CN116716321B - Hmx1及其编码基因在提高酿酒酵母木糖发酵性能和乙酸耐受性上的应用 - Google Patents
Hmx1及其编码基因在提高酿酒酵母木糖发酵性能和乙酸耐受性上的应用 Download PDFInfo
- Publication number
- CN116716321B CN116716321B CN202310967527.0A CN202310967527A CN116716321B CN 116716321 B CN116716321 B CN 116716321B CN 202310967527 A CN202310967527 A CN 202310967527A CN 116716321 B CN116716321 B CN 116716321B
- Authority
- CN
- China
- Prior art keywords
- hmx1
- saccharomyces cerevisiae
- strain
- xylose
- acetic acid
- 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
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 title claims abstract description 64
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 title claims abstract description 59
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 title claims abstract description 32
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 title claims abstract description 32
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 27
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 title claims abstract description 27
- 238000000855 fermentation Methods 0.000 title claims abstract description 26
- 230000004151 fermentation Effects 0.000 title claims abstract description 26
- 108090000623 proteins and genes Proteins 0.000 title abstract description 26
- 102100029019 Homeobox protein HMX1 Human genes 0.000 title description 2
- 101000986308 Homo sapiens Homeobox protein HMX1 Proteins 0.000 title description 2
- 230000002018 overexpression Effects 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 11
- 230000008569 process Effects 0.000 claims abstract description 7
- 239000013604 expression vector Substances 0.000 claims description 4
- 230000006872 improvement Effects 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 39
- 239000003112 inhibitor Substances 0.000 abstract description 11
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 239000001913 cellulose Substances 0.000 abstract description 3
- 229920002678 cellulose Polymers 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 12
- 239000001963 growth medium Substances 0.000 description 12
- 239000013598 vector Substances 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 10
- 239000002609 medium Substances 0.000 description 9
- 239000003242 anti bacterial agent Substances 0.000 description 8
- 229940088710 antibiotic agent Drugs 0.000 description 8
- 230000012010 growth Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 239000006228 supernatant Substances 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
- 238000010276 construction Methods 0.000 description 5
- 238000001976 enzyme digestion Methods 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 108010042407 Endonucleases Proteins 0.000 description 4
- 102000004533 Endonucleases Human genes 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 238000009630 liquid culture Methods 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 108700040099 Xylose isomerases Proteins 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 150000002772 monosaccharides Chemical class 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000011426 transformation method Methods 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 241000235070 Saccharomyces Species 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000000446 fuel Substances 0.000 description 2
- -1 furan aldehydes Chemical class 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000004127 xylose metabolism Effects 0.000 description 2
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- PYMYPHUHKUWMLA-VPENINKCSA-N aldehydo-D-xylose Chemical compound OC[C@@H](O)[C@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-VPENINKCSA-N 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
- 230000003042 antagnostic effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 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 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000025938 carbohydrate utilization Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000003245 coal Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000002803 fossil fuel Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000003208 gene overexpression Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000004108 pentose phosphate pathway Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000004144 purine metabolism Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
- C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
-
- 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
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
- C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
-
- 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
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
-
- 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
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明属于酿酒酵母技术领域,涉及HMX1及其编码基因在提高酿酒酵母木糖发酵性能和乙酸耐受性上的应用。本发明首次公开过表达HMX1基因可显著提高酿酒酵母的木糖发酵性能及乙酸耐受性,这为解决木质纤维素乙醇发酵过程中微生物木糖利用率低、抑制物耐受性弱的瓶颈问题提供了技术指导,为纤维素乙醇工业生产中的菌种优化贡献了有效元件。
Description
技术领域
本发明属于酿酒酵母技术领域,涉及HMX1及其编码基因在提高酿酒酵母木糖发酵性能和乙酸耐受性中应用,具体涉及酿酒酵母HMX1基因过表达菌株的构建及过表菌株在同时改善酿酒酵母木糖发酵性能及乙酸耐受性中的应用。
背景技术
能源是人类赖以生存和发展的物质基础,其中包括不可再生资源和可再生资源。不可再生资源包括煤炭、石油等矿产资源,随着人类生活水平的提高及工业的迅速发展,不可再生资源的持续开发利用也带来了很多弊端,比如环境污染,资源短缺,温室效应等,因此,寻找可替代的绿色可再生能源变得尤为重要。目前,以木质纤维素为原料生产的二代燃料乙醇已成为不可再生类化石燃料的有力替代品。
木质纤维素所具备的来源广泛、廉价易得及可再生的优点极大程度上推动了二代燃料乙醇的全球产业化生产。全球植被平均每年以光合作用被固定的CO2大约为1230亿吨,相当于人类一年能源消耗的十倍,其中木质纤维素总量约占光合作用合成生物质总量的60%-80%。木质纤维素由高分子聚合物纤维素、半纤维素和木质素组成,由于此类聚合物内部化学键比较复杂,所以微生物难以将其直接利用,需要经过预处理及水解过程,将其分解成可发酵单糖后才能用于微生物乙醇的发酵生产。木质纤维素水解后的可发酵单糖主要有葡萄糖木糖两种,葡萄糖可被微生物直接利用,而多数微生物对木质纤维素水解单糖中含量仅次于葡萄糖的木糖的利用能力非常低或者无法利用。另一方面,木质纤维素在预处理过程中会产生包括酸类、呋喃醛、过氧化物及酚类等多种小分子化合物,这些小分子化合物我们统称为发酵抑制物,发酵抑制物可通过抑制微生物的生长及代谢过程影响发酵效率。综上所述,酿酒酵母的木糖发酵性能及对发酵抑制物的耐受性是限制以木质纤维素为原料的二代乙醇生产效率的两大因素。
为充分利用水解液中含量丰富的木糖组分,研究人员通过改造木糖转运相关蛋白的性能、外源引入木糖代谢途径、调节木糖代谢下游磷酸戊糖途径相关基因的表达水平等遗传学手段提高酿酒酵母的木糖发酵性能。另有多项研究成果报道,利用基因工程手段对转录因子、氧化还原及嘌呤代谢等途径中的关键基因进行遗传改造,可显著提高菌株对木质纤维素预处理液中小分子抑制物的耐受性。作为二代乙醇生产的常用菌株,酿酒酵母的木糖发酵性能与抑制物耐受性之间往往存在拮抗关系,即菌株木糖发酵能力的提升可能伴随其对发酵抑制物耐受性的下降,反之亦然。基于以木质纤维素水解液为原料发酵生产二代乙醇的微生物菌株不仅要具备高效的五碳糖、六碳糖利用能力,还需具有较强的抑制物耐受性,利用基因工程手段同时改善微生物以上两方面性能为实现木质纤维素乙醇的产业化、经济化及可持续化提供了有力保障。
发明内容
为了解决木质纤维素乙醇发酵过程木糖利用能力低、抑制物耐受性弱的难题,本发明公开了一株HMX1过表达菌株的构建方法,并证明该基因的过表达可同时提高菌株的木糖发酵性能及对发酵抑制物乙酸的耐受性。
本发明的技术方案如下:
HMX1在提高酿酒酵母乙酸耐受性上的应用,所述的HMX1的序列如SEQ ID No.1所示。
同时保护,HMX1在提高酿酒酵母木糖发酵性能上的应用。
进一步HMX1在优化木质纤维素乙醇生产酿酒酵母菌株性能上的应用。
所述的应用通过过表达HMX1,得到过表达HMX1的酿酒酵母菌株。
过表达HMX1的过程为:采用的过表达载体为pJFE3-K-HMX1,将过表达载体转入酿酒酵母菌株中,得到过表达HMX1的酿酒酵母菌株。
本发明首先构建了过表达HMX1基因的载体pJFE3-K-HMX1,然后将重组载体转入BSPC039菌株中,最后对过表达HMX1基因的BSPC039-HMX1菌株进行了木糖发酵性能及乙酸耐受性的检测。
具体的技术步骤如下:
(1)过表达HMX1菌株的构建
为了获得HMX1基因序列,以已有的BSPC039基因组DNA为模板(BSPC039菌株由山东大学鲍晓明老师提供,具体可参考Metabolic Engineering14(2012)9–18),利用引物HMX1-A(碱基序列如SEQ ID No.4所示,具体为5’-CGCGGATCCCGCAAAAATGGAGGACAGTAGC-3’)和引物HMX1-B(碱基序列如SEQ ID No.5所示,具体为5’-ACGCGTCGACCTCTGCTGTTTTTCCTTCCCTATTC-3’)进行PCR扩增,得到HMX1基因的编码区序列。其中HMX1-A引物的5’端携带BamH I核酸内切酶识别位点(5’-GGATCC-3’),HMX1-B引物的3’端携带Sal Ⅰ核酸内切酶识别位点(5’-GTCGAC-3’)。pJFE3-K载体携带KanMX4抗性标记基因,启动子为TEF1p,多克隆位点中包含BamH I和Sal Ⅰ内切酶识别序列。我们同时使用BamH I和Sal Ⅰ两个内切酶对扩增得到的HMX1基因序列和pJFE3-K载体分别进行酶切消化,随后使用T4连接酶将酶切后的pJFE3-K载体和HMX1基因序列进行连接,连接后的载体转入大肠杆菌后经体内复制及菌落PCR鉴定获得连接成功的重组pJFE3-K-HMX1质粒。菌落PCR鉴定使用的引物为HMX1-C(碱基序列如SEQID No.6所示,具体为5’-CTCTTTCGATGACCTCCCA-3’)和HMX1-D(碱基序列如SEQ ID No.7所示,具体为5’-GGATGGGGAAAGAGAAAAG-3’)。将菌落PCR鉴定正确的质粒pJFE3-K-HMX1转入BSPC039菌株获得BSPC039-HMX1菌株,同时将载体pJFE3-K转入BSPC039菌株获得BSPC039-P菌株。
(2)菌株的木糖发酵性能检测
为了使酿酒酵母具有一定的木糖代谢能力,我们向BSPC039-P、BSPC039-HMX1菌株中转入能够过表达木糖异构酶(XI)的pJFE3-XI质粒,获得BSPC039X-P、BSPC039X-HMX1菌株。从固体平板上划取少量BSPC039X-P、BSPC039X-HMX1的菌体置于添加有200 mg/L G418抗生素的Sc-Ura+G液体培养基中,于200 rpm 摇床中30℃过夜培养24 h。将上述菌液用添加有150 mg/L G418抗生素的Sc-Ura+X培养基稀释至OD600为0.2,于200 rpm 摇床中30℃培养,每隔12 h取样一次,使用液相色谱仪检测木糖消耗量、乙醇产量。结果发现,过表达HMX1基因的菌株木糖消耗速率及乙醇产率明显高于对照组。
(3)菌株的乙酸耐受性的检测
从固体平板取少量BSPC039X-P、BSPC039X-HMX1菌体于添加有200 mg/L G418抗生素的Sc-Ura+G液体培养中,于200 rpm 摇床中30℃过夜培养。将上述菌液用相同培养基稀释至OD600为0.2,于200 rpm 摇床中30℃培养,每隔3 h取样一次并测取OD600数值。由生长曲线可得,过表达HMX1基因的菌株乙酸耐受性明显好于对照组。
本发明的有益效果
本发明首次公开过表达HMX1基因可显著提高酿酒酵母的木糖发酵性能及乙酸耐受性,这为解决木质纤维素乙醇发酵过程中微生物木糖利用率低、抑制物耐受性弱的瓶颈问题提供了技术指导,为纤维素乙醇工业生产中的菌种优化贡献有效元件。
附图说明
图1为pJFE3-K-HMX1载体图谱;
图2为过表达HMX1基因前后菌株在以木糖为唯一碳源的液体培养基中的限氧发酵性能,实线代表木糖消耗速率,虚线代表乙醇产生速率;
图3为过表达HMX1基因前后菌株的生长曲线;(A):未添加乙酸培养条件下的菌株生长曲线;(B):添加50 mM乙酸培养条件下的菌株生长曲线。
具体实施方式
实施例1培养基种类及配方
YPD培养基:20 g/L蛋白胨,10 g/L酵母粉,固体培养基中添加20 g/L琼脂粉,115℃灭菌15 min,继续向培养基中添加20 g/L无菌葡萄糖溶液,制成YPD培养基。
SC-Ura培养基:1.7 g/L Yeast Nitrogen Base,5 g/L硫酸铵,0.77 g/L CSM-Ura,固体培养基添加20 g/L琼脂粉,115 ℃灭菌15 min。使用前加入20 g/L葡萄糖制Sc-Ura+G培养基,或添加20 g/L木糖制成Sc-Ura+X培养基。
LB培养基:蛋白胨10 g/L,5 g/L Yeast Nitrogen Base,10 g/L NaCl,pH 7.0,固体培养基添加20 g/L琼脂粉,121℃高压灭菌15 min。
实施例2:过表达HMX1菌株的构建
以上构建过表达HMX1菌株所涉及的具体实验方法如下:
(1)提取BSPC039菌株的基因组DNA
向1.5 mL EP管中加入200 μL的DNA提取液,从固体平板中刮取适量BSPC039菌体于DNA提取液中,制成菌悬液。向菌悬液中加入1.0 g玻璃珠,随后加入200 μL苯酚:氯仿:异戊醇(25:24:1)混合液,上下充分颠倒混匀,之后置于离心机中12000 g离心5 min。将离心后的上清液转移到新的1.5 mL EP管中,并向该管中加入1 mL无水乙醇,上下轻轻颠倒混匀,静置15 min后于离心机中12000 g离心10 min。去除上清并于室温充分干燥,随后向干燥后的DNA中加入50 μL的去离子水制成DNA溶液。
DNA提取液配制:100 mM NaCl,2% Triton X-100,1 mM EDTA,1% SDS,10 mMTris-Cl,pH为8.0。
(2)PCR扩增获得HMX1基因序列
以BSPC039基因组DNA为模板,利用引物HMX1-A和引物HMX1-B进行PCR扩增得到HMX1基因序列,HMX1基因序列如SEQ ID No .1所示。其中HMX1-A引物的5’端携带BamH I核酸内切酶识别位点(5’-GGATCC-3’),HMX1-B引物的3’端携带Sal Ⅰ核酸内切酶识别位点(5’-GTCGAC-3’)。取1μL PCR产物,使用琼脂糖凝胶电泳检测DNA条带大小是否符合预期。
PCR扩增体系见表1,PCR扩增程序见表2。
表1 KOD PCR扩增体系
表2 PCR扩增程序
(3)对DNA片段和载体进行酶切、连接
使用DNA纯化试剂盒对扩增得到的HMX1基因序列进行纯化,同时利用BamH I和Sal Ⅰ两个核酸内切酶对纯化后的HMX1基因序列和pJFE3-K载体分别进行酶切消化,酶切条件为37℃水浴锅中消化12 h,pJFE3-K载体序列如表1中SEQ ID No .2所示,下划线标记序列分别为BamHI和Sal Ⅰ的酶切位点。随后使用T4连接酶将酶切后的pJFE3-K载体和HMX1基因序列进行连接(图1),连接条件为4℃过夜。
酶切体系见表3,连接体系见表4。
表3酶切体系
表4连接体系
(4)将重组质粒转化至大肠杆菌AH109菌株并进行阳性克隆的鉴定
将连接后的载体转入大肠杆菌AH109菌株中,转化条件为:将载体混合液全部转入50 μL大肠杆菌感受态溶液中,之后于42℃水浴锅中热击90 s,之后将离心管立刻插到冰上冷却5 min,将菌液涂至添加有氨苄抗生素的LB平板中,于37℃培养过夜。第二天挑取阳性克隆至于PCR管中,并向PCR管中添加PCR扩增溶液,使用引物HMX1-C和HMX1-D对阳性克隆进行PCR鉴定,获得连接正确的质粒pJFE3-K-HMX1,其序列见SEQ ID No .3。将鉴定正确的克隆于添加有50 mg/L氨苄抗生素的LB液体培养基培养37℃,200 rpm摇床中培养过夜。第二天,利用质粒提取试剂盒提取菌液中的质粒pJFE3-K-HMX1。PCR扩增体系见表1,扩增程序见表2。
(5)将提取的质粒转入酿酒酵母菌株
将质粒pJFE3-K-HMX1和pJFE3-K通过醋酸锂转化法分别转入BSPC039菌株,获得BSPC039-HMX1和BSPC039-P菌株。具体转化方法如下:
将BSPC039菌株接种到YPD液体培养基中,30 ℃培养至对数生长期,5000 g离心5min收集菌体,去除上清后用去离子水重悬菌体,5000 g离心1 min再次收集菌体并用0.1 M的LiAC溶液重悬菌体,将菌悬液后转移到1.5 mL EP管中,5000 g离心1 min,去除上清,随后向菌管中加入240 μL 50% PEG溶液,充分混匀后继续向其中加入36 μL 1M的LiAC溶液,15 μL 4 mg/mL的鲑鱼精DNA,1 μL质粒,充分混匀,随后将离心管置于30 ℃水浴锅中孵育30 min,之后转移至42 ℃水浴锅中孵育20 min。5000g离心1min收集菌体,去除上清液后使用200 μL无菌水重悬菌体,吸取50 μL菌液涂板至添加有200 mg/L G418抗生素的YPD固体平板上,30 ℃培养3天,挑取单菌落转化子。
实施例3:对过表达HMX1的菌株进行木糖发酵性能检测
为了使酿酒酵母具有一定的木糖代谢能力,我们使用醋酸锂转化法向BSPC039-P、BSPC039-HMX1菌株中转入能够过表达木糖异构酶(XI)的pJFE3-XI质粒,并使用添加有200mg/L G418抗生素的Sc-Ura+G固体平板筛选阳性克隆,获得BSPC039X-P、BSPC039X-HMX1菌株,具体转化方法参考实施例2。将获取的转化菌株置于添加有200 mg/L G418抗生素的Sc-Ura+G液体培养基中,摇床中30℃,200 rpm过夜培养24 h。将上述菌液用添加有200 mg/LG418抗生素的Sc-Ura+X培养基稀释至OD600为0.2,于200 rpm 摇床中30℃培养,每隔12 h取样一次,每次取1 mL菌液,于离心机中12000 g离心15 min。使用0.22 μm的滤膜过滤离心后的上清液,并将过滤液注入样品瓶,利用高效液相色谱仪和Aminex HPX-87H离子交换柱对样品瓶中的过滤液进行木糖和乙醇含量的测定。离子交换柱的柱温控制在45 ℃,使用5 mMH2SO4作为流动相,其流速设定为0.6 mL/min,采用示差折光器进行参数测定,以下是样品消耗或生成速率的计算公式。
注:r为取样点m到n这段时间处的检测对象比利用或生成速率;A、B及t分别为取样时间点n、i和m处的代谢物浓度,生物质浓度和时间。
使用液相色谱仪检测木糖消耗量、乙醇产量。结果发现,过表达HMX1基因的菌株木糖消耗速率及乙醇产率明显高于对照组(图2)。
实施例4:对过表达HMX1的菌株进行乙酸耐受性检测
取适量BSPC039X-P、BSPC039X-HMX1菌体于添加有200 mg/L G418抗生素的Sc-Ura+G液体培养中摇菌,200 rpm 摇床中30℃过夜培养。将上述菌液用相同培养基稀释至OD600为0.2并平均分为两等份,其中一份加入乙酸至终浓度为50 mM,另一份作为对照组。将上述菌液放置于200 rpm 摇床中30℃培养,每隔3 h取样一次并测取OD600数值绘制成生长曲线。由生长曲线可得,在未添加乙酸的液体培养基中,过表达菌株跟对照生长没有太大差别(图3A),而在添加50 mM乙酸的液体培养基中,过表达HMX1基因菌株的乙酸耐受性明显好于对照组(图3B)。
Claims (3)
1.HMX1在提高酿酒酵母BSPC039乙酸耐受性上的应用,其特征在于,所述的HMX1的序列如SEQ ID No.1所示,所述的应用通过过表达HMX1,得到过表达HMX1的酿酒酵母BSPC039菌株。
2.HMX1在提高酿酒酵母BSPC039木糖发酵性能上的应用,其特征在于,所述的HMX1的序列如SEQ ID No.1所示,所述的应用通过过表达HMX1,得到过表达HMX1的酿酒酵母BSPC039菌株。
3.根据权利要求1或2所述的应用,其特征在于,过表达HMX1的过程为:采用的过表达载体为pJFE3-K-HMX1,将过表达载体转入酿酒酵母BSPC039菌株中,得到过表达HMX1的酿酒酵母BSPC039菌株。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310967527.0A CN116716321B (zh) | 2023-08-03 | 2023-08-03 | Hmx1及其编码基因在提高酿酒酵母木糖发酵性能和乙酸耐受性上的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310967527.0A CN116716321B (zh) | 2023-08-03 | 2023-08-03 | Hmx1及其编码基因在提高酿酒酵母木糖发酵性能和乙酸耐受性上的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116716321A CN116716321A (zh) | 2023-09-08 |
CN116716321B true CN116716321B (zh) | 2023-12-05 |
Family
ID=87868198
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310967527.0A Active CN116716321B (zh) | 2023-08-03 | 2023-08-03 | Hmx1及其编码基因在提高酿酒酵母木糖发酵性能和乙酸耐受性上的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116716321B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101942435A (zh) * | 2009-12-16 | 2011-01-12 | 安徽丰原发酵技术工程研究有限公司 | 一种发酵木糖产乙醇的重组酿酒酵母及其构建方法 |
CN113980993A (zh) * | 2021-11-17 | 2022-01-28 | 齐鲁工业大学 | Mal33基因缺失在提高酿酒酵母对木质纤维素水解液抑制物耐受性中的应用 |
WO2022103318A1 (en) * | 2020-11-11 | 2022-05-19 | Olena Ishchuk | A genetically modified yeast cell for hemoglobins production |
-
2023
- 2023-08-03 CN CN202310967527.0A patent/CN116716321B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101942435A (zh) * | 2009-12-16 | 2011-01-12 | 安徽丰原发酵技术工程研究有限公司 | 一种发酵木糖产乙醇的重组酿酒酵母及其构建方法 |
WO2022103318A1 (en) * | 2020-11-11 | 2022-05-19 | Olena Ishchuk | A genetically modified yeast cell for hemoglobins production |
CN113980993A (zh) * | 2021-11-17 | 2022-01-28 | 齐鲁工业大学 | Mal33基因缺失在提高酿酒酵母对木质纤维素水解液抑制物耐受性中的应用 |
Non-Patent Citations (2)
Title |
---|
Engel,S.R..Hmx1p [Saccharomyces cerevisiae S288C],NCBI Reference Sequence: NP_013306.2 .NCBI Genbank database.2023,全文. * |
Exploring metal ion metabolisms to improve xylose fermentation in Saccharomyces cerevisiae;Palermo, GCD;MICROBIAL BIOTECHNOLOGY;第14卷(第5期);全文 * |
Also Published As
Publication number | Publication date |
---|---|
CN116716321A (zh) | 2023-09-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR20190018724A (ko) | 피치아 파스토리스 발현 재조합 단백질의 발효 공법 | |
CN101631864A (zh) | 使用酵母菌通过丁酰-CoA作为中间体制备丁醇的方法 | |
US10053712B2 (en) | Use of enzymes which catalyze pyruvate synthesis from formate and acetyl-CoA and bacteria expressing same | |
CN106995794B (zh) | 一种提高丁二酸产量的产琥珀酸放线杆菌工程菌株及其构建方法与用途 | |
CN101748069A (zh) | 一种重组蓝藻 | |
CN116716321B (zh) | Hmx1及其编码基因在提高酿酒酵母木糖发酵性能和乙酸耐受性上的应用 | |
CN109706104B (zh) | sll0528基因在提高集胞藻PCC6803乙醇耐受性中的应用 | |
CN115806922B (zh) | 运动发酵单胞菌的基因工程菌株及其应用 | |
CN1191369C (zh) | 一种用代谢工程菌生产腺苷甲硫氨酸的方法 | |
Wang et al. | Carbon-economic biosynthesis of poly-2-hydrobutanedioic acid driven by nonfermentable substrate ethanol | |
CN111826372B (zh) | 利用木糖生产丁醇的工程菌株及其构建方法和应用 | |
CN104403956B (zh) | 木糖醇高温高产工程菌株的构建及应用 | |
CN106434700B (zh) | 一种提高乙醇产率的酿酒酵母spt15定点饱和基因突变方法 | |
CN116731136B (zh) | H3k23a组蛋白点突变在提高酿酒酵母乙酸耐受性及木糖发酵性能中的应用 | |
CN115058350B (zh) | 一种引入钾离子转运体提高s-腺苷甲硫氨酸产量的方法 | |
CN114015634B (zh) | 高产琥珀酸的重组大肠杆菌及其构建方法和应用 | |
CN117417874B (zh) | 一种工程菌株hc6-mt及其在低温生产海藻糖中的应用 | |
CN103088434A (zh) | 树干毕赤酵母大片段dna基因组文库的构建方法及其应用 | |
CN112779174B (zh) | 一株敲除Cln3基因的酿酒酵母基因工程菌及其构建方法和应用 | |
KR101411828B1 (ko) | 에탄올 생산용 재조합 방선균 및 이를 이용한 에탄올 생산방법 | |
CN116478898A (zh) | 一种经CRISPR技术改造hrcA的重组菌株及其构建方法与应用 | |
CN116376732A (zh) | 一株高产甘油三酯和甘油二酯的酿酒酵母工程菌株、构建方法及其应用 | |
CN116496917A (zh) | 一种高效共利用木糖和葡萄糖的重组酿酒酵母及其应用 | |
CN117844837A (zh) | 产琥珀酸的大肠杆菌基因工程菌的构建方法与应用 | |
CN117946878A (zh) | 一株耐酸的重组马克斯克鲁维酵母菌株及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |