JP5585952B2 - Ethanol production method - Google Patents
Ethanol production method Download PDFInfo
- Publication number
- JP5585952B2 JP5585952B2 JP2009278555A JP2009278555A JP5585952B2 JP 5585952 B2 JP5585952 B2 JP 5585952B2 JP 2009278555 A JP2009278555 A JP 2009278555A JP 2009278555 A JP2009278555 A JP 2009278555A JP 5585952 B2 JP5585952 B2 JP 5585952B2
- Authority
- JP
- Japan
- Prior art keywords
- yeast
- strain
- msn2
- gene
- ethanol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims description 85
- 238000004519 manufacturing process Methods 0.000 title claims description 27
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 54
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 52
- 108090000623 proteins and genes Proteins 0.000 claims description 31
- 235000020083 shōchū Nutrition 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 description 40
- 101100520819 Arabidopsis thaliana At5g66631 gene Proteins 0.000 description 19
- 101100184274 Candida albicans (strain SC5314 / ATCC MYA-2876) MNL1 gene Proteins 0.000 description 19
- 101100078102 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) MSN2 gene Proteins 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 18
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 16
- 101150060051 MSN2 gene Proteins 0.000 description 16
- 238000000855 fermentation Methods 0.000 description 16
- 230000004151 fermentation Effects 0.000 description 16
- 235000019992 sake Nutrition 0.000 description 16
- 108091023040 Transcription factor Proteins 0.000 description 11
- 102000040945 Transcription factor Human genes 0.000 description 11
- 239000003550 marker Substances 0.000 description 11
- 101150069333 MSN4 gene Proteins 0.000 description 10
- 101100078103 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) MSN4 gene Proteins 0.000 description 10
- 230000003938 response to stress Effects 0.000 description 9
- 239000001569 carbon dioxide Substances 0.000 description 8
- 229910002092 carbon dioxide Inorganic materials 0.000 description 8
- 238000010353 genetic engineering Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 101100459439 Caenorhabditis elegans nac-2 gene Proteins 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 241000209094 Oryza Species 0.000 description 6
- 235000007164 Oryza sativa Nutrition 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 235000009566 rice Nutrition 0.000 description 6
- 230000035882 stress Effects 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 241000235070 Saccharomyces Species 0.000 description 5
- 230000006378 damage Effects 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000007222 ypd medium Substances 0.000 description 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 4
- 235000013379 molasses Nutrition 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 235000013405 beer Nutrition 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000006353 environmental stress Effects 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- 235000014101 wine Nutrition 0.000 description 3
- 210000005253 yeast cell Anatomy 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 235000013334 alcoholic beverage Nutrition 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000005498 polishing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 101150033839 4 gene Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 108091007916 Zinc finger transcription factors Proteins 0.000 description 1
- 102000038627 Zinc finger transcription factors Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000006860 carbon metabolism Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000004665 defense response Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- -1 liquor such as sake Chemical compound 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000008723 osmotic stress Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
本発明は、エタノールの製造方法に関する。さらに詳しくは、高発酵性酵母を用いた、清酒、ビール、ワイン等の酒類やバイオエタノール等を含むエタノールの製造方法に関する。 The present invention relates to a method for producing ethanol. More specifically, the present invention relates to a method for producing ethanol including liquor such as sake, beer and wine, bioethanol and the like using highly fermentable yeast.
酒類や燃料用のバイオエタノールに含まれているエタノールは、一般に、ブドウ糖などの糖類をサッカロミセス属に属する酵母により発酵させて製造する。 In general, ethanol contained in bioethanol for liquor and fuel is produced by fermenting sugars such as glucose with yeast belonging to the genus Saccharomyces.
したがって、エタノール発酵中の酵母細胞は、高濃度のエタノールにさらされることになる。酵母細胞は、エタノール等の環境ストレスに速やかに応答して遺伝子発現を変化させ、酵母細胞の生命を維持するための分子機構を備えている。このシステムの中心的役割を果たすのが、ストレス応答転写因子Ｍｓｎ２ｐ及びＭｓｎ４ｐである。 Thus, yeast cells undergoing ethanol fermentation are exposed to high concentrations of ethanol. Yeast cells have a molecular mechanism for rapidly changing gene expression in response to environmental stresses such as ethanol and maintaining the life of yeast cells. The stress-responsive transcription factors Msn2p and Msn4p play a central role in this system.
Ｍｓｎ２ｐ及びＭｓｎ４ｐは互いに相同性を有するジンク・フィンガー型転写因子であり、いずれも熱ショック、酸化ストレス、浸透圧ストレス、エタノールストレス等の様々な環境ストレス下において、細胞質から核内に移行し、様々な遺伝子のプロモーター領域に結合し、下流の遺伝子の転写を活性化するものである。Ｍｓｎ２ｐ及びＭｓｎ４ｐによって発現調節を受ける遺伝子は、炭素代謝関連、タンパク質の保護・分解関連、細胞防御反応関連、細胞内シグナル伝達関連等多岐にわたっており、Ｍｓｎ２ｐ及びＭｓｎ４ｐは、これらの遺伝子の発現を総合的に変化させることによって、環境ストレスに対する耐性に寄与すると考えられている。したがって、これらのストレス応答転写因子の機能が異常になると、ストレス条件下での生命維持に影響が現れ、例えば、非特許文献１では、ＭＳＮ２／ＭＳＮ４遺伝子二重破壊株は、様々なストレス条件下での生存率が低下することが報告されている。
Msn2p and Msn4p are zinc-finger transcription factors that are homologous to each other, and all migrate from the cytoplasm into the nucleus under various environmental stresses such as heat shock, oxidative stress, osmotic stress, and ethanol stress. It binds to the promoter region of a gene and activates transcription of a downstream gene. Genes whose expression is regulated by Msn2p and Msn4p are diverse, such as those related to carbon metabolism, protein protection / degradation, cell defense response, intracellular signal transduction, etc. Msn2p and Msn4p comprehensively express the expression of these genes It is thought that it contributes to tolerance to environmental stress by changing to. Therefore, when the functions of these stress response transcription factors become abnormal, life-sustaining under stress conditions is affected. For example, in Non-Patent
酵母を用いたエタノール生産において、エタノール生産速度を上げることは、エタノール発酵の生産性を高めるために極めて重要であるが、酵母のストレス応答転写因子に変異を導入することにより改変し、高発酵性を獲得した例は知られていない。 In ethanol production using yeast, increasing the rate of ethanol production is extremely important to increase the productivity of ethanol fermentation, but it can be modified by introducing mutations into the stress-responsive transcription factor of yeast, resulting in high fermentability. No example of winning is known.
本発明の課題は、酵母を用いて、生産性高くエタノールを製造する方法を提供することにある。 An object of the present invention is to provide a method for producing ethanol with high productivity using yeast.
本発明者らは、従来の知見から、酵母によるエタノール発酵には正常なストレス応答機構が機能することが必要であると考え、そのことを証明するために、実験室酵母又は清酒酵母のストレス応答転写因子の遺伝子を破壊した形質転換酵母を用いて清酒製造試験又はエタノール製造試験を行い、遺伝子破壊がエタノール発酵に与える影響を解析した。その結果、意外にも、ストレス応答転写因子の遺伝子を破壊した形質転換酵母はいずれも、高い発酵性を示すことが判明した。そこで、本発明者らは、この現象は酵母のエタノール発酵性を改善させるために利用可能であると考え、ストレス応答転写因子の遺伝子の破壊とエタノール発酵についてさらに詳細な研究を行うことにより、本発明を完成するに至った。 From the conventional knowledge, the present inventors consider that a normal stress response mechanism is required for ethanol fermentation by yeast, and in order to prove that, the stress response of laboratory yeast or sake yeast A sake production test or an ethanol production test was conducted using transformed yeast in which the gene of the transcription factor was disrupted, and the effect of the gene disruption on ethanol fermentation was analyzed. As a result, it was surprisingly found that any transformed yeast in which the gene of the stress response transcription factor was disrupted showed high fermentability. Therefore, the present inventors consider that this phenomenon can be used to improve the ethanol fermentability of yeast, and by conducting further detailed research on the destruction of the gene of stress response transcription factor and ethanol fermentation, The invention has been completed.
即ち、本発明の要旨は、
ストレス応答転写因子をコードする遺伝子が破壊されてなる酵母の遺伝子破壊株を用いた、エタノールの製造方法、に関する。
That is, the gist of the present invention is as follows.
The present invention relates to a method for producing ethanol using a gene-disrupted strain of yeast obtained by disrupting a gene encoding a stress response transcription factor.
本発明により、酵母のエタノール発酵において、速いエタノール生産速度でエタノールを生産することができる。 According to the present invention, ethanol can be produced at a high ethanol production rate in ethanol fermentation of yeast.
本発明のエタノールの製造方法は、酵母を用いてエタノールを製造するものであって、ストレス応答転写因子（例えばＭｓｎ２ｐ及びＭｓｎ４ｐ）をコードする遺伝子のうち少なくとも１種が破壊されている酵母の遺伝子破壊株を用いることに、一つの特徴を有する。 The method for producing ethanol according to the present invention comprises producing yeast using yeast, wherein at least one of genes encoding a stress response transcription factor (for example, Msn2p and Msn4p) is disrupted. Using a strain has one feature.
ストレス応答転写因子をコードする遺伝子としては、ＭＳＮ２及びＭＳＮ４が挙げられる。これらの遺伝子は、ＳＧＤ（Ｓａｃｃｈａｒｏｍｙｃｅｓ Ｇｅｎｏｍｅ Ｄａｔａｂａｓｅ）のホームページ（http://genome-www.stanford.edu/Saccharomyces）に配列が記載されており、かかる配列に基づいて、ＰＣＲプライマーを設計して遺伝子破壊や部位特異的変異を行うことができる。例えば、ＭＳＮ２を破壊するために用いるＤＮＡのプライマーとしては、ＭＳＮ２−ＤＦ（配列表の配列番号１）、ＭＳＮ４−ＤＲ（配列表の配列番号２）、ＭＳＮ４を破壊するために用いるＤＮＡのプライマーとしては、ＭＳＮ４−ＤＦ（配列表の配列番号３）、ＭＳＮ４−ＤＲ（配列表の配列番号４）等が例示される。 Examples of genes encoding a stress response transcription factor include MSN2 and MSN4. The sequences of these genes are described on the SGD (Saccharomyces Genome Database) website (http://genome-www.stanford.edu/Saccharomyces). Based on such sequences, PCR primers are designed to disrupt the gene. And site-specific mutations can be made. For example, as a primer for DNA used for destroying MSN2, as a primer for DNA used for destroying MSN2-DF (SEQ ID NO: 1 in the sequence listing), MSN4-DR (SEQ ID NO: 2 in the sequence listing), and MSN4 Examples include MSN4-DF (SEQ ID NO: 3 in the sequence listing), MSN4-DR (SEQ ID NO: 4 in the sequence listing), and the like.
本発明における酵母とは、サッカロミセス属に属するエタノール発酵に用いることのできる酵母であって、例えば、実験室酵母、清酒酵母、ワイン酵母、ビール酵母、焼酎酵母、パン酵母、及びバイオエタノール酵母が挙げられる。これらの酵母は一種類を単独で用いてもよく、複数種の酵母を用いてもよい。実験室酵母としては、サッカロミセス・セレビシエに属するＸ２１８０−１Ａ株が好ましい。清酒酵母としては、サッカロミセス・セレビシエに属するきょうかい７号株が好ましい。 The yeast in the present invention is a yeast that can be used for ethanol fermentation belonging to the genus Saccharomyces, and includes, for example, laboratory yeast, sake yeast, wine yeast, beer yeast, shochu yeast, baker's yeast, and bioethanol yeast. It is done. One kind of these yeasts may be used alone, or a plurality of kinds of yeasts may be used. As the laboratory yeast, the X2180-1A strain belonging to Saccharomyces cerevisiae is preferable. As the sake yeast, the No. 7 strain belonging to Saccharomyces cerevisiae is preferred.
本明細書において、「破壊」とは、破壊される遺伝子の機能を喪失させたり、失活させたりするような遺伝子操作のことをいい、目的遺伝子領域の全遺伝子を喪失させる遺伝子操作や正常な機能に必要な部位を欠失又は変異させる遺伝子操作のことをいう。具体的には、ストレス応答転写因子をコードするＭＳＮ２又はＭＳＮ４遺伝子を全喪失させる遺伝子操作が例示される。かかる遺伝子操作を行う方法としては、特に限定はなく公知の方法を用いることができ、例えば、変異剤処理による突然変異、遺伝子工学を用いた遺伝子破壊、遺伝子工学を用いた部位特異的変異などが挙げられる。これらの遺伝子破壊株の作成方法については、Saccharomyces Genome Deletion Projectのホームページ（http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html）を参考にすることができる。なお、本発明においては、自然突然変異により破壊が生じた遺伝子破壊株も用いることができる。 In this specification, “disruption” refers to genetic manipulation that causes the function of the disrupted gene to be lost or inactivated, such as genetic manipulation that causes loss of all genes in the target gene region or normal operation. This refers to genetic manipulation that deletes or mutates a site necessary for function. Specifically, genetic manipulation that completely loses the MSN2 or MSN4 gene encoding a stress response transcription factor is exemplified. A method for performing such genetic manipulation is not particularly limited, and a known method can be used. For example, mutation by treatment with a mutation agent, gene disruption using genetic engineering, site-specific mutation using genetic engineering, etc. Can be mentioned. About the production method of these gene-disrupted strains, the Saccharomyces Genome Deletion Project website (http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html) can be referred to. In the present invention, a gene disrupted strain in which disruption has occurred due to spontaneous mutation can also be used.
なお、上記遺伝子操作を行う際には、アミノ酸などの栄養要求マーカーや、薬剤に対する耐性マーカーなどを選択マーカーとして使用してもよい。 When performing the above-described genetic manipulation, an auxotrophic marker such as an amino acid, a drug resistance marker, or the like may be used as a selection marker.
ストレス応答転写因子の遺伝子破壊株の代表株については、Ｘ２１８０−１Ａ ｍｓｎ２、Ｘ２１８０−１Ａ ｍｓｎ４、Ｘ２１８０−１Ａ ｍｓｎ２／４、及びＫ７ ｍｓｎ２と命名、表示され、独立行政法人製品評価技術基盤機構特許微生物寄託センターに、それぞれ、ＮＩＴＥ Ｐ−８１３、ＮＩＴＥ Ｐ−８１４、ＮＩＴＥ Ｐ−８１５、及びＮＩＴＥ Ｐ−８１６として寄託されている。 The representative strains of the stress-responsive transcription factor gene-disrupted strains are named and displayed as X2180-1A msn2, X2180-1A msn4, X2180-1A msn2 / 4, and K7 msn2, and are independent administrative corporation product evaluation technology infrastructure mechanism patent microorganism NITE P-813, NITE P-814, NITE P-815, and NITE P-816 are deposited at the depository centers, respectively.
本発明は、上記で得られた酵母の遺伝子破壊株を用いてエタノールを製造するが、酵母が特定のものである以外は、公知の方法を用いて、エタノールを製造することができる。 Although this invention manufactures ethanol using the yeast gene-disrupted strain obtained above, ethanol can be manufactured using a well-known method except that yeast is a specific thing.
本発明の製造方法は、例えば、清酒、ビール、ワイン等の酒類やバイオエタノール等の製造に適用することができるが、好ましくは、清酒又はバイオエタノールの製造に適用することができる。 The production method of the present invention can be applied, for example, to the production of alcoholic beverages such as sake, beer, wine, and bioethanol, but is preferably applicable to the production of sake or bioethanol.
以下、本発明を実施例に基づいて説明するが、本発明はこれらの実施例等によりなんら制限されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, this invention is not restrict | limited at all by these Examples.
実施例１
サッカロミセス・セレビシエＸ２１８０−１Ａ株のＭＳＮ２遺伝子及びＭＳＮ４遺伝子のいずれか１種又は両者を破壊した株を作成し、清酒製造試験を行った。なお、Ｘ２１８０−１Ａ株は、Ａｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎ（ＡＴＣＣ）から、ＡＴＣＣ２６７８６として入手可能である。
Example 1
A Saccharomyces cerevisiae X2180-1A strain in which one or both of the MSN2 gene and the MSN4 gene were disrupted was prepared, and a sake production test was conducted. The X2180-1A strain is available as ATCC 26786 from the American Type Culture Collection (ATCC).
〔遺伝子破壊株の作成〕
Ｘ２１８０−１Ａ株の遺伝子破壊は、マーカー遺伝子としてｎａｔ１及びｋａｎＭＸを用いて行った。まず、ｎａｔ１を含むプラスミドｐＡＧ２５（ＥＵＲＯＳＣＡＲＦから入手可能、http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html）を鋳型として、プライマーＭＳＮ２−ＤＦ（配列表の配列番号１）及びＭＳＮ２−ＤＲ（配列表の配列番号２）を用いてＰＣＲを行い、マーカーであるｎａｔ１の両側に、ＭＳＮ２のＯＲＦに隣接する上流部分及び下流部分のＤＮＡ配列を持つＤＮＡを作成した。このＤＮＡを用いてＸ２１８０−１Ａ株を形質転換して、ノーセオスリシン含有培地で増殖した酵母を、形質転換体１とした。形質転換体１からゲノムＤＮＡを抽出し、プライマーＭＳＮ２−Ｆ（配列表の配列番号５）及びＭＳＮ２−Ｒ（配列表の配列番号６）を用いてＰＣＲを行い、得られた産物のサイズを測定することによって、ＭＳＮ２が遺伝子破壊されていることを確認し、これをＸ２１８０−１Ａ株のＭＳＮ２遺伝子破壊株（Ｘ２１８０−１Ａ ｍｓｎ２）とした。
[Generation of gene disruption strain]
The gene disruption of the X2180-1A strain was performed using nat1 and kanMX as marker genes. First, using a plasmid pAG25 containing nat1 (available from EUROS CARF, http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html) as a template, primer MSN2-DF (SEQ ID NO: 1 in the sequence listing) ) And MSN2-DR (SEQ ID NO: 2 in the sequence listing), and DNA having DNA sequences of upstream and downstream portions adjacent to the ORF of MSN2 on both sides of the marker nat1 was prepared. A
次に、ｋａｎＭＸを含むプラスミドｐＵＧ６（ＥＵＲＯＳＣＡＲＦから入手可能、http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html）を鋳型として、プライマーＭＳＮ４−ＤＦ（配列表の配列番号３）及びＭＳＮ４−ＤＲ（配列表の配列番号４）を用いてＰＣＲを行い、マーカーであるｋａｎＭＸの両側に、ＭＳＮ４のＯＲＦに隣接する上流部分及び下流部分のＤＮＡ配列を持つＤＮＡを作成した。このＤＮＡを用いてＸ２１８０−１Ａ株及びＭＳＮ２遺伝子破壊株を形質転換して、ジェネティシン含有培地で増殖した酵母を、それぞれ形質転換体２及び３とした。形質転換体２及び３からゲノムＤＮＡを抽出し、プライマーＭＳＮ４−Ｆ（配列表の配列番号７）及びＭＳＮ４−Ｒ（配列表の配列番号８）を用いてＰＣＲを行い、得られた産物のサイズを測定することによって、ＭＳＮ４が遺伝子破壊されていることを確認し、これらをそれぞれ、Ｘ２１８０−１Ａ株のＭＳＮ４遺伝子破壊株（Ｘ２１８０−１Ａ ｍｓｎ４）及びＭＳＮ２／ＭＳＮ４遺伝子二重破壊株（Ｘ２１８０−１Ａ ｍｓｎ２／４）とした。
Next, using the plasmid pUG6 containing kanMX (available from EUROSCARF, http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html) as a template, primer MSN4-DF (SEQ ID NO: SEQ ID NO: PCR was performed using 3) and MSN4-DR (SEQ ID NO: 4 in the Sequence Listing) to prepare DNA having DNA sequences of upstream and downstream portions adjacent to the ORF of MSN4 on both sides of the marker kanMX.
〔清酒の製造〕
親株であるＸ２１８０−１Ａと、上記で得られたＭＳＮ２遺伝子破壊株、ＭＳＮ４遺伝子破壊株、及びＭＳＮ２／ＭＳＮ４遺伝子二重破壊株を用いて、以下に示す方法で清酒を製造した。
[Production of sake]
Using the parent strain X2180-1A and the MSN2 gene disrupted strain, MSN4 gene disrupted strain, and MSN2 / MSN4 gene double disrupted strain obtained above, sake was produced by the method described below.
掛米８０ｇ、麹米２０ｇ、水１６０ｍＬ、９０％乳酸４０μＬ混合による一段仕込を実施した。掛米として精米歩合７０％のアルファー化米、麹米として精白歩合７０％の乾燥麹を用いた。各酵母は、ＹＰＤ培地（酵母エキス１％、ペプトン２％、ブドウ糖２％含有）において一晩振とう培養した後、酵母数が２×１０7ｃｅｌｌｓ／ｍＬになるように仕込時に添加した。発酵温度は１５℃とした。仕込試験は各株について３回ずつ繰り返した。仕込後、毎日重量を測定することにより、重量減少分から発酵に伴う二酸化炭素発生量を求め、各株について平均二酸化炭素発生量を算出した。結果を図１に示す。また、仕込後２０日目には、遠心分離によって回収した清酒におけるエタノール濃度を測定し、各株について平均エタノール濃度を算出した。また、得られた平均エタノール濃度について、親株における濃度に対する遺伝子破壊株における濃度の有意差検定も同時に行った。なお、エタノール濃度の測定は、アルコール濃度計（理研計器社製アルコメイトＡＬ−３）を用いて行った。 One-stage charging was performed by mixing 80 g of rice, 20 g of glutinous rice, 160 mL of water, and 40 μL of 90% lactic acid. Alpha rice with a 70% polishing rate was used as the rice, and dried rice with a 70% polishing rate was used as the polished rice. Each yeast was cultured overnight in a YPD medium (containing 1% yeast extract, 2% peptone, and 2% glucose), and then added at the time of charging so that the number of yeasts was 2 × 10 7 cells / mL. The fermentation temperature was 15 ° C. The preparation test was repeated three times for each strain. After the preparation, the weight was measured every day to obtain the carbon dioxide generation amount accompanying fermentation from the weight loss, and the average carbon dioxide generation amount was calculated for each strain. The results are shown in FIG. On the 20th day after the preparation, the ethanol concentration in sake collected by centrifugation was measured, and the average ethanol concentration was calculated for each strain. Moreover, about the obtained average ethanol concentration, the significant difference test of the density | concentration in a gene disruption strain | stump | stock with respect to the density | concentration in a parent strain was also performed simultaneously. The ethanol concentration was measured using an alcohol concentration meter (Alcomate AL-3 manufactured by Riken Keiki Co., Ltd.).
いずれの遺伝子破壊株も、親株に比べて二酸化炭素発生量が多く（図１）、エタノール発酵速度が速いこと、また、最終的なエタノール濃度も、親株が１５．４±０．２％であるのに対し、ＭＳＮ２遺伝子破壊株が１６．０±０．１％、ＭＳＮ４遺伝子破壊株が１５．７±０．１％、ＭＳＮ２／ＭＳＮ４遺伝子二重破壊株が１６．０±０．２％と高く、エタノール生産性に優れることが分かった。エタノール濃度について平均値の差の検定を行った結果、いずれの遺伝子破壊株も親株と比較して５％未満の危険率で有意であった。 All of the gene-disrupted strains generate more carbon dioxide than the parent strain (FIG. 1), have a fast ethanol fermentation rate, and the final ethanol concentration is 15.4 ± 0.2% for the parent strain. In contrast, the MSN2 gene disruption strain was 16.0 ± 0.1%, the MSN4 gene disruption strain was 15.7 ± 0.1%, and the MSN2 / MSN4 gene double disruption strain was 16.0 ± 0.2%. It was found to be high and excellent in ethanol productivity. As a result of testing the difference in mean values for the ethanol concentration, all of the gene-disrupted strains were significant at a risk rate of less than 5% compared to the parent strain.
実施例２
サッカロミセス・セレビシエ二倍体清酒酵母きょうかい７号株の２個のＭＳＮ２遺伝子を両者共に破壊した株を作成し、清酒製造試験を行った。なお、きょうかい７号株は、財団法人日本醸造協会より入手可能である。
Example 2
A strain in which both MSN2 genes of Saccharomyces cerevisiae diploid sake yeast No. 7 were disrupted together was tested for sake production. Kyokai No. 7 is available from the Japan Brewing Association.
〔遺伝子破壊株の作成〕
きょうかい７号株の遺伝子破壊は、マーカー遺伝子としてｎａｔ１及びｋａｎＭＸを用いて行った。きょうかい７号株は二倍体であることから、まず、１コピー目のＭＳＮ２遺伝子をｎａｔ１をマーカーとして遺伝子破壊した。ｎａｔ１を含むプラスミドｐＡＧ２５（ＥＵＲＯＳＣＡＲＦから入手可能http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html）を鋳型として、プライマーＭＳＮ２−ＤＦ（配列表の配列番号１）及びＭＳＮ２−ＤＲ（配列表の配列番号２）を用いてＰＣＲを行い、マーカーであるｎａｔ１の両側に、ＭＳＮ２のＯＲＦに隣接する上流部分及び下流部分のＤＮＡ配列を持つＤＮＡを作成した。このＤＮＡを用いてきょうかい７号株を形質転換して、ノーセオスリシン含有培地で増殖した酵母を、形質転換体４とした。形質転換体４からゲノムＤＮＡを抽出し、プライマーＭＳＮ２−Ｆ（配列表の配列番号５）及びＭＳＮ２−Ｒ（配列表の配列番号６）を用いてＰＣＲを行い、得られた産物のサイズを測定することによって、１コピー目のＭＳＮ２が遺伝子破壊されていることを確認した。
[Generation of gene disruption strain]
The gene disruption of Kyokai No. 7 strain was performed using nat1 and kanMX as marker genes. Since the Kyokai No. 7 strain is diploid, the first copy of the MSN2 gene was first disrupted using nat1 as a marker. Using plasmid pAG25 containing nat1 (available from EUROSCARF http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html) as a template, primers MSN2-DF (SEQ ID NO: 1 in the sequence listing) and MSN2 PCR was performed using -DR (SEQ ID NO: 2 in the sequence listing) to prepare DNA having DNA sequences of the upstream and downstream portions adjacent to the ORF of MSN2 on both sides of the marker nat1.
次に、２コピー目のＭＳＮ２をｋａｎＭＸをマーカーとして遺伝子破壊した。ｋａｎＭＸを含むプラスミドｐＵＧ６（ＥＵＲＯＳＣＡＲＦから入手可能、http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html）を鋳型として、プライマーＭＳＮ２−ＤＦ（配列表の配列番号１）及びＭＳＮ２−ＤＲ（配列表の配列番号２）を用いてＰＣＲを行い、マーカーであるｋａｎＭＸの両側に、ＭＳＮ２のＯＲＦに隣接する上流部分及び下流部分のＤＮＡ配列を持つＤＮＡを作成した。このＤＮＡを用いて、上記で得られた形質転換体４を形質転換して、ジェネティシン含有培地で増殖した酵母を形質転換体５とした。形質転換体５からゲノムＤＮＡを抽出し、プライマーＭＳＮ２−Ｆ（配列表の配列番号５）及びＭＳＮ２−Ｒ（配列表の配列番号６）を用いてＰＣＲを行い、得られた産物のサイズを測定することによって、２コピー分のＭＳＮ２が遺伝子破壊されていることを確認し、これを、きょうかい７号株のＭＳＮ２遺伝子破壊株（Ｋ７ ｍｓｎ２）とした。
Next, the second copy of MSN2 was gene disrupted using kanMX as a marker. Using plasmid pUG6 containing kanMX (available from EUROSCARF, http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html) as a template, primer MSN2-DF (SEQ ID NO: 1 in the sequence listing) and PCR was performed using MSN2-DR (SEQ ID NO: 2 in the sequence listing) to prepare DNA having DNA sequences of the upstream and downstream portions adjacent to the ORF of MSN2 on both sides of the marker kanMX. Using this DNA,
〔エタノールの製造〕
親株であるきょうかい７号株と、上記で得られたＭＳＮ２遺伝子破壊株を用いて、以下に示す方法でエタノールを製造した。
[Production of ethanol]
Ethanol was produced by the following method using the parent strain, Kyoto No. 7 strain, and the MSN2 gene disruption strain obtained above.
廃糖蜜培地（ケーンモラセス原液を蒸留水にてＢｒｉｘ２５％に調整後、０．０２５％硫酸アンモニウムを添加したもの）及び高濃度ブドウ糖含有ＹＰＤ培地（酵母エキス１％、ペプトン２％、ブドウ糖２０％含有）を用いたエタノール発酵試験を実施した。各酵母は、ＹＰＤ培地（酵母エキス１％、ペプトン２％、ブドウ糖２％含有）において一晩振とう培養した後、酵母数が２×１０6ｃｅｌｌｓ／ｍＬになるように両培地に添加し、７２時間静置培養した。発酵温度は３０℃とした。発酵試験は各株について３回以上繰り返した。培養開始後、発酵モニター装置（アトー株式会社製ファーモグラフII）を用いて発酵に伴う二酸化炭素発生量を求め、各株について平均二酸化炭素発生量を算出した。結果を図２に示す。
Waste molasses medium (Ken molasses stock solution adjusted to
ＭＳＮ２遺伝子破壊株は、廃糖蜜培地及びＹＰＤ培地のいずれの条件においても、親株に比べて二酸化炭素発生量が多く（図２）、エタノール発酵速度が速く、エタノール生産性に優れることが分かった。 It was found that the MSN2 gene-disrupted strain had a larger amount of carbon dioxide generation than the parent strain (FIG. 2), the ethanol fermentation rate was high, and the ethanol productivity was excellent in both the molasses medium and the YPD medium.
本発明の製造方法は、清酒、ビール、ワイン、焼酎等の酒類や、バイオエタノール等を含むエタノールの製造に好適に用いられる。 The production method of the present invention is suitably used for producing alcoholic beverages such as sake, beer, wine, shochu, and ethanol including bioethanol.
配列表の配列番号１は、ＭＳＮ２破壊用ＤＮＡ作成用プライマーである。
配列表の配列番号２は、ＭＳＮ２破壊用ＤＮＡ作成用プライマーである。
配列表の配列番号３は、ＭＳＮ４破壊用ＤＮＡ作成用プライマーである。
配列表の配列番号４は、ＭＳＮ４破壊用ＤＮＡ作成用プライマーである。
配列表の配列番号５は、ＭＳＮ２増幅用プライマーである。
配列表の配列番号６は、ＭＳＮ２増幅用プライマーである。
配列表の配列番号７は、ＭＳＮ４増幅用プライマーである。
配列表の配列番号８は、ＭＳＮ４増幅用プライマーである。
Claims (5)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2009278555A JP5585952B2 (en) | 2009-12-08 | 2009-12-08 | Ethanol production method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2009278555A JP5585952B2 (en) | 2009-12-08 | 2009-12-08 | Ethanol production method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2011120486A JP2011120486A (en) | 2011-06-23 |
| JP5585952B2 true JP5585952B2 (en) | 2014-09-10 |
Family
ID=44285089
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2009278555A Active JP5585952B2 (en) | 2009-12-08 | 2009-12-08 | Ethanol production method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5585952B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102287810B1 (en) | 2014-07-28 | 2021-08-09 | 삼성전자주식회사 | Genetically engineered and stress resistant yeast cell with enhanced activity of MSN2 and method for producing lactate using the same |
| KR20210032972A (en) * | 2018-06-27 | 2021-03-25 | 베링거 인겔하임 에르체파우 게엠베하 운트 코 카게 | Means and methods for increasing protein expression using transcription factors |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006280268A (en) * | 2005-03-31 | 2006-10-19 | Chiba Univ | Method for improving alcohol production efficiency by promoting cell proliferation |
| EP2407541A1 (en) * | 2006-12-07 | 2012-01-18 | Massachusetts Institute of Technology | Global transcription machinery engineering |
| JP2009240185A (en) * | 2008-03-28 | 2009-10-22 | Toyota Central R&D Labs Inc | Highly productive transformant of exogenous protein and its use |
| KR20100048224A (en) * | 2008-10-30 | 2010-05-11 | 삼성전자주식회사 | Gene increasing metabolic availability of galactose, recombinant vector and recombinant microorganism containing the same |
| KR20100117465A (en) * | 2009-04-24 | 2010-11-03 | 삼성전자주식회사 | Isolated polynucleotides increasing alcohol tolerance, vector and host cell containing the same, and method of producing alcohol using the same |
-
2009
- 2009-12-08 JP JP2009278555A patent/JP5585952B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2011120486A (en) | 2011-06-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lian et al. | Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains | |
| Filho et al. | Isolation by genetic and physiological characteristics of a fuel-ethanol fermentative Saccharomyces cerevisiae strain with potential for genetic manipulation | |
| Hubmann et al. | Quantitative trait analysis of yeast biodiversity yields novel gene tools for metabolic engineering | |
| Hubmann et al. | Identification of multiple interacting alleles conferring low glycerol and high ethanol yield in Saccharomyces cerevisiae ethanolic fermentation | |
| JP2000509988A (en) | Stable recombinant yeast for fermenting xylose to ethanol | |
| Zhang et al. | Adaptive evolution relieves nitrogen catabolite repression and decreases urea accumulation in cultures of the Chinese rice wine yeast strain Saccharomyces cerevisiae XZ-11 | |
| Wang et al. | The combination of glycerol metabolic engineering and drug resistance marker-aided genome shuffling to improve very-high-gravity fermentation performances of industrial Saccharomyces cerevisiae | |
| He et al. | Engineering industrial Saccharomyces cerevisiae strain with the FLO1-derivative gene isolated from the flocculating yeast SPSC01 for constitutive flocculation and fuel ethanol production | |
| CA2968726C (en) | Causative genes conferring acetic acid tolerance in yeast | |
| JP2020501595A (en) | Xylitol-producing Methinicobia species | |
| Kuanyshev et al. | Domesticating a food spoilage yeast into an organic acid‐tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii | |
| EP2900690B1 (en) | Mutant yeast strain with decreased glycerol production | |
| De Valk et al. | Engineering proton-coupled hexose uptake in Saccharomyces cerevisiae for improved ethanol yield | |
| Zheng et al. | Highly efficient rDNA‐mediated multicopy integration based on the dynamic balance of rDNA in Saccharomyces cerevisiae | |
| JP5585952B2 (en) | Ethanol production method | |
| US20100190223A1 (en) | Yeast for transformation, transformation method, and method for producing substance | |
| Pribylova et al. | The salt tolerant yeast Zygosaccharomyces rouxii possesses two plasma-membrane Na+/H+-antiporters (ZrNha1p and ZrSod2–22p) playing different roles in cation homeostasis and cell physiology | |
| Štafa et al. | Novel Approach in the Construction of Bioethanol-Producing Saccharomyces cerevisiae Hybrids § | |
| Pribylova et al. | Tools for the genetic manipulation of Zygosaccharomyces rouxii | |
| Gao et al. | Global transcription engineering of brewer’s yeast enhances the fermentation performance under high-gravity conditions | |
| Karpel et al. | Analysis of the major hexose transporter genes in wine strains of Saccharomyces cerevisiae | |
| JP5828447B2 (en) | Ethanol production method | |
| BR112021001577A2 (en) | increased alcohol production from yeast that produces an increased amount of active crz1 protein | |
| US20220348966A1 (en) | System and method for increased alcohol tolerance and production in yeast | |
| EP3162896B1 (en) | Transformant, method for producing same, and method for producing c4-dicarboxylic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20121102 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20140417 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20140612 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20140627 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20140714 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 5585952 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |