JP6697780B2 - Stress responsive promoter - Google Patents

Description

  The present invention relates to a stress responsive promoter artificially designed to more efficiently express endogenous and foreign genes, and more particularly to a stress responsive promoter applicable in the production of useful substances using yeast.

  The production of useful substances using microorganisms represented by fermentation technology is derived from fermented foods such as sake, shochu, awamori, soy sauce, and miso, and has been accumulated as a technology unique to Japan. In particular, as a mobile fuel application, first-generation biomass ethanol, which has already been commercialized in Brazil, the United States, China, etc., uses food products such as starch and sugar liquid as raw materials and contains microorganisms such as yeast and ethanol-producing bacteria. The fermentability of ethanol possessed is used as a basic technology. However, with the explosive growth of the world population, as food and livestock feed, which are essential to our lives, are depleted, non-edible biomass, rather than industrial production of first-generation biomass ethanol that directly competes for their use, For example, non-edible biomass resources such as rice culm, wheat culm, corncobs and foliage, non-edible parts of agricultural products such as sugarcane juice residue (bagasse), food residues, thinned wood, construction waste, etc. It is required to establish an industrial production technology for cellulosic biomass ethanol (second generation biomass ethanol).

  The first elemental technology required for the production of cellulosic biomass ethanol is a saccharifying enzyme that decomposes cellulose, which is the main component of the plant cell wall, into the hexasaccharide Glucose that can be used by microorganisms. Most organisms can utilize Glucose as the most preferred carbon source, and the microorganisms that perform Ethanol fermentation are no exception. The second elemental technology is that non-edible biomass contains a considerable amount of carbohydrates such as hemicellulose and lignin in addition to cellulose. Not only Glucose derived from cellulose but also pentoses such as Xylose and Arabinose derived from hemicellulose, which could not be used until now, can be used for ethanol production, resulting in more economical and high ethanol production efficiency. Can be expected. These elemental technologies are indispensable for establishing the industrial production technology of cellulosic biomass ethanol.

  The budding yeast Saccharomyces cerevisiae (hereinafter referred to as yeast) is one of the microorganisms familiar to us known as baker's yeast, and alcoholic beverages in Japan such as sake, shochu, and awamori produce alcohol. However, although yeast are good at producing Ethanol from Glucose, they are basically unable to utilize pentoses except for some microorganisms. Therefore, by using gene recombination technology, technology development that enables Ethanol fermentation from Xylose by advancing the enzyme required for Xylose metabolism as a foreign gene into yeast that cannot originally utilize Xylose is being advanced in each country. Is coming.

The production process of cellulosic biomass Ethanol can be roughly divided into two types. One is to perform saccharification and fermentation as separate processes. This is a process in which solid biomass is saccharified in advance, and the produced sugar solution is used as a substrate for microbial ethanol fermentation in a liquid (Saccharified Hydrolysate Fermentation, SHF, saccharified solution fermentation). The other is a process of simultaneously performing saccharification and fermentation. This is a process in which saccharifying enzyme and yeast are allowed to act at the same time by using solid biomass as a raw material to simultaneously advance the saccharification reaction and the fermentation reaction in one container (Simultaneous Saccharification and Fermentation, SSF, simultaneous saccharification fermentation). SSF is considered to be more economical than SHF because it allows two processes to run concurrently in the same vessel in parallel. It is called simultaneous saccharification and fermentation. In the former case, the saccharified solution obtained by separating and purifying the residue may be used or may be used as it is. The YPDX simulated saccharified solution is used when investigating the Ethanol fermentation performance of a cellulosic biomass ethanol-producing strain. This is made by mixing reagents whose composition is clear, instead of the biomass saccharified solution, and is one of SHF as a fermentation process. Further, in the latter case, when an enzyme such as xylanase that produces Xylose from Xylan is included as a saccharifying enzyme for cellulosic biomass, both Glucose and Xylose are supplied, so that Glucose is added to the simultaneous reaction of saccharification and fermentation. And Xylose complex fermentation process (Simultaneous Saccharification and Co-fermentation, SSCF, simultaneous saccharification parallel fermentation). Furthermore, a process has been devised, in which not only saccharification and fermentation are performed at the same time, but also a saccharifying enzyme is produced by a microorganism, which is called CBP (Consolidate BioProcessing).

  Up to now, studies on imparting Xylose-metabolizing ability to microorganisms that cannot originally utilize Xylose have been conducted by introducing various Xylose-metabolizing genes using many microorganisms as hosts. Examples of genes involved in Xylose metabolism include Xylose reductase (XR), Xylitol dehydrogenase (XDH), and Xylose isomerase (XI). There are also many reports on gene mutations that improve enzyme activity. However, in the industrial production of cellulosic biomass ethanol, if the enzyme activity cannot be improved or efficient gene expression cannot be achieved even if the enzyme is robust and can withstand various environmental conditions. Halve.

  In eukaryotes, genes located on the chromosome are transcribed as mRNA from the base sequence upstream of the translation region. It depends greatly on the situation and environment in which the cells are placed. As described above, in the production process of useful substances such as ethanol by microorganisms, the concentration of nutrients such as sugars in the outside world, growth inhibitory substances, etc. changes, so that the state of cells also changes moment by moment. That is, it is known that the behavior of gene expression can greatly change even if the microorganism is the same cell depending on the surrounding conditions. Anaerobic conditions are desirable for efficient ethanol fermentation. Microbial ethanol production is a reaction that takes in assimilable carbohydrates such as Glucose, reduces it to acetic acid, Acetaldehyde, and Ethanol via a glycolytic system, pyruvic acid. Ethanol is one of the available carbon sources for yeast, and when available sugars are exhausted under aerobic conditions, gluconeogenesis breaks down and utilizes ethanol to survive. Therefore, the production efficiency of Ethanol cannot be sufficiently improved under aerobic conditions. It is known that gene expression in yeast is significantly different in the presence of sufficient oxygen and under oxygen limitation. That is, a glycolytic gene promoter that provides strong gene expression in ideal growth conditions, which is usually supplied with sufficient oxygen and Glucose and the necessary nutrients, is never optimal in the Ethanol fermentation process. Gene expression device Not enough. However, industrial production of cellulosic biomass ethanol requires a promoter capable of expressing an exogenous Xylose metabolic gene, which is particularly suitable for the fermentation process. It is not known if the microorganism has a promoter with such properties, and such may not exist. The same can be said not only in the production of cellulosic biomass ethanol, but also in the production of useful substances using all microorganisms. Maximize the production efficiency of the target product for the first time by dynamically optimizing the expression of all enzyme genes involved in metabolism of the target product in a certain bioprocess in spatiotemporal (expression timing) and quantity (expression intensity) You can

  A method using the yeast endogenous promoter HSP12 for the expression of the Xylose-metabolizing enzyme gene for imparting Xylose-metabolizing ability to Saccharomyces cerevisiae is known. As can be seen from the name of HSP (Heat Shock Protein), it is one of the family genes encoding a protein whose gene expression is enhanced in response to heat stress. HSP family genes change their gene expression states in response to various stresses as well as heat stress. In addition, there are a large number of overlapping genes in the family, and individual gene products are devices for protecting the life of microorganisms under conditions where excessive stress is applied, that is, in the critical situation of the microorganism body. It can be said that Indeed, stress-responsive genes such as HSP12 transiently increase in expression upon heat stress and Glucose depletion, but their expression does not last long. Although it is an example of Wine yeast, gene expression of many HSP family genes is known even in cells in the stationary phase where Glucose is depleted and growth is stopped. However, although it is a member of the HSP family, there is a report that HSP12 cannot be detected when Glucose is depleted. On the other hand, it has been reported that genes such as HSP30 and HSP26 have absolute and relatively strong gene expression in the Stationary phase. Therefore, the process of producing ethanol by utilizing xylose under high temperature, high osmotic pressure, anaerobic (oxygen-limited), and glucose depletion conditions, such as fermentation production of cellulosic biomass ethanol, is extremely large for the host microorganism. However, under such conditions, there is a demand for a promoter that is a strong and sustainable gene expression device. An artificial promoter designed with gene expression characteristics is considered to be one of the ways to solve such problems.

JP, 2005-033342, A

  To provide an artificially designed stress-responsive promoter that can express endogenous and foreign genes more efficiently even under stress conditions such as high temperature, high osmotic pressure, anaerobic (oxygen limitation), and glucose deprivation conditions. It is an issue.

In view of the above problems, the inventors focused on the use of an artificially designed stress responsive promoter consisting of a novel nucleotide sequence, designed a stress responsive promoter consisting of 480 bp, and named it pSynSR480. It has been shown that this nucleotide sequence has a stronger promoter activity than the PGK1 promoter, which is conventionally known as a strong promoter. Specifically, as an actual ethanol production process, the simultaneous fermentation of glucose and xylose using the YPDX pseudo-saccharified solution showed a marked improvement in xylose metabolism, and the simultaneous simultaneous saccharification and double fermentation using sodium hydroxide-treated sugarcane bagasse. It was shown that excellent ethanol fermentation production is possible even in the process. This has led to the solution of this problem.
That is, the present invention is as described below:

One aspect of the present invention is
[1]
(I) at least one heat shock element HSE,
(Ii) STRE, which is at least one stress response element,
(Iii) a stress-responsive promoter containing a cis element different from the HSE and STRE, which is bound by a transcriptional activator whose expression is increased under stress conditions (wherein the stress-responsive promoter is Glucose presence). It does not include a cis element to which a transcription factor that acts suppressively below and a cis element to which a transcription factor involved in gluconeogenesis are bound).
Further, the stress responsive promoter of the present invention, in one embodiment,
[1]'
(I) at least one heat shock element HSE,
(Ii) STRE, which is at least one stress response element,
(Iii) A stress-responsive promoter comprising a cis element different from the HSE and STRE, the cis element being bound by a transcriptional activator whose expression is enhanced under stress conditions,
The stress-responsive promoter is characterized in that it does not contain a cis element to which a transcription factor that acts suppressively in the presence of Glucose or a transcription factor involved in gluconeogenesis is bound.

Here, the stress responsive promoter of the present invention is, in one embodiment,
[2] It is characterized in that it enhances transcription activity in response to high temperature, high osmotic pressure, oxygen limitation, or Glucose starvation stress.
Further, the stress responsive promoter of the present invention, in one embodiment,
[3] The stress responsive promoter according to the above [1] or [2],
At least two or more of (i) the heat shock element HSE and (ii) the stress response element STRE,
The HSE and the STRE are alternately arranged.
Further, the stress responsive promoter of the present invention, in one embodiment,
[4] The stress responsive promoter according to any one of [1] to [3] above,
The transcriptional activator whose expression is enhanced under the stress condition is at least one transcriptional activator selected from the group consisting of Met31/32, Rgt1, Cad1, Cin5/Yap4, and Yap1.
In one embodiment of the stress responsive promoter of the present invention,
It includes all cis elements to which transcriptional activators of Met31/32, Rgt1, Cad1, Cin5/Yap4, and Yap1 bind.
Further, the stress responsive promoter of the present invention, in one embodiment,
[5] The stress responsive promoter according to any one of [1] to [4] above,
The (i) HSE is a cis element containing the consensus sequence represented by SEQ ID NO: 16, and the (ii) STRE is a cis element containing the consensus sequence represented by SEQ ID NO: 17. To do.
Further, the stress responsive promoter of the present invention, in one embodiment,
[6] The stress-responsive promoter according to any one of [1] to [5] above,
The cis element to which the transcription activator (iii) binds is a cis element containing a consensus sequence selected from the group consisting of consensus sequences represented by SEQ ID NOs: 18 to 22.

Further, the stress responsive promoter of the present invention, in one embodiment,
[7] The stress responsive promoter according to any one of [1] to [6] above,
The transcription factor that acts suppressively in the presence of Glucose and the transcription factor involved in gluconeogenesis are Rap1, Nrg1, Gln3, Hap4/5, Adr1, Mig1, Mot3, and Snf1.
Further, the stress responsive promoter of the present invention, in one embodiment,
[8] The stress-responsive promoter according to any one of [1] to [7] above,
A cis element to which a transcription factor that suppressively acts in the presence of Glucose or a cis element to which a transcription factor involved in gluconeogenesis binds contains a consensus sequence represented by any of SEQ ID NOs: 23 to 47. It is characterized by being an element.
In addition, the present invention, in another aspect,
[9] (i) HSE which is at least one heat shock element, comprising the consensus sequence shown in SEQ ID NO: 16, and
(Ii) STRE, which is at least one stress response element, and which comprises the consensus sequence shown in SEQ ID NO:17;
(Iii) A stress-responsive promoter (including a cis element containing a consensus sequence represented by SEQ ID NOS: 18 to 22) which is a cis element to which a transcription activator whose expression is increased under stress conditions is bound (however, The stress responsive promoter does not include cis elements including the consensus sequences shown in SEQ ID NOs: 23 to 48 and nucleotide sequences similar to them.
Further, the stress responsive promoter of the present invention, in one embodiment,
[10] The stress responsive promoter according to any one of [1] to [9] above,
Further, (iv) 5'-untranslated region has a Kozak sequence represented by SEQ ID NO: 49.
Further, the stress responsive promoter of the present invention, in one embodiment,
[11] A stress-responsive promoter having the sequence represented by SEQ ID NO: 48.

In addition, the present invention, in another aspect,
[12] A vector including the stress-responsive promoter according to any one of [1] to [11] above.
Further, the vector of the present invention, in one embodiment,
[13] The vector according to [12] above, which can be a shuttle vector between Escherichia coli or a eukaryotic cell, a 2-micron plasmid vector, and a replication origin such as a centromere and an autonomously replicating sequence of a eukaryotic cell. It is characterized by having a nucleotide sequence.
The “vector that can be a shuttle vector” is, for example, a vector that can be a shuttle vector between Escherichia coli and a eukaryotic cell (Saccharomyces cerevisiae if limited) such as pUC19 vector. As a vector having a 2-micron plasmid vector and a nucleotide sequence that can serve as an origin of replication such as a centromere of eukaryotic cells and an autonomously replicating sequence, for example, pUG35 having a chromosome 6 centromere and an autonomously replicating sequence 4 CEN6/ARS4 is used. Can be mentioned.
Further, the vector of the present invention, in one embodiment,
[14] The vector according to [12] or [13] above, which comprises a gene operably linked downstream of the stress-responsive promoter.
Further, the vector of the present invention, in one embodiment,
[15] The vector according to [14] above, wherein the gene is an Xylose isomerase gene.

In addition, the present invention, in another aspect,
[16] A transformant in which a part or all of the vector according to any of [12] to [15] above is introduced into a chromosome.
In addition, the present invention, in another aspect,
[17] A microorganism for producing bioethanol using Xylose as a substrate,
The present invention relates to a microorganism containing part or all of the vector described in [15] above.

In addition, the present invention, in another aspect,
[18] A method for producing bioethanol using Xylose as a substrate,
(A) A method comprising a step of culturing the microorganism according to [17] above in a culture solution containing Xylose.
Here, the method for producing bioethanol of the present invention,
[19] The method described in [18] above,
The step (a) is characterized in that it is carried out simultaneously with the saccharification treatment carried out in the presence of xylan and xylanase.

  According to the stress responsive promoter of the present invention, endogenous and foreign genes can be expressed more efficiently. This enhances the gene expression necessary for the production of the useful substance, and improves the target product amount and production rate.

[Structure of site predicted to bind transcription factor contained in stress response promoter pSynSR480] Of 480 bp, predicted transcription start point is A at position -61. It is considered that the region of -480 to -62 (419 bp) functions as a transcription control, and -61 to -1 (61 bp) functions as a 5'-untranslated region (5'-UTR) that is transcribed but not translated. Kozak sequences thought to be involved in translation efficiency. 8 Msn2/4 binding sites (STRE), 6 Hsf1 binding sites (HSE), 2 Cad1 binding sites, 2 Yap1 binding sites, 2 Rgt1 binding sites, Met31/32 binding sites, and Cin5/Yap4 binding sites Each has one place. The TATA box indicates the site presumed to be the TATA-box required for transcription initiation, and the 5'-UTR indicates the untranslated region of transcribed mRNA. [Result of homology search Constitution of site predicted to bind transcription factor contained in known base sequence most similar to pSynSR480] As a result of BLAST search, pSynSR480 419 bp base sequence excluding 5'-UTR, Among all the nucleotide sequences of all known organisms, the most similar nucleotide sequence was 404 bp in the yeast HSP26 promoter region. However, in this sequence, there was only one Hsf1 binding site (HSE) except for the TATA box, which is the binding site for the basic transcription factor. [Physical Map of Plasmid Vector pAUR101-pHSP12-CpXI-CYC1t-XKS1-pPGK1 (Plasmid No. 1)] A plasmid vector that expresses CpXI by the HSP12 promoter in the pAUR101 vector (Takara Bio Inc.) and simultaneously expresses XKS1 by the PGK1 promoter. . In this plasmid vector, one CYC1t was used as a terminator for two sugar metabolism enzyme genes CpXI and XKS1. In order to remove CYC1t and modify the transcription of each gene so that it is terminated by a different terminator, as a template for linearization by performing inverse-PCR using PK98 and PK106 shown in the figure. Using. AUR1-C: Aureobasidin A resistance gene (included in pAUR101 plasmid vector), MRP17 and BYE1 partial ORF: upstream and downstream genes of AUR1 gene on yeast chromosome (included in pAUR101 plasmid vector), AmpR: Ampicillin resistance gene ( (included in pAUR101 plasmid vector), ori: ColE1 origin required for replication in E. coli (included in pAUR101 plasmid vector), pPGK1: yeast PGK1 promoter, XKS1: yeast Xylulokinase gene, CYC1t: yeast CYC1 terminator, CpXI: Clostridium Xylose isomerase gene mutant from phytofermentans, pHSP12: yeast HSP12 promoter [Physical map of plasmid vector pAUR101-pHSP12-CpXI-ADH1t-TEF1t-XKS1-pPGK1 (plasmid number 2)] CYC1t of plasmid vector pAUR101-pHSP12-CpXI-CYC1t-XKS1-pPGK1 (plasmid number 1) is converted to ADH1t and TEF1t Replaced. A TEF1t fragment was amplified using PK99 and PK100, an ADH1t fragment was amplified using PK107 and PK108, and this vector was constructed by substituting CYC1t by an In-Fusion reaction. ADH1t: yeast ADH1 terminator, TEF1t: yeast TEF1 terminator. Other abbreviations are the same as in FIG. [Physical map of plasmid vector pAUR101-pPGK1-CpXI-ADH1t-TEF1t-ScXKS1-pPGK1 (plasmid number 3)] pHSP12 of plasmid vector pAUR101-pHSP12p-CpXI-ADH1t-TEF1t-ScXKS1-pPGK1 (plasmid number 2) to pPGK1 Replaced. In order to remove pHSP12 using PK114 and PK116, inverse-PCR was performed, a pPGK1 fragment was amplified using PK97 and PK115 in a linearized plasmid vector, and this vector was constructed by an In-Fusion reaction. KEF205 is a strain in which this plasmid vector was linearized with the restriction enzyme StuI (894) in the AUR1-C gene and inserted into the AUR1 site of the yeast chromosome. The abbreviations are the same as in FIGS. 2 and 3. The restriction enzyme name and the base number of the cleavage site are shown in parentheses. [Physical map of plasmid vector pAUR101-pSynSR480-CpXI-ADH1t-TEF1t-ScXKS1-pPGK1 (plasmid number 4)] pHS12 of plasmid vector pAUR101-pHSP12p-CpXI-ADH1t-TEF1t-ScXKS1-pPGK1 (plasmid number 2) was added to pSynSR480. Replaced. In order to remove pHSP12 using PK114 and PK116, inverse-PCR was performed, and a pSynSR480 fragment was amplified using PK117 and PK118 in a linearized plasmid vector, and this vector was constructed by an In-Fusion reaction. KEF206 is a strain in which this plasmid vector was linearized with the restriction enzyme StuI (894) in the AUR1-C gene and inserted into the AUR1 site of the yeast chromosome. pSynSR480: Stress response promoter. Other abbreviations are the same as in FIGS. 2 and 3. The restriction enzyme name and the base number of the cleavage site are shown in parentheses. FIG. 7 shows the results of a fermentation test using a simulated saccharified solution performed under slightly aerobic conditions. In addition, Table 6 is illustrated. ◆: Glucose, ■: Xylose, ▲: Xylitol, ×: Glycerol, *: Acetate, ●: Ethanol concentration (g/L). FIG. 8 shows the results of a simultaneous simultaneous saccharification and double fermentation test using sugar cane bagasse treated with sodium hydroxide, which was carried out under oxygen limitation. In addition, Table 10 is illustrated. The upper panel shows the results for the KEF205 strain and the lower panel for the KEF206 strain. 1X to 4X is the amount of saccharifying enzyme used, and 1X was defined as the use of 8.7 mg of enzyme per 1 g of dried sodium hydroxide-treated sugarcane bagasse. The horizontal axis indicates Glucose, Xylose, Glycerol, Xylitol, Acetate, and Ethanol from the left. The vertical axis represents the concentration of each component (g/L). The oligo DNA (primer) information used in the following examples is shown.

  The present invention relates to a promoter nucleotide sequence artificially designed to enable long-term gene expression in the presence of stress, and a microorganism having an Xylose isomerase gene by a gene expressed using the promoter or its use. .

One aspect of the present invention is: (i) at least one heat shock element, HSE;
(Ii) at least one stress response element STRE, and (iii) a cis element different from the HSE and STRE, which binds to a transcriptional activator whose expression is enhanced under stress conditions, A stress-responsive promoter (however, the stress-responsive promoter does not include a cis element to which a transcription factor that suppressively acts in the presence of Glucose binds, and a cis element to which a transcription factor involved in gluconeogenesis binds) provide.
In the present specification, (i) HSE (heat shock element), which is a heat shock element, means that when a cell is exposed to a stress condition such as heat, its expression is increased by the stress. Transcription factor (HSF) refers to a specific region on DNA that binds. The sequence of HSE is known, and the nucleotide sequence is not particularly limited as long as it binds to HSF and controls transcription on the cis sequence. In one example, the HSE comprises the consensus sequence shown in SEQ ID NO:16.
In the present specification, (ii) a stress response element, STRE (stress response element), is a transcription factor whose expression is increased by the stress when cells are exposed to stress conditions such as hyperosmotic pressure (for example, It refers to a specific region on DNA to which transcription factors such as MSN2 and MSN4 are included). The STRE sequence is publicly known, and the base sequence is not particularly limited as long as the above transcription factor binds and controls transcription on the cis sequence. In one example, the STRE comprises the consensus sequence shown in SEQ ID NO:17.
In the present specification, (iii) a cis element different from HSE and STRE, which is bound by a transcriptional activator whose expression is increased under stress conditions, means that cells have high temperature, high osmotic pressure, oxygen limitation, Alternatively, it refers to a specific region (excluding HSE and STRE) on DNA to which a transcription factor whose expression increases when exposed to stress conditions such as Glucose starvation conditions is excluded. Examples of the transcription factor whose expression increases in response to stress include MET31, MET32, RGT1, CAD1, CIN5, YAP4, YAP1 and the like. The base sequences of cis elements for these transcription factors are known, and the base sequences are not particularly limited as long as the transcription factors bind to control transcription on the cis sequence. In one embodiment, the cis element for the transcription factor is at least one cis element selected from the group consisting of cis elements containing consensus sequences shown in SEQ ID NOs: 18-22 (see Table 3). As for the cis element (iii) above, a plurality of cis elements having different consensus sequences may be selected, and a plurality of cis elements having the same consensus sequence may be selected. In a more preferred embodiment, the cis element for the transcription factor comprises all cis elements selected from the group consisting of cis elements containing the consensus sequences shown in SEQ ID NOs: 18-22.

In the stress responsive promoter of the present invention, when the stress response element of (i) to (iii) "includes a consensus sequence represented by a specific sequence number", a transcription factor that should originally bind can bind to the stress response element, And, as long as the transcription on the cis sequence can be controlled, the consensus sequence has mutations (substitution, insertion, deletion) in one or several (2, 3, 4, etc.) base sequences. Good.
In one embodiment of the stress-responsive promoter of the present invention, (i) the number of HSE that is a heat shock element is not limited as long as it enhances downstream gene expression in response to stress. It can be about ten (two, three, four, five, six, seven, eight, nine, ten).
Further, in one embodiment of the stress responsive promoter of the present invention, (ii) the number of STRE that is a stress response element is not limited as long as it enhances downstream gene expression in response to stress. It can be about ten (two, three, four, five, six, seven, eight, nine, ten).
Further, in one embodiment of the stress responsive promoter of the present invention, (iii) the number of stress response elements other than HSE and STRE is not limited as long as it enhances downstream gene expression in response to stress, for example, It can be about 2 to 10 (two, three, four, five, six, seven, eight, nine, ten).

Here, the stress-responsive promoter of the present invention is characterized by not containing a cis element to which a transcription factor that acts suppressively in the presence of Glucose and a cis element to which a transcription factor involved in gluconeogenesis are bound. ..
Transcription factors that act suppressively in the presence of Glucose, or as transcription factors involved in gluconeogenesis, include, but are not limited to, for example, RAP1, NRG1, GLN3, HAP4, HAP5, ADR1, MIG1, MOT3, SNF1 etc. Can be mentioned. A cis element to which a transcription factor that acts suppressively in the presence of Glucose or a transcription factor involved in gluconeogenesis is bound is known. For example, the cis element to which RAP1, NRG1, GLN3, HAP4, HAP5, ADR1, MIG1, MOT3, and SNF1 bind can be a cis element containing the consensus sequence shown in SEQ ID NOs: 23 to 47.
Here, the stress-responsive promoter of the present invention does not include a cis element having a nucleotide sequence similar to the consensus sequences shown in SEQ ID NOs: 23 to 48. A base sequence similar to the consensus sequence represented by a specific sequence number is a mutation (substitution, deletion, insertion) of 1 to several bases in the consensus sequence, and is a transcription factor to be originally bound. Is capable of binding to the base sequence after mutation and controlling transcription on the cis sequence. In one embodiment, at least two or more, preferably three or more, and more preferably four or more mutations are introduced into the consensus sequence shown in SEQ ID NOs: 23 to 47, thereby binding the transcription factor to the consensus sequence. Refers to what has been lost.
In addition, in one embodiment, the stress-responsive promoter of the present invention does not include the consensus sequences shown in SEQ ID NOs: 23 to 47.

In the stress-responsive promoter of the present invention, (i) HSE, which is a heat shock element, and (ii) STRE, which is a stress-responsive element, can be arranged at intervals of 1 bp or more and 100 bp or less.
In the stress responsive promoter of the present invention, it is preferable that (i) heat shock element HSE and (ii) stress response element STRE are alternately arranged. It is preferable that the HSEs or STREs are alternately arranged at a certain distance, as compared with the case where the HSEs or STREs are continuously arranged, because the transcription can be further improved by the stress response.
Further, in the Kozak sequence in the stress responsive promoter of the present invention, it is preferable that the base at position -3 is A. Further, it is more preferable that the Kozak sequence is 5'-untranslated region and 5'-AATATTAAAA-3' (SEQ ID NO: 49) is present at positions -1 to -10. By using these Kozak sequences, the translation activity can be improved.

  The stress-responsive promoter of the present invention, in one embodiment, enhances transcriptional activity in response to high temperature, hyperosmolarity, oxygen limitation, or Glucose starved stress. That is, the activity of downstream transcription can be improved in response to either stress. Further, the stress-responsive promoter of the present invention has a high temperature, a high osmotic pressure, an oxygen limitation, and even under a condition in which two or more stresses are combined in the Glucose starvation state, in the downstream of the response to the combined stress. The gene expression can be improved. In a more preferred embodiment, the stress-responsive promoter of the present invention enhances transcriptional activity in response to the stress even under high temperature, hyperosmolarity, oxygen limitation, and Glucose starvation stress conditions. Is.

Moreover, this invention provides the vector containing a stress response promoter as another aspect.
The vector is not limited as long as it retains the stress-responsive promoter described above, for example, a vector that can be a shuttle vector between E. coli or eukaryotic cells, 2-micron plasmid vector, eukaryotic centromere and autonomous replication sequence, etc. Examples include a vector having a nucleotide sequence that can serve as the origin of replication of Those skilled in the art can design and manufacture these vectors according to the intended use and the host cell of the recipient, based on the present specification and common general knowledge.
In addition, the vector of the present invention, in one embodiment, can include a gene operably linked downstream of a stress-responsive promoter. As a result, the expression of the gene is enhanced under stress conditions by controlling the stress-responsive promoter. The gene operably linked to the downstream of the stress response promoter is not particularly limited. Preferably, cells are genes that are desired to have improved expression even under stress conditions, for example, saccharifying enzyme genes for bioethanol production (cellulase, xylanase, etc.) and fermentation enzyme genes (Xylose reductase (XR), Xylitol dehydrogenase). (XDH), Xylose isomerase (XI) and the like).

The present invention also provides, as another aspect, a transformant or a microorganism containing a part (a region containing a stress-responsive promoter and a gene operably linked downstream thereof) or all of the above vector.
The method for introducing the vector containing the stress responsive promoter into the host cell can be performed by a known method. The vector containing a stress responsive promoter may be introduced into a host cell in the form of a plasmid or the like, or may be integrated into genomic DNA. It may be replaced with the original gene existing on the genomic DNA. Further, the copy number to be introduced does not matter in the case of insertion into a plasmid or the genome. The host cell to be transformed is not limited to the following, but examples thereof include species included in the genus Saccharomyces of the genus Saccharomyces, the genus Schizosaccharomyces of the fission yeast, and the genus Kluyveromyces of the thermostable yeast.
Further, the present invention, as another aspect, a microorganism for bioethanol production using Xylose as a substrate, wherein a part of a vector having an Xylose isomerase gene operably linked downstream of a stress response gene Provided is a microorganism containing (a region containing a stress response promoter and a Xylose isomerase gene) or all. Also provided is a method for producing bioethanol using the microorganism. The method for producing bioethanol using the microorganism can be performed according to known methods and conditions.
In one embodiment, the method for producing bioethanol using Xylose as a substrate comprises (a) a part or all of a vector containing the stress-responsive promoter of the present invention and an Xylose isomerase gene operably linked downstream thereof. And culturing the microorganism containing X in a culture solution containing Xylose. In another embodiment, step (a) can be performed simultaneously with the saccharification treatment performed in the presence of xylan and xylanase.

  The present invention is described in detail below, but the present invention is not limited to these examples.

(1. Medium and culture conditions)
YPD medium was used for yeast growth. YPD medium, Bacto yeast extract (BD Bioscience, USA) 10g, Bacto peptone (BD Bioscience) 20g, D-(+)-Glucose (hereinafter referred to as Glucose. Sigma-Aldrich, USA) 20g purified water Included in 1L. In the Ethanol fermentation test using the simulated saccharified liquid, YPDX medium was used as a liquid medium that mimics the sugar composition of the biomass saccharified liquid. YPDX medium, Bacto yeast extract 10g, Bacto peptone 20g, Glucose (Sigma Aldrich) 85g/L, D-(+)-Xylose (hereinafter referred to as Xylose. Sigma Aldrich) 35g/L purified water
Included in 1L. For maintenance and management of the wild-type yeast strain, a plate medium prepared by adding 20 g of Bacto agar (BD Bioscience) to 1 L of the above YPD medium was used. In addition, for the construction, maintenance and management of yeast strains by genetic engineering, the antibiotics described below were appropriately added to the medium of the above composition so as to have respective concentrations. Geneticin (registered trademark) (hereinafter referred to as G418. Thermo Fisher Scientific Co., Ltd., USA) 200 ug/mL, Zeocin (registered trademark) (Thermo Fisher Scientific Co., Ltd.) 200 ug/mL, Aureobasidin A (Takara Bio Co., Ltd.) Company) 0.5ug/mL. For the growth of Escherichia coli DH5α strain (Nippon Gene Co., Ltd.), Difco LB, Miller medium (BD Bioscience) was used as a prepared LB medium. DifcoLB Agar, Miller medium (BD Bioscience) was used for maintenance and management of the wild-type Escherichia coli strain. For the construction, maintenance and management of genetically engineered transformants using the Escherichia coli DH5α strain as a host, a selection medium in which the antibiotic Ampicillin (Wako Pure Chemical Industries, Ltd.) 100 ug/mL was added to the LB medium was used. I was there. The yeast strain was cultured at 30° C. for 1 to 3 days depending on the growth using a shaker. The culture of Escherichia coli was carried out at 36° C. by standing overnight.

(2. DNA extraction, PCR, transformation, determination of nucleotide sequence)
When extracting a plasmid from transformed E. coli, QIAprep Spin Miniprep Kit (Qiagen, Germany) was used and extraction and purification were performed according to the attached protocol. When the genomic DNA was extracted from the yeast strain, Gen Torukun (for yeast) High Recovery (Takara Bio Inc.) was used for extraction and purification according to the attached protocol. Amplification of a specific DNA fragment, a pair of base sequences that match the target DNA fragment was synthesized using the gene analysis software SnapGene (GSL Biotech, USA) and Genetyx (Genetyx Co., Ltd.) as an oligo DNA (Eurofin Genomics and Thermo Fisher Scientific). The oligo DNA used in this example is shown in FIG. PCR was performed according to the attached reaction protocol using the synthesized oligo DNA as a primer and KOD-Plus-Neo (Toyobo Co., Ltd.) as the DNA polymerase. Specifically, as a PCR reaction solution, 1x PCR buffer for KOD-Plus-Neo, 0.2mM dNTPs, 1.5mM MgSO4, 0.32uM Forward/Reverse primer, template DNA 1ug, KOD-Plus-Neo (1U/50uL). The solution containing 50-100 uL was prepared in a 200 uL PCR tube. Pre-denature: 94°C, 2 minutes, Denature: 98°C, 10 seconds, Annealing: (Tm)°C, 30 seconds, Extension: 68°C, 30 seconds/kb 3 step PCR reaction using a thermal cycler (Veriti Thermal Cycler, The reaction was performed for 25 to 30 cycles using Applied Bioscience/Thermo Fisher. At that time, the annealing temperature was determined based on the Tm of the oligo DNA designed by SnapGene software, and the extension time was determined according to the length of the PCR product. The plasmid vector was constructed by linearizing the plasmid vector serving as a backbone by the PCR reaction, using amplified and purified DNA fragments to be inserted, and using In-Fusion HD Cloning Kit (Takara Bio Inc.), It was carried out by seamlessly ligating via a 15 bp overlapping sequence according to the attached reaction protocol. Specifically, prepare 1x In-Fusion HD Enzyme Premix, 100 ng/reaction linearized vector, and In-Fusion reaction solution of the same number of inserted DNA fragments, and heat to 50°C for 15 minutes using a thermal cycler. The reaction was carried out.

(3. Analysis of promoter possessed by yeast stress response gene)
Prior to the design of the stress response promoter, we focused on the stress response gene of yeast and analyzed its properties. Since the yeast strain used was the industrial yeast strain IR-2, a difference from the genomic nucleotide sequence of the laboratory yeast strain was considered. Therefore, it was examined including the comparison of homologous regions of both strains. It is known that under the strong stress of Glucose starvation for yeast, the transcription level of all genes in cells is lowered, that is, the transcriptional activity of almost all genes expressed in the presence of Glucose is reduced. However, yeast is known to express a specific gene group even under such circumstances. These genes are called stress response genes. Most of them have various functions of gene products such as proteins for quality control of proteins and enzymes for promoting degradation of denatured proteins, but their expression levels transiently and strongly respond. Examples of the stress response gene include HSP12, HSP26, HSP30, HSP42, HSP60, HSP70/SSA1, HSP70/SSA2, HSP70/SSA3, HSP70/SSA4, HSP78, HSP104, SDP1, CPS1, YGP1, ECM10 and the like. Based on the genome database (SGD, Saccharomyces Genome Database, URL http://www.yeastgenome.org/) of the reference strain yeast strain S288c, a non-translated region containing a promoter existing upstream of the ORF encoding these proteins (intergenic Region), the consensus base sequence (HSE, STRE) predicted to bind to a transcription factor called a stress response element contained in the base sequence (HSE, STRE), an incomplete sequence that is similar but does not match the consensus sequence (HSEL, STREL), TATA box required for transcription initiation, putative transcription initiation point and ribosome binding sequence during translation (Kozak sequence, translation initiation point is +1 and one nucleotide upstream is -1 to -1 to- Table 1 shows a summary of 6 bases.

(表１は、「Glucose飢餓ストレス応答遺伝子プロモーターに存在するストレス応答要素の種類及び数」を示す。実験室酵母S288c株においてストレス応答を示すと考えられる代表的な遺伝子として、Yeast Promoter Atlas(YPA、http://ypa.csbb.ntu.edu.tw/)に登録されている15遺伝子、HSP12、HSP26、HSP30、HSP42、HSP60、HSP70/SSA1、HSP70/SSA2、HSP70/SSA3、HSP70/SSA4、HSP78、HSP104、SDP1、CPS1、YGP1、ECM10を選抜し、プロモーター領域に含まれるシスエレメントの種類と数を比較した。また、Kozak配列として、各遺伝子の翻訳開始点(メチオニンをコードするATG)の直前の６塩基を抽出した。Gene、遺伝子名；Length、推定されるプロモーター領域の塩基長(bp)；HSE、熱ショック応答エレメント；HSEL、HSE コンセンサス配列に類似するが、完全には一致しない配列；STRE、ストレス応答エレメント；STREL、ストレス応答エレメントのコンセンサス配列に類似するが、完全には一致しない配列；TATA-box、必須要素であるTATA-boxの位置；TSS、推定転写開始点；Kozak、Kozak配列(-1〜-6)。翻訳効率は-3位(太字)の塩基により影響を受けるが、酵母の場合Aが最も優れたものとされる。Others、各プロモーター塩基配列内に結合すると考えられるストレス応答以外の転写因子)

(Table 1 shows "the type and number of stress response elements present in the Glucose starvation stress response gene promoter". Yeast Promoter Atlas (YPA) is a representative gene considered to exhibit a stress response in the laboratory yeast S288c strain. , 15 genes registered in http://ypa.csbb.ntu.edu.tw/), HSP12, HSP26, HSP30, HSP42, HSP60, HSP70/SSA1, HSP70/SSA2, HSP70/SSA3, HSP70/SSA4, HSP78, HSP104, SDP1, CPS1, YGP1, and ECM10 were selected, and the type and number of cis elements contained in the promoter region were compared.In addition, Kozak sequences of the translation initiation point (ATG encoding methionine) of each gene were selected. The immediately preceding 6 bases were extracted: Gene, gene name; Length, putative promoter region base length (bp); HSE, heat shock response element; HSEL, sequence similar to HSE consensus sequence but not completely matched STRE, stress response element; STREL, a sequence that is similar to the consensus sequence of stress response element but does not completely match; TATA-box, position of essential element TATA-box; TSS, putative transcription start point; Kozak, Kozak sequence (-1 to -6) The translation efficiency is affected by the base at position -3 (bold), but in yeast, A is the best. (Transcription factors other than possible stress response)

  As shown in Table 1, a group of genes known to undergo some kind of transient response to various stresses was selected. The number of HSE and STRE bound by transcriptional regulatory factors HSF1 and MSN2/MSN4 involved in stress response was the highest in HSP70, and three in each. Some genes, such as CPS1 and ECM10, have a promoter region that does not exist, including HSEL and STREL. From the above, it is not possible to conclude that the promoter responds to stress simply by the presence of cis elements that are involved in stress response, such as HSE and STRE, contained in the promoter region. In addition, it is impossible to predict the function of a promoter, which is the timing and the strength of gene expression in response to stress, from the base sequence alone.

(4. Difference in nucleotide sequence of stress-responsive gene promoter between laboratory yeast S288c and industrial yeast IR-2)
Next, regarding the HSP family genes, the nucleotide sequences were compared in order to confirm whether the nucleotide sequences of the promoter regions of the genes are different. Table 2 shows a summary of the results.

(表２は、ストレス応答遺伝子プロモーターの実験室酵母S288c株・工業用酵母IR-2株間の差異を示す。実験室酵母S288c株と工業用酵母株IR-2におけるストレス応答遺伝子HSP12, HSP26, HSP30, HSP42, HSP78, HSP104, HSP70/SSA4, HSP70/SSA1の推定プロモーター塩基配列の差異。Lengthは推定プロモーター領域の塩基数、塩基置換・塩基挿入に記載の数字は、酵母基準株S288cと工業用酵母IR-2の間で異なる塩基の数を示している。NDは完全に一致し、差が無いことを示している)

(Table 2 shows the difference in the stress response gene promoter between the laboratory yeast S288c strain and the industrial yeast IR-2 strain. Stress response genes HSP12, HSP26, HSP30 in the laboratory yeast S288c strain and the industrial yeast strain IR-2. , HSP42, HSP78, HSP104, HSP70/SSA4, HSP70/SSA1 differences in the putative promoter base sequence, Length is the number of bases in the putative promoter region, the numbers in base substitution/base insertion are the yeast reference strain S288c and industrial yeast. (Indicates the number of bases that differ between IR-2. ND is a perfect match, indicating no difference.)

  Some were exactly the same, some were different in bases, some had insertions and substitutions, etc., but the HSE, STRE, HSEL and STREL deduced in Table 1 above were all conserved. None of the elements had mutations. In addition, due to the difference in the nucleotide sequence found between the S288c strain and the IR-2 strain in the gene promoter, new ones corresponding to HSE, STRE sequence and other known cis element sequences in IR-2 newly appear. It never happened.

(表３は結合すると予測される転写因子の遺伝子名とそのコンセンサス配列を示す。転写因子HSF1はHSEに、MSN2/MSN4はSTREにそれぞれ結合すると考えられる)
（７）Glucose存在下で抑制的に作用する、また糖新生に関与すると考えられている転写因子Rap1, Nrg1, Gln3, Hap4/5, Adr1, Mig1, Mot3, Snf1の結合するシスエレメントのコンセンサス配列(表４)が生じないように、類似配列も含めて置換あるいは欠損により削除する。

(表４は結合すると予測される転写因子の名称とそのコンセンサス配列を示す)
（８）推定される転写開始点とタンパク質のコード領域との間に、予期せぬ翻訳開始アミノ酸であるメチオニンをコードするATG配列が含まれないようにする。もし、不測のATGが生成された場合には、置換あるいは欠損により削除する。
（９）改変の仕方は、塩基・領域の置換(replacement)、挿入(insertion)、削除(deletion)、移動(move)の４方法によるものとする。
この戦略に従って、37塩基・領域の置換37箇所、挿入9箇所、削除33箇所、移動1箇所の合計80ステップの改変(表５)によって、ストレス応答プロモーターを設計した。

(表５は、酵母のHSP26、HSP42、HSP78、HSP104の部分塩基配列を元に、replacement(塩基置換)、insertion(挿入)、deletion(削除)、move(移動)の改変ステップを行うことによって設計された新規ストレス応答プロモーター塩基配列の、設計過程の各段階の履歴を示す。Position/Regionは当該塩基の位置(翻訳開始点メチオニンをコードするATGのAを1位、その一つ上流を-1位とする)；Before、改変前の塩基；After、改変後の塩基。) (5. Design of stress response promoter)
From the above results, it was not possible to find a relationship with the stress response intensity and duration regarding the arrangement and number of HSE and STRE. In addition, an artificial promoter in which HSE and STRE are designed in a tandem repeat form is known as a core promoter, but it is known that its transcriptional activity and stress response are not high. Among the HSE and STRE sequences, it was hypothesized that the cis elements internal to the HSP104, HSP78, HSP42 promoters are important for long-term stress response properties. The 5'-untranslated region of the gene located downstream of the promoter (hereinafter referred to as 5'-UTR) may be an endogenous one, but the 5'-UTR end should determine the translation activity. Is known and called the Kozak array. In the case of yeast, the Kozak sequence has the highest translational activity when the base at the -3 position is A, and the sequence having the highest translational activity is 5'-AATATTAAAA-3' (SEQ ID NO: 49). We also hypothesized that it is important that HSE and STRE function as scaffolds for transcription mediators in order to maintain transcriptional activity and gene expression under long-term stress. In addition, in addition to Glucose starvation, yeast can avoid critical situations by gluconeogenesis, utilizing carbohydrates that are not normally used as energy sources, such as Ethanol and acetic acid. Since gluconeogenesis is not the production from Glucose but is the consumption of the product that tries to make sugar from the substance produced in the opposite, it is desirable to delete the cis element activated by gluconeogenesis from the artificial promoter base sequence. . In light of the above, the process of producing useful substances by microorganisms results in Glucose starvation as a result of high concentration of substrate, suboptimal temperature for host microorganisms, various inhibitory factors, and competition for Glucose, which is a major carbon source. In consideration of the fact that the producing microorganisms are inevitably loaded, the following is a basic strategy for designing artificial promoters capable of performing strong gene expression in such situations. did.
(1) The starting point is a base sequence of pHSP26, pHSP42, pHSP78, and pHSP104, in which the peripheral base sequences including the HSE and STRE base sequences are aligned to form 500 bp.
(2) It was hypothesized to be important for long-term stress response characteristics. All incomplete HSE and STRE are replaced with complete HSE and STRE sequences.
(3) The HSE and STRE nucleotide sequences are arranged upstream of the TATA-box, and each cis element is not directly adjacent (tandem repeat), but is arranged at intervals of 1 bp or more and 100 bp or less.
(4) HSE and STRE are arranged alternately as much as possible. (5) In order to improve translational activity, 5'-UTR downstream of the promoter is HSP26, but it is not necessary. However, the nucleotide sequence from position -1 to -10 is replaced with the optimal Kozak sequence 5'-AATATTAAAA-3', which is considered to have the highest translation efficiency in yeast (6). A consensus sequence (Table 3) of cis elements binding to transcription factors Met31/32, Rgt1, Cad1, Cin5/Yap4, and Yap1, which are considered to be certain activators, is added.

(Table 3 shows the gene names of the transcription factors predicted to bind and their consensus sequences. It is considered that the transcription factor HSF1 binds to HSE and MSN2/MSN4 binds to STRE.)
(7) Consensus sequence of cis elements that bind to transcription factors Rap1, Nrg1, Gln3, Hap4/5, Adr1, Mig1, Mot3, Snf1 that are thought to be inhibitory in the presence of Glucose and involved in gluconeogenesis To prevent (Table 4) from occurring, the similar sequences are deleted by substitution or deletion.

(Table 4 shows the names of transcription factors predicted to bind and their consensus sequences)
(8) The ATG sequence encoding methionine, which is an unexpected translation initiation amino acid, should not be included between the putative transcription initiation point and the coding region of the protein. If an unexpected ATG is generated, delete it by replacing or deleting it.
(9) The method of modification is based on the four methods of base/region substitution (replacement), insertion (deletion), deletion (deletion), and movement (move).
According to this strategy, a stress-responsive promoter was designed by modifying 37 bases/regions at 37 positions for replacement, 9 positions for insertion, 33 positions for deletion, and 1 position for migration (Table 5) for a total of 80 steps.

(Table 5 is designed based on partial base sequences of yeast HSP26, HSP42, HSP78, and HSP104 by performing modification steps of replacement (base substitution), insertion (deletion), move (movement)) The history of each stage of the design process of the identified novel stress-responsive promoter nucleotide sequence is shown in Position/Region, where the position of the nucleotide is (position A of ATG encoding the translation initiation point methionine is position 1, and one upstream thereof is -1 Position); Before, base before modification; After, base after modification.)

(表６は、設計された新規ストレス応答プロモーターpSynSR480の塩基配列を示す。非翻訳領域(5’-UTR)は厳密にはプロモーターではないが、下流の遺伝子発現には、転写開始点と翻訳開始点の間に何らかの5’-UTRが必要である。5’-UTRは、本来、下流の遺伝子配列の一部でありまた遺伝子に固有のものであるが、発現させる遺伝子が外来遺伝子である場合、特に細菌等の原核生物の場合、酵母のような真核生物で機能しうる5’-URTが存在しない。また、5’-UTRには、転写制御因子が含まれる可能性もあるため、ストレス応答プロモーターpSynSR480の設計上、無視することはできない。そのため、改変のベースとして用いた酵母HS26, HSP42, HSP78, HSP104遺伝子のうち、HSP26遺伝子の5’-UTRを含めて塩基配列を設計した。すなわち、HSP26遺伝子の予測転写開始点である-64位のAの上流-480〜-62(419 bp)がプロモーター領域として転写調節の機能を、-61〜-1(61 bp)が5’-UTRとしての機能を有すると考えられる。ただし、表５の改変履歴にあるように、Kozak配列の最適化を含め5’-UTRの中にある不要な塩基配列も削除・改変している) A 480 bp nucleotide sequence containing a 419 bp transcriptional regulatory region and 61 bp 5'-UTR was designated as pSynSR480 and synthesized by an artificial gene (Eurofin Genomics). In pSynSR480, the structure of the site predicted to bind the transcription factor is shown in FIG. 1, and the nucleotide sequence is shown in Table 6.

(Table 6 shows the designed nucleotide sequence of the novel stress-responsive promoter pSynSR480. Although the untranslated region (5'-UTR) is not strictly a promoter, it has a transcription start point and a translation start for downstream gene expression. There must be some 5'-UTR between the points, which is originally part of the downstream gene sequence and is unique to the gene, but when the gene to be expressed is a foreign gene. , Especially in the case of prokaryotes such as bacteria, there is no 5′-URT that can function in eukaryotes such as yeast, and because 5′-UTR may contain transcription factors, Since the stress response promoter pSynSR480 cannot be ignored in the design, the nucleotide sequence was designed including the 5'-UTR of the HSP26 gene among the yeast HS26, HSP42, HSP78, and HSP104 genes used as the base for modification. That is, the predicted transcription initiation point of the HSP26 gene-upstream of A at position -64-480 to -62 (419 bp) functions as a promoter region for transcriptional regulation, and -61 to -1 (61 bp) is 5'-. It is considered to have a function as a UTR.However, as shown in the modification history of Table 5, unnecessary nucleotide sequences in the 5'-UTR are deleted and modified, including optimization of Kozak sequences.)

(6. Search for a nucleotide sequence most similar to the stress response promoter pSynSR480)
In order to verify whether a sequence similar to the nucleotide sequence designed above exists in nature, a 419 bp nucleotide sequence involved in transcriptional control of pSynSR480 excluding 61 bp 5'-UTR was used as a query sequence, BLAST program (NCBI: National Center for Biotechnology Information, https://blast.ncbi.nlm.nih.gov/Blast.cgi) was searched for all the nucleotide sequences registered in the database, the top 50, All of them corresponded to the genomic DNA of Saccharomyces cerevisiae strains, but the similar portions were localized at both ends.

  The sequence showing the highest similarity (Score 118 bits) to the longest part of the query sequence, 169 bp, was 130 bp (77%) and contained a 23 bp (14%) gap. Next, since the candidates were narrowed down to yeast, the same WU-BLAST search (SGD) was performed on the S288c strain, and only 3 results were obtained.

  The identities for the result with the highest score (Score 81.1 bits) included gaps of 265 bp/419 bp (63%) and 34 bp/419 bp (8%). This region was the region corresponding to 404 bp in the promoter of the HSP26 gene, which is the basis of the design. The predicted transcription factor binding site in pHSP26 (404 bp) was only one HSE (Fig. 2).

(7. Construction of plasmid vector)
Using the stress-responsive promoter pSynSR480 designed above, a highly active mutant (CpXImut2mut20) of Clostridium phytofermentans-derived Xylose isomerase gene (CpXI; accession number ABX41597) that we have already obtained is homologously recombined into AUR1 chromosome. A plasmid vector for introduction into the site was established by the following procedure.
(1) Using the pAUR101 vector (Takara Bio) having the yeast AUR1 chromosomal region and AUR1-C (Aureobasidin A resistance gene, dominant) as a template, PK98 and PK106 were used to amplify a linearized DNA fragment by PCR reaction And purified. A plasmid vector pAUR101-pHSP12-CpXI-CYC1t-XKS1-pPGK1 for expressing Xylose isomerase and yeast-derived xylulose phosphatase ScXKS1 was used (plasmid No. 1, FIG. 3).
(2) The above plasmid vector uses a CYC1 terminator that can act bidirectionally. In order to replace the terminator of the ScXKS1 gene with that of the TEF1 terminator so as to obtain a more stable mRNA, IR-2idA30a Using the genomic DNA extracted from the strain as a template, PK99 and PK100 were used to amplify a DNA fragment by a PCR reaction and purification was performed.
(3) Similarly, in order to replace the terminator of CpXI with that of ADH1, using genomic DNA extracted from the IR-2idA30a strain as a template, PK107 and PK108 were used to amplify a DNA fragment by PCR reaction and purification. went.
(4) The two fragments of (2) to (3) were cloned into the linear plasmid vector of (1) by In-Fusion reaction, and circular plasmid vector pAUR101-pHSP12-CpXI-ADH1t-TEF1t-XKS1-pPGK1 (plasmid No. 2, FIG. 4) was constructed.
(5) After the In-Fusion reaction, Escherichia coli DH5α was transformed using this reaction solution, and cultured overnight in LB plate medium containing Ampicillin to obtain a transformant clone. This clone was cultured overnight in LB medium containing Ampicillin, and pAUR101-pHSP12-CpXI-ADH1t-TEF1t-XKS1-pPGK1 plasmid DNA was extracted. Moreover, it was confirmed by the Sanger sequence method that the nucleotide sequence of the obtained plasmid vector was correct.
(6) pAUR101-pHSP12-CpXI-ADH1t-TEF1t-XKS1-pPGK1 plasmid DNA as a template, PCR reaction and purification using P97 and P107 to obtain a linear DNA fragment lacking the promoter region of CpXI gene did.
(7) As a promoter for expressing the Xylose isomerase gene mutant CpXI, the conventionally used strong promoter 3-phosphoglycerate kinase 1 gene promoter pPGK1 and the novel stress response promoter pSynSR480 designed by the above are used as Xylose isomerase. In order to insert it upstream of the gene, PCR reaction was carried out using PK97 and PK115 and PK117 and PK118, respectively, to obtain a promoter DNA fragment.
(8) The promoter DNA fragments pPGK1 and pSynSR480 obtained in (7) were each cloned into the linear plasmid vector of (6) by In-Fusion reaction.
(9) After the In-Fusion reaction, Escherichia coli DH5α was transformed using these reaction solutions, and cultured overnight in LB plate medium containing Ampicillin to obtain a transformant clone. This clone was cultured overnight in LB medium containing ampicillin, and pAUR101-pPGK1-CpXI-ADH1t-TEF1t-XKS1-pPGK1 (plasmid number 3, FIG. 5) plasmid DNA and pAUR101-pSynSR480-CpXI-ADH1t-TEF1t- XKS1-pPGK1 (plasmid number 4, FIG. 6) plasmid DNA was extracted. The nucleotide sequence of the obtained plasmid vector was confirmed to be correct by the Sanger sequencing method (Eurofingenomics).
(10) The plasmid vectors of plasmid numbers 3 and 4 were linearized with a restriction enzyme StuI existing at one site in the AUR1-C gene, separated by agarose gel electrophoresis, and the band was cut out, and then the QIAquick Gel Extraction Kit (Qiagen). Using 1 ug of the purified DNA, the yeast KEF180 strain (MATahoΔ::loxP gre3Δ::kanMX-loxP, Table 7: strain number 2) was transformed. The transformation was performed by the lithium acetate method, and the cells were cultured in an Aureobasidin A-added YPD plate medium at 30° C. for 3 days to select a transformant clone. These strains were named KEF205 (control) and KEF206. The only difference between these two strains is whether the promoter for expressing the Xylose isomerase gene CpXI is pPGK1 or pSynSR480.

(表７は本実施例で使用した株とその遺伝型を示したものである。IR-2idA30Aは２倍体であるIR-2からホモタリズムを欠如し、１倍体を維持するためにHO遺伝子をノックアウトした後に、分離したものである。KEF180は、IR-2idA30aを親株とし、内在性のXylose reductase遺伝子(GRE3)を欠損させたものである。GRE3はXyloseをXylitolに変換するXylose reductase酵素をコードする遺伝子である。内在性のGRE3遺伝子が取り込まれたXyloseを基質として生産するXylitolはXylose異性化酵素を阻害すると考えられる。また、Xylitolの生産はEthanol発酵における副産物であり、Ethanol収率を低下させるため、あらかじめ欠損させた。KEF205、KEF206はXylose異性化酵素を発現させるためのプロモーターとしてpPGK1(KEF205)、pSynSR480(KEF206)を構成したプラスミドベクターを染色体上のAUR1部位に挿入したものである。Xylose異性化酵素以外に、酵母では発現が低く、また機能が不十分であると考えられているxyluloseをリン酸化するxylulokinase遺伝子(XKS1)をpPGK1により発現させている。 Table 7 shows the names and genotypes of the industrial yeast strains used in this example.

(Table 7 shows the strains used in this example and their genotypes. IR-2idA30A lacks homothalism from diploid IR-2, and the HO gene is required to maintain haploid. KEF180 was isolated after knocking out the gene, KEF180 was the parent strain of IR-2idA30a, and the endogenous Xylose reductase gene (GRE3) was deleted. Xylitol, which uses the endogenous GRE3 gene incorporated as a substrate to produce Xylose as a substrate, inhibits the Xylose isomerase, and Xylitol production is a by-product in Ethanol fermentation, KEF205 and KEF206 were inserted into the AUR1 site on the chromosome of a plasmid vector constructed with pPGK1(KEF205) and pSynSR480(KEF206) as promoters for expressing Xylose isomerase. In addition to Xylose isomerase, the expression is low in yeast, and the xylulokinase gene (XKS1) that phosphorylates xylulose, which is considered to have insufficient function, is expressed by pPGK1.

(8. Fermentation test using YPDX simulated saccharified solution)
In order to evaluate the transcriptional activity of the stress response promoter pSynSR480, a KEF206 strain expressing a Xylose isomerase mutant having high Xylose isomerization activity in yeast (hereinafter, CpXI) by the promoter, conventionally, a strong gene expression as a control is used. Using the KEF205 strain that also expresses CpXI by the enabled PGK1 promoter, an Ethanol fermentation test using a YPDX pseudo saccharified solution was performed according to the following procedure.

(1) Preparation of inoculated cells KEF205 and KEF206 strains, which had been developed in advance on Aureobasidin A-containing YPD plate medium, were inoculated into 2 mL of Aureobasidin A-containing YPD medium (14 mL test tube), and at 30°C, 150 rpm for 1 day. Shaking culture was performed (pre-preculture). 20 mL of YPD medium (100 mL baffled flask) was inoculated with the whole amount of the pre-pre-preculture medium and cultivated with shaking at 135° C. and 135 rpm for 1 day (pre-culture). The total amount of the preculture solution was collected by centrifugation (3,000 rpm, 5 minutes, 4° C.) to collect the yeast cells, resuspended in 10 mL of YPDX pseudo saccharified solution, and the absorbance A600 was measured (Smart Spec, Bio-Rad). .. Dilution was performed based on this value, and the final cell concentration was adjusted to OD _A600 =30.

(2) Fermentation test
To a 100 mL flask, 60 mL of the pseudo saccharification solution YPDX was added in advance, and 7 mL of the cell suspension prepared in (1) was inoculated. The total volume of the culture solution was 70 mL, and the cell concentration at the start of the fermentation test was ODA600=3. This fermentation test is conducted under microaerobic conditions to maximize the production of Ethanol, and in order to exhaust gases such as carbon dioxide generated during sampling and fermentation, a sampling injection needle (Terumo Co., 21G , 90 mm) and an aeration needle (Terumo Co., 21G) penetrating a soft silicone stopper (non-vented), the flask was closed and at 38° C., 135 rpm, in a gas phase incubator (Titech Co., BR-40LF). Shaking culture was performed for 72 hours.

(3) Sampling A three-way stopcock (Terumo Co., Ltd.) was connected to the upper part of the sampling needle, the cock was opened only at the time of sampling, and a 2.5 mL syringe was used to start culture 1, 3, 6, About 0.5 mL of each sample was aseptically sampled at 13, 27, 36, 48, 60 and 72 hours. The collected culture solution was immediately ice-cooled, and centrifuged at 15,000 rpm, 5 minutes, 4°C using a high-speed centrifuge with a cooler, and about 400 uL of the supernatant was added so as not to contain precipitated bacterial cells. Was transferred to a new Eppendorf tube and frozen and stored in a -30°C medical freezer until HPLC measurement.

(9. Determination of sugar, sugar alcohol, organic acid and alcohol by HPLC)
In order to quantify the concentrations of Glucose, Xylose, Xylitol, Glycerol, Acetate and Ethanol in the sampled culture medium, measurement was performed using high performance liquid chromatography (HPLC). Aminex HPX-87C column (Bio-Rad) and Cation H Refill Guard column (Bio-Rad) and HPLC LC-2000Plus series (JASCO Corporation) equipped with a differential refractometer (JASCO Corporation) are used for measurement. I was there. Using 5 mM H ₂ SO ₄ as a mobile phase, each component was separated under the conditions of a flow rate of 0.6 mL/min, a temperature of 65° C., and an input sample amount of 25 uL. Glucose, Xylose, Xylitol, Glycerol, Acetate and Ethanol were mixed at concentrations of 2% (w/v), 1% (w/v), 0.1% (w/v) and 0.01% (w/v), respectively. The same measurement was performed using the standard substance solution, and a calibration curve was obtained from the peak areas of the respective components. The concentration of each substance contained in the culture solution sample was calculated from the area obtained by the HPLC measurement using a calibration curve. In order to reduce the measurement error of volatile components such as Acetate and Ethanol, the standard solution was prepared by dispensing the required volume into an Eppendorf tube, which was frozen and stored in a -30°C medical freezer.

(表８は、pPGK1によりXylose異性化酵素を発現する株KEF205の微好気条件下における擬似糖化液を用いて行った発酵試験の結果、発酵溶液に含まれる各時間における各種成分のHPLC分析の結果を示す。hr: 発酵開始からの時間、Glucose, Xylose, Xylitol, Glycerol, Acetate及びEthanolの数値は各時間における各成分の濃度(g/L)。n.d.は、HPLCの検出限界以下であることを示す。)

(表９は、pSynSR480によりXylose異性化酵素を発現する株KEF205の微好気条件下における擬似糖化液を用いて行った発酵試験の結果、発酵溶液に含まれる各時間における各種成分のHPLC分析の結果を示す。hr: 発酵開始からの時間、Glucose, Xylose, Xylitol, Glycerol, Acetate及びEthanolの数値は各時間における各成分の濃度(g/L)。n.d.は、HPLCの検出限界以下であることを示す。) (10. Results of YPDX simulated saccharified liquid fermentation test)
The results of the fermentation test using the YPDX pseudo-saccharified solution containing Glucose (85 g/L) and Xylose (35 g/L) imitating the cellulosic biomass saccharified solution are shown in FIG. 7, Table 8 and Table 9.

(Table 8 shows the results of a fermentation test carried out using a pseudo-saccharified solution of the strain KEF205 that expresses Xylose isomerase by pPGK1 under microaerobic conditions, as a result of HPLC analysis of various components contained in the fermentation solution at each time. The results are shown: hr: The time from the start of fermentation, the values of Glucose, Xylose, Xylitol, Glycerol, Acetate and Ethanol are the concentrations (g/L) of each component at each time.nd is below the detection limit of HPLC. Is shown.)

(Table 9 shows the results of the fermentation test conducted using a pseudo-saccharified solution under microaerobic conditions of the strain KEF205 expressing the Xylose isomerase by pSynSR480, showing the results of HPLC analysis of various components contained in the fermentation solution at each time. The results are shown: hr: The time from the start of fermentation, the values of Glucose, Xylose, Xylitol, Glycerol, Acetate and Ethanol are the concentrations (g/L) of each component at each time.nd is below the detection limit of HPLC. Is shown.)

  As is clear from comparison between FIGS. 7A and 7B, the Xylose consumption rate of the KEF206 strain expressing the Xylose isomerase gene CpXI using the newly designed stress responsive promoter pSynSR480 was found to express the same gene using the PGK1 promoter. It was significantly faster than the Xylose consumption rate of the KEF205 strain. Also, Ethanol productivity was correspondingly improved. At 72 hours of fermentation, the KEF206 strain consumed all 36.24 g/L of the initial amount of Xylose (Table 9), but the KEF205 strain could not consume all the amount, leaving 7.20 g/L (Table 8). Similarly, the 72-hour Ethanol productivity of the KEF206 strain was 0.783 g/L/h (Table 9), whereas the KEF205 strain was 0.702 g/L/h (Table 8). This corresponds to a 12% increase in Ethanol productivity. The KEF206 strain had an Ethanol conversion efficiency per input sugar of 91% (Table 9), whereas the KEF205 strain had 82% (Table 8). This corresponds to an increase in the Ethanol yield of 11%. On the other hand, the KEF206 strain had an ethanol conversion efficiency of 91% per consumed sugar (Table 9), whereas the KEEF205 strain had 90% (Table 8). The conversion efficiency of Ethanol per consumed sugar, that is, the efficiency of converting the sugar taken up by the yeast and consumed to Ethanol, is almost the same, and therefore, the activity of metabolic enzymes other than Xylose isomerase involved in Xylose metabolism is Suggests that there is no change. From the above results, it is considered that the improvement in Ethanol production performance in the KEF206 strain is due to the increase in the amount of Xylose consumed as a result of the improvement in Xylose consumption performance. The increase in the consumption rate of Xylose is controlled by the first Xylose metabolic gene, Xylose isomerase in the Xylose metabolic pathway, and the transcriptional activity of the Xylose isomerase gene was improved by the stress-responsive promoter. It was concluded that it was due to the increased amount, and it was revealed that it has a higher transcriptional activity suitable for the Ethanol fermentation production process than the yeast PGK1 promoter, which has been capable of expressing the highest gene in the past. ..

Ethanol productivity (g/L/h) is the concentration of Ethanol produced divided by fermentation time and is given by equation [1]. Here, the values at 72 hours of producing maximum Ethanol are shown.
Formula [1] Ethanol productivity (g/L/h) = Ethanol concentration at the fermentation time (g/L) ÷ fermentation time (h)
The Ethanol yield (g/g) in the fermentation test is given by the formula [2].
Formula [2] Ethanol yield (g/g) = Mass of Ethanol produced (g) / Total mass of input sugar (g)
Ethanol conversion efficiency (%) is the ratio of the Ethanol yield (g/g) obtained in the actual fermentation test to the theoretical Ethanol yield (g/g), which is the theoretical maximum value of the Ethanol yield, multiplied by 100. Which is given by equation [3]. The theoretical Ethanol yield (g/g) from Glucose to Ethanol and from Xylose to Ethanol is 0.51.
Formula [3] Ethanol conversion efficiency (%) = Ethanol yield (g/g) ÷ theoretical Ethanol yield (g/g) × 100

(11. Simultaneous simultaneous saccharification and parallel fermentation test using sugar cane bagasse treated with sodium hydroxide)
Fermentation test using the above YPDX simulated saccharified solution is a saccharified solution fermentation process (SHF) that is composed of laboratory reagents whose component composition such as sugar mimics the saccharified solution of biomass produced from cellulosic biomass. Met. On the other hand, as described above, in the industrial production of cellulosic biomass ethanol, the simultaneous saccharification and parallel multiple fermentation process (SSCF) is an industrial process that simultaneously performs the saccharification reaction and the fermentation reaction, in addition to the saccharified liquid fermentation process. In order to show that the stress-responsive promoter can improve the expression of the Xylose isomerase gene, which leads to high Xylose assimilation performance and improvement of Ethanol productivity in the fermentation process using cellulosic biomass as a substrate, The following experiment was conducted.

(1) Substrate It is considered that non-edible parts of various plants such as corn, sugar cane and palm can be used as a substrate for cellulosic biomass. Here, as a typical substrate, a pulverized product of sugarcane bagasse was used, which was subjected to hydrothermal treatment with sodium hydroxide and then washed with water to separate solid components (JGC Corporation). This solid component contained 53.50% cellulose, 18.60% hemicellulose, 35.95% lignin and other ash, and the water content was 72.61%. Assuming that Glucose is produced from cellulose (glucan) and Xylose from hemicellulose (xylan), when these are fully saccharified by saccharifying enzymes (cellulases, hemicellulases), Glucose and Xylose from the dry solid biomass are about 141 g/x. L It was decided to use the amount of substrate supplied. This is nothing but supplying sugar corresponding to the sugar concentration of 120 g/L used in the YPDX pseudo-saccharified liquid fermentation test in the case of 85% saccharification.

(2) Saccharifying enzyme As the saccharifying enzyme, JN24 containing hemicellulase capable of producing Xylose from hemicellulose (xylan) was used in addition to cellulase producing Glucose from cellulose (glucan) (Kao Corporation). An enzyme solution containing 8.7 mg of protein per mass of dry cellulose/hemicellulose was used as 1X.

(3) Preparation of inoculated cells
The KEF205 and KEF206 strains were inoculated into 2 mL of Aureobasidin A-containing YPD medium (14 mL test tube), and shake culture was performed at 30° C. and 150 rpm for 1 day (pre-preculture). 20 mL of YPD medium (100 mL baffled flask) was inoculated with the whole amount of the pre-pre-preculture medium, and cultured at 30° C. with 135 rpm shaking for 1 day (pre-culture). The total amount of the preculture solution was collected by centrifugation (3,000 rpm, 5 minutes, 4°C) to collect the yeast cells, washed with 50 mL of sterilized water, centrifuged, and resuspended in 10 mL of sterilized water to obtain the absorbance A600. Measured (Smart Spec, Bio-Rad).

(4) Simultaneous simultaneous saccharification and double fermentation test Using an infrared moisture meter (FD-720, Ketsuto Scientific Laboratory Co., Ltd.), measure the moisture content (%) on the day of the experiment of the sugarcane bagasse pretreated product, and subtract the moisture content from 100. The solid content rate (%) was calculated from the measured values. From this solid content, the sugarcane bagasse pretreatment product in an amount capable of supplying 141 g/L sugar at the time of complete saccharification was weighed from the solid content, and a plastic cell culture flask (cap with a hydrophobic filter, AGC Techno Glass Co., Ltd.), then, sterilized water, saccharifying enzyme (1X ~ 4X), 25 mM dilute sulfuric acid (SIGMA) was added for pH adjustment, finally, the cell concentration at the start of the fermentation test OD The cell solution was added so that the amount became 3, and the mixture was stirred so as to be uniform. From the above, setup was performed so that the total solution amount was 20 mL, the slurry concentration was about 20% (w/v), and the initial pH was 5. In order to perform oxygen limitation, these flasks were doubly placed in an oxygen-impermeable vinyl bag with a chuck (Mitsubishi Gas Chemical Co., Inc.), and after sealing, a 38°C gas phase incubator (TAITEC, BR- 40LF) was carried out at the same time. In order to prevent the vinyl bag from inflating and bursting due to gas such as carbon dioxide generated by cell growth or Ethanol fermentation, degassing was appropriately performed.

(5) Sampling
Ethanol fermentation is known to significantly reduce the production amount and productivity in the presence of oxygen. Therefore, in order to prevent oxygen inflow due to sampling as much as possible, sampling was performed only once per flask for 120 hours of fermentation. At each time, about 1.0 mL of the fermentation solution containing the fermentation residue was collected, transferred to an Eppendorf tube, and immediately cooled with ice, using a high-speed centrifuge with a cooler, 15,000 rpm, 30 minutes, 4°C. The supernatant was transferred to a new Eppendorf tube so as not to contain the precipitated biomass residue, and stored frozen in a -30°C medical freezer until HPLC measurement. The quantification of sugars, sugar alcohols, organic acids and alcohol components contained in the fermentation solution by HPLC was carried out in the same manner as in 9 above.

pPGK1によりXylose異性化酵素を発現するKEF205株とpSynSR480によってXylose異性化酵素を発現するKEF206株を比較すると、発酵120時間のEthanol生産量はどの酵素濃度においても、KEF206株がKEF205株を上回っていた。最大エタノール生産は、4Xの糖化酵素を用いた場合のKEF206株で、Ethanol濃度は61.19g/Lであった。推定糖化率98.4％、推定変換効率90.7％、SSF効率は89.2％であった。1X〜4Xの各酵素濃度の発酵性能を詳細に比較すると、推定変換効率は、KEF206株はKEF205株より、それぞれ5.2％、3.5％、3.0％、0.6％高く、また、推定糖化率は、KEF206株はKEF205株より、それぞれ5.0％、5.1％、2.4％、2.1％高かった。その結果、バイオエタノール生産の指標となるSSF効率は、KEF206株はKEF205株より、それぞれ10.5％、8.7％、5.6％、2.9％高かった。すなわち、YPDX培地を用いたYPDX擬似糖化液発酵試験と同じく、水酸化ナトリウム処理サトウキビバガスを用いた同時糖化併行複発酵試験においても、ストレス応答プロモーターpSynSR480はpPGK1より強くXylose異性化酵素遺伝子を発現しており、その結果として、エタノール生産量の増大につながったと考えられる。同時糖化併行複発酵においては、糖化反応が律速になりうる。すなわち糖が供給されなければ、酵母は発酵することができないため、エタノール生産は頭打ちになる。糖化酵素に含まれる各酵素は、自身の酵素活性によって生成した単糖やオリゴ糖によって、生成物阻害を受けることが知られている。同時糖化併行複発酵プロセスでは、酵母が効率よく糖を消費することによって生成物(糖)の濃度が減少することによって、糖化酵素はリサイクルされ、より効率的に糖化反応が進むという好循環が生まれると考えられる。本実験では、酵母固有の性能である推定変換効率及び推定糖化率のいずれにおいても、KEF206株のほうがKEF205株を上回るという結果が得られた。この結果は、KEF206で用いられているpSynSR480は、単により多くのXylose異性化酵素を発現するだけでなく、KEF206株がより早く糖を消費することができ、その結果、糖化率の向上にも寄与しうることを示唆している。これらの結果をまとめると、発酵プロセスに適したストレス応答プロモーターpSynSR480は、高いXylose異性化酵素発現を通じて、従来強いとされてきたpPGK1を用いるよりも、高い効率でバイオエタノール生産が可能であることを示している。 (12. Results of simultaneous saccharification and parallel multiple fermentation test using sugar cane bagasse treated with sodium hydroxide)
The results of the simultaneous saccharification and parallel multiple fermentation test using the KEF205 and KEF206 strains are shown in FIG. 8 and Table 10.

Comparing the KEF205 strain expressing Xylose isomerase with pPGK1 and the KEF206 strain expressing Xylose isomerase with pSynSR480, the KEF206 strain exceeded the KEF205 strain at all enzyme concentrations for 120 hours fermentation. . The maximum ethanol production was KEF206 strain using 4X saccharifying enzyme, and the Ethanol concentration was 61.19 g/L. The estimated saccharification rate was 98.4%, the estimated conversion efficiency was 90.7%, and the SSF efficiency was 89.2%. Comparing the fermentation performances of each enzyme concentration of 1X to 4X in detail, the estimated conversion efficiency of the KEF206 strain was 5.2%, 3.5%, 3.0%, 0.6% higher than that of the KEF205 strain, respectively, and the estimated saccharification rate was KEF206. The strains were 5.0%, 5.1%, 2.4% and 2.1% higher than the KEF205 strain, respectively. As a result, the SSF efficiencies, which are indicators of bioethanol production, were higher in KEF206 strain than in KEF205 strain by 10.5%, 8.7%, 5.6% and 2.9%, respectively. That is, similarly to the YPDX pseudo-saccharified solution fermentation test using YPDX medium, also in the simultaneous saccharification parallel multiple fermentation test using sodium hydroxide-treated sugarcane bagasse, the stress response promoter pSynSR480 expresses the Xylose isomerase gene stronger than pPGK1. As a result, it is thought that this has led to an increase in ethanol production. In the simultaneous simultaneous saccharification and parallel fermentation, the saccharification reaction can be rate-determining. That is, without sugar, yeast cannot ferment and ethanol production peaks. It is known that each enzyme contained in a saccharifying enzyme is subject to product inhibition by monosaccharides and oligosaccharides produced by its own enzymatic activity. In the simultaneous simultaneous saccharification and parallel multiple fermentation process, the yeast efficiently consumes sugar, and the concentration of the product (sugar) decreases, so that the saccharifying enzyme is recycled and a virtuous cycle in which the saccharification reaction proceeds more efficiently is created. it is conceivable that. In this experiment, the KEF206 strain was superior to the KEF205 strain in both the estimated conversion efficiency and the estimated saccharification rate, which are performances unique to yeast. This result indicates that the pSynSR480 used in KEF206 not only expresses more Xylose isomerase, but also that the KEF206 strain can consume sugar faster, and as a result, it also improves the saccharification rate. It suggests that it can contribute. Taken together, these results show that the stress-responsive promoter pSynSR480 suitable for the fermentation process can produce bioethanol with higher efficiency than pPGK1 which has been conventionally considered to be strong, through high Xylose isomerase expression. Showing.

In the simultaneous simultaneous saccharification and parallel fermentation test, it is difficult to directly measure the saccharification rate. This is because the fermentation solution containing the residue is not only unsaccharified cellulose/hemicellulose, but also a miscellaneous secondary component derived from biomass, and also a yeast cell. Therefore, the amount of unsaccharified sugar contained in this fermentation solution cannot be directly quantified. Therefore, the ratio of the amount of sugar produced from the input cellulose/hemicellulose, that is, the saccharification rate, is contained in the total carbon amount contained in the biomass input to the flask and the sugar/organic acid/alcohol etc. measured by HPLC. It can be calculated as a ratio of total carbon content and is given by the following equation [4].
Formula [4] Estimated saccharification rate = (carbon content in metabolites in the fermentation solution) / (carbon content in input biomass)
The efficiency of the simultaneous simultaneous saccharification and parallel multiple fermentation process (SSCF efficiency) is divided by the theoretical maximum Ethanol amount that can be maximally produced from the input biomass, and is given by the equation [5].
Formula [5] SSCF efficiency = amount of produced Ethanol ÷ theoretical maximum amount of Ethanol The estimated conversion efficiency, which is the ethanol-specific fermentation performance of Ethanol, is given by the following formula [6].
Formula [6] Estimated Ethanol conversion efficiency = SSCF efficiency ÷ Estimated saccharification rate

Claims (16)

(I) at least one heat shock element HSE,
(Ii) STRE, which is at least one stress response element,
(Iii) the A HSE and cis elements different from said STRE, viewed contains a cis element that binds the transcription activator expression under stress conditions is enhanced,
At least two or more of (i) the heat shock element HSE and (ii) the stress response element STRE,
A stress responsive promoter in which the HSE and the STRE are alternately arranged (however, the stress responsive promoter is involved in gluconeogenesis and a cis element to which a transcription factor that acts suppressively in the presence of Glucose binds. A cis element to which a transcription factor binds, and does not include a cis element containing the consensus sequence shown in any of SEQ ID NOs: 23 to 47 ). (I) at least one heat shock element HSE,
(Ii) STRE, which is at least one stress response element,
(Iii) a cis element different from the above HSE and STRE, which binds to a transcriptional activator whose expression is enhanced under stress conditions
Including,
The transcriptional activator whose expression is enhanced under the stress condition is at least one transcriptional activator selected from the group consisting of Met31/32, Rgt1, Cad1, Cin5/Yap4, and Yap1, a stress-responsive promoter (however, The stress-responsive promoter is a cis element to which a transcription factor that acts suppressively in the presence of Glucose binds, and a cis element to which a transcription factor involved in gluconeogenesis binds, which is any one of SEQ ID NOs: 23 to 47. Does not include cis elements containing the consensus sequence shown in . (I) at least one heat shock element HSE,
(Ii) STRE, which is at least one stress response element,
(Iii) a cis element different from the above HSE and STRE, which binds to a transcriptional activator whose expression is enhanced under stress conditions
Including,
A stress responsive promoter, wherein (i) HSE is a cis element containing the consensus sequence represented by SEQ ID NO: 16 and (ii) STRE is a cis element containing the consensus sequence represented by SEQ ID NO: 17. (However, the stress-responsive promoter is a cis element to which a transcription factor that acts suppressively in the presence of Glucose binds, and a cis element to which a transcription factor involved in gluconeogenesis binds. It does not contain cis-elements containing the consensus sequence shown by either) . (I) at least one heat shock element HSE,
(Ii) STRE, which is at least one stress response element,
(Iii) a cis element different from the above HSE and STRE, which binds to a transcriptional activator whose expression is enhanced under stress conditions
Including,
A stress-responsive promoter (provided that the stress-responsive promoter is a cis element to which the transcription activator (iii) binds, which is a cis element containing a consensus sequence selected from the group consisting of consensus sequences represented by SEQ ID NOS: 18 to 22 Is a cis element to which a transcription factor that suppressively acts in the presence of Glucose binds, and a cis element to which a transcription factor involved in gluconeogenesis binds, and is a consensus sequence represented by any of SEQ ID NOs: 23 to 47. Not including cis elements) . The stress responsive promoter according to any one of claims 1 to 5 ,
A stress-responsive promoter in which the transcription factors that act suppressively in the presence of Glucose and the transcription factors involved in gluconeogenesis are Rap1, Nrg1, Gln3, Hap4/5, Adr1, Mig1, Mot3, and Snf1. (I) at least one heat shock element HSE comprising the consensus sequence shown in SEQ ID NO: 16;
(Ii) STRE, which is at least one stress response element, and which comprises the consensus sequence shown in SEQ ID NO:17;
(Iii) A stress-responsive promoter (including a cis element containing a consensus sequence represented by SEQ ID NOS: 18 to 22) which is a cis element to which a transcription activator whose expression is increased under stress conditions is bound (however, wherein the stress-responsive promoter does not contain the cis-element containing the consensus sequence shown in SEQ ID nO: 23-4 7). The stress responsive promoter according to any one of claims 1 to 7 ,
Furthermore, (iv) a stress responsive promoter having a Kozak sequence represented by SEQ ID NO: 49 in the 5'-untranslated region.   A stress responsive promoter consisting of the sequence represented by SEQ ID NO: 48. Vector comprising a stress-responsive promoter according to any one of claims 1-8. The vector according to claim 9 , which is a shuttle vector between Escherichia coli or a eukaryotic cell,
2-micron plasmid vector or vector containing origin of replication. A vector according to claim 9 or 10 , comprising a gene operably linked downstream of the stress responsive promoter. The vector according to claim 11 , wherein the gene is the Xylose isomerase gene. Transformants part or all is introduced into the chromosome of a vector according to any one of claims 9-12. A microorganism for producing bioethanol using Xylose as a substrate,
A microorganism comprising part or all of the vector according to claim 12 . A method for producing bioethanol using Xylose as a substrate,
(A) A method comprising a step of culturing the microorganism according to claim 14 in a culture solution containing Xylose. The method of claim 15 , wherein
The method wherein the step (a) is performed simultaneously with the saccharification treatment performed in the presence of xylan and xylanase.

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