a schematic diagram of the transformant produced in Example 1.
- a transformant was prepared by introducing a gene in which oprI gene 1 and GFP gene 20 were linked into a cellulose-synthesizing bacterium.
- GFP21 is expressed on the cell surface layer by the oprI protein 11 .
- the method for producing the transformant and the test results using the transformant are specifically described below.
- pTE105-gfp Construction of plasmid> (pTE105-gfp)
- pTE105 was amplified by PCR using pTE105 (obtained from Addgene) as a template and pTE105-KpnI-F1 (SEQ ID NO: 13) and pTE105-SpeI-R1 (SEQ ID NO: 14) as primers.
- the PCR conditions were 95° C. for 10 s, (95° C. for 10 s, 70° C. for 30 s, 72° C. for 4 min) ⁇ 35 times, 72° C. for 2 min.
- gfp (sequence No. 3) was amplified by PCR.
- the PCR conditions were 95° C. for 10 s, (95° C.
- PCR-amplified fragments were purified by gel extraction, mixed so that the purified products of each amplified fragment were equimolar, mixed with NEBuilder HiFi DNA Assembly Master Mix (NEB) so that they were equimolar, and incubated at 55°C for 15 minutes. Incubate and obtain a reactant. Escherichia coli DH5 ⁇ was transformed by the heat shock method using 5 ⁇ L of the obtained reaction product.
- NEB NEBuilder HiFi DNA Assembly Master Mix
- the transformed Escherichia coli DH5 ⁇ was cultured on LB agar medium (containing 20 ⁇ g/mL tetracycline) to obtain a transformant having the sequence of pTE105-gfp (SEQ ID NO: 3).
- a plasmid was extracted from the transformant using MagExtractor-Plasmid- (Toyobo) and confirmed to be pTE105-gfp of the objective plasmid by electrophoresis.
- pTE105-oprI-gfp pTE105 was amplified by PCR using pTE105 (obtained from Addgene) as a template and pTE105-KpnI-F1 (SEQ ID NO: 13) and pTE105-SpeI-R1 (SEQ ID NO: 14) as primers.
- the PCR conditions were 95° C. for 10 s, (95° C. for 10 s, 70° C. for 30 s, 72° C., 4 min) ⁇ 35 times, 72° C. for 2 min.
- gfp (SEQ ID NO: 3) was amplified by PCR using pGreenTIR (Gene 191:149-153 (1997)) as a template and gfp-F2 (SEQ ID NO: 19) and gfp-KpnI-R1 (SEQ ID NO: 16) as primers. .
- the PCR conditions were 95° C. for 10 s, (95° C. for 10 s, 70° C. for 30 s, 72° C. for 4 min) ⁇ 35 times, 72° C. for 2 min.
- PCR-amplified fragments were purified by gel extraction, mixed so that the purified products of each amplified fragment were equimolar, mixed with NEBuilder HiFi DNA Assembly Master Mix (NEB) so that they were equimolar, and incubated at 55°C for 15 minutes. Incubate and obtain a reactant. Transformation of E. coli DH5 ⁇ was performed by the heat shock method using 5 ⁇ L of the reaction product. After completion of transformation, the transformed E. coli DH5 ⁇ was cultured on LB agar medium (containing 20 ⁇ g/mL tetracycline) to obtain a transformant having the sequence pTE105-oprI-gfp (SEQ ID NO: 7). A plasmid was extracted from the transformant using MagExtractor-Plasmid- (Toyobo) and confirmed to be pTE105-oprI-gfp of the desired plasmid by electrophoresis.
- MagExtractor-Plasmid-
- Methylorubrum extorquens AM1 JCM 2805, obtained from RIKEN BioResource Research Center
- the pTE105, ⁇ 1-1 Transformation was performed using pTE105-gfp or pTE105-oprI-gfp constructed in Plasmid Construction>. Transformation was performed by the electroporation method (FEMS microbiol. Lett. 166(1):1-7 (1998)).
- extorquens AM1 (pTE105-oprI-gfp).
- a plasmid was extracted from each transformant obtained using MagExtractor-Plasmid- (Toyobo) and electrophoresis was performed to confirm that it was the target plasmid pTE105-gfp or pTE105-oprI-gfp.
- the composition of the MP medium containing 1% by volume of methanol is as follows.
- the cells After recovering the cells from the pre-cultured culture medium, the cells were washed with physiological saline three times. The cells were inoculated into 50 mL of the MP medium so that the culture solution turbidity (OD 600 ) at a wavelength of 600 nm was 0.1, and main culture was carried out at 30° C. and a shaking speed of 150 rpm for 5 days. After recovering the cells from 1 mL of the main culture medium, the cells were washed with physiological saline three times. After washing, the cells were resuspended in 1 mL of 100 mM sodium phosphate buffer (pH 7.0) or the buffer supplemented with 1 mg/mL proteinase K. After incubation at 37° C.
- FIG. 3 is a schematic diagram of a transformant prepared in Example 2.
- Example 2 the oprI gene 1 and ⁇ -glucosidase gene 2, exoglucanase gene 3, and endoglucanase gene 4, which are cellulase expression genes, were linked via a linker 5.
- a transformant introduced into a cellulose-synthesizing bacterium was prepared.
- ⁇ -glucosidase 12, exoglucanase 13 and endoglucanase 14 are expressed on the cell surface layer by the oprI protein 11. Therefore, the cellulose produced from glucose inside the cell by the transformant is decomposed by cellulase expressed on the surface of the cell, and glucose is produced outside the cell.
- the method for producing the transformant and the test results using the transformant are specifically described below.
- pTE105-oprI-bglC-cex-cenA pTE105-oprI-gfp prepared in Example 1 as a template and pTE105-KpnI-F1 (SEQ ID NO: 13) and oprI-R2 (SEQ ID NO: 26) as primers
- pTE105-oprI was amplified by PCR.
- the PCR conditions were 95° C. for 10 s, (95° C. for 10 s, 70° C. for 30 s, 72° C. for 4 min) ⁇ 35 times, 72° C. for 2 min.
- bglC sequence number 4 was amplified by PCR.
- the PCR conditions were 95° C. for 10 s, (95° C. for 10 s, 60° C. for 30 s, 72° C. for 4 min) ⁇ 35 times, 72° C. for 2 min.
- Amplify cex (SEQ ID NO: 5) by PCR using genomic DNA of Cellulomonas fimi NBRC15513 (obtained from National Institute of Technology and Evaluation) as a template and cex-F1 (SEQ ID NO: 22) and cex-R1 (SEQ ID NO: 23) as primers. bottom.
- the PCR conditions were 95° C. for 10 s, (95° C. for 10 s, 60° C. for 30 s, 72° C. for 4 min) ⁇ 35 times, 72° C. for 2 min.
- cenA (SEQ ID NO: 6) was amplified by PCR.
- the PCR conditions were 95° C. for 10 s, (95° C. for 10 s, 60° C. for 30 s, 72° C. for 4 min) ⁇ 35 times, 72° C. for 2 min.
- PCR amplified fragments were purified by gel extraction, mixed so that the purified products of each amplified fragment were equimolar, mixed with NEBuilder HiFi DNA Assembly Master Mix (NEB) so that they were equimolar, and incubated at 55° C. for 15 min. Incubate and obtain a reactant. Transformation of E. coli DH5 ⁇ was performed by the heat shock method using 5 ⁇ L of the reaction product. After completion of transformation, the transformed E. coli DH5 ⁇ was cultured on LB agar medium (containing 20 ⁇ g/mL tetracycline) to obtain a transformant having the sequence oprI-bglC-cex-cenA (SEQ ID NO: 8). rice field.
- the plasmid was extracted from the transformant using MagExtractor-Plasmid- (Toyobo), and electrophoresis was performed to obtain the target plasmid pTE105-oprI-bglC-cex-cenA (hereinafter also referred to as pTE105-oprI-bcc). It was confirmed.
- M. extorquens AM1 JCM 2805, obtained from RIKEN BioResource Research Center
- the transformants M.extorquens AM1 (pTE105) and M.extorquens AM1 (pTE105-oprI) were obtained by culturing on MP agar medium containing 1% by volume methanol (containing 20 ⁇ g/mL tetracycline). -bcc).
- the composition of the MP medium containing 1% by volume of methanol is ⁇ 1-2. It is the same as MP medium containing 1% by volume methanol used in Production of Recombinants>.
- M. extorquens AM1 (pTE105-oprI-bcc) was inoculated into 50 mL of MP medium containing 1% by volume methanol supplemented with 20 ⁇ g/mL tetracycline.
- M. extorquens AM1 wild strain was inoculated into a medium supplemented with 0.3 U/mL ⁇ -glucosidase (Toyobo) and 2 U/mL cellulase (Nacalai Tesque) in 50 mL of MP medium containing 1 vol% methanol. . After inoculating the medium with M.
- extorquens AM1 pTE105-oprI-bcc
- M. extorquens AM1 wild strain
- the medium was precultured at 30° C. and a shaking speed of 150 rpm for 5 days.
- the cells were washed with physiological saline three times.
- the cells were inoculated into 50 mL of a medium having the same composition as the pre-culture so that the culture turbidity (OD 600 ) at 600 nm was 0.1, and incubated at 30° C. and a shaking speed of 150 rpm for 10 or 13 days. cultured.
- 0.3 U/mL ⁇ -glucosidase (Toyobo) and 2 U/mL cellulase (Nacalai Tesque) were added to the culture medium every two days.
- Glucose was quantified using Glucose CII-Test Wako (Wako Pure Chemical Industries). After mixing 2 ⁇ L of the culture supernatant and 300 ⁇ L of the coloring reagent, the mixture was allowed to react at room temperature for 15 minutes, and the absorbance at a wavelength of 505 nm was measured. Methanol was quantified using a gas chromatograph GC-2025 system (Shimadzu Corporation). Column: MCI GEL CRS10W (Mitsubishi Chemical), detector: FID detector, mobile phase: nitrogen gas, flow rate: 0.5 mL/min, column temperature: 50°C, detector temperature: 50°C, inlet temperature: 250°C and The measurement results are shown in FIGS. 4 and 5.
- FIG. 4 and 5 The measurement results are shown in FIGS. 4 and 5.
- FIG. 4 shows the results using the wild strain
- Figure 5 shows the results using M.extorquens AM1 (pTE105-oprI-bcc). From FIG. 5, it was found that M. extorquens AM1 (pTE105-oprI-bcc) can produce glucose without adding ⁇ -glucosidase and cellulase. Furthermore, as shown in FIGS. 4 and 5, the wild strain increased in dry cell weight over time, but M. extorquens AM1 (pTE105-oprI-bcc) did not increase in cell weight. Therefore, it can be seen that M.
- extorquens AM1 (pTE105-oprI-bcc) has a higher glucose productivity (specific productivity) per cell than the wild strain, and can produce glucose at a high yield.
- M. extorquens AM1 (pTE105-oprI-bcc) in FIG. 5 consumes methanol in a shorter time and produces glucose in a shorter time compared to the wild strain in FIG. Therefore, it was found that M.extorquens AM1 (pTE105-oprI-bcc) is also superior in glucose production rate.
- the wild strain was inoculated into 50 mL of MP medium containing 1 vol% methanol, and the M. extorquens AM1 (pTE105) and M. extorquens AM1 (pTE105-oprI-bcc) were added to 50 mL of MP medium containing 1 vol% methanol (20 ⁇ g). /mL tetracycline added) and pre-cultured at 30°C, shaking speed 150 rpm for 5 days. After recovering the cells from the pre-cultured culture medium, the cells were washed with physiological saline three times.
- Cells were inoculated into 50 mL of the medium having the same composition as in the pre-culture so as to have a concentration of 1% by volume, and main culture was carried out at 30° C. and a shaking speed of 150 rpm for 5 days. After recovering the cells from 1 mL of the main culture medium, the cells were washed with physiological saline three times. After washing, the cells were resuspended in 1 mL of 100 mM sodium phosphate buffer (pH 7.0) or the buffer supplemented with 1 mg/mL proteinase K. The same cell suspension was used to measure the activity of three types of cellulase ( ⁇ -glucosidase, endoglucanase, exoglucanase).
- M. extorquens AM1 wild type was inoculated in 50 mL of MP medium containing 1 vol% methanol supplemented with 0.3 U/mL ⁇ -glucosidase (Toyobo) and 2 U/mL cellulase (Nacalai Tesque).
- M. extorquens AM1 pTE105-oprI-bcc was inoculated in 50 mL of MP medium containing 1% by volume methanol supplemented with 20 ⁇ g/mL tetracycline. After inoculating the medium with M. extorquens AM1 (wild type) or M.
- the medium was pre-cultured at 30° C. and a shaking speed of 150 rpm for 5 days. After recovering the cells from the pre-culture medium, the cells were washed with physiological saline three times and suspended in 50 mL of physiological saline. 50 mL of the medium having the same composition as the preculture was inoculated with the above cell suspension to 1% by volume, and main culture was carried out at 30° C. and a shaking speed of 150 rpm for 6 days. After completion of the culture, changes over time in the activity of each enzyme in the main culture medium were measured. The method for measuring each enzyme activity is as follows.
- the cell suspension or culture medium to which 300 ⁇ L of 40% potassium sodium tartrate aqueous solution was added was centrifuged, and the supernatant was collected.
- the amount of glucose produced was determined by measuring absorbance at a wavelength of 540 nm for the collected supernatant. Enzyme activity was calculated from the amount of glucose produced.
- the cell suspension or culture medium to which 300 ⁇ L of 40% potassium sodium tartrate aqueous solution was added was centrifuged, and the supernatant was collected.
- the amount of glucose produced was determined by measuring absorbance at a wavelength of 540 nm for the collected supernatant. Enzyme activity was calculated from the amount of glucose produced.
- FIG. 6 shows the results of cellulase activity measurement for the wild strain and each transformant.
- the amount of enzyme that produces 1 ⁇ mol of product per minute of reaction time was defined as 1 U.
- FIG. 7 shows the measurement results of changes in cellulase activity over time in the wild strain and M.extorquens AM1 (pTE105-oprI-bcc).
- the culture solution inoculated with cellulase-expressing M. extorquens AM1 showed clear activity for any cellulase.
- proteinase K was added, the cellulase activity of the culture medium decreased, indicating that each cellulase was expressed on the cell surface layer of M. extorquens AM1 (pTE105-oprI-bcc).
- the cellulase activity in the culture medium of the wild strain decreased over time. This is thought to be reduced by the protease produced by the wild-type strain.
- M.extorquens AM1 pTE105-oprI-bcc
- M. extorquens AM1 pTE105-oprI-bcc
- Example 3 Synthesis of Glucose Using an Assimilable Substrate] ⁇ 3-1. Production of glucose from substrates other than methanol> As substrates, 1% by mass of sodium L-malate, sodium L-lactate, disodium succinate, methylamine hydrochloride, or disodium fumarate, or 1% by volume of ethanol, and 20 ⁇ g/mL tetracycline were added. 50 mL of MP medium was inoculated with M. extorquens AM1 (pTE105-oprI-bcc).
- ⁇ -glucosidase with 1% by mass of sodium L-malate, sodium L-lactate, disodium succinate, methylamine hydrochloride, or disodium fumarate, or 1% by volume of ethanol
- M. extorquens AM1 wild strain was inoculated into 50 mL of MP medium supplemented with (Toyobo) and 2 U/mL cellulase (Nacalai Tesque).
- the medium inoculated with M. extorquens AM1 (pTE105-oprI-bcc) or M. extorquens AM1 (wild strain) was precultured at 30° C.
- FIG. 8 shows the results using the wild strain
- FIG. 9 shows the results using M. extorquens AM1 (pTE105-oprI-bcc). Note that descriptions of “sodium” and “hydrochloride” are omitted for each substrate in FIGS.
- graphs with the vertical axis representing Cell growth (OD 600 ) show the measurement results of the culture solution turbidity
- the culture medium turbidity and glucose concentration were sufficiently high when any substrate was used. Therefore, it was found that both the wild strain and M.extorquens AM1 (pTE105-oprI-bcc) are capable of producing glucose from substrates other than methanol.
- FIG. 10 is a schematic diagram of transformants produced in Example 4.
- Example 4 among the genes introduced into the transformant of Example 2, only the ⁇ -glucosidase gene was not introduced. As a result, the transformant of Example 4 produces cellobiose instead of glucose.
- pTE105-oprI-cex-cenA pTE105-oprI-cex-cenA
- pTE105-oprI-bcc cex-F2
- oprI-R2 SEQ ID NO: 26
- pTE105-oprI-cex-cenA was amplified by PCR.
- the PCR conditions were 95° C. for 10 s, (95° C. for 10 s, 70° C. for 30 s, 72° C. for 4 min) ⁇ 35 times, 72° C. for 2 min.
- This PCR-amplified fragment was purified by gel extraction, mixed with NEBuilder HiFi DNA Assembly Master Mix (NEB) in an equal volume, and incubated at 55° C. for 15 minutes. Transformation of E. coli DH5 ⁇ was performed by the heat shock method using 5 ⁇ L of the reaction product. After completion of transformation, the transformed E. coli DH5 ⁇ was cultured on LB agar medium (containing 20 ⁇ g/mL tetracycline) to obtain a transformant having the sequence of oprI-cex-cenA (SEQ ID NO: 9).
- the plasmid was extracted from the transformant using MagExtractor-Plasmid- (Toyobo), and electrophoresis was performed to identify the target plasmid as pTE105-oprI-cex-cenA (hereinafter also referred to as pTE105-oprI-cc). confirmed.
- extorquens AM1 (pTE105-oprI-cc) was inoculated into 50 mL of MP medium containing 1% by volume methanol (20 ⁇ g/mL tetracycline was added) and pre-cultured for 5 days at 30° C. and shaking speed of 150 rpm. After recovering the cells from the pre-cultured preculture, the cells were washed three times with physiological saline. The cells were inoculated into 50 mL of the MP medium so that the culture solution turbidity (OD 600 ) at 600 nm was 0.1, and main culture was carried out at 30° C. and a shaking speed of 150 rpm for 5 days.
- the cellobiose production amount was measured using the HPLC system Prominence (Shimadzu Corporation).
- the column was Aminex HPX-87H (Bio-Rad), the mobile phase was 5 mM sulfuric acid, the flow rate was 0.4 mL/min, the column oven temperature was 65°C, and an RI detector was used. The measurement results are shown in FIGS. 11 and 12.
- FIG. 11 The measurement results are shown in FIGS. 11 and 12.
- FIG. 11 when pTE105-oprI-cc was introduced, glucose was not detected from the culture medium, but cellobiose was detected.
- FIG. 12 also shows the presence of cellobiose in the culture medium. Therefore, it was shown that the transformant according to one embodiment of the present invention can produce not only glucose but also cellobiose.
- the present invention can be suitably used to produce cellobiose and glucose.
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Abstract
Description
本発明の一実施形態に係る形質転換体(以下、本形質転換体とも称する)は、セルロース合成微生物に、セルラーゼ遺伝子、および細胞表層タンパク質遺伝子が導入されてなる。
(2)配列番号1の塩基配列と90%以上の相同性を有し、かつ細胞表層タンパク質であるポリペプチドをコードするDNA;
(3)配列番号1の塩基配列と相補的な塩基配列からなる遺伝子とストリンジェントな条件でハイブリダイズし、かつ細胞表層タンパク質であるポリペプチドをコードするDNA;
(4)配列番号2のアミノ酸配列からなるポリペプチドをコードするDNA;
(5)配列番号2のアミノ酸配列と90%以上の同一性を有し、かつ細胞表層タンパク質であるポリペプチドをコードするDNA;
(6)配列番号2のアミノ酸配列において、1個以上のアミノ酸が欠失、置換、挿入または付加されたアミノ酸配列からなり、かつ細胞表層タンパク質であるポリペプチドをコードするDNA。
(2)配列番号5の塩基配列と90%以上の相同性を有し、エキソグルカナーゼ活性を有するタンパク質をコードするDNA;
(3)配列番号5の塩基配列からなる遺伝子とストリンジェントな条件でハイブリダイズし、かつエキソグルカナーゼ活性を有するタンパク質をコードするDNA;
(4)配列番号11のアミノ酸配列からなるポリペプチドをコードするDNA;
(5)配列番号11のアミノ酸配列と90%以上の同一性を有し、かつエキソグルカナーゼ活性を有するタンパク質をコードするDNA;
(6)配列番号11のアミノ酸配列において、1個以上のアミノ酸が欠失、置換、挿入または付加されたアミノ酸配列からなり、かつエキソグルカナーゼ活性を有するタンパク質をコードするDNA。
(2)配列番号6の塩基配列と90%以上の相同性を有し、エンドグルカナーゼ活性を有するタンパク質をコードするDNA;
(3)配列番号6の塩基配列からなる遺伝子とストリンジェントな条件でハイブリダイズし、かつエンドグルカナーゼ活性を有するタンパク質をコードするDNA;
(4)配列番号12のアミノ酸配列からなるポリペプチドをコードするDNA;
(5)配列番号12のアミノ酸配列と90%以上の同一性を有し、かつエンドグルカナーゼ活性を有するタンパク質をコードするDNA;
(6)配列番号12のアミノ酸配列において、1個以上のアミノ酸が欠失、置換、挿入または付加されたアミノ酸配列からなり、かつエンドグルカナーゼ活性を有するタンパク質をコードするDNA。
(2)配列番号4の塩基配列と90%以上の相同性を有し、β-グルコシダーゼ活性を有するタンパク質をコードするDNA;
(3)配列番号4の塩基配列からなる遺伝子とストリンジェントな条件でハイブリダイズし、かつβ-グルコシダーゼ活性を有するタンパク質をコードするDNA;
(4)配列番号10のアミノ酸配列からなるポリペプチドをコードするDNA;
(5)配列番号10のアミノ酸配列と90%以上の同一性を有し、かつβ-グルコシダーゼ活性を有するタンパク質をコードするDNA;
(6)配列番号10のアミノ酸配列において、1個以上のアミノ酸が欠失、置換、挿入または付加されたアミノ酸配列からなり、かつβ-グルコシダーゼ活性を有するタンパク質をコードするDNA。
本発明の一実施形態に係るグルコースまたはセロビオースの製造方法は、本形質転換体を当該形質転換体が資化可能な炭素源を用いて培養する培養工程を含む。前記製造方法によれば、セルラーゼ等の添加が不要であるため、従来よりも効率的にグルコースまたはセロビオースを製造することができる。また、本形質転換体は従来よりも細胞(菌体)当たりの生産性(比生産性)が高く、より収率良くグルコース、またはセロビオースを製造可能である。
本発明の一実施形態に係る組成物の製造方法は、前記製造方法によりグルコースまたはセロビオースを製造する工程を含む。すなわち、前記組成物は前記製造方法により得られたグルコースまたはセロビオースを含む。このような組成物としては例えば、グルコースまたはセロビオース、および本形質転換体を含む培養物、本形質転換体以外のグルコースまたはセロビオースを栄養源とする細菌、あるいは形質転換体を、本形質転換体と共培養することによって得られる培養物等が挙げられる。前記グルコースまたはセロビオースを含む組成物は、さらにメタノール、エタノール、L-リンゴ酸、L-乳酸、コハク酸、メチルアミン、フマル酸、ギ酸、酢酸、ベタイン等およびこれらの塩の資化可能な炭素源を含んでいてもよい。
本発明の一実施形態に係る物品の製造方法は、前記製造方法によりグルコースまたはセロビオースを製造する工程を含む。前記物品としては、グルコースまたはセロビオースを含む物品、またはグルコースまたはセロビオースを使用して製造される物品であれば特に限定されるものではない。前記物品としては、例えば、食料、飼料、微生物の栄養源、または化学品の原料などが挙げられる。
<その他>
<1>
セルロース合成微生物に、セルラーゼ遺伝子、および細胞表層タンパク質遺伝子が導入されてなる形質転換体。
<2>
前記セルロース合成微生物が、セルロース合成細菌である、<1>に記載の形質転換体。
<3>
前記セルロース合成細菌が、Methylobacteriaceae科細菌である、<2>に記載の形質転換体。
<4>
前記Methylobacteriaceae科細菌が、Methylorubrum属細菌である、<3>に記載の形質転換体。
<5>
前記Methylorubrum属細菌が、Methylorubrum extorquensである、<4>に記載の形質転換体。
<6>
前記細胞表層タンパク質遺伝子がoprI遺伝子である、<1>~<5>のいずれかに記載の形質転換体。
<7>
Methylobacteriaceae科細菌にoprI遺伝子が導入されてなる形質転換体。
<8>
前記Methylobacteriaceae科細菌が、Methylorubrum属細菌である、<7>に記載の形質転換体。
<9>
前記Methylorubrum属細菌が、Methylorubrum extorquensである、<8>に記載の形質転換体。
<10>
前記oprI遺伝子が、下記(1)~(6)からなる群より選択されるいずれかのDNAである、<6>~<9>のいずれかに記載の形質転換体。
(1)配列番号1の塩基配列からなるDNA;
(2)配列番号1の塩基配列と90%以上の相同性を有し、かつ細胞表層タンパク質であるポリペプチドをコードするDNA;
(3)配列番号1の塩基配列と相補的な塩基配列からなる遺伝子とストリンジェントな条件でハイブリダイズし、かつ細胞表層タンパク質であるポリペプチドをコードするDNA;
(4)配列番号2のアミノ酸配列からなるポリペプチドをコードするDNA;
(5)配列番号2のアミノ酸配列と90%以上の同一性を有し、かつ細胞表層タンパク質であるポリペプチドをコードするDNA;
(6)配列番号2のアミノ酸配列において、1個以上のアミノ酸が欠失、置換、挿入または付加されたアミノ酸配列からなり、かつ細胞表層タンパク質であるポリペプチドをコードするDNA。
<11>
さらにセルラーゼ遺伝子が導入されてなる、<7>~<10>のいずれかに記載の形質転換体。
<12>
前記セルラーゼ遺伝子として、エンドグルカナーゼ遺伝子およびエキソグルカナーゼ遺伝子から選択される少なくとも1種類を含む、<1>~<6>、<11>のいずれかに記載の形質転換体。
<13>
前記エキソグルカナーゼ遺伝子が下記(1)~(6)からなる群より選択される1種類以上のDNAである、<12>に記載の形質転換体。
(1)配列番号5の塩基配列からなるDNA;
(2)配列番号5の塩基配列と90%以上の相同性を有し、エキソグルカナーゼ活性を有するタンパク質をコードするDNA;
(3)配列番号5の塩基配列からなる遺伝子とストリンジェントな条件でハイブリダイズし、かつエキソグルカナーゼ活性を有するタンパク質をコードするDNA;
(4)配列番号11のアミノ酸配列からなるポリペプチドをコードするDNA;
(5)配列番号11のアミノ酸配列と90%以上の同一性を有し、かつエキソグルカナーゼ活性を有するタンパク質をコードするDNA;
(6)配列番号11のアミノ酸配列において、1個以上のアミノ酸が欠失、置換、挿入または付加されたアミノ酸配列からなり、かつエキソグルカナーゼ活性を有するタンパク質をコードするDNA。
<14>
前記エンドグルカナーゼ遺伝子が下記(1)~(6)からなる群より選択される1種類以上のDNAである、<12>または<13>に記載の形質転換体。
(1)配列番号6の塩基配列からなるDNA;
(2)配列番号6の塩基配列と90%以上の相同性を有し、エンドグルカナーゼ活性を有するタンパク質をコードするDNA;
(3)配列番号6の塩基配列からなる遺伝子とストリンジェントな条件でハイブリダイズし、かつエンドグルカナーゼ活性を有するタンパク質をコードするDNA;
(4)配列番号12のアミノ酸配列からなるポリペプチドをコードするDNA;
(5)配列番号12のアミノ酸配列と90%以上の同一性を有し、かつエンドグルカナーゼ活性を有するタンパク質をコードするDNA;
(6)配列番号12のアミノ酸配列において、1個以上のアミノ酸が欠失、置換、挿入または付加されたアミノ酸配列からなり、かつエンドグルカナーゼ活性を有するタンパク質をコードするDNA。
<15>
前記セルラーゼ遺伝子として、β-グルコシダーゼ遺伝子をさらに含む、<12>~<14>のいずれかに記載の形質転換体。
<16>
前記β-グルコシダーゼ遺伝子が下記(1)~(6)からなる群より選択される1種類以上のDNAである、<15>に記載の形質転換体。
(1)配列番号4の塩基配列からなるDNA;
(2)配列番号4の塩基配列と90%以上の相同性を有し、β-グルコシダーゼ活性を有するタンパク質をコードするDNA;
(3)配列番号4の塩基配列からなる遺伝子とストリンジェントな条件でハイブリダイズし、かつβ-グルコシダーゼ活性を有するタンパク質をコードするDNA;
(4)配列番号10のアミノ酸配列からなるポリペプチドをコードするDNA;
(5)配列番号10のアミノ酸配列と90%以上の同一性を有し、かつβ-グルコシダーゼ活性を有するタンパク質をコードするDNA;
(6)配列番号10のアミノ酸配列において、1個以上のアミノ酸が欠失、置換、挿入または付加されたアミノ酸配列からなり、かつβ-グルコシダーゼ活性を有するタンパク質をコードするDNA。
<17>
<1>~<16>のいずれかに記載の形質転換体を、当該形質転換体が資化可能な炭素源を用いて培養する培養工程を含む、グルコースまたはセロビオースの製造方法。
<18>
<17>に記載の製造方法によりグルコースまたはセロビオースを製造する工程を含む、物品の製造方法。
<19>
<17>に記載の製造方法により得られたグルコースまたはセロビオースを含む組成物。
図1は実施例1において作製した形質転換体の模式図である。図1に示すように、実施例1ではoprI遺伝子1とGFP遺伝子20とを結合させた遺伝子をセルロース合成細菌に導入した形質転換体を作製した。当該形質転換体においては、oprIタンパク質11によってGFP21が菌体表層に発現している。以下に、当該形質転換体の作製方法、および当該形質転換体を用いた試験結果を具体的に記載する。
(pTE105-gfp)
鋳型にpTE105(Addgeneから入手)、プライマーにpTE105-KpnI-F1(配列番号13)とpTE105-SpeI-R1(配列番号14)を用いてpTE105をPCRにより増幅した。PCR条件は95℃を10s、(95℃を10s、70℃を30s、72℃を4min)×35回、72℃を2minとした。鋳型としてpGreenTIR(Gene 191(2):149-153 (1997))、プライマーとしてgfp-SpeI-RBS-F1(配列番号15)とgfp-KpnI-R1(配列番号16)とを用いてgfp(配列番号3)をPCRにより増幅した。PCR条件は95℃を10s、(95℃を10s、70℃を30s、72℃を4min)×35回、72℃を2minとした。これらのPCR増幅断片をゲル抽出により精製し、各増幅断片の精製物が等モルとなるように混合後、NEBuilder HiFi DNA Assembly Master Mix(NEB)と等量になるように混ぜて55℃、15minインキュベーションし、反応物を得た。得られた反応物を5μL用いて、ヒートショック法にてEscherichia coli DH5αの形質転換を行った。形質転換終了後、LB寒天培地(20μg/mLテトラサイクリン添加)にて形質転換させたEscherichia coli DH5αを培養し、pTE105-gfp(配列番号3)の配列を有する形質転換体を得た。MagExtractor -Plasmid-(Toyobo)を用いて形質転換体からプラスミドを抽出し、電気泳動によって目的のプラスミドのpTE105-gfpであることを確認した。
鋳型にpTE105(Addgeneから入手)、プライマーにpTE105-KpnI-F1(配列番号13)とpTE105-SpeI-R1(配列番号14)を用いてpTE105をPCRにより増幅した。PCR条件は95℃を10s、(95℃を10s、70℃を30s、72℃、4min)×35回、72℃、2minとした。鋳型にpVUB3(Gene 221:25-34(1998))、プライマーにoprI-SpeI-RBS-F1(配列番号17)とoprI-R1(配列番号18)とを用いてoprI(配列番号1)をPCRにより増幅した。PCR条件は95℃を10s、(95℃を10s、60℃を30s、72℃を4min)×35回、72℃を2minとした。鋳型としてpGreenTIR(Gene 191:149-153 (1997))、プライマーとしてgfp-F2(配列番号19)とgfp-KpnI-R1(配列番号16)を用いてgfp(配列番号3)をPCRにより増幅した。PCR条件は95℃を10s、(95℃を10s、70℃を30s、72℃を4min)×35回、72℃を2minとした。これらのPCR増幅断片をゲル抽出により精製し、各増幅断片の精製物が等モルとなるように混合後、NEBuilder HiFi DNA Assembly Master Mix(NEB)と等量になるように混ぜて55℃、15minインキュベーションし、反応物を得た。5μLの反応物を用いてヒートショック法にてE. coli DH5αの形質転換を行った。形質転換終了後、LB寒天培地(20μg/mLテトラサイクリン添加)にて形質転換させたE.coli DH5αを培養し、pTE105-oprI-gfp(配列番号7)の配列を有する形質転換体を得た。MagExtractor-Plasmid-(Toyobo)を用いて形質転換体からプラスミドを抽出し、電気泳動によって目的のプラスミドのpTE105-oprI-gfpであることを確認した。
Methylorubrum extorquens AM1(JCM 2805、理化学研究所バイオリソース研究センターから入手)に対して、前記pTE105、<1-1.プラスミドの構築>において構築したpTE105-gfp、またはpTE105-oprI-gfpをそれぞれ用いて形質転換を行った。形質転換はエレクトロポレーション法(FEMS microbiol. Lett.166(1):1-7(1998))により行った。形質転換終了後、1容量%メタノール入りMP寒天培地(20μg/mLテトラサイクリン添加)にて培養を行うことにより、形質転換体であるMethylorubrum extorquens AM1(pTE105)、M.extorquens AM1(pTE105-gfp)、およびM.extorquens AM1(pTE105-oprI-gfp)を得た。得られた各形質転換体からプラスミドをMagExtractor -Plasmid-(Toyobo)を用いて抽出し、電気泳動によって目的のプラスミドのpTE105-gfp、あるいはpTE105-oprI-gfpであることを確認した。なお、前記1容量%メタノール入りMP培地の組成は以下の通りである。1Lあたり:10mL メタノール、9.1g PIPES、0.3g K2HPO4、0.3g NaH2PO4・2H2O、0.1g MgCl2・6H2O、2.6g (NH4)2SO4、2.2mg CaCl2、13.4mg クエン酸ナトリウム・2H2O、0.3mg ZnSO4・7H2O、0.2mg MnCl2・4H2O、5mg FeSO4・7H2O、2.5mg (NH4)6Mo7O24・4H2O、0.3mg CuSO4・5H2O、0.5mg CoCl2・6H2O、0.1mg Na2WO4・2H2O)(PLoS ONE 8(4):e62957(2013))。
前記<1-2.遺伝子組み換え体の作製>において得られた形質転換体であるMethylorubrum extorquens AM1(pTE105)、M.extorquens AM1(pTE105-gfp)、M.extorquens AM1(pTE105-oprI-gfp)をそれぞれ、50mLの1容量%メタノール入りMP培地(20μg/mLテトラサイクリン、0.3U/mLのβ-グルコシダーゼ(Toyobo)、2U/mLのセルラーゼ(ナカライテスク)を添加)に接種し、30℃、振盪速度150rpmにて、5日間前培養した。前培養した培養液から菌体を回収した後、生理食塩水を用いて菌体を3回洗浄した。50mLの前記MP培地に前記菌体を、波長600nmにおける培養液濁度(OD600)が0.1となるように接種し、30℃、振盪速度150rpmにて、5日間本培養した。1mLの本培養液から菌体を回収した後、生理食塩水を用いて菌体を3回洗浄した。洗浄後、1mLの100mMリン酸ナトリウム緩衝液(pH7.0)、または1mg/mLプロテイナーゼKを添加した当該緩衝液に菌体を再懸濁した。37℃、18hインキュベーションした後、菌体を回収し、生理食塩水を用いて菌体を3回洗浄した。得られた集菌体を1mLの100mMリン酸ナトリウム緩衝液(pH7.0)に再懸濁した。蛍光光度計FP6500(日本分光)を用いて、励起波長490nmと蛍光波長510nmにて各菌体懸濁液の蛍光強度を調べた。結果を図2に示す。
図3は実施例2において作製した形質転換体の模式図である。図3に示すように、実施例2においては、oprI遺伝子1と、セルラーゼ発現遺伝子であるβ-グルコシダーゼ遺伝子2、エキソグルカナーゼ遺伝子3、エンドグルカナーゼ遺伝子4とをリンカー5を介して結合させた遺伝子をセルロース合成細菌に導入した形質転換体を作製した。当該形質転換体においては、oprIタンパク質11によってβ-グルコシダーゼ12、エキソグルカナーゼ13、エンドグルカナーゼ14が菌体表層に発現している。そのため、当該形質転換体によって菌体内部のグルコースから生産されたセルロースは、菌体表層に発現させたセルラーゼによって分解され、菌体外にグルコースが生産される。以下に、当該形質転換体の作製方法、および当該形質転換体を用いた試験結果を具体的に記載する。
(pTE105-oprI-bglC-cex-cenA)
鋳型として実施例1において作製したpTE105-oprI-gfp、プライマーとしてpTE105-KpnI-F1(配列番号13)とoprI-R2(配列番号26)とを用いてpTE105-oprIをPCRにより増幅した。PCR条件は95℃を10s、(95℃を10s、70℃を30s、72℃を4min)×35回、72℃を2minとした。鋳型にpGV3-bglC(J.Biosci.Bioeng.127(4):441-446(2019))、プライマーにbglC-F1(配列番号20)とbglC-R1(配列番号21)を用いてbglC(配列番号4)をPCRにより増幅した。PCR条件は95℃を10s、(95℃を10s、60℃を30s、72℃を4min)×35回、72℃を2minとした。鋳型としてCellulomonas fimi NBRC15513(製品評価技術基盤機構より入手)のゲノムDNA、プライマーとしてcex-F1(配列番号22)とcex-R1(配列番号23)を用いてcex(配列番号5)をPCRにより増幅した。PCR条件は95℃を10s、(95℃を10s、60℃を30s、72℃を4min)×35回、72℃を2minとした。鋳型にC. fimi NBRC15513のゲノムDNA、プライマーにcenA-F1(配列番号24)とcenA-R1(配列番号25)を用いてcenA(配列番号6)をPCRにより増幅した。PCR条件は95℃を10s、(95℃を10s、60℃を30s、72℃を4min)×35回、72℃を2minとした。これらのPCR増幅断片をゲル抽出により精製し、各増幅断片の精製物が等モルとなるように混合後、NEBuilder HiFi DNA Assembly Master Mix(NEB)と等量になるように混ぜ、55℃、15minインキュベーションし、反応物を得た。5μLの反応物を用いてヒートショック法にてE. coli DH5αの形質転換を行った。形質転換終了後、LB寒天培地(20μg/mLテトラサイクリン添加)にて形質転換させたE.coli DH5αを培養し、oprI-bglC-cex-cenA(配列番号8)の配列を有する形質転換体を得た。MagExtractor -Plasmid-(Toyobo)を用いて形質転換体からプラスミドを抽出し、電気泳動によって目的のプラスミドのpTE105-oprI-bglC-cex-cenA(以下、pTE105-oprI-bccとも記載する。)であることを確認した。
M.extorquens AM1(JCM 2805、理化学研究所バイオリソース研究センターから入手)に対して、前記pTE105、または<2-1.プラスミドの構築>において構築したpTE105-oprI-bccをそれぞれ用いて形質転換を行った。形質転換はエレクトロポレーション法(FEMS microbiol.Lett.166(1):1-7(1998))により行った。形質転換終了後、1容量%メタノール入りMP寒天培地(20μg/mLテトラサイクリン添加)にて培養を行うことにより、形質転換体であるM.extorquens AM1 (pTE105)、およびM.extorquens AM1 (pTE105-oprI-bcc)を得た。なお、前記1容量%メタノール入りMP培地の組成は<1-2.遺伝子組み換え体の作製>において使用した1容量%メタノール入りMP培地と同一である。
M.extorquens AM1(pTE105-oprI-bcc)を50mLの1容量%メタノール入りMP培地に20μg/mLテトラサイクリンを添加した培地に接種した。また、M.extorquens AM1(野生株)を50mLの1容量%メタノール入りMP培地に0.3U/mLのβ-グルコシダーゼ(Toyobo)および2U/mLのセルラーゼ(ナカライテスク)を添加した培地に接種した。M.extorquens AM1(pTE105-oprI-bcc)あるいはM.extorquens AM1(野生株)を培地に接種した後、30℃、振盪速度150rpm、5日間前培養した。前培養した培養液から菌体を回収した後、生理食塩水を用いて菌体を3回洗浄した。50mLの前培養時と同じ組成の培地に前記菌体を、600nmにおける培養液濁度(OD600)が0.1となるように接種し、30℃、振盪速度150rpm、10日間あるいは13日間本培養した。ただし、野生株に対しては2日おきに、培養液に0.3U/mLのβ-グルコシダーゼ(Toyobo)と2U/mLのセルラーゼ(ナカライテスク)を添加した。
前記野生株を50mLの1容量%メタノール入りMP培地に接種し、前記M.extorquens AM1(pTE105)、およびM.extorquens AM1 (pTE105-oprI-bcc)を50mLの1容量%メタノール入りMP培地(20μg/mLテトラサイクリン添加)に接種し、30℃、振盪速度150rpm、5日間前培養した。前培養した培養液から菌体を回収した後、生理食塩水を用いて菌体を3回洗浄した。50mLの前培養時と同じ組成の培地に菌体を濃度1容量%となるように接種し、30℃、振盪速度150rpm、5日間本培養した。1mLの本培養液から菌体を回収した後、生理食塩水を用いて菌体を3回洗浄した。洗浄後、1mLの100mMリン酸ナトリウム緩衝液(pH7.0)、または1mg/mLのプロテイナーゼKを添加した当該緩衝液に菌体を再懸濁した。3種類のセルラーゼ(β-グルコシダーゼ、エンドグルカナーゼ、エキソグルカナーゼ)の活性測定には同じ菌体懸濁液を用いた。
50mMリン酸ナトリウム緩衝液(pH7.0)に溶解した500μLの10mM p-ニトロフェニル-β-D-グルコピラノシドを、10μLの菌体懸濁液もしくは培養液に添加し、30℃でインキュベーションした。インキュベーション後、菌体懸濁液もしくは培養液を100μL分取して、速やかに1mLの2M炭酸ナトリウム水溶液を加えて反応を停止させた。反応停止後、菌体懸濁液もしくは培養液を遠心分離して、上清を採取した。採取した上清について、波長400nmの吸光度を測定することによって、生成したp-ニトロフェノール量を求めた。p-ニトロフェノールの400nmでのモル吸光係数を18.5mM-1として酵素活性を算出した。
50mMリン酸ナトリウム緩衝液(pH7.0)に溶解した100μLの1%カルボキシルメチルセルロースを100μLの菌体懸濁液もしくは培養液に加えて、30℃にてインキュベーションした。インキュベーション後、菌体懸濁液もしくは培養液を100μL分取して、速やかに1%3,5-ジニトロサリチル酸溶液(10g/L、3,5-ジニトロサリチル酸溶液、10g/L水酸化ナトリウム、2g/Lフェノール、0.5g/L硫酸水素ナトリウム)を300μL添加した。添加後、菌体懸濁液もしくは培養液を100℃、15min加熱し反応を停止させた。反応停止後、300μLの40%酒石酸カリウムナトリウム水溶液を加えた菌体懸濁液もしくは培養液を遠心分離し、上清を採取した。採取した上清について、波長540nmの吸光度を測定することによって、生成したグルコース量を求めた。生成したグルコース量から酵素活性を算出した。
50mMリン酸ナトリウム緩衝液(pH7.0)に溶解した100μLの1%微結晶セルロース(Avicel PH-101)を100μLの菌体懸濁液もしくは培養液に加えて、30℃でインキュベーションした。インキュベーション後、菌体懸濁液もしくは培養液を100μL分取して、速やかに1%3,5-ジニトロサリチル酸溶液(10g/L3,5-ジニトロサリチル酸、10g/L水酸化ナトリウム、2g/Lフェノール、0.5g/L硫酸水素ナトリウム)を300μL添加した。添加後、菌体懸濁液もしくは培養液を100℃、15minで加熱し反応を停止させた。反応停止後、300μLの40%酒石酸カリウムナトリウム水溶液を加えた菌体懸濁液もしくは培養液を遠心分離し、上清を採取した。採取した上清について、波長540nmの吸光度を測定することにより、生成したグルコース量を求めた。生成したグルコース量から酵素活性を算出した。
<3-1.メタノール以外の基質からのグルコースの生産>
基質として、L-リンゴ酸ナトリウム、L-乳酸ナトリウム、コハク酸二ナトリウム、メチルアミン塩酸塩、もしくはフマル酸二ナトリウムを1質量%、またはエタノールを1容量%と、20μg/mLテトラサイクリンとを添加した50mLのMP培地に、M.extorquens AM1(pTE105-oprI-bcc)を接種した。また、L-リンゴ酸ナトリウム、L-乳酸ナトリウム、コハク酸二ナトリウム、メチルアミン塩酸塩、もしくはフマル酸二ナトリウムを1質量%、またはエタノールを1容量%と、0.3U/mLのβ-グルコシダーゼ(Toyobo)および2U/mLのセルラーゼ(ナカライテスク)とを添加した50mLのMP培地に、M.extorquens AM1(野生株)を接種した。M.extorquens AM1(pTE105-oprI-bcc)あるいはM.extorquens AM1(野生株)を接種した前記培地を30℃、振盪速度150rpm、5日間前培養した。前培養した培養液から菌体を回収した後、生理食塩水を用いて菌体を3回洗浄し、50mLの生理食塩水に懸濁した。50mLの前培養時と同じ組成の培地に上記菌体懸濁液を1容量%となるように接種し、30℃、振盪速度150rpm、5日間本培養した。グルコースの定量は実施例2と同様に行った。測定結果を図8および図9に示す。
図10は実施例4において作製した形質転換体の模式図である。図10に示すように、実施例4においては、実施例2の形質転換体に導入した遺伝子のうち、β-グルコシダーゼ遺伝子のみを導入しなかった。その結果、実施例4の形質転換体からは、グルコースではなくセロビオースが製造される。
(pTE105-oprI-cex-cenA)
鋳型として前記pTE105-oprI-bcc、プライマーとしてcex-F2(配列番号27)とoprI-R2(配列番号26)とを用いてpTE105-oprI-cex-cenAをPCRにより増幅した。PCR条件は95℃を10s、(95℃を10s、70℃を30s、72℃を4min)×35回、72℃を2minとした。このPCR増幅断片をゲル抽出により精製し、NEBuilder HiFi DNA Assembly Master Mix(NEB)と等量になるように混ぜ、55℃、15minインキュベーションした。5μLの反応物を用いてヒートショック法にてE. coli DH5αの形質転換を行った。形質転換終了後、LB寒天培地(20μg/mLテトラサイクリン添加)にて形質転換させたE.coli DH5αを培養し、oprI-cex-cenA(配列番号9)の配列を有する形質転換体を得た。MagExtractor-Plasmid-(Toyobo)を用いて形質転換体からプラスミドを抽出し、電気泳動によって目的のプラスミドのpTE105-oprI-cex-cenA(以下、pTE105-oprI-ccとも記載する。)であることを確認した。
M.extorquens AM1(JCM 2805、理化学研究所バイオリソース研究センターから入手)に対して、<4-1.プラスミドの構築>において構築したプラスミドを用いて形質転換を行った。形質転換はエレクトロポレーション法(FEMS microbiol.Lett.166(1):1-7(1998))により行った。形質転換終了後、1容量%メタノール入りMP寒天培地(20μg/mLテトラサイクリン添加)にて培養し、形質転換体であるM.extorquens AM1(pTE105-oprI-cc)を得た。なお、前記1容量%メタノール入りMP培地の組成は<1-2.遺伝子組み換え体の作製>において使用した1容量%メタノール入りMP培地と同一である。
M.extorquens AM1(pTE105-oprI-cc)を、50mLの1容量%メタノール入りMP培地(20μg/mLテトラサイクリンを添加)に接種し、30℃、振盪速度150rpmにて、5日間前培養した。前培養した前培養液から菌体を回収した後、生理食塩水を用いて菌体を3回洗浄した。50mLの前記MP培地に前記菌体を、600nmにおける培養液濁度(OD600)が0.1となるように接種し、30℃、振盪速度150rpmにて、5日間本培養した。
2 β-グルコシダーゼ遺伝子
3 エキソグルカナーゼ遺伝子
4 エンドグルカナーゼ遺伝子
5 リンカー
11 oprIタンパク質
12 β-グルコシダーゼ
13 エキソグルカナーゼ
14 エンドグルカナーゼ
20 GFP遺伝子
21 GFP
Claims (19)
- セルロース合成微生物に、セルラーゼ遺伝子、および細胞表層タンパク質遺伝子が導入されてなる形質転換体。
- 前記セルロース合成微生物が、セルロース合成細菌である、請求項1に記載の形質転換体。
- 前記セルロース合成細菌が、Methylobacteriaceae科細菌である、請求項2に記載の形質転換体。
- 前記Methylobacteriaceae科細菌が、Methylorubrum属細菌である、請求項3に記載の形質転換体。
- 前記Methylorubrum属細菌が、Methylorubrum extorquensである、請求項4に記載の形質転換体。
- 前記細胞表層タンパク質遺伝子がoprI遺伝子である、請求項1に記載の形質転換体。
- Methylobacteriaceae科細菌にoprI遺伝子が導入されてなる形質転換体。
- 前記Methylobacteriaceae科細菌が、Methylorubrum属細菌である、請求項7に記載の形質転換体。
- 前記Methylorubrum属細菌が、Methylorubrum extorquensである、請求項8に記載の形質転換体。
- 前記oprI遺伝子が、下記(1)~(6)からなる群より選択されるいずれかのDNAである、請求項6または7に記載の形質転換体。
(1)配列番号1の塩基配列からなるDNA;
(2)配列番号1の塩基配列と90%以上の相同性を有し、かつ細胞表層タンパク質であるポリペプチドをコードするDNA;
(3)配列番号1の塩基配列と相補的な塩基配列からなる遺伝子とストリンジェントな条件でハイブリダイズし、かつ細胞表層タンパク質であるポリペプチドをコードするDNA;
(4)配列番号2のアミノ酸配列からなるポリペプチドをコードするDNA;
(5)配列番号2のアミノ酸配列と90%以上の同一性を有し、かつ細胞表層タンパク質であるポリペプチドをコードするDNA;
(6)配列番号2のアミノ酸配列において、1個以上のアミノ酸が欠失、置換、挿入または付加されたアミノ酸配列からなり、かつ細胞表層タンパク質であるポリペプチドをコードするDNA。 - さらにセルラーゼ遺伝子が導入されてなる、請求項7に記載の形質転換体。
- 前記セルラーゼ遺伝子として、エンドグルカナーゼ遺伝子およびエキソグルカナーゼ遺伝子から選択される少なくとも1種類を含む、請求項1または11に記載の形質転換体。
- 前記エキソグルカナーゼ遺伝子が下記(1)~(6)からなる群より選択される1種類以上のDNAである、請求項12に記載の形質転換体。
(1)配列番号5の塩基配列からなるDNA;
(2)配列番号5の塩基配列と90%以上の相同性を有し、エキソグルカナーゼ活性を有するタンパク質をコードするDNA;
(3)配列番号5の塩基配列からなる遺伝子とストリンジェントな条件でハイブリダイズし、かつエキソグルカナーゼ活性を有するタンパク質をコードするDNA;
(4)配列番号11のアミノ酸配列からなるポリペプチドをコードするDNA;
(5)配列番号11のアミノ酸配列と90%以上の同一性を有し、かつエキソグルカナーゼ活性を有するタンパク質をコードするDNA;
(6)配列番号11のアミノ酸配列において、1個以上のアミノ酸が欠失、置換、挿入または付加されたアミノ酸配列からなり、かつエキソグルカナーゼ活性を有するタンパク質をコードするDNA。 - 前記エンドグルカナーゼ遺伝子が下記(1)~(6)からなる群より選択される1種類以上のDNAである、請求項12に記載の形質転換体。
(1)配列番号6の塩基配列からなるDNA;
(2)配列番号6の塩基配列と90%以上の相同性を有し、エンドグルカナーゼ活性を有するタンパク質をコードするDNA;
(3)配列番号6の塩基配列からなる遺伝子とストリンジェントな条件でハイブリダイズし、かつエンドグルカナーゼ活性を有するタンパク質をコードするDNA;
(4)配列番号12のアミノ酸配列からなるポリペプチドをコードするDNA;
(5)配列番号12のアミノ酸配列と90%以上の同一性を有し、かつエンドグルカナーゼ活性を有するタンパク質をコードするDNA;
(6)配列番号12のアミノ酸配列において、1個以上のアミノ酸が欠失、置換、挿入または付加されたアミノ酸配列からなり、かつエンドグルカナーゼ活性を有するタンパク質をコードするDNA。 - 前記セルラーゼ遺伝子として、β-グルコシダーゼ遺伝子をさらに含む、請求項12に記載の形質転換体。
- 前記β-グルコシダーゼ遺伝子が下記(1)~(6)からなる群より選択される1種類以上のDNAである、請求項15に記載の形質転換体。
(1)配列番号4の塩基配列からなるDNA;
(2)配列番号4の塩基配列と90%以上の相同性を有し、β-グルコシダーゼ活性を有するタンパク質をコードするDNA;
(3)配列番号4の塩基配列からなる遺伝子とストリンジェントな条件でハイブリダイズし、かつβ-グルコシダーゼ活性を有するタンパク質をコードするDNA;
(4)配列番号10のアミノ酸配列からなるポリペプチドをコードするDNA;
(5)配列番号10のアミノ酸配列と90%以上の同一性を有し、かつβ-グルコシダーゼ活性を有するタンパク質をコードするDNA;
(6)配列番号10のアミノ酸配列において、1個以上のアミノ酸が欠失、置換、挿入または付加されたアミノ酸配列からなり、かつβ-グルコシダーゼ活性を有するタンパク質をコードするDNA。 - 請求項1または11に記載の形質転換体を、当該形質転換体が資化可能な炭素源を用いて培養する培養工程を含む、グルコースまたはセロビオースの製造方法。
- 請求項17に記載の製造方法によりグルコースまたはセロビオースを製造する工程を含む、物品の製造方法。
- 請求項17に記載の製造方法により得られたグルコースまたはセロビオースを含む組成物。
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