CN115418322B - 低产酸解脂耶氏酵母选育及在赤藓糖醇发酵中的应用方法 - Google Patents
低产酸解脂耶氏酵母选育及在赤藓糖醇发酵中的应用方法 Download PDFInfo
- Publication number
- CN115418322B CN115418322B CN202211046538.7A CN202211046538A CN115418322B CN 115418322 B CN115418322 B CN 115418322B CN 202211046538 A CN202211046538 A CN 202211046538A CN 115418322 B CN115418322 B CN 115418322B
- Authority
- CN
- China
- Prior art keywords
- yarrowia lipolytica
- fermentation
- erythritol
- yield
- strain
- 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
- 239000004386 Erythritol Substances 0.000 title claims abstract description 23
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 title claims abstract description 23
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 title claims abstract description 23
- 229940009714 erythritol Drugs 0.000 title claims abstract description 23
- 235000019414 erythritol Nutrition 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims abstract description 15
- 238000000855 fermentation Methods 0.000 title claims description 46
- 230000004151 fermentation Effects 0.000 title claims description 46
- 241000235015 Yarrowia lipolytica Species 0.000 title abstract description 30
- 238000009395 breeding Methods 0.000 title abstract description 4
- 238000004519 manufacturing process Methods 0.000 claims abstract description 11
- 241000235013 Yarrowia Species 0.000 claims abstract description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 12
- 238000009629 microbiological culture Methods 0.000 claims description 3
- 230000002906 microbiologic effect Effects 0.000 claims 1
- 239000002253 acid Substances 0.000 abstract description 7
- 230000001488 breeding effect Effects 0.000 abstract description 2
- -1 sodium ions Chemical class 0.000 description 8
- 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 7
- 229960001031 glucose Drugs 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- 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 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 210000005253 yeast cell Anatomy 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 239000001257 hydrogen Substances 0.000 description 5
- 229910052739 hydrogen Inorganic materials 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical class [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 description 4
- 229910052782 aluminium Inorganic materials 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000029142 excretion Effects 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 239000008055 phosphate buffer solution Substances 0.000 description 3
- 230000035790 physiological processes and functions Effects 0.000 description 3
- GRLPQNLYRHEGIJ-UHFFFAOYSA-J potassium aluminium sulfate Chemical compound [Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRLPQNLYRHEGIJ-UHFFFAOYSA-J 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 108091006112 ATPases Proteins 0.000 description 2
- 102000057290 Adenosine Triphosphatases Human genes 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 159000000013 aluminium salts Chemical class 0.000 description 2
- 229910000329 aluminium sulfate Inorganic materials 0.000 description 2
- 229940103272 aluminum potassium sulfate Drugs 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 235000013615 non-nutritive sweetener Nutrition 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 150000005846 sugar alcohols Chemical class 0.000 description 2
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 2
- 239000007222 ypd medium Substances 0.000 description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- 230000002407 ATP formation Effects 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 239000005696 Diammonium phosphate Substances 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 102000003697 P-type ATPases Human genes 0.000 description 1
- 108090000069 P-type ATPases Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 239000002696 acid base indicator Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940089206 anhydrous dextrose Drugs 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 1
- 235000019838 diammonium phosphate Nutrition 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- XJRPTMORGOIMMI-UHFFFAOYSA-N ethyl 2-amino-4-(trifluoromethyl)-1,3-thiazole-5-carboxylate Chemical compound CCOC(=O)C=1SC(N)=NC=1C(F)(F)F XJRPTMORGOIMMI-UHFFFAOYSA-N 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 235000021474 generally recognized As safe (food) Nutrition 0.000 description 1
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 231100000001 growth retardation Toxicity 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000037427 ion transport Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- YPJKMVATUPSWOH-UHFFFAOYSA-N nitrooxidanyl Chemical compound [O][N+]([O-])=O YPJKMVATUPSWOH-UHFFFAOYSA-N 0.000 description 1
- 235000003715 nutritional status Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 238000011197 physicochemical method Methods 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000012799 strong cation exchange Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
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
- 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
- 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
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes 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
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Botany (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明涉及一种定向选育解脂亚罗酵母（Yarrowia lipolytica）低产酸变异株的方法，应用该方法获得的解脂亚罗酵母CGMCC 25430菌种显示出更低的产酸趋势和赤藓糖醇的产率，可以使菌种的赤藓糖醇的转化率从原始的55%提高到65%以上，具有良好的应用价值。
Description
技术领域
本发明涉及工业微生物的改造和应用，属于生物工程技术领域。
背景技术
解脂耶氏酵母(Yarrowia lipolytica)是一种非常规酵母菌种。由于其非发酵、无致病性及分泌特征显著等特点，在酶制剂、有机酸和合成香料等产物的生产领域获得广泛应用，而且其作为外源基因的表达底盘菌方面的应用也越来越广。然而在一些逆境的应用场合下，如温度、渗透压及营养状态的改变容易诱发解脂耶氏酵母的应激和多形性转变，造成细胞生理状态的剧变，使得应用过程参数劣化，引起诸如生长迟滞、代谢紊乱、产品产量降低甚至酵母细胞死亡。为此对该酵母的代谢生理过程的控制，改善其发酵品质具有重要的意义。其中以控制酵母涉膜离子流性质是稳定细胞生理状态的重要手段，也成为改善发酵性能，如降低副产有机酸的一个途径，对提高解脂耶氏酵母这种酵母菌种的应用性能具有重要的意义。
近年来，众多的研究表明高热量的食糖摄入会对健康带来诸多不利影响，而食用低热量甜味剂成为满足人们对甜味需求的重要途径，并为食品、饮料生产企业和广大消费者所普遍接受。在低热量甜味剂中糖醇类化合物，其中的赤藓糖醇是所谓的填充型、零热量甜味剂的最突出的代表，由于其具有良好的甜感和生物耐受性而备受业界的重视，具有良好的市场容量和发展前景。目前，可用于赤藓糖醇生产的菌种中解脂耶氏酵母是唯一的被国际公认安全(GRAS)的菌种，对该菌种的赤藓糖醇发酵过程研究和改造受到了普遍的重视，而提高赤藓糖醇的发酵性能的研究也是其生物工艺学研究的重要领域。
由于解脂耶氏酵母的糖醇发酵是在高渗透压环境下进行的，发酵条件对其生长和代谢都是不利的，而高渗的逆境除了能诱导菌种产生多元醇等相容性基质外，还会诱导跨细胞膜的离子流的产生，阴离子有氯离子等，而阳离子则包括钠离子、钾离子和氢离子等。由定向离子流所产生的跨膜离子梯度又会带来跨膜物质传输的变化。对于赤藓糖醇发酵过程，会因为胞内氢离子的外排造成发酵基质的酸性升高，从而会限制菌种的生长和发酵，同时由于氢离子的外排，使得胞内pH的升高，因此又诱导解脂耶氏酵母细胞形态从比表面积更大、发酵活力更强的酵母形态转变为拉长的发酵活力减弱的菌丝细胞形态。以往为了克服因胞内氢离子外排造成的基质的pH变化，通常采用在发酵过程中连续添加中和剂来抵消pH的变化来解决。但会造成发酵液中难处理的电解质杂质的增加，而且会造成生产成本的提高。
研究表明，P型ATP酶是直接造成胞内氢离子外排的膜结合复合体，其与pH梯度耦合的磷酸化过程对酵母细胞的生命过程的ATP合成具有重要的作用。通过对P型磷酸酶系统功能的遗传学改造，可对这一重要系统的合成效率及氢离子转运能力产生调制作用的新酵母菌种，对解脂耶氏酵母的应用可产生有益的影响。
铝是一个两性的三价元素，较高浓度的铝离子可与ATP酶结合，并影响底物的对抗离子状态河跨膜的pH梯度，因此可以作为生物细胞ATP酶结构和功能变异的筛选因子。本发明通过从经理化方法诱变结合高浓铝离子抗性富集，选育得到低产酸的解脂耶氏酵母变异株，从而改善菌种的发酵性能，并将该方法用于赤藓糖醇的发酵生产。
发明内容
本发明的目的是提供一种低产酸解脂耶氏酵母变异株的筛选的方法，并利用该方法选育适合赤藓糖醇发酵的新菌株，以提高菌种的发酵效率和产品纯度，以降低生产成本。
为实现上述目的，本发明采用如下技术方案。
一、本发明的解脂耶氏酵母菌种改造技术，是以普通的野生型的解脂耶氏酵母为出发株，经过理化、分子生物学方法使其发生遗传变异，并利用铝盐抗性性状从中筛选出低产酸的变异菌株。变异株之一寄存于中国微生物菌种保藏管理委员会普通微生物中心，分类名称：解脂耶氏酵母(Yarrowia lipolytica)，保藏编号：CGMCC No.25430，保藏地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏日期：2022年7月29日。
进一步的，该操作按如下步骤进行：
(1)利用EMS、UV等化学及物理诱变方法和其他分子诱变方法处理野生型的解脂耶氏酵母细胞，使之产生随机性的基因突变，然后接种含较高浓度的铝盐的完全培养基上富集培养。
(2)将经过富集培养的酵母细胞涂布含酸碱指示剂的筛选平板挑选快速生长、低产酸变异株CGMCC 25430。
二、通过发酵试验验证变异株的赤藓糖醇发酵性能。
进一步，具体步骤如下：
(1)菌种活化
将变异株在AlYPDA固体培养基平板上划线，30±1℃培养72小时。
(2)种子培养
无菌条件下，挑取步骤(1)平板上的单菌落，接种发酵培养基，30±1℃摇床培养24小时，获得种子液。
(3)发酵培养
将种子液以0.1-20％接种量接种到发酵培养基中，30±1℃摇床培养96小时，获得发酵液。
其中，上述步骤(1)中所述AlYPDA培养基配方为：1L水中加入酵母浸粉3g，蛋白胨5g，葡萄糖100g，硫酸铝钾4g，琼脂20g，115℃灭菌30分钟。
上述步骤(2)中所述种子培养基配方为：1L水中加入酵母浸粉3g，蛋白胨5g，葡萄糖100g，115℃灭菌30分钟。
上述步骤(3)中所述发酵培养基配方为：1L水中加入葡萄糖300g，酵母粉5g，柠檬酸铵5g，磷酸二氢钾1g，硫酸镁0.5g，115℃灭菌30分钟。
(4)发酵过程葡萄糖和赤藓糖醇检测方法
将发酵液稀释并经0.22μm滤膜过滤后，采用HPLC的方式检测过滤液中葡萄糖和赤藓糖醇的含量。色谱条件如下：检测器，示差检测器；色谱柱，钙型强阳离子交换柱；柱温，80℃；流动相，重蒸水；流速，0.7mL/min；进样体积，10μL。
本发明具有的突出特点是：
(1)本发明成功通过采用随机基因突变结合高浓度铝盐抗性为初筛条件，获得低产酸，高转化的解脂耶氏酵母的突变株，具有筛选指向性强，菌种高产性能稳定的等特点。
(2)经过发酵条件的优化，菌种CGMCC No.25430的赤藓糖醇的发酵转化率可达到65％以上，比出发株提高约15％。
附图说明
图1随机挑选的解脂耶氏酵母变异株与耐10mM硫酸铝钾变异株的抗性筛选变异株的赤藓糖醇发酵情况比较。
图2解脂耶氏酵母铝盐抗性株的发酵终点pH及赤藓糖醇发酵的转化率
具体实施方式
下面通过实施例进一步阐明本发明，实施例将有助于理解本发明，但不仅限于此。
实施例1.解脂耶氏酵母铝盐抗性变异株的获得
(1)接种活化的解脂耶氏酵母到YPD培养基中，摇瓶培养24小时后，将酵母细胞用pH6.0的磷酸缓冲溶液洗涤3次，再用缓冲液调整OD600达到约为1.0后，用2％(v/v)的EMS处理30分钟，然后1000g离心10min除去上清液中的诱变剂以终止反应，沉淀酵母细胞用pH6.0的磷酸缓冲溶液洗涤3次，然后接种到100毫升的YPD培养基中摇瓶培养16小时，适当稀释后涂布到Al-YPDA固体培养基平板上，同时涂布YPDA作为对照，于30℃培养3天。
所述的YPD培养基配方为：酵母浸粉3.0g，蛋白胨5.0g，无水葡萄糖50g，加水到1.0升。
所述的Al-YPDA培养基配方为：酵母浸粉3.0g，蛋白胨5.0g，无水葡萄糖50g，硫酸铝钾5g，琼脂粉20g，加水到1.0升。
所述的EMS是指甲基磺酸乙酯。
所述的磷酸缓冲溶液是指用浓NaOH溶液调整50mM的磷酸溶液至pH6.0获得的溶液。
(2)将步骤(1)培养3天的Al-YPDA平板表面覆盖了浸透0.01％溴甲酚紫的绒布的圆形工具轻轻溻湿，5分钟后，挑选生长菌落周围无黄色透明圈且直径较大的酵母单菌落，转接到YPD液体培养基中进行扩培，并进行发酵试验，结果见附图1所示。可见具有铝盐抗性的变异株的解脂耶氏酵母具有更快的发酵速度和转化率。变异株中之一寄存于中国微生物菌种保藏管理委员会普通微生物中心，北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，编号为CGMCC No.25430。
实施例2.解脂耶氏酵母CGMCC No.25430的赤藓糖醇发酵
(1)为了验证解脂耶氏酵母变异株CGMCC No.25430的赤藓糖醇发酵性能，将该菌株与数十株实施例1(2)中获得的变异株及出发菌株在相同条件下进行摇瓶发酵，在500ml锥形瓶中装25毫升发酵培养基，培养基组成为：葡萄糖30％(w/v)，酵母粉1％(w/v)，磷酸氢二铵0.2％(w/v)，柠檬酸铵0.2％(w/v)，硫酸镁0.05％(w/v)，磷酸二氢钾0.1％(w/v)；接种量为10％(v/v)；摇瓶转速250rpm，在30±1℃下摇瓶发酵90h，得到赤藓糖醇发酵液。
(2)利用HPLC分析发酵液中赤藓糖醇的转化率，表明解脂耶氏酵母变异株CGMCCNo.25430的转化率达到65％以上，而出发对照株的转化率为50％，结果见附图2所示。
实施例3.解脂耶氏酵母CGMCC No.25430的发酵罐发酵验证
为了验证解脂耶氏酵母变异株CGMCC No.25430在赤藓糖醇发酵过程中的低产酸性能，进行了220m3发酵罐的发酵试验，结果表明CGMCC No.25430菌株的发酵产杂酸比对照菌显著降低，转化率提高非常显著。
解脂耶氏酵母CGMCC 25430与对照菌的发酵杂酸和pH比较
| 阴离子种类 | 单位 | 对照菌 | CGMCC 25430 |
| 乳酸 | mg/l | 22.584 | 20.606 |
| 乙酸 | mg/l | 18.72 | 11.923 |
| 丙酮酸 | mg/l | 89.996 | 46.046 |
| 氯离子 | mg/l | 112.202 | 105.289 |
| 硝酸根 | mg/l | 2.457 | 0.755 |
| 苹果酸 | mg/l | 433.981 | 130.037 |
| 硫酸根 | mg/l | 318.136 | 36.038 |
| 草酸 | mg/l | 16.49 | 6.46 |
| 富马酸 | mg/l | 6.895 | 16.054 |
| 磷酸 | mg/l | 61.125 | 57.517 |
| 柠檬酸 | mg/l | 2873.81 | 2559.213 |
| pH | / | 3.36 | 4.7 |
| 转化率 | ％ | 52.3 | 66.2 |
Claims (2)
1.一种低产酸解脂耶氏酵母(Yarrowia lipolytica)，其特征在于，该菌种寄存于中国微生物菌种保藏管理委员会普通微生物中心，北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，其保藏编号为CGMCC No.25430。
2.权利要求1所述低产酸解脂耶氏酵母(Yarrowia lipolytica)在发酵生产赤藓糖醇中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211046538.7A CN115418322B (zh) | 2022-08-30 | 2022-08-30 | 低产酸解脂耶氏酵母选育及在赤藓糖醇发酵中的应用方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211046538.7A CN115418322B (zh) | 2022-08-30 | 2022-08-30 | 低产酸解脂耶氏酵母选育及在赤藓糖醇发酵中的应用方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115418322A CN115418322A (zh) | 2022-12-02 |
| CN115418322B true CN115418322B (zh) | 2024-02-27 |
Family
ID=84200108
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211046538.7A Active CN115418322B (zh) | 2022-08-30 | 2022-08-30 | 低产酸解脂耶氏酵母选育及在赤藓糖醇发酵中的应用方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115418322B (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103374534A (zh) * | 2013-07-05 | 2013-10-30 | 上海交通大学 | 解脂亚罗酵母菌株及其用于合成赤藓糖醇的方法 |
| CN113980813A (zh) * | 2021-10-19 | 2022-01-28 | 中国食品发酵工业研究院有限公司 | 一种耐高温解脂亚罗酵母变异菌株的选育方法及其在赤藓糖醇发酵中的应用 |
| CN114456959A (zh) * | 2022-01-26 | 2022-05-10 | 山东星光首创生物科技有限公司 | 一种耐高渗的解脂亚罗酵母菌株、选育方法及应用 |
-
2022
- 2022-08-30 CN CN202211046538.7A patent/CN115418322B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103374534A (zh) * | 2013-07-05 | 2013-10-30 | 上海交通大学 | 解脂亚罗酵母菌株及其用于合成赤藓糖醇的方法 |
| CN113980813A (zh) * | 2021-10-19 | 2022-01-28 | 中国食品发酵工业研究院有限公司 | 一种耐高温解脂亚罗酵母变异菌株的选育方法及其在赤藓糖醇发酵中的应用 |
| CN114456959A (zh) * | 2022-01-26 | 2022-05-10 | 山东星光首创生物科技有限公司 | 一种耐高渗的解脂亚罗酵母菌株、选育方法及应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115418322A (zh) | 2022-12-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11279961B2 (en) | Aspergillus oryzae BLCY-006 strain and application thereof in preparation of galactooligosaccharide | |
| CN109207373B (zh) | 一株高产柠檬酸的微生物菌株及其发酵淀粉糖质生产柠檬酸的方法 | |
| CN111676166B (zh) | 一种乳酸菌及其在液态醋酿造中的应用 | |
| CN113980813A (zh) | 一种耐高温解脂亚罗酵母变异菌株的选育方法及其在赤藓糖醇发酵中的应用 | |
| CN107267422B (zh) | 睾丸酮丛毛单胞菌hhala-001及采用该菌株生产l-丙氨酸的方法 | |
| CN110564580B (zh) | 一种微生物共培养发酵生产含有吡咯喹啉醌食醋的方法 | |
| CN113321580B (zh) | 一种生产苹果酸的方法 | |
| CN107760623A (zh) | 一株产中性生淀粉酶的阿氏芽孢杆菌 | |
| CN111826308B (zh) | 一株海洋沉积物来源的几丁质高效降解菌及其应用 | |
| CN115418322B (zh) | 低产酸解脂耶氏酵母选育及在赤藓糖醇发酵中的应用方法 | |
| CN117143933A (zh) | 一种发酵生产色氨酸的方法 | |
| CN106282145B (zh) | 一种腺苷酸脱氨酶的液态发酵方法 | |
| CN109266578B (zh) | 大肠埃希氏菌ACThr1032及其在发酵生产L-苏氨酸中的应用 | |
| CN107058263A (zh) | 一种新型β‑淀粉酶的高效制备方法 | |
| CN104046572B (zh) | 一株能降低黄酒中生物胺的酿酒酵母及其构建方法与应用 | |
| WO2008092297A1 (fr) | Nouveau procédé de préparation d'acide abscissique naturel | |
| CN112746026B (zh) | 一株维斯假丝酵母及其应用 | |
| CN108587923B (zh) | 一种改善苹果酸发酵性能的方法 | |
| CN112592945A (zh) | 一种腺苷发酵工艺 | |
| CN105154360A (zh) | 一种睾丸酮丛毛单胞菌hy-08d的培养方法 | |
| CN106191157B (zh) | 一种低聚乳果糖的制备方法 | |
| CN104357347B (zh) | 一株氧化葡萄糖酸杆菌及其在发酵产vc前体中的应用 | |
| US7846699B2 (en) | Process for gibberellic acid production with “Fusarium moniliforme” strains | |
| CN106282146B (zh) | 一种腺苷酸脱氨酶的固态发酵方法 | |
| CN116286513B (zh) | 一株希氏乳杆菌FR-1012及其工业化生产γ-氨基丁酸的方法 |
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 |