JP2013165673A - Extracellular expression enhancer of sugar hydrolase and use of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an enhancer of cell surface presentation or extracellular secretion of a hydrolase for obtaining the hydrolase suitable for the cell surface presentation or extracellular secretion in a eukaryotic cell.SOLUTION: In an enhancer of extracellular expression of a sugar hydrolase in a eukaryotic cell, a peptide having a specific amino acid sequence or one or more amino acid sequences that have 55% or higher identity with the amino acid sequence is used by fusing to the sugar hydrolase.

Description

  The disclosure of the present specification relates to an enhancer of extracellular expression of sugar hydrolase and use thereof.

  At present, various attempts have been made to use biomass raw materials as resources. For example, there is CBP (Consolidated Bioprocessing). CBP aims at producing a useful substance by saccharifying a biomass raw material and fermenting the saccharified product with yeast or the like.

  In this case, it is necessary to secrete a large amount of a sugar hydrolase having a high sugar-degrading activity in yeast or to present it on the cell surface. However, it is said that it is difficult to secrete foreign proteins out of the cells or present them on the cell surface in yeast. From such a background, for example, it is described that the secretion of the lipase is promoted by fusing the cellulose binding domain (CBD) of endoglucanase II of Trichoderma harzianum and a linker (Non-patent Document 1). .

J. K. Jun et al., Appl. Microbiol. Biotechnol (2004) 64, 833-839

  Non-patent document 1 mentions that both the CBD of the endoglucanase of T. harzianum and the linker may promote secretion of the fusion protein, and that these combinations are useful for promoting secretion in S. cerevisiae. .

  However, there is no further description in Non-Patent Document 1, and there is no description or suggestion about further improvement in secretion.

  Accordingly, the disclosure of the present specification provides an enhancer of extracellular expression of a sugar hydrolase and a use thereof that can obtain a sugar hydrolase suitable for extracellular expression in a eukaryotic cell. One purpose.

  The inventors of the present invention have tried various modifications for sugar hydrolase, and found that the addition of a certain amino acid sequence enhances the extracellular expression level of sugar hydrolase in eukaryotic cells. Got. Moreover, the knowledge that the effect | action increases further by multiply adding such an amino acid sequence was acquired. The disclosure herein is provided based on these findings.

  According to the disclosure of the present specification, an enhancer of extracellular expression of a sugar hydrolase in a eukaryotic cell is provided. This enhancer is a peptide fused to the sugar hydrolase, and the first amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 55% or more identity with this amino acid sequence. A peptide having one or more.

  The enhancer may have two or more of the first amino acid sequences, and the first amino acid sequence is a linker sequence between a cellulose-binding domain and a catalytic domain in a Trichoderma endoglucanase. May be. Furthermore, the sugar hydrolase may be a cellulase, and the cellulase may be an endoglucanase or a cellobiohydrolase. Further, the eukaryotic cell may be a yeast.

  According to the disclosure of the present specification, an enhancer of extracellular expression of a sugar hydrolase in a eukaryotic cell is provided. This enhancer is a peptide fused to the sugar hydrolase, and the first amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 55% or more identity with this amino acid sequence. A polynucleotide encoding a peptide having one or more.

  According to the disclosure herein, fusion proteins are also provided. This fusion protein comprises an amino acid sequence represented by SEQ ID NO: 1 or a peptide having one or more first amino acid sequences that are 55% or more identical to this amino acid sequence, a sugar hydrolase, It is equipped with.

  In the fusion protein, the sugar hydrolase comprises a substrate binding domain and a catalytic domain, and one or more of the first amino acid sequences are provided between the substrate binding domain and the catalytic domain. it can.

  According to the disclosure herein, a polynucleotide is also provided. This polynucleotide encodes the fusion protein.

  According to the disclosure of the present specification, a eukaryotic cell that retains DNA encoding the fusion protein and is capable of expressing the fusion protein extracellularly is provided.

  According to the disclosure of the present specification, there is provided a method for producing a sugar hydrolase, wherein the eukaryotic cell is cultured to express the fusion protein extracellularly.

  According to the disclosure of the present specification, the first amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 55% or more identity with this amino acid sequence is represented by 1 Alternatively, a method of modifying the extracellular expression of the sugar hydrolase by providing two or more is provided.

  According to the disclosure of the present specification, one or two or more linker sequences selected from the linker sequences of the substrate binding domain and the catalytic domain of the same or different type of sugar hydrolase with respect to the sugar hydrolase A step of preparing a eukaryotic cell capable of extracellularly expressing a test fusion protein fused with a test amino acid sequence having one or two or more, and a step of measuring the extracellular expression level of the test fusion protein by the prepared eukaryotic cell And a method for screening for sugar hydrolase.

  According to the disclosure of the present specification, there is provided a method for producing a degradation product of cellulose, which is a cellulase, an amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having 55% identity or more with this amino acid sequence. Holding a DNA encoding a fusion protein with a peptide having one or more amino acid sequences, and contacting a cellulase produced by a eukaryotic cell capable of extracellular expression of the fusion protein with cellulose. A production method is provided.

  According to the disclosure of the present specification, the first amino acid is a method for producing a useful substance, which is an amino acid sequence represented by SEQ ID NO: 1 with cellulase or an amino acid sequence having 55% or more identity with this amino acid sequence. A eukaryotic cell that retains DNA encoding a fusion protein with a peptide having one or more sequences and is capable of expressing the fusion protein extracellularly and having the ability to produce the useful substance in the presence of cellulose. A production method comprising the step of culturing.

It is a figure which shows the outline | summary of the multiple connection method of a linker. It is a figure which shows the secretory expression vector in yeast. It is a figure which shows a ThCBD1-PcCD secretion expression vector. It is a figure which shows the ThCBD1-PcCD fragment which connected one or more linkers. It is a figure which shows the PSC degradation activity evaluation result of the yeast culture supernatant transformed with the ThCBD1-PcCD fragment which connected one or more linkers. It is a figure which shows the evaluation result of the secretory expression level in the yeast transformed with the ThCBD1-PcCD fragment which connected one or more linkers. It is a figure which shows a ThCBD1-TrEG2 secretion expression vector. It is a figure which shows the ThCBD1-TrEG2 fragment which linked one or more linkers. It is a figure which shows the PSC degradation activity evaluation result of the yeast culture supernatant transformed with the ThCBD1-TrEG2 fragment which connected one or more linkers. It is a figure which shows the evaluation result of the secretory expression level in the yeast transformed with the ThCBD1-TrEG2 fragment which connected one or more linkers. It is a figure which shows the change of the secretory expression level by linker multiplexing of wild type PcCBH2 and wild type TrEG2, and the evaluation result of PSC degradation activity. It is a figure which shows the comparison result of the amino acid sequence of the linker of wild type ThEG2 and wild type TrEG2. It is a figure which shows the homology search result by the BLAST analysis of the linker of wild type ThEG2. FIG. 3 shows a ThCBD1-TvLinker-PcCD fragment into which a ThCBD1-PcCD fragment and Trichoderma viride endoglucanase II linker are inserted. It is a figure which shows the evaluation result of the PSC degradation activity in the yeast each transformed with the ThCBD1-TvLinker-PcCD fragment which inserted the ThCBD1-PcCD fragment and the endoglucanase II linker of Trichoderma viride.

  The disclosure of the present specification relates to an agent for enhancing extracellular expression of a sugar hydrolase in a eukaryotic cell and use thereof. The enhancer disclosed in the present specification is a peptide used by being fused to a sugar hydrolase, and the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 55% or more identity with this amino acid sequence Is a peptide having one or more first amino acid sequences.

  By using this enhancer, when the hydrolase of sugar is produced in a eukaryotic cell, its extracellular expression, that is, for example, cell surface presentation or extracellular secretion can be enhanced. Therefore, it is useful when a sugar hydrolase is desired to function extracellularly using eukaryotic cells, or when a sugar hydrolase is desired to be recovered extracellularly using eukaryotic cells. Furthermore, it is useful for fermentation in which sugars are decomposed extracellularly using such eukaryotic sugar hydrolase and the degradation products are metabolized to eukaryotic cells to produce useful substances.

  Conventionally, enhancement of extracellular expression of a foreign protein involves modification of a host cell or modification of a secretory expression system or a cell surface display system. According to the disclosure of the present specification, sugar hydrolysis is performed. Modifications that enhance extracellular expression on the enzyme side were realized.

  Hereinafter, the enhancer disclosed in the present specification and use thereof, that is, a production method of a fusion protein, a eukaryotic cell, a sugar hydrolase, a method of producing a cellulose degradation product, and a method of producing a useful substance using the enhancer Etc. will be described in detail.

(Enhancer)
The enhancer disclosed in the present specification is an enhancer of extracellular expression of sugar hydrolase in eukaryotic cells. In the present specification, extracellular expression is not particularly limited, and examples include forms presented on the surface of eukaryotic cells and forms secreted extracellularly.

(Sugar hydrolase)
In this specification, sugar hydrolase refers to an enzyme that hydrolyzes polysaccharides, oligosaccharides, trisaccharides, disaccharides, and the like. The sugar to be decomposed is not particularly limited. Typical examples include hemicelluloses such as cellulose, xylan, arabinoxylan, xyloglucan, mannan, glucomannan, and glucunoxylan, and polysaccharides such as chitin, chitosan, agarose, pectin, amylose, and amylopectin. Examples of polysaccharides include cellulose, hemicellulose, agarose, pectin, and chitin from the viewpoint of effective utilization of biomass resources. As the sugar hydrolase, an enzyme that hydrolyzes such a sugar is preferable. Typically, cellobiohydrolase, endoglucanase, cellulase such as β-glucosidase (for cellulose degradation), xylanase, galactanase and other hemicellulases (for hemicellulose degradation), α-amylase, β-amylase, glucoamylase (starch) For decomposition).

  Examples of cellobiohydrolase include cellobiohydrolase I (GHF7) and cellobiohydrolase II (GHF6) derived from Phanerochaete chrysosporium. Moreover, cellobiohydrolase of other origins other than Phanerochaete chrysosporium is also mentioned.

  Examples of endoglucanases include endoglucanases belonging to GHF5. Among endoglucanases classified as GHF5, preferably, endoglucanase derived from Trichoderma reesei, endoglucanase derived from Aspergillus oryzae, and endoglucanase derived from Aspergillus niger can be preferably used. More preferred is an endoglucanase derived from Trichoderma reesei endoglucanase Aspergillus niger. The endoglucanase classified as GHF5 can be used by appropriately combining one or more selected from such endoglucanases.

  Examples of endoglucanases include endoglucanases belonging to GHF12. Among these, endoglucanase derived from Trichoderma reesei, endoglucanase derived from Aspergillus niger, and endoglucanase derived from Aspergillus oryzae can be mentioned. One or more selected from these endoglucanases can be used in appropriate combination. The endoglucanase may be an endoglucanase belonging to GHF7 and GHF45. Among these, an endoglucanase derived from Trichoderma reesei can be preferably used.

  The sugar hydrolase may be a natural sugar hydrolase or an appropriately modified one. As for the sugar hydrolase, those skilled in the art can obtain the amino acid sequence of the enzyme (protein) and the base sequence encoding the amino acid sequence according to the purpose, and can also obtain mutants.

(Eukaryotic cells)
In the present specification, eukaryotic cells include eukaryotic microorganisms such as yeast and bacilli, but are not particularly limited. The yeast is not particularly limited, and yeast of the genus Saccharomyces such as Saccharomyces cerevisiae, yeast of the genus Schizosaccharomyces pombe such as Schizosaccharomyces pombe, Candida shehatae, etc. Yeast of the genus Candida, yeast of the genus Pichia such as Pichia stipitis, yeast of the genus Hansenula, yeast of the genus Trichosporon, yeast of the genus Brettanomyces, the genus Pachysolen And yeast of the genus Yamadazyma, yeasts of the genus Kluyveromyces such as Kluyveromyces marxianus and Kluveromyces lactis. Of these, Saccharomyces yeasts are preferable from the viewpoint of industrial availability. Of these, Saccharomyces cerevisiae is preferable.

  In addition, aspergillus oryzae, Aspergillus oryzae, Aspergillus aculeatus, Aspergillus niger, Aspergillus nidulans, Aspergillus soya (Aspergillus soya) Aspergillus kawachii), Aspergillus awamori, Aspergillus saitoi and the like.

  Other eukaryotic microorganisms include Trichoderma genus bacteria such as Trichoderma reesei, which are cellulase-producing microorganisms, and wood rot fungi including white rot fungi. By using a cellulase-producing microorganism as a host, the present protein can be produced simultaneously with cellulase, hemicellulase and the like that can be produced by these microorganisms.

  The enhancer is a peptide having one or more amino acid sequences represented by SEQ ID NO: 1 or one or more first amino acid sequences that are 55% or more identical to this amino acid sequence.

  The amino acid sequence represented by SEQ ID NO: 1, which is a component of the first amino acid sequence, is located between the cellulose-binding domain (CBD) and the catalytic domain (CD) of Trichoderma harsianum endoglucanase II. Linker sequence.

  The first amino acid sequence may also be an amino acid sequence having 55% or more identity with the amino acid sequence represented by SEQ ID NO: 1. As shown in the following table, when a known sequence is searched for the amino acid sequence represented by SEQ ID NO: 1 using a BLAST search described later, an amino acid sequence having 55% or more identity can be mentioned. The present inventors have obtained a linker sequence (SEQ ID NO: 4) between CBD and CD in the endoglucanase II of Trichoderma reesei having 64% identity with the amino acid sequence represented by SEQ ID NO: 1 and 55% of the same. The enhancing effect of the linker sequence (SEQ ID NO: 7) between CBD and CD in the endoglucanase II of Trichoderma viride having identity has also been confirmed.

  As used herein, identity or similarity is a relationship between two or more proteins or two or more polynucleotides determined by comparing sequences, as is known in the art. In the art, “identity” means between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of such sequences. Means the degree of sequence invariance. Similarity is also the correlation between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of partial sequences. Means the degree of More specifically, it is determined by sequence identity and conservation (substitutions that maintain specific amino acids in the sequence or physicochemical properties in the sequence). The similarity is referred to as Similarity in the BLAST sequence homology search result described later. The method for determining identity and similarity is preferably a method designed to align the longest between the sequences to be compared. Methods for determining identity and similarity are provided as programs available to the public. For example, BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (Eg Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschyl SF, Madden TL, Schaffer AA, Zhang J, Miller W, Lipman DJ., Nucleic Acids Res. 25: p3389-3402 (1997)). The conditions for using software such as BLAST are not particularly limited, but it is preferable to use default values.

  An embodiment in which one or several amino acids are deleted, substituted or added within the range of 55% identity to the amino acid sequence represented by SEQ ID NO: 1 is exemplified. Substituting and adding amino acids include (serine, threonine), (glycine, alanine) (valine, isoleucine, leucine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine), etc. In addition to substitution, substitution or addition with serine (S) and threonine (T) is preferred.

Although the disclosure of the present specification is not limited, it can be said that the first amino acid sequence has one or more of the following characteristics.
(1) The ratio of serine (S) to threonine (T) is 55% or more in the entire amino acid sequence.
(2) Does not contain aspartic acid (N).
(3) It is derived from Trichoderma endoglucanase and is a linker sequence of CBD and CD.

  Although it is an inference and does not constrain the disclosure of the present specification, such a feature makes it possible for the first amino acid sequence to avoid or suppress N-type sugar chain modification via aspartic acid (N), or to serine. It is considered that only O-type sugar chain modification via (S) or threonine (T) is possible.

  The enhancer has one or more such first amino acid sequences. Preferably, two or more are provided. By having two or more first amino acid sequences repeatedly (tandem repeat), the extracellular expression level of a sugar hydrolase can be increased as compared with the case of having one amino acid sequence. A plurality of first amino acid sequences are provided so that the amount of extracellular expression can be increased. A suitable amount of the first amino acid sequence can be obtained by assigning 1 or 2 or more to the first amino acid sequence to the target sugar hydrolase and evaluating the extracellular expression level. The first amino acid sequence is, for example, more preferably 3 or more and 6 or less, and still more preferably 3 or more and 5 or less.

  In the case of having two or more first amino acid sequences, the two or more amino acid sequences may be the same or different. Moreover, when it has 2 or more of 1st amino acid sequences, it does not need to provide the amino acid residue by an amide bond between adjacent 1st amino acid sequences, and the amino acid sequence which consists of 1 or 2 or more amino acids. You may have. For example, in order to obtain a peptide in which a plurality of first amino acid sequences are linked by genetic engineering, a base sequence that can be translated into amino acids with respect to DNA consisting of a base sequence encoding the first amino acid sequence is In some cases, a base sequence containing a restriction enzyme sequence is provided as a linking site. In addition, when connecting two or more 1st amino acid sequences, it is preferable that the said connection part does not contain an amino acid sequence.

  The peptide having one or more first amino acid sequences is a peptide that is used by being fused (linked) to a sugar hydrolase. By linking such a peptide to a sugar hydrolase, the extracellular expression level can be improved when the sugar hydrolase is expressed extracellularly using a known extracellular expression system (described later). .

  The fusion site of such a peptide to a sugar hydrolase (in the case of functioning only in the catalytic domain, may be only the domain) is not particularly limited, and may be the N-terminal or the C-terminal. There may be. Considering the activity of sugar hydrolase and the like, the N-terminal side is preferred. The same applies to the case where the sugar hydrolase has a substrate binding domain such as CBD and a catalytic domain (CD), such as cellulase. It is preferable to have one or more first amino acid sequences in between. Typically, it is N-terminal to the catalytic domain and C-terminal to the substrate binding domain. The N terminal is preferred.

  Such a fusion protein can be obtained by a person skilled in the art based on genetic engineering or protein engineering if the amino acid sequence of the sugar hydrolase or the base sequence encoding the amino acid sequence and the first amino acid sequence can be known. .

  The enhancer disclosed herein may also be a polynucleotide encoding a peptide having one or more first amino acid sequences. Such enhancers are useful for transformation of eukaryotic cells. The polynucleotide may be DNA or RNA. The construct for transformation is preferably used in the form of DNA. The codon usage encoding the peptide is appropriately determined according to the type of eukaryotic cell used.

  The enhancer in the form of a polynucleotide may be in the form of a polynucleotide fragment such as a DNA fragment, or in a form inserted into various vectors, and the form is not particularly limited. The enhancer in the form of polynucleotides can take the form of an expression vector intended primarily for transformation of suitable host cells. Depending on the transformation method and the retention form of the polynucleotide in the host cell (form introduced into the chromosome, form retained outside the chromosome, etc.), components other than the coding region are appropriately determined. Typically, under the control of a promoter operable in a transformed cell such as a eukaryotic cell, a base sequence encoding an enhancer is linked singly or together with a base sequence encoding a sugar hydrolase, and a terminator or the like. Are preferably linked.

(Fusion protein)
The fusion protein disclosed in the present specification is a fusion protein of a peptide having one or more first amino acid sequences and a sugar hydrolase. The fusion protein has activity as a sugar hydrolase, and is improved in extracellular secretion and cell surface presentation in a known extracellular expression system.

  For the sugar hydrolase and the first amino acid sequence in the fusion protein, the contents described in the embodiment of the enhancer can be applied as they are. In addition, the fusion form of the first amino acid sequence and the sugar hydrolase is as already described in the embodiment of the enhancer.

  This fusion protein can also be obtained by those skilled in the art by genetic engineering or chemical synthesis methods. Preferably, the eukaryotic cell is transformed into the eukaryotic cell so that the DNA encoding the fusion protein can be expressed using the extracellular expression in the eukaryotic cell, as described later. Produce a fusion protein.

  A preferred fusion protein is a cellulase in which the sugar hydrolase is cellobiohydrolase or endoglucanase and has a cellulose binding domain as a substrate binding domain and a catalytic domain. And 1 or 2 or more of the 1st amino acid sequence is arrange | positioned between the substrate binding domain and the catalytic domain.

(Polynucleotide encoding fusion protein)
The polynucleotide disclosed herein encodes the fusion protein. The polynucleotide may be DNA or RNA, but is preferably DNA in consideration of transformation. The codon usage for encoding the fusion protein is appropriately determined according to the type of eukaryotic cell used. The enhancer in the form of a polynucleotide may be in the form of a polynucleotide fragment such as a DNA fragment, or in a form inserted into various vectors, and the form is not particularly limited. In the form inserted into the vector, it is preferably linked under the control of a promoter operable in a transformed cell such as a eukaryotic cell, and further a terminator or the like is preferably linked.

(Eukaryotic cells holding DNA encoding this fusion protein)
The eukaryotic cells disclosed herein are eukaryotic cells that retain the DNA encoding the fusion protein and are capable of extracellular expression. According to the present eukaryotic cell, a sugar hydrolase excellent in extracellular expression can be produced and expressed extracellularly. The eukaryotic cell can be obtained by transforming a eukaryotic cell serving as a host with a vector containing a DNA encoding the fusion protein by a known method. In order to actually impart extracellular secretion properties and cell surface presentation properties to the fusion protein, known secretion signals and surface layer presentation systems can be used. For example, a secretory signal, an aggregating protein, or a partial amino acid sequence thereof is given. Examples of the secretion signal include secretion signals of glucoamylase genes of Rhizopus oryzae and C. albicans, yeast invertase leader, α factor leader and the like. Examples of the aggregating protein include peptides consisting of 320 amino acid residues in the 5 ′ region of the SAG1 gene encoding α-agglutinin. Polypeptides and techniques for displaying a desired protein on the cell surface are disclosed in WO01 / 79483, JP2003-235579, WO2002 / 042483, WO2003 / 016525, and JP2006. No. 136223, Fujita et al. (Fujita et al., 2004. Appl Environ Microbiol 70: 1207-1212 and Fujita et al., 2002. Appl Environ Microbiol 68: 5136-5141.), Murai et al., 1998. Appl Environ Microbiol 64: 4857 -4861.

  The eukaryotic cell is preferably capable of expressing two or more sugar hydrolases extracellularly in the form of the fusion protein. This is because in the degradation of polysaccharides and the like, in many cases, two or more sugar hydrolases act in cooperation to enable efficient degradation. When the polysaccharide is cellulose, it is preferable that the eukaryotic cell can be expressed extracellularly using two or more selected from cellobiohydrolase, endoglucanase and β-glucosidase as a fusion protein. When the polysaccharide is starch, it is preferable that the eukaryotic cell can be expressed extracellularly as a fusion protein of two or more selected from α-amylase, β-amylase and glucoamylase.

(Method for producing sugar hydrolase)
The method for producing a sugar hydrolase disclosed in the present specification can include a step of culturing the eukaryotic cell and presenting the sugar hydrolase to the cell surface layer or secreting it out of the cell. According to this production method, sugar hydrolase can be produced efficiently, and the amount of enzyme preparation used can be reduced or avoided. Such a production process is also carried out as a decomposition process in a cellulose decomposition method to be described later or a decomposition process in a useful substance production method.

(Method for modifying sugar hydrolase)
The method for producing a sugar hydrolase disclosed in the present specification is the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 55% or more identity with this amino acid sequence with respect to the sugar hydrolase. It is a method of modifying the extracellular expression of the sugar hydrolase in eukaryotic cells by adding one or more first amino acid sequences.

  This method is a method for enhancing the extracellular expression of a known sugar hydrolase, and is also a production method (acquisition method) of a sugar hydrolase modified to enhance the extracellular expression. . Conventionally, in order to improve the extracellular expression of sugar hydrolase, etc., it is conceivable to modify the host-side gene or to modify the secretion signal, etc. By modifying the enzyme itself, extracellular expression can be improved while using a conventional extracellular expression system.

  The modification can be performed as follows, for example. Production of eukaryotic cells capable of expressing several fusion proteins in which one or two or more first amino acid sequences are fused to any position of the sugar hydrolase for the desired sugar hydrolase Then, the secretory expression level of the fusion protein, the sugar hydrolyzing activity of the culture supernatant, etc. are evaluated, and the number and position of the first amino acid sequence linked to the desired sugar hydrolase are determined based on the evaluation. be able to.

(Screening method for sugar hydrolase)
The sugar hydrolase screening method disclosed in the present specification is selected from the linker sequences of the substrate binding domain and the catalytic domain of the same or different sugar hydrolase with respect to the sugar hydrolase. Preparing a eukaryotic cell capable of expressing a test fusion protein fused with a test amino acid sequence having one or two or more linker sequences, and extracellular expression of the test fusion protein by the prepared eukaryotic cell Measuring the amount. According to this screening method, other known linker sequences useful for the desired sugar hydrolase, the number thereof, and their fused (linked) forms can be screened.

  The test fusion protein to be screened is designed as follows. First, one or more linker sequences can be selected from linker sequences known in sugar hydrolases. A test amino acid sequence is designed by linking one or more selected linker sequences, and an amino acid sequence of the test fusion protein linked to any position of the amino acid sequence of the sugar hydrolase is designed.

  A vector holding DNA encoding the designed amino acid sequence is constructed, a eukaryotic cell is transformed with the vector, a culture experiment of the eukaryotic cell is performed, and the extracellular expression level of the test fusion protein is measured. When the fusion protein is designed to be secreted and expressed outside the cell, the amount of extracellular expression is measured so that the amount of fusion protein in the culture supernatant is measured and presented to the cell surface For example, sugar hydrolysis activity using cultured cells is measured.

  Based on the extracellular expression level, it is possible to determine the effective linker sequence and the number of linker sequences and the position of the linker sequence linked to the hydrolase.

(Production method of cellulose degradation products)
Cellulase and a peptide having one or more amino acid sequences represented by SEQ ID NO: 1 or an amino acid sequence having 55% or more identity with this amino acid sequence A cellulase produced by a eukaryotic cell that retains DNA encoding a fusion protein (cellulase variant) and that can express the fusion protein extracellularly, and cellulose. According to this production method, cellulose can be decomposed by cellulase expressed extracellularly.

  In this contact step, when the eukaryotic cell is constructed so as to secrete and express the cellulase variant, the culture supernatant and the cellulose-containing material may be contacted. In this case, the eukaryotic cell may be cultured in the presence of a cellulose-containing material. In this case, eukaryotic cells can proliferate while hydrolyzing cellulose. Furthermore, when the eukaryotic cell is constructed so that a cellulase variant can be presented on the cell surface, the eukaryotic cell can be cultured in the presence of a cellulose-containing material.

  As cellulase, it is preferable to use cellobiohydrolase and endoglucanase. As a synergistic action of these, cellulose can be decomposed efficiently. Both of these cellulases are preferably expressed extracellularly, and it is preferable to co-express two types in one eukaryotic cell, but other embodiments may be used, and either one may be It may be supplied from outside the cell.

  Conditions such as treatment temperature and time in the contacting step can be appropriately set depending on the type of cellulase used and the supply form of cellulose.

  In the present specification, the cellulose may be supplied as a cellulose that has undergone a separation step from other components from biomass, or biomass that has been subjected to untreated or partial pretreatment in which lignin, hemicellulose, and / or pectin coexist. For example, it may be supplied as a material containing cellulose. As a material containing cellulose, waste resources such as rice straw, wheat straw, bagasse, and dead grass, as well as unused resources may be used, and there is no particular limitation as long as cellulose is contained. Moreover, in this specification, as a cellulose degradation product, what is necessary is just the low molecular weight thing of a cellulose, and glucose, its oligomer, cellobiose etc. are mentioned.

(Useful substance production method)
The method for producing a useful substance disclosed in the present specification comprises: 1 or 1 of a first amino acid sequence which is an amino acid sequence represented by SEQ ID NO: 1 with cellulase or an amino acid sequence having 55% or more identity with this amino acid sequence A eukaryotic cell that retains DNA encoding a fusion protein (cellulase variant) with a peptide having two or more, can express the fusion protein extracellularly, and has the ability to produce the useful substance in the presence of cellulase. Culturing in a step.

  According to this production method, since the eukaryotic cell effectively expresses the cellulase variant extracellularly, cellulase can be efficiently saccharified, and the eukaryotic cell metabolizes the degradation product as it is. A useful substance can be produced based on the fermentation ability (the ability to produce a useful substance).

  According to this production method, a so-called simultaneous saccharification / fermentation process (CBP) can be efficiently performed. The eukaryotic cells are preferably constructed so that both cellobiohydrolase and endoglucanase necessary for cellulose degradation are expressed extracellularly, but when only one of them is expressed. May supply the other from the outside.

  As the eukaryotic cells used in the culturing step, the eukaryotic cells already described are used, but ethanol-producing microorganisms such as yeast and eukaryotic microorganisms such as Neisseria gonorrhoeae can be preferably used. The eukaryotic microorganism may be an artificially obtained microorganism. For example, it may be genetically engineered so that it can produce a compound that is not the original metabolite obtained by replacing or adding one or more enzymes in the metabolic system from glucose by genetic recombination. Good. By using such eukaryotic microorganisms, for example, the production of fine chemicals (coenzyme Q10, vitamins and their raw materials, etc.) by adding an isoprenod synthesis pathway, the production of glycerin by modification of glycolysis, the production of plastics and chemical raw materials It can be applied to biorefinery technology. Although it does not specifically limit as a useful substance, The thing which can produce | generate microorganisms using glucose is preferable, and as above-mentioned, the substance over the biorefinery technique can be made into object.

  Any of the embodiments disclosed in the present specification described above can be carried out in accordance with the method described in Molecular Cloning 3rd Edition, Current Protocols in Molecular Biology and the like. Vectors for the transformation of host cells such as eukaryotic microorganisms and methods for their construction are well known to those skilled in the art and disclosed in the above-mentioned books and the like. In addition, for transformation, various conventionally known methods such as transformation method, transfection method, conjugation method, protoplast method, electroporation method, lipofection method, lithium acetate method and the like can be used as well. Well known in the art.

  The technical elements described in this specification or the drawings exhibit technical usefulness alone or in various combinations, and are not limited to the combinations described in the claims at the time of filing. In addition, the technology exemplified in this specification or the drawings can achieve a plurality of objects at the same time, and has technical usefulness by achieving one of the objects.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples. The gene recombination operation described below was performed according to the previously described Molecular Cloning.

(Construction of a DNA fragment (linker multiple linked fragment) in which multiple linkers are linked)
In this example, the linker multiple ligation fragment shown in FIG. 4 was prepared by the method shown in FIG. Specifically, an oligonucleotide comprising a 102 bp base sequence (SEQ ID NO: 8) encoding a Trichoderma harzianum endoglucanase II linker (SEQ ID NO: 1) (depending on codon usage in yeast) and this oligonucleotide Complementary oligonucleotides (5 ′ end is phosphorylated modified) were synthesized with 9 bp shifted at both ends. The shifted 9 bp was designed in a complementary sequence so that it would be a sticky end during ligation.

  The synthesized oligonucleotide was heat denatured at 95 ° C. for 5 minutes and then annealed by slowly lowering the temperature to room temperature to form a double strand. Next, the prepared double-stranded oligonucleotide was ligated in multiple by ligation using a DNA ligation kit Ver2.1, and after agarose electrophoresis, a fragment having a length of 500 bp or more was cut out from the gel. Furthermore, various multilinker vectors were prepared by subcloning into pUC19 using In-Fusion Advantage PCR Cloning Kit (Takara Bio).

  PCR is performed with two types of primer pairs using a multi-linker vector as a template, and linker fragments of various lengths are prepared, and then inserted using the In-Fusion Advantage PCR Cloning Kit to link between any CBD-CD. A DNA construct into which multiple ligation fragments were inserted was constructed.

(Construction of a vector carrying a ThCBD1-PcCD fragment containing multiple linking linkers)
As a model gene for cellobiohydrolase, a DNA fragment (ThCBD1-PcCD) in which CBD of cellobiohydrolase II (PcCBH2) of Phanerochaete chrysosporium was replaced with CBD of endoglucanase II of Trichoderma harzianum was used. As a secretory expression vector in S. cerevisiae, pAUR112-GAPSSRG (FIG. 2) in which a TDH3 promoter-Rhizopus orizae glucoamylase signal sequence (MQLFNLPLKVSFFLVLSYFSLLVSAA) -CYC terminator was inserted into the SmaI site of pAUR112 (Takara Bio). . pAUR112-GAPSSRG was treated with restriction enzymes with SphI and ClaI, and subcloned using the In-Fusion Advantage PCR Cloning Kit so that ThCBD1-PcCD was located downstream of the signal sequence derived from glucoamylase of Rhizopus oryzae. The pAUR112-GAPSSRG-ThCBD1-PcCD shown was made. At this time, by inserting a tetracystine tag (TC: Cys-Cys-Pro-Gly-Cys-Cys) into the C-terminus of the inserted DNA fragment, using TC-FLASH Expression Anaysis Detection kits (Iinvitrogen), It was designed so that the amount of enzyme in the culture supernatant could be measured.

  Next, by using the technique of Example 1, a Trichoderma harzianum endoglucanase II linker was ligated between CBD-CDs of pAUR112-GAPSSRG-ThCBD1-PcCD to prepare a vector in which a multiplex linker fragment was inserted. The prepared vector was transformed into BJ5465 strain using Frozen-EZ Yeast Transforamation II Kit (Zymo Research).

(Secret expression of ThCBD1-PcCD having various linker lengths in S. cerevisiae and evaluation of degrading activity of PSC (phosphate-swelling cellulose) using the culture supernatant Culture was performed using a 96-well deep well plate. The grown bacterial colonies were inoculated into 500 μl of SD-URA liquid medium (1.7 g of Yeast Nitorogen Base without amino acid without sulfate, 10 g of casamino acid, 0.77 g of -URA amino acid mix, 10 g of glucose, 1000 ml of deionized water) The bacterial solution cultured at 30 ° C. for 20 hours was used as a pre-culture solution, 100 μl of the pre-culture solution was newly cultivated on 500 μl of SD-URA liquid medium, and cultured at 30 ° C. for 20 hours for main culture. Cellulose degradation tests were performed using the culture supernatant obtained by the separation as a crude enzyme solution.

  As a substrate, 0.5% PSC was used. 15 μl of the culture supernatant derived from each DNA fragment was added to 200 μl of the substrate, and after 24 hours of reaction at 40 ° C., the amount of reducing sugar in the centrifuged supernatant was measured by the TZ assay method. All measurements were done in triplicate. The results are shown in FIG.

  As shown in FIG. 5, it was found that the PSC degradation activity was improved according to the linker length. It was also found that the maximum activity was exhibited when the number of insertion linkers was 5.

(Evaluation of secreted expression of ThCBD1-PcCD with various linker lengths in S. cerevisiae)
The amount of ThCBD1-PcCD contained in the culture supernatant whose activity was measured in Example 3 was measured using TC-FLASH Expression Anaysis Detection kits. The results are shown in FIG. The expression level was quantified by zone decimetry from the image after SDS-PAGE, and it was shown as a relative ratio when the case where no linker was inserted was 1. As shown in FIG. 6, the secretory expression level in yeast was improved according to the number of linkers, and the peak expression level was when the number of insertion linkers was 5. This result was correlated with the PSC degradation activity shown in Example 3.

(Construction of a vector carrying a ThCBD1-TrEG2CD fragment containing multiple linking linkers)
As a model gene for endoglucanase, a DNA fragment (ThCBD1-TrEGIICD) in which CBD of Trichoderma reesei endoglucanase II (TrEG2) was replaced with CBD of Trichoderma harzianum endoglucanase II was used. As in Example 2, the base vector shown in FIG. 7 was constructed, and then a Trichoderma harzianum endoglucanase II linker (SEQ ID NO: 1) was used between CBD-CDs using the same method as in Example 1. A DNA fragment in which 0, 1, 3, 4, and 5 nucleotide sequences (SEQ ID NO: 8) encoding) were inserted was constructed (FIG. 8). The constructed vector was transformed into BJ5465 strain using Frozen-EZ Yeast Transformation II Kit (Zymo Research).

(Secret expression of ThCBD1-TrEGIICD with various linker lengths in S. cerevisiae and evaluation of PSC (phosphate-swelling cellulose) degradation activity using culture supernatant)
Pre-culture and main culture were performed in the same manner as in Example 3. Using the culture supernatant obtained by centrifugation of the main culture as the crude enzyme solution, a cellulose degradation test was conducted in the same manner as in Example 3. The results are shown in FIG.

  As shown in FIG. 9, it was found that not only PcCBH2 but also TrEG2, the PSC degradation activity in yeast was improved according to the linker length. It was also found that the maximum activity was exhibited when the number of insertion linkers was 4.

(Evaluation of secreted expression level of ThCBD1-TrEG2 with various linker lengths in S. cerevisiae)
Using the same method as in Example 4, the secretory expression level was measured. The results are shown in FIG. As shown in FIG. 10, the secretory expression level in yeast was improved according to the number of linkers, and the peak expression level was when the number of insertion linkers was 5.

(Evaluation of linker multiplexing effect of wild type PcCBH2 and TrEGII)
In the present example, wild-type PcCBH2 and TrEGII have these inherently provided linkers (amino acid sequence: SEQ ID NO: 15, 4, base sequence: SEQ ID NO: 16 (however, depending on codon usage in yeast), 11 (however, According to the codon usage in yeast.))), An extended DNA fragment was constructed by multiplexing two or three by the inverse PCR method, a vector was constructed in the same manner as in Example 2, and the BJ5465 strain was similarly constructed. Transformed. Furthermore, the PSC decomposition activity and secretory expression level of the culture supernatant were measured using the same method as in Examples 3 and 4. The results are shown in FIG.

  As shown in FIG. 11, even when the linker of PcCBH2 was multiplexed, neither the degradation activity nor the secretory expression level increased, but rather it tended to decrease. In contrast, the TrEG2 linker also improved the expression level and PSC degradation activity in response to linker multiplexing.

(Sequence comparison of various linkers)
The linker amino acid sequences between CBD-CD of ThEG2, TrEG2, and PcCBH2 were compared. The results are shown in FIG. As shown in FIG. 12, the ThEG2 and TrEG2 sequences showed very high identity, whereas the PcCBH2 linker had low identity to the other two species. Further, FIG. 13 shows the results of homology search with the ThEG2 linker by BLAST search. As shown in FIG. 13, the linker sequence between CBD-CD of Trichoderma genus endoglucanase has high identity, and the amount of secretory expression in yeast by being inserted between CBD-CD of other cellulases in a multiplexed manner. It was found that the activity can be improved.

(Improvement of expression level by Trichoderma viride endoglucanase II linker)
In Example 9, the endoglucanase II linker of Trichoderma viride, which was 55% homologous to the endoglucanase II linker of Trichoderma harzianum, was inserted between the CDs of ThCBD1 and PcCBH2 to improve the secreted expression level in yeast. Confirmed whether or not. A DNA fragment ThCBD1-TvLinker-PcCD provided with a Linker between ThCBD1 and PcCD was prepared and subcloned into pAUR112-GAPSSRG as shown in FIG. In the same manner as in Examples 2 and 3, after transformation into yeast, the PSC degradation activity was measured and compared with that before insertion. The results are shown in FIG.

  As shown in FIG. 15, in ThCBD1-TvLinker-PcCD inserted with TvLinker, the PSC degradation activity is improved by about 1.2 times compared to ThCBD1-PcCD before insertion, and TvLinker also increases the amount of secretion. I was able to confirm.

Claims (21)

An enhancer of extracellular expression of sugar hydrolase in eukaryotic cells,
A peptide fused to the sugar hydrolase,
An enhancer, which is a peptide having one or more amino acid sequences represented by SEQ ID NO: 1 or a first amino acid sequence that is 55% or more identical to the amino acid sequence.   The enhancer according to claim 1, which has two or more of the first amino acid sequences.   The enhancer according to claim 1 or 2, wherein the first amino acid sequence is a linker sequence of a cellulose binding domain and a catalytic domain in an endoglucanase of the genus Trichoderma.   The enhancer according to any one of claims 1 to 3, wherein the sugar hydrolase is a cellulase.   The enhancer according to any one of claims 1 to 4, wherein the cellulase is one of endoglucanase and cellobiohydrolase.   The enhancer according to any one of claims 1 to 5, wherein the eukaryotic cell is yeast. An enhancer of extracellular expression of sugar hydrolase in eukaryotic cells,
A peptide fused to the sugar hydrolase, having one or more amino acid sequences represented by SEQ ID NO: 1 or an amino acid sequence having 55% or more identity with this amino acid sequence An enhancer, which is a polynucleotide encoding a peptide.   A fusion protein of a sugar hydrolase and a peptide having one or more amino acid sequences represented by SEQ ID NO: 1 or a first amino acid sequence that is 55% or more identical to this amino acid sequence.   The fusion protein according to claim 8, which has two or more of the first amino acid sequences.   The fusion protein according to claim 8 or 9, wherein the first amino acid sequence is a linker sequence between a cellulose binding domain and a catalytic domain in an endoglucanase of the genus Trichoderma.   The fusion protein according to any one of claims 8 to 10, wherein the sugar hydrolase is a cellulase.   The fusion protein according to any one of claims 8 to 11, wherein the cellulase is one of endoglucanase and cellobiohydrolase.   The sugar-hydrolyzing enzyme comprises a substrate binding domain and a catalytic domain, and comprises the first amino acid sequence between the substrate binding domain and the catalytic domain. Fusion protein.   A polynucleotide encoding the fusion protein according to any one of claims 8 to 13.   A eukaryotic cell which retains the DNA encoding the fusion protein according to any one of claims 8 to 13 and is capable of expressing the fusion protein extracellularly.   A method for producing a sugar hydrolase, wherein the eukaryotic cell according to claim 15 is cultured and the fusion protein is expressed extracellularly.   Giving 1 or 2 or more of the first amino acid sequence which is the amino acid sequence represented by SEQ ID NO: 1 or the amino acid sequence having 55% or more identity to the sugar hydrolase, A method for modifying the extracellular expression of a sugar hydrolase.   A test amino acid sequence having one or two or more linker sequences selected from a linker sequence of a substrate binding domain and a catalytic domain of a same or different kind of sugar hydrolase with respect to a sugar hydrolase And a step of preparing a eukaryotic cell capable of extracellularly expressing the test fusion protein fused with the above, and a step of measuring the extracellular expression level of the test fusion protein by the prepared eukaryotic cell. Screening method. A method for producing a degradation product of cellulose,
DNA encoding a fusion protein of a cellulase and a peptide having one or more amino acid sequences represented by SEQ ID NO: 1 or a first amino acid sequence which is 55% or more identical to this amino acid sequence Holding the cellulase produced by a eukaryotic cell capable of extracellular expression of the fusion protein and cellulose;
A production method comprising: A method for producing useful substances,
Holds DNA encoding a fusion protein of cellulase and the amino acid sequence represented by SEQ ID NO: 1 or a peptide having one or more first amino acid sequences that are 55% or more identical to this amino acid sequence And culturing eukaryotic cells capable of extracellular expression of the fusion protein and capable of producing the useful substance in the presence of cellulose,
A production method comprising:   The production method according to claim 20, wherein the eukaryotic cell is yeast.