CN107429221B - 高效乙醇发酵菌 - Google Patents
高效乙醇发酵菌 Download PDFInfo
- Publication number
- CN107429221B CN107429221B CN201480083830.3A CN201480083830A CN107429221B CN 107429221 B CN107429221 B CN 107429221B CN 201480083830 A CN201480083830 A CN 201480083830A CN 107429221 B CN107429221 B CN 107429221B
- Authority
- CN
- China
- Prior art keywords
- ethanol
- strain
- xylose
- medium
- guilliermondii
- 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.)
- Expired - Fee Related
Links
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 104
- 230000004151 fermentation Effects 0.000 claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 26
- 241000894006 Bacteria Species 0.000 claims abstract description 12
- 244000005700 microbiome Species 0.000 claims abstract description 7
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 84
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 44
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 44
- 230000002255 enzymatic effect Effects 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 28
- 241000235048 Meyerozyma guilliermondii Species 0.000 claims description 25
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 24
- 235000007164 Oryza sativa Nutrition 0.000 claims description 14
- 235000009566 rice Nutrition 0.000 claims description 14
- 239000010902 straw Substances 0.000 claims description 14
- 229910021529 ammonia Inorganic materials 0.000 claims description 12
- 239000003471 mutagenic agent Substances 0.000 claims description 5
- 231100000707 mutagenic chemical Toxicity 0.000 claims description 5
- 230000003505 mutagenic effect Effects 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 3
- 150000002972 pentoses Chemical class 0.000 claims description 3
- 150000002402 hexoses Chemical class 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 14
- 235000000346 sugar Nutrition 0.000 abstract description 14
- 108090000623 proteins and genes Proteins 0.000 abstract description 13
- 229910052799 carbon Inorganic materials 0.000 abstract description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 30
- 239000002609 medium Substances 0.000 description 21
- 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 20
- 239000008103 glucose Substances 0.000 description 20
- 240000008042 Zea mays Species 0.000 description 16
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 16
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 16
- 235000005822 corn Nutrition 0.000 description 16
- 239000010907 stover Substances 0.000 description 15
- 241000209094 Oryza Species 0.000 description 13
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000000769 gas chromatography-flame ionisation detection Methods 0.000 description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 5
- 239000002028 Biomass Substances 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 239000002002 slurry Substances 0.000 description 4
- ZAQJHHRNXZUBTE-WUJLRWPWSA-N D-xylulose Chemical compound OC[C@@H](O)[C@H](O)C(=O)CO ZAQJHHRNXZUBTE-WUJLRWPWSA-N 0.000 description 3
- 241000235648 Pichia Species 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 241001019659 Acremonium <Plectosphaerellaceae> Species 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 2
- 229920002488 Hemicellulose Polymers 0.000 description 2
- FUSGACRLAFQQRL-UHFFFAOYSA-N N-Ethyl-N-nitrosourea Chemical compound CCN(N=O)C(N)=O FUSGACRLAFQQRL-UHFFFAOYSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 229940106157 cellulase Drugs 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 230000034659 glycolysis Effects 0.000 description 2
- 239000002029 lignocellulosic biomass Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004108 pentose phosphate pathway Effects 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 102000040811 transporter activity Human genes 0.000 description 2
- 108091092194 transporter activity Proteins 0.000 description 2
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 1
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- HEBKCHPVOIAQTA-QWWZWVQMSA-N D-arabinitol Chemical compound OC[C@@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-QWWZWVQMSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000588901 Zymomonas Species 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 238000005915 ammonolysis reaction Methods 0.000 description 1
- 150000008209 arabinosides Chemical class 0.000 description 1
- 150000007514 bases Chemical class 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- SFDJOSRHYKHMOK-UHFFFAOYSA-N nitramide Chemical class N[N+]([O-])=O SFDJOSRHYKHMOK-UHFFFAOYSA-N 0.000 description 1
- 150000002832 nitroso derivatives Chemical class 0.000 description 1
- 238000011085 pressure filtration Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
-
- 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
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供在不导入外来基因的情况下得到乙醇产生效率高的高效乙醇发酵菌。高效乙醇发酵菌是由五碳糖和六碳糖有效地产生乙醇的发酵菌，其以保藏号NITE BP‑01962被保藏于专利微生物保藏中心。
Description
技术领域
本发明涉及在使用木质纤维素类生物质生成生物乙醇的过程中用于发酵糖化溶液的微生物。
特别涉及在使用木质纤维素类生物质生成生物乙醇的过程中能够由五碳糖(下文中也称为C5糖)以及六碳糖(下文中也称为C6糖)有效地生成乙醇的微生物。
背景技术
生物乙醇作为由生物质生成的不会发生枯竭的可再生资源而倍受期待。另外，由于燃烧生物乙醇而生成的二氧化碳为碳中性的，因而据信通过促进生物乙醇的利用可抑制作为地球温室化的主要原因的二氧化碳的增加。
生物乙醇是将生物质发酵并进行蒸馏提纯乙醇制得的。为了提高生物乙醇的收率，需要由糖化溶液中生成大多数的醇。在生物乙醇生成的过程中通常使用的酵母无法将木糖、阿拉伯糖等五碳糖转换为醇，因而作为发酵原料，仅使用了六碳糖。
尽管根据原料而不同，但在代表性的生物质中被认为含有35～45％的纤维素、25～40％的半纤维素、15～30％的木质素。因而，在不仅可利用六碳糖聚合而成的纤维素作为底物、而且还可利用主要含有作为五碳糖的木糖等的半纤维素作为底物时，可有效地产生乙醇。
木糖被认为是在生物质中大量含有的糖，其含量仅次于葡萄糖，有效地利用五碳糖是生物乙醇生产中的一大课题。
目前为止公开了少量地利用了木糖的技术，其是通过经基因重组而赋予木糖利用能力、或者利用木糖产生乙醇的微生物的利用等而实现的。
在专利文献1中公开了下述发明：将具有木糖转运体活性的基因导入到宿主细胞中，从而将木糖(C5糖)转换为木酮糖，整合在糖酵解系统的戊糖磷酸途径中，在发酵中加以利用。
在专利文献2中公开了利用被赋予了阿拉伯糖转运体的酵母来生成醇的技术。与专利文献1同样地、将阿拉伯糖(C5糖)经阿拉伯糖醇、木酮糖而整合在糖酵解系统的戊糖磷酸途径中，在发酵中加以利用。
在非专利文献1中公开了通过将源自大肠杆菌的木糖同化基因重组到发酵单胞菌中而赋予木糖同化能力的内容。
在非专利文献2中记载了毕赤属酵母可利用木糖生产乙醇的内容。
现有技术文献
专利文献
专利文献1：日本特开2012-170422号公报
专利文献2：美国专利申请公开第2013/189788号说明书
非专利文献
非专利文献1：Zhang,M.,et al.,Science,1995.Vol.267,pp.240-243.
非专利文献2：Bicho,P.A.,et al.,Appl.Environ.Microbiol.,1988,Vol.54,pp.50-54.
发明内容
发明所要解决的问题
但是，专利文献1的发明是将源自季也蒙假丝酵母(Candiida guilliermondii)的具有木糖转运体活性的蛋白质导入到作为宿主的酿酒酵母(Saccharomyces cerevisiae)中。即导入了外来基因。另外，专利文献2的发明，尽管转运基因不同，但也是向宿主中导入不同种基因的发明。
另外，非专利文献1所记载的技术是导入木糖同化基因的技术，与上述专利文献1和2的技术思想不同，但导入外来基因这一点不变。
因此，上述专利文献1和2、非专利文献1中记载的发明均需要采取用于实施联合国所采纳的“关于生物多样性公约的卡塔赫纳生物安全议定书”的遵循在日本施行的卡塔赫纳法的封锁对策。从而需要用于保证生物安全的设施，因而利用该细菌生成乙醇从成本方面考虑是不利的。
另外，根据非专利文献2记载的技术，关于毕赤属酵母的利用，由于野生型的毕赤属酵母的木糖利用性低，因而乙醇生成效率并不那么高。
本发明的课题在于在不导入外来基因的情况下得到乙醇产生效率高的高效乙醇发酵菌。
用于解决课题的手段
为了解决上述课题，本发明的高效乙醇发酵菌是一种由五碳糖和六碳糖有效地产生乙醇的发酵菌，其特征在于以保藏号NITE BP-01962被保藏于专利微生物保藏中心。
野生型季也蒙毕赤酵母(Meyerozyma guilliermondii)虽说具备葡萄糖同化能力以及木糖同化能力，但是并不是说其具备对于生产生物乙醇为充分的木糖利用能力。与此相对地，本发明的高效乙醇发酵菌(下文也称作BP-01962菌株。)是能够通过对以季也蒙毕赤酵母(Meyerozyma guilliermondii)N菌株作为亲代菌株进行菌株育种，选择出木糖利用效率高的菌而得到的。
其结果，本发明的高效乙醇发酵菌在不导入外来基因的情况下，能够得到比亲代菌株高的乙醇产生效率。
本发明的高效乙醇发酵菌是在向源自氨处理的稻秆的酶糖化液中添加诱变剂而得到的培养基中对季也蒙毕赤酵母(Meyerozyma guilliermondii)N菌株进行驯化培养，通过选择出在该培养基中生长的菌来提高乙醇发酵性能。
附图说明
图1是示出木糖同化性能与乙醇收率的图表，A表示N菌株，B表示BP-01962菌株。
图2是示出涉及源自氨处理的稻秆的酶糖化液的BP-01962菌株的葡萄糖和木糖的消化量，以及乙醇的生成量的经时性变化的图表。
图3是示出涉及源自BP-01962菌株和N菌株的稀硫酸处理玉米秸秆的酶糖化液的糖浓度与发酵收率的关系的图表。
图4是示出涉及源自BP-01962菌株和N菌株的稀硫酸处理玉米秸秆的酶糖化液的乙醇生成量的经时性变化的图表。
具体实施方式
下文，参照附图对本发明的实施方式进行更详细的说明。
作为子囊菌系酵母的季也蒙毕赤酵母(Meyerozyma guilliermondii)的野生菌株不仅具备葡萄糖同化能力而且还具备木糖利用能力，但其木糖利用能力对于生物乙醇生产而言并不能说是充分。因此，本实施方式的高效乙醇发酵菌是通过下述方式得到的变异菌株：将作为子囊菌系酵母的季也蒙毕赤酵母(Meyerozyma guilliermondii)N菌株作为亲代菌株，在向源自氨处理的稻秆的酶糖化液中添加诱变剂而得到的培养基中进行驯化培养，选择出在该培养基中生长的菌，从而得到变异菌株。
本实施方式的高效乙醇发酵菌由申请人保藏于日本独立行政法人日本国家技术评估学会专利微生物保藏中心(日本〒292-0818千叶县木更津市上总镰足2-5-8 122号室)。保藏日为2014年11月19日，保藏号为NITE BP-01962。下文中也会将本实施方式的高效乙醇发酵菌称为BP-01962菌株。
所述源自氨处理的稻秆的酶糖化液，例如可以是使用下述方式得到的酶糖化液。首先，将日本埼玉县熊谷市产的稻秆浸入在等量的25质量％氨水中，于30℃浸渍3小时后，将氨解吸出，由此进行前处理。接着，对已经施以所述前处理的稻秆进行pH调节后，添加糖化酶(Meiji Seika Pharma公司制，商品名：支顶孢属纤维素酶)，于50℃保持72小时进行酶糖化，得到含有酶糖化液的浆液。接着，通过压滤对所述浆液进行固液分离，将所回收的液体成分作为所述源自氨处理的稻秆的酶糖化液。所述源自氨处理的稻秆的酶糖化液，例如含有3～15质量％的葡萄糖和1～10质量％的木糖。
作为所述诱变剂，可以使用例如N-乙基-N-亚硝基脲(ENU)、甲磺酸乙酯(EMS)等乙基化剂、5-溴-2'-脱氧尿嘧啶核苷(BrdU)等碱类似化合物、硝基胺、亚硝基胍等亚硝基化合物等。
作为本实施方式的高效乙醇发酵菌的BP-01962菌株是通过以下方式获得的变异菌株：向所述源自氨处理的稻秆的酶糖化液中添加诱变剂而得到的培养基，在该培养基中驯化培养所述亲代菌株，并反复选择出在该培养基中生长的菌。因此，BP-01962菌株与季也蒙毕赤酵母(Meyerozyma guilliermondii)的野生菌株或N菌株相比，在不导入外来基因的情况下提高了木糖同化性和乙醇发酵性能。
接着，针对季也蒙毕赤酵母(Meyerozyma guilliermondii)N菌株和BP-01962菌株的木糖同化性和乙醇收率进行比较。
首先，向含有葡萄糖97.6g/L、木糖41.5g/L、酵母提取物10.0g/L、蛋白胨(peptone)20.0g/L的pH4.7的合成培养基中添加季也蒙毕赤酵母(Meyerozymaguilliermondii)N菌株的培养液，以使得培养基的OD600达到2.0，于30℃培养168小时。然后，每隔规定时间采集所述培养基，分别通过HPLC(东曹公司制，商品名：LC-8020)测定葡萄糖和木糖的浓度、通过GC-FID(GL Sciences公司制，商品名：GC390B)测定乙醇的浓度。木糖浓度和乙醇浓度的测定结果示于图1A。
接着，以26质量％的源自氨处理的稻秆的酶糖化液作为培养基，向该培养基中添加BP-01962菌株的培养液，以使得培养基的OD600达到2.0，于30℃进行168小时培养。所述源自氨处理的稻秆的酶糖化液含有葡萄糖112g/L、木糖40.6g/L，且为pH4.5。并且，每隔规定时间采集所述培养基，分别通过HPLC(东曹公司制，商品名：LC-8020)测定葡萄糖和木糖的浓度、通过GC-FID(GL Sciences公司制，商品名：GC390B)测定乙醇的浓度。木糖浓度和乙醇浓度的测定结果示于图1B。
由图1A、1B可知，在季也蒙毕赤酵母(Meyerozyma guilliermondii)N菌株中的木糖浓度几乎不发生变化，乙醇浓度也不超过40g/L，与此相对，BP-01962菌株的培养时间越长木糖浓度越低，乙醇浓度超过40g/L。
因此，根据BP-01962菌株明显可知，其具备比N菌株更优异的木糖同化性能和乙醇发酵性能。
接着，以26质量％的源自氨处理的稻秆的酶糖化液作为培养基，向该培养基中添加BP-01962菌株的培养液，以使得培养基的OD600达到2.0，于30℃培养120小时。所述源自氨处理的稻秆的酶糖化液含有葡萄糖73.8g/L、木糖28.3g/L，且为pH5.8。并且，每隔规定时间采集所述培养基，分别通过HPLC(东曹公司制，商品名：LC-8020)测定葡萄糖和木糖的浓度、通过GC-FID(GL Sciences公司制，商品名：GC390B)测定乙醇的浓度。结果示于图2。
由图2可知，从培养开始起120小时后，葡萄糖和木糖的全部量被消化，乙醇浓度随着培养时间越长而变得越高。此外，从培养开始起48小时后，葡萄糖浓度几乎变为零，但其后木糖浓度也下降，乙醇浓度继续增加，因此明显可知，BP-01962菌株在将葡萄糖的全部量消化后，以木糖作为底物进行乙醇发酵。
接着，使用源自稀硫酸处理的玉米秸秆的酶糖化液，对BP-01962菌株和N菌株的发酵收率进行比较。
所述源自稀硫酸处理的玉米秸秆的酶糖化液是使用下述方式得到的酶糖化液。首先，将美国爱荷华州产的玉米秸秆浸入2倍量的3.7质量％硫酸中,于170℃浸渍10分钟后，恢复至常温，由此进行前处理。接着，向已经施以所述前处理的玉米秸秆添加NaOH水溶液从而调节至pH4后，添加糖化酶(Meiji Seika Pharma公司制，商品名：支顶孢属纤维素酶)，于50℃保持72小时进行酶糖化，得到含有酶糖化液的浆液。接着，通过离心分离对所述浆液进行固液分离，将所回收的液体成分用NaOH水溶液调节至pH6，作为所述源自稀硫酸处理的玉米秸秆的酶糖化液。所述源自稀硫酸处理的玉米秸秆的酶糖化液例如含有3～15质量％的葡萄糖、和1～10质量％的木糖。
接着，以15质量％的源自稀硫酸处理的玉米秸秆的酶糖化液作为培养基，向该培养基中添加BP-01962菌株的培养液，以使得培养基的OD600达到0.5，于30℃培养100小时。所述源自稀硫酸处理的玉米秸秆的酶糖化液含有葡萄糖45g/L、木糖38g/L，且为pH6。并且，进行所述培养之后，采集所述培养基，通过GC-FID(GL Sciences公司制，商品名：GC390B)测定乙醇的浓度，通过下式(1)算出发酵收率。结果示于图3。
发酵收率＝生成乙醇浓度/(葡萄糖浓度+木糖浓度)/0.5114···(1)
(葡萄糖浓度和木糖浓度为培养开始前的初始浓度)
接着，以26质量％的源自稀硫酸处理的玉米秸秆的酶糖化液作为培养基，向该培养基中添加季也蒙毕赤酵母(Meyerozyma guilliermondii)N菌株的培养液，以使得培养基的OD600达到0.5，于30℃培养100小时。所述源自稀硫酸处理的玉米秸秆的酶糖化液含有葡萄糖64g/L、木糖48g/L，且为pH6。并且，在所述培养后采集所述培养基，通过GC-FID(GLSciences公司制，商品名：GC390B)测定乙醇的浓度，通过式(1)算出发酵收率。结果示于图3。
由图3明显可知，BP-01962菌株与N菌株相比，对于低浓度的源自所述稀硫酸处理的玉米秸秆的酶糖化液，具备比N菌株更优异的乙醇发酵性能。
接着，以20质量％的源自稀硫酸处理的玉米秸秆的酶糖化液作为培养基，分别向该培养基中添加BP-01962菌株和N菌株的培养液，以使得培养基的OD600达到2.0，于30℃培养120小时。所述源自稀硫酸处理的玉米秸秆的酶糖化液含有葡萄糖58.8g/L、木糖33.8g/L，且为pH6。并且，每隔规定时间采集所述培养基，通过GC-FID(GL Sciences公司制，商品名：GC390B)测定乙醇的浓度。结果示于图4。
由图4可知，BP-01962菌株在同浓度的所述源自稀硫酸处理的玉米秸秆的酶糖化液中，具备比N菌株更优异的乙醇发酵性能。
另外，将BP-01962菌株由作为实验室水平规模的1L改变为5000L来进行发酵时，在菌体增殖、葡萄糖同化、木糖同化、乙醇产生量中的任一方面均能够得到几乎同等的结果。因此，BP-01962菌株在工业生产方面也是有用的菌株。
附图标记说明
无标记。
Claims (1)
1.一种高效乙醇发酵菌，其是由五碳糖和六碳糖有效地产生乙醇的发酵菌，该发酵菌的特征在于，
其是通过在向源自氨处理的稻秆的酶糖化液中添加诱变剂而得到的培养基中对季也蒙毕赤酵母(Meyerozyma guilliermondii)N菌株进行驯化培养、选择出在该培养基中生长的菌而得到的与季也蒙毕赤酵母(Meyerozyma guilliermondii)N菌株相比木糖同化性和乙醇发酵性得到提高的菌，
其以保藏号NITE BP-01962被保藏于专利微生物保藏中心。
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/JP2014/082331 WO2016088274A1 (ja) | 2014-12-05 | 2014-12-05 | 高効率エタノール発酵菌 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107429221A CN107429221A (zh) | 2017-12-01 |
| CN107429221B true CN107429221B (zh) | 2020-08-18 |
Family
ID=56091245
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201480083830.3A Expired - Fee Related CN107429221B (zh) | 2014-12-05 | 2014-12-05 | 高效乙醇发酵菌 |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US10119146B2 (zh) |
| EP (1) | EP3228700B1 (zh) |
| JP (1) | JP6145582B2 (zh) |
| CN (1) | CN107429221B (zh) |
| BR (1) | BR112017011495A2 (zh) |
| WO (1) | WO2016088274A1 (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4472501A (en) * | 1981-08-12 | 1984-09-18 | Kyowa Hakko Kogyo Co., Ltd. | Process for manufacturing alcohol by fermentation |
| CN102918161A (zh) * | 2010-04-14 | 2013-02-06 | 乐斯福公司 | 能够由至少一种戊糖生产乙醇的工业酵母 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7846712B2 (en) | 2006-06-01 | 2010-12-07 | Alliance For Sustainable Energy, Llc | L-arabinose fermenting yeast |
| JP2011050359A (ja) | 2009-09-04 | 2011-03-17 | Musashino Chemical Laboratory Ltd | 新規微生物および該微生物由来の酵素、ならびにこれらを用いた糖化液の製造方法 |
| JP2012170422A (ja) | 2011-02-23 | 2012-09-10 | Kyoto Institute Of Technology | キシローストランスポーター活性を有する新規タンパク質および当該タンパク質をコードするポリヌクレオチド、並びにそれらの利用 |
| JP2014158437A (ja) * | 2013-02-19 | 2014-09-04 | Nippon Shokubai Co Ltd | リグノセルロース系バイオマスの糖化液、及びその製造方法と使用方法 |
| US10059965B2 (en) * | 2014-12-05 | 2018-08-28 | Honda Motor Co., Ltd. | Highly efficient ethanol-fermentative yeast |
-
2014
- 2014-12-05 WO PCT/JP2014/082331 patent/WO2016088274A1/ja active Application Filing
- 2014-12-05 BR BR112017011495A patent/BR112017011495A2/pt not_active Application Discontinuation
- 2014-12-05 EP EP14907557.4A patent/EP3228700B1/en active Active
- 2014-12-05 CN CN201480083830.3A patent/CN107429221B/zh not_active Expired - Fee Related
- 2014-12-05 JP JP2016562190A patent/JP6145582B2/ja active Active
- 2014-12-05 US US15/532,801 patent/US10119146B2/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4472501A (en) * | 1981-08-12 | 1984-09-18 | Kyowa Hakko Kogyo Co., Ltd. | Process for manufacturing alcohol by fermentation |
| CN102918161A (zh) * | 2010-04-14 | 2013-02-06 | 乐斯福公司 | 能够由至少一种戊糖生产乙醇的工业酵母 |
Non-Patent Citations (2)
| Title |
|---|
| Efficient ethanol production from corncob residues by repeated fermentation of an adapted yeast;C. Fan et al.;《Bioresource Technology》;20130313;第136卷;第309-315页 * |
| Production of ethanol from soybean hull hydrolysate by osmotolerant Candida guilliermondii NRRL Y-2075;A.C. Schirmer-Michel et al.;《Bioresource Technology》;20080207;第99卷(第8期);第2898-2904页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| US20170342425A1 (en) | 2017-11-30 |
| EP3228700A4 (en) | 2018-05-23 |
| JP6145582B2 (ja) | 2017-06-14 |
| BR112017011495A2 (pt) | 2018-04-10 |
| EP3228700A1 (en) | 2017-10-11 |
| US10119146B2 (en) | 2018-11-06 |
| CN107429221A (zh) | 2017-12-01 |
| WO2016088274A1 (ja) | 2016-06-09 |
| JPWO2016088274A1 (ja) | 2017-05-25 |
| EP3228700B1 (en) | 2019-07-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sheng et al. | Direct hydrogen production from lignocellulose by the newly isolated Thermoanaerobacterium thermosaccharolyticum strain DD32 | |
| US10294497B2 (en) | Highly efficient ethanol-fermentation yeast | |
| CN107429219B (zh) | 高效乙醇发酵菌 | |
| CN107429221B (zh) | 高效乙醇发酵菌 | |
| Thanonkeo et al. | The batch ethanol fermentation of jerusalem artichoke using Saccharomyces cerevisiae | |
| CN107429218B (zh) | 高效乙醇发酵菌 | |
| JP6240344B2 (ja) | 高効率エタノール発酵菌 | |
| KR101599997B1 (ko) | 신규한 고온 효모 피키아 길리에르몬디 y-2 및 이의 용도 | |
| Dénes et al. | Bioethanol fermentation of Jerusalem artichoke using mixed culture of Saccharomyces cerevisiae and Kluyveromyces marxianus | |
| JP6327822B2 (ja) | 糸状菌を用いる木質系バイオマスからのエタノールの製造方法 | |
| Kim et al. | Bioalcohol production with M. aeruginosa as a novel resource | |
| JP2015181392A (ja) | アルコールの製造方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200818 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |