CN113755420A - 一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用 - Google Patents
一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用 Download PDFInfo
- Publication number
- CN113755420A CN113755420A CN202111125496.1A CN202111125496A CN113755420A CN 113755420 A CN113755420 A CN 113755420A CN 202111125496 A CN202111125496 A CN 202111125496A CN 113755420 A CN113755420 A CN 113755420A
- Authority
- CN
- China
- Prior art keywords
- nox
- alss
- acetoin
- butanedione
- psip409
- 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.)
- Withdrawn
Links
- ROWKJAVDOGWPAT-UHFFFAOYSA-N Acetoin Chemical compound CC(O)C(C)=O ROWKJAVDOGWPAT-UHFFFAOYSA-N 0.000 title claims abstract description 66
- QSJXEFYPDANLFS-UHFFFAOYSA-N Diacetyl Chemical compound CC(=O)C(C)=O QSJXEFYPDANLFS-UHFFFAOYSA-N 0.000 title claims abstract description 64
- 235000013618 yogurt Nutrition 0.000 title claims abstract description 38
- 241000894006 Bacteria Species 0.000 title claims abstract description 34
- GFAZHVHNLUBROE-UHFFFAOYSA-N hydroxymethyl propionaldehyde Natural products CCC(=O)CO GFAZHVHNLUBROE-UHFFFAOYSA-N 0.000 title claims abstract description 33
- 238000010353 genetic engineering Methods 0.000 title abstract description 13
- 244000199866 Lactobacillus casei Species 0.000 claims abstract description 23
- 235000013958 Lactobacillus casei Nutrition 0.000 claims abstract description 23
- 229940017800 lactobacillus casei Drugs 0.000 claims abstract description 23
- 239000013612 plasmid Substances 0.000 claims abstract description 19
- 101150050280 alsD gene Proteins 0.000 claims abstract description 14
- 108010007843 NADH oxidase Proteins 0.000 claims abstract description 12
- WTLNOANVTIKPEE-UHFFFAOYSA-N 2-acetyloxypropanoic acid Chemical compound OC(=O)C(C)OC(C)=O WTLNOANVTIKPEE-UHFFFAOYSA-N 0.000 claims abstract description 11
- 101150081618 nox gene Proteins 0.000 claims description 11
- 101150037395 alsS gene Proteins 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 238000000855 fermentation Methods 0.000 claims description 3
- 230000004151 fermentation Effects 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 14
- 108090000790 Enzymes Proteins 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 14
- 108010084631 acetolactate decarboxylase Proteins 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 description 11
- 239000000796 flavoring agent Substances 0.000 description 10
- 230000015572 biosynthetic process Effects 0.000 description 9
- 235000019634 flavors Nutrition 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- 101100072559 Bacillus subtilis (strain 168) alsS gene Proteins 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000004186 co-expression Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 235000013305 food Nutrition 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 241000186660 Lactobacillus Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000012239 gene modification Methods 0.000 description 3
- 229940039696 lactobacillus Drugs 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- KJCVRFUGPWSIIH-UHFFFAOYSA-N 1-naphthol Chemical compound C1=CC=C2C(O)=CC=CC2=C1 KJCVRFUGPWSIIH-UHFFFAOYSA-N 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 2
- 241000194033 Enterococcus Species 0.000 description 2
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 2
- 241000194036 Lactococcus Species 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 230000005017 genetic modification Effects 0.000 description 2
- 235000013617 genetically modified food Nutrition 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- SJWFXCIHNDVPSH-UHFFFAOYSA-N octan-2-ol Chemical compound CCCCCCC(C)O SJWFXCIHNDVPSH-UHFFFAOYSA-N 0.000 description 2
- 229940093430 polyethylene glycol 1500 Drugs 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 description 1
- 241000186672 Lactobacillus delbrueckii subsp. bulgaricus Species 0.000 description 1
- 241000208822 Lactuca Species 0.000 description 1
- 241000194019 Streptococcus mutans Species 0.000 description 1
- 241000194020 Streptococcus thermophilus Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 230000009134 cell regulation Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 235000016213 coffee Nutrition 0.000 description 1
- 235000013353 coffee beverage Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229960003624 creatine Drugs 0.000 description 1
- 239000006046 creatine Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- VWWQXMAJTJZDQX-UYBVJOGSSA-N flavin adenine dinucleotide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1CO[P@](O)(=O)O[P@@](O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UYBVJOGSSA-N 0.000 description 1
- 235000019162 flavin adenine dinucleotide Nutrition 0.000 description 1
- 239000011714 flavin adenine dinucleotide Substances 0.000 description 1
- 229940093632 flavin-adenine dinucleotide Drugs 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000010855 food raising agent Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003988 headspace gas chromatography Methods 0.000 description 1
- 238000001319 headspace solid-phase micro-extraction Methods 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229940004208 lactobacillus bulgaricus Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229960002363 thiamine pyrophosphate Drugs 0.000 description 1
- 235000008170 thiamine pyrophosphate Nutrition 0.000 description 1
- 239000011678 thiamine pyrophosphate Substances 0.000 description 1
- YXVCLPJQTZXJLH-UHFFFAOYSA-N thiamine(1+) diphosphate chloride Chemical compound [Cl-].CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N YXVCLPJQTZXJLH-UHFFFAOYSA-N 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 235000014101 wine Nutrition 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
- C12Y106/03—Oxidoreductases acting on NADH or NADPH (1.6) with oxygen as acceptor (1.6.3)
- C12Y106/03001—NAD(P)H oxidase (1.6.3.1), i.e. NOX1
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
- C12Y202/01—Transketolases and transaldolases (2.2.1)
- C12Y202/01006—Acetolactate synthase (2.2.1.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01005—Acetolactate decarboxylase (4.1.1.5)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用。本发明的基因工程菌是一种包含pSIP409‑nox‑alsS质粒并在敲除α‑乙酰乳酸脱羧酶基因(alsD)的干酪乳杆菌上表达的干酪乳杆菌，所述的pSIP409‑nox‑alsS质粒能够表达NADH氧化酶和α‑乙酰乳酸合成酶，不能够表达α‑乙酰乳酸脱羧酶。本发明的基因工程菌较野生型干酪乳杆菌具有更高的酶活，且在发酵酸奶中的丁二酮和乙偶姻的产量分别是野生型菌株TCS的2.38倍、11.19倍，本发明为干酪乳杆菌生物合成丁二酮和乙偶姻提供了新的思路，为今后优化酸奶风味生产提供了理论依据，具有广阔的应用前景。
Description
技术领域
本发明涉及一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用，属于生物工程技术领域。
背景技术
丁二酮和乙偶姻是酸奶中公认的关键香气物质，它们为酸奶贡献了牛奶和黄油的味道，这对于酸奶的典型风味是必不可少的。丁二酮和乙偶姻常用作各种乳制品、咖啡、酒类等食品的增香剂，用于增加产品的风味，也用于各种香精配方中。目前丁二酮和乙偶姻多作为食品添加剂生产，在酸奶发酵过程中很少作为风味化合物生产。而目前有通过内源生成而非外源添加来增强食品风味的趋势，更加符合当代社会对绿色天然食品的要求。因此现采用微生物发酵法，利用内源增香方式通过基因工程的手段来促进丁二酮和乙偶姻的积累。
由于酸奶的风味主要是由乳酸菌代谢产生的，目前常用于食品的乳酸菌主要集中于双歧杆菌属(Bifidobacterium)、链球菌属(Streptococcus)、肠球菌属(Enterococcus)、乳杆菌属(Lactobacillus)、乳球菌属(Lactococcus)等，这些菌属在酸奶生产过程中已显示出具有生产丁二酮的潜力。在我们前期的研究中，我们鉴定了一株具有较高丁二酮生产能力的菌株-干酪乳杆菌TCS(Lacticaseibacillus casei TCS)(专利申请号：CN201810414559.7)。它可以作为亲本菌株进行遗传改造和辅助培养，以此来提高酸奶中的奶香味。
目前，对菌株的单基因遗传操作技术已经比较完善，但随着代谢工程研究的深入，经常需要同时在一个宿主菌中改造多个基因以实现对细胞的综合调节，从而提高目标代谢产物的产量。利用基因工程手段实现多个基因在同一个宿主菌中表达的技术称为共表达，其转化策略包括利用含单基因的载体进行多重转化，或利用含多基因载体一次共表达多个基因。这种基因工程的内源增香方式可以更好的提高代谢产物的含量，可以提高干酪乳杆菌在酸奶生产中丁二酮和乙偶姻的产量。
发明内容
本发明所要解决的技术问题是：针对目前传统发酵剂菌种嗜热链球菌、保加利亚乳杆菌发酵酸奶的丁二酮和乙偶姻的产量比较低，难以满足酸奶风味的需求，提供一种用于提高酸奶中丁二酮和乙偶姻产量的基因工程菌。
为了解决上述问题，本发明提供了一种提高酸奶中丁二酮和乙偶姻产量的基因工程菌，是一种将pSIP409-nox-alsS质粒导入敲除了alsD基因的干酪乳杆菌中所得的工程菌，所述的工程菌中包含pSIP409-nox-alsS质粒，不包含alsD基因，所述的pSIP409-nox-alsS质粒能够表达NADH氧化酶和α-乙酰乳酸合成酶。
优选地，所述的pSIP409-nox-alsS质粒包含串联表达的nox基因和alsS基因。
本发明还提供了一种细胞，为一种包含pSIP409-nox-alsS质粒的E.coli DH5α感受态细胞，所述的pSIP409-nox-alsS质粒能够表达NADH氧化酶和α-乙酰乳酸合成酶。
本发明还提供了一种细胞，为一种敲除alsD基因的干酪乳杆菌感受态细胞。
本发明还提供了上述的提高酸奶中丁二酮和乙偶姻产量的基因工程菌在酸奶和/或酸奶制品的发酵中的应用。
与现有技术相比，本发明的有益效果在于：
1.本发明主要是对NADH氧化酶基因(nox)、α-乙酰乳酸合成酶基因(alsS)和α-乙酰乳酸脱羧酶基因(alsD)三个基因进行研究，采用过表达和基因敲除策略，通过多个基因共表达的基因修饰手段来确定这些基因对丁二酮和乙偶姻产量的影响；本发明为干酪乳杆菌生物合成丁二酮和乙偶姻提供了新的思路，为今后优化酸奶风味生产提供了理论依据；
2.本发明的基因工程菌较野生型干酪乳杆菌TCS具有更高的酶活(NADH氧化酶和α-乙酰乳酸合成酶酶活)，本发明的基因工程菌在发酵酸奶中的丁二酮和乙偶姻的产量分别是野生型菌株TCS的2.38倍、11.19倍。
附图说明
图1为丁二酮和乙偶姻的生物合成途径(a)和工程菌的重组质粒图谱(b)；
图2为重组质粒pSIP409-nox-alsS导入敲除alsD基因的干酪乳杆菌验证产物，其中图中的M表示DNA Marker；
图3为野生型菌和工程菌发酵酸奶中丁二酮和乙偶姻的含量。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下。
实施例中所用的干酪乳杆菌来自实验室前期鉴定的一株具有较高丁二酮生产能力的菌株-干酪乳杆菌TCS(Lacticaseibacillus casei TCS)，其公开在申请号为CN201810414559.7的专利中。
实施例1
产丁二酮和乙偶姻工程菌的构建：
1.表达载体pSIP409-nox-alsS的构建
根据变异链球菌Streptococcus mutans的nox基因序列和干酪乳杆菌TCS全基因组中的alsS基因序列，为了在单一载体中共表达nox和alsS基因，在alsS基因的5'端增加一个核糖体结合位点，利用Premier 5.0和Vector NTI软件设计引物nox-F、nox-R和alsS-F、alsS-R，并利用一步法克隆试剂盒将nox和alsS片段连接到载体pSIP409上。其中在引物nox-F的5'端引入NcoI酶切位点，在引物alsS-R的5’端引入XhoI酶切位点。PCR产物经纯化后与同样酶切位点双酶切的表达载体pSIP409连接，转化E.coli DH5α感受态细胞(VazymeBiotech Co.,Ltd.,Nanjing,China)，挑取转化子，经酶切验证获得pSIP409-nox-alsS，其序列如SEQ ID NO：1所示。
2.干酪乳杆菌感受态细胞的制备
将利用CRISPR基因编辑系统构建的敲除α-乙酰乳酸脱羧酶基因(alsD)的干酪乳杆菌进行活化(具体构建方法和活化方法参照论文“干酪乳杆菌中双乙酰的合成机制初步解析及应用，刘晗”中的方法)，将活化好的干酪乳杆菌菌液按合适比例接种到100mL的MRS液体培养基中，于37℃下静置培养至OD600值为0.5-0.8左右；将培养液在冰上冰浴20min，于4℃，4,000g，离心5min收集菌体，再用预冷的电击缓冲液聚乙二醇1500缓慢吹打菌体，冰浴10min，同等条件下离心收集菌体；重复该步骤4-5次；将菌体小心悬浮在1/100体积的聚乙二醇1500缓冲液中，于-80℃保存备用。
3.构建在敲除α-乙酰乳酸脱羧酶基因(alsD)的干酪乳杆菌上共表达nox和alsS基因的工程菌并鉴定
将带有nox和alsS基因的表达质粒pSIP409-nox-alsS电转化到敲除alsD基因的干酪乳杆菌感受态细胞上，涂布在带有红霉素抗性的MRS平板上，于37℃倒置培养48-72h，挑取单菌落进行菌落PCR和质粒提取验证，分别用引物403-F、403-R和nox-R、alsS-F通过PCR验证重组质粒pSIP409-nox-alsS是否成功导入干酪乳杆菌中。重组菌株经测序验证，如图2的泳道得出一个大小2-3kb的条带，符合nox片段(1374bp)和alsS片段(1524bp)之和，成功构建出将nox和alsS基因串联表达在敲除alsD基因的干酪乳杆菌并命名重组工程菌为TCS-ΔalsD-nox-alsS，该工程菌的重组质粒图谱如图1b所示。
以上所涉及的引物序列如表1所示。
表1引物序列
实施例2
利用野生型菌TCS和工程菌TCS-ΔalsD-nox-alsS发酵酸奶：
将待用的TCS、TCS-ΔalsD-nox-alsS菌株分别接种于MRS肉汤中，37℃常规培养12～16h，于室温下6000×g离心10min收集菌体，无菌水洗涤2次后用等体积的无菌水重悬，悬浮液被用作发酵剂待用。用12％(w/v)脱脂奶粉制备酸奶，95℃灭菌10min，待灭菌脱脂乳冷却至40℃左右，将上述得到的菌悬液按照4％(v/v)的比例接种于灭菌脱脂乳中，并对诱导型重组菌TCS-ΔalsD-nox-alsS中加入终浓度为25ng/mL的IP-673进行诱导。之后在37℃下静置发酵至pH约为4.6，再置于4℃冰箱后熟12-24h，即得到酸奶样品。
实施例3
工程菌的酶活测定：
对野生菌株TCS和工程菌TCS-ΔalsD-nox-alsS菌株进行酶活测定，分别测定其中的NADH氧化酶和α-乙酰乳酸合成酶酶活含量。
(1)NADH氧化酶酶活测定：采用NADH氧化酶活性检测试剂盒(Solarbio Science&Technology，北京，中国)测定NOX活性。酶活力单位(U)定义为反应体系中每1万个细菌在反应体系中每分钟A600变化0.01定义为一个酶活力单位。
(2)α-乙酰乳酸合成酶酶活测定：ALS活性是根据Holtzclaw(Holtzclaw和Chapman1975)描述的方法测定的，并作了一些修改。在1mM焦磷酸硫胺、5mM MgCl2、0.1mM黄素腺嘌呤二核苷酸、100mM丙酮酸钠和300μL粗酶液中进行测定。反应在37℃下进行1h，之后立即加入200μL的3M H2SO4终止反应。60℃水浴脱羧15min后，加入8.5mL显色液(5％α-萘酚、0.5％肌酸、5％氢氧化钠、去离子水)。该混合液在37℃下孵育30min，使乙酰乳酸酸水解为乙偶姻，最后用分光光度计在520nm处测定吸光度。酶活单位定义为37℃下，在pH为7.0的反应条件下，每分钟合成1nmol乙酰乳酸所用的酶量。
表2 NADH氧化酶和α-乙酰乳酸合成酶酶活
由表2可知，经过基因工程共表达改造后的工程菌，NADH氧化酶和α-乙酰乳酸合成酶的酶活能力都比野生型菌株TCS有所提高。由此说明，基因工程的共表达改造可以有效提升丁二酮和乙偶姻合成途径中关键酶的能力，从而进一步可以提升丁二酮和乙偶姻的生产能力。
实施例4
酸奶中丁二酮和乙偶姻的检测：
利用顶空固相微萃取并借助气相色谱测定酸奶中丁二酮和乙偶姻的含量：
称取5.00g后熟24h的酸奶样品于加有转子的15mL萃取瓶中，并加入10μL 2-辛醇(内标，220mg/L)，于55℃的磁力恒温水浴锅中平衡5min，然后插入萃取头富集顶空中的挥发性物质40min；色谱条件如下：HP-INNOWAX色谱柱(60m×0.25mm×0.25mm)；进样口温度：230℃；升温程序：40℃恒温保持3min，再以4℃/min速率升温至120℃，最后以5℃/min速率升温至230℃，并保持10min；载气：氦气；流速：1mL/min；进样方式：采用不分流进样。
测试结果表明本发明构建的工程菌TCS-ΔalsD-nox-alsS发酵酸奶中丁二酮的产量达到7.28μg/kg，乙偶姻产量达到92.39μg/kg。而野生型干酪乳杆菌TCS发酵酸奶中丁二酮产量只有3.06μg/kg，乙偶姻产量为8.26μg/kg。由此对比发现，工程菌TCS-ΔalsD-nox-alsS发酵酸奶中丁二酮和乙偶姻的产量分别是野生型菌株TCS的2.38倍、11.19倍。由此可以看出当nox、alsS和alsD这3个基因共同修饰时，多种遗传修饰对风味增强具有协同作用，对酸奶中丁二酮和乙偶姻的产量提升有明显的影响。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 上海应用技术大学
<120> 一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 8790
<212> DNA
<213> 人工合成
<400> 1
caaggagatt ttaggaattc ggtaccccgg gttcgaaggc gccaagcttc aaattacagc 60
acgtgttgct ttgattgata gccaaaaagc agcagttgat aaagcaatta ctgatattgc 120
tgaaaaattg taatttataa ataaaaatca ccttttagag gtggtttttt tatttataaa 180
ttattcgttt gatttcgctt tcgatagaac aatcaaagcg agaataagga agataaatcc 240
cataagggcg ggagcagaat gtccgagact aattcatgag atcgattttt tattaaaacg 300
tctcaaaatc gtttctgaga cgttttagcg tttatttcgt ttagttatcg gcataatcgt 360
taaaacaggc gttatcgtag cgtaaaagcc cttgagcgta gcgtgctttg cagcgaagat 420
gttgtctgtt agattatgaa agccgatgac tgaatgaaat aataagcgca gcgtccttct 480
atttcggttg gaggaggctc aagggagttt gagggaatga aattccctca tgggtttgat 540
tttaaaaatt gcttgcaatt ttgccgagcg gtagcgctgg aaaaattttt gaaaaaaatt 600
tggaatttgg aaaaaaatgg ggggaaagga agcgaatttt gcttccgtac tacgaccccc 660
cattaagtgc cgagtgccaa tttttgtgcc aaaaacgctc tatcccaact ggctcaaggg 720
tttgaggggt ttttcaatcg ccaacgaatc gccaacgttt tcgccaacgt tttttataaa 780
tctatattta agtagcttta ttgttgtttt tatgattaca aagtgataca ctaattttat 840
aaaattattt gattggagtt ttttaaatgg tgatttcaga atcgaaaaaa agagttatga 900
tttctctgac aaaagagcaa gataaaaaat taacagatat ggcgaaacaa aaaggttttt 960
caaaatctgc ggttgcggcg ttagctatag aagaatatgc aagaaaggaa tcagaataaa 1020
aaaaataagc gaaagctcgc gtttttagaa ggatacgagt tttcgctact tgtttttgat 1080
aaggtaatat atcatggcta ttaaatacta aagctagaaa ttttggattt ttattatatc 1140
ctgactcaat tcctaatgat tggaaagaaa aattagagag tttgggcgta tctatggctg 1200
tcagtccttt acacgatatg gacgaaaaaa aagataaaga tacatggaat agtagtgatg 1260
ttatacgaaa tggaaagcac tataaaaaac cacactatca cgttatatat attgcacgaa 1320
atcctgtaac aatagaaagc gttaggaaca agattaagcg aaaattgggg aatagttcag 1380
ttgctcatgt tgagatactt gattatatca aaggttcata tgaatatttg actcatgaat 1440
caaaggacgc tattgctaag aataaacata tatacgacaa aaaagatatt ttgaacatta 1500
atgattttga tattgaccgc tatataacac ttgatgaaag ccaaaaaaga gaattgaaga 1560
atttactttt agatatagtg gatgactata atttggtaaa tacaaaagat ttaatggctt 1620
ttattcgcct taggggagcg gagtttggaa ttttaaatac gaatgatgta aaagatattg 1680
tttcaacaaa ctctagcgcc tttagattat ggtttgaggg caattatcag tgtggatata 1740
gagcaagtta tgcaaaggtt cttgatgctg aaacggggga aataaaatga caaacaaaga 1800
aaaagagtta tttgctgaaa atgaggaatt aaaaaaagaa attaaggact taaaagagcg 1860
tattgaaaga tacagagaaa tggaagttga attaagtaca acaatagatt tattgagagg 1920
agggattatt gaataaataa aagcccccct gacgaaagtc gaagggggct tttattttgg 1980
tttgatgttg cgattaatag caatacgatt gcaataaaca aaatgatccc cttagaagca 2040
aacttaagag tgtgttgata gtgcattatc ttaaaatttt gtataatagg aattgaagtt 2100
aaattagatg ctaaaaatag gaattgaagt taaattagat gctaaaaatt tgtaattaag 2160
aaggagggat tcgtcatgtt ggtattccaa atgcgtaatg tagataaaac atctactgtt 2220
ttgaaacaga ctaaaaacag tgattacgca gataaataaa tacgttagat taattcctac 2280
cagtgactaa tcttatgact ttttaaacag ataactaaaa ttacaaacaa atcgtttaac 2340
ttcaggagag attacatgaa caaaaatata aatatctcaa actttttaac gagtgaaaaa 2400
gtactcaacc aaataataaa acaattgaat ttaaaagaaa ccgataccgt ttacgaaatt 2460
ggaacaggta aagggcattt aacgacgaaa ctggctaaaa taagtaaaca ggtaacgtct 2520
attgaattag acagtcatct attcaactta tcgtcagaaa aattaaaact gaatactcgt 2580
gtcactttaa ttcaccaaga tattctacag tttcaattcc ctaacaaaca gaggtataaa 2640
attgttggga atattcctta caatttaagc acacaaatta ttaaaaaagt ggtttttgaa 2700
agccgtgcgt ctgacatcta tctgactgtt gaagaaggat tctacaagcg taccttggat 2760
attcaccgaa cactagggtt gctcttgcac actcaagtct cgattcagca attgcttaag 2820
ctgccagcgg aatgctttca tcctaaacca aaagtaaaca gtgtcttaat aaaacttacc 2880
cgccatacca cagatgttcc agataaatat tggaagctat ataagtactt tgtttcaaaa 2940
tgggtcaatc gagaatatcg tcaactgttt actaaaaatc agtttcgtca agcaatgaaa 3000
cacgccaaag taaacaattt aagtaccatt acttatgagc aagtattgtc tatttttaat 3060
agttatctat tatttaacgg gaggaaataa ttctatgagt cgctttttta aatttggaaa 3120
gttacacgtt actaaaggga atggagaccg gggtcgaccc ttcaatagag ttcttaacgt 3180
taatccgaaa aaaactaacg ttaatattaa aaaataagat ccgcttgtga attatgtata 3240
atttgattag actaaagaat aggagaaagt atgatgatat ttaaaaaact ttctcgttaa 3300
gataggttgt tggtgagcat gttatatacg gatgtatcgg tttccttaat gcaaaatttt 3360
gttgctatct tattaatttt tctattatat agatatattc aaagaaagat aacatttaaa 3420
cggatcatat tagatatttt aatagcgatt attttttcaa tattatatct gtttatttca 3480
gatgcgtcat tacttgtaat ggtattaatg cgattagggt ggcattttca tcaacaaaaa 3540
gaaaataaga taaaaacgac tgatacagct aatttaattc taattatcgt gatccagtta 3600
ttgttagttg cggttgggac tattattagt cagtttacca tatcgattat caaaagtgat 3660
ttcagccaaa atatattgaa caatagtgca acagatataa ctttattagg tattttcttt 3720
gctgttttat ttgacggctt gttctttata ttattgaaga ataagcggac tgaattacaa 3780
catttaaatc aagaaatcat tgaattttcg ttagaaaaac aatattttat atttatattt 3840
attttattta tagtaataga aattatttta gcagttggga atcttcaagg agtaacagcc 3900
acgatattat taaccattat cattattttt tgtgtcctta tcgggatgac tttttggcaa 3960
gtgatgcttt ttttgaaggc ttattcgatt cgccaagaag ccaatgacca attggtccgg 4020
aatcaacaac ttcaagatta tctagtcaat atcgaacagc agtacaccga attacggcga 4080
tttaagcatg attatcaaaa catcttatta tcgttggaga gttttgccga aaagggcgat 4140
cagcaacagt ttaaggcgta ttaccaagaa ttattagcac aacggccaat tcaaagtgaa 4200
atccaagggg cagtcattgc acaactcgac tacttgaaaa atgatcctat tcgaggatta 4260
gtcattcaaa agtttttggc agccaaacag gctggtgtta ctttaaaatt cgaaatgacc 4320
gaaccaatcg aattagcaac cgctaatcta ttaacggtta ttcggattat cggtatttta 4380
ttagacaatg cgattgaaca agccgttcaa gaaaccgatc aattggtgag ttgtgctttc 4440
ttacaatctg atggtttaat cgaaattacg attgaaaata cggccagtca agttaagaat 4500
ctccaagcat tttcagagtt aggctattca acgaaaggcg ctggtcgggg gactggttta 4560
gctaatgtgc aggatttgat tgccaaacaa accaatttat tcttagaaac acagattgaa 4620
aatagaaagt tacgacagac attgatgatt acggaggaaa cttaatttgt atcccgttta 4680
tttattagag gatgatttac agcaacaagc gatttatcag caaattatcg cgaatacgat 4740
tatgattaac gaatttgcaa tgactttaac atgcgctgcc agtgatactg agacattgtt 4800
ggcggcaatt aaggatcagc aacgaggttt attctttttg gatatggaaa ttgaggataa 4860
ccgccaagcc ggtttagaag tggcaactaa gattcggcag atgatgccgt ttgcgcaaat 4920
tgtcttcatt acaacccacg aggaactgac attattaacg ttagaacgaa aaatagcgcc 4980
tttagattac attctcaagg accaaacaat ggctgaaatc aaaaggcaat tgattgatga 5040
tctattgtta gctgagaagc aaaacgaggc ggcagcgtat caccgagaaa atttatttag 5100
ttataaaata ggtcctcgct ttttctcatt accattaaag gaagttgttt atttatatac 5160
tgaaaaagaa aatccgggtc atattaattt gttagccgtt accagaaagg ttacttttcc 5220
aggaaattta aatgcgctgg aagcccaata tccaatgctc tttcggtgtg ataaaagtta 5280
cttagttaac ctatctaata ttgccaatta tgacagtaaa acacggagtt taaaatttgt 5340
agatggcagt gaggcaaaag tctcgttccg gaaatcacgg gaactagtgg ccaaattaaa 5400
acaaatgatg tagcgcctgc aggcacgcca aatgatccca gtaaaaagcc acccgcatgg 5460
cgggtggctt tttattagcc ctagaagggc ttcccacacg catttcagcg ccttagtgcc 5520
ttagtttgtg aatcataggt ggtatagtcc cgaaataccc gtctaaggaa ttgtcagata 5580
ggcctaatga ctggctttta taatatgaga taatgccgac tgtacttttt acagtcggtt 5640
ttctaatgtc actaacctgc cccgttagtt gaagaaggtt tttatattac agctccagat 5700
ctaccggttt aatttgaaaa ttgatattag cgtttaacag ttaaattaat acgttaataa 5760
tttttttgtc tttaaatagg gatttgaagc ataatggtgt tatagcgtac ttagctggcc 5820
agcatatatg tattctataa aatactatta caaggagatt ttagccatgg ctatgagtaa 5880
aatcgttatt gttggagcta accatgcagg tacagctgcc attaatacta ttctagataa 5940
ttacggtagt gaaaacgaag ttgtcgtttt tgaccaaaat tctaatattt cattcttggg 6000
ttgtggaatg gcactttgga ttggaaaaca aatatcaggc cctcaaggtc ttttttatgc 6060
tgacaaggaa tcgttagaag caaaaggtgc taaaatttat atggaatcgc cagtgacagc 6120
cattgattat gatgctaaga gggttactgc tttggtcaat ggtcaagaac atgttgaaag 6180
ctatgagaag cttattttgg caacaggatc aacaccaatc ttaccaccta tcaaaggtgc 6240
agctatcaaa gaaggtagtc gtgattttga agcaactttg aaaaatcttc aatttgttaa 6300
attgtatcaa aatgcagaag atgttattaa taaattacag gataagagtc aaaatctgaa 6360
tcgtattgct gttgttggtg ctggttatat tggtgtagaa cttgctgaag cctttaaacg 6420
cctcggaaaa gaagtgattc ttattgatgt tgttgacact tgcttagctg gttattatga 6480
tcaggatctt tcagaaatga tgcgtcaaaa tttggaagat catggtattg aattagcatt 6540
cggagaaact gtcaaagcca ttgaaggtga tggtaaagtc gaacgtattg taactgataa 6600
agcgagccat gatgtggata tggttatttt agctgtcggt ttccgtccta atactgcact 6660
tggcaacgct aaactcaaaa ccttccgtaa tggtgctttc cttgttgata aaaaacaaga 6720
gacaagtatt cctgacgttt atgccatcgg cgattgcgcg actgtttatg acaacgctat 6780
taatgatacc aattatattg ccttagcttc aaacgctctt cgctcaggta ttgtagctgg 6840
tcataatgca gcagggcata aattggaatc tcttggtgtt caaggttcaa atggtatttc 6900
aatttttggt ctcaatatgg tttcaactgg gttaacgcaa gaaaaagcaa agcgttttgg 6960
ctataatcca gaagtcactg catttacaga ttttcagaag gctagtttta ttgaacatga 7020
taattatcct gttacactta aaattgtcta tgataaggat aaccgactgg ttcttggtgc 7080
acaaatggca tctaaagaag atatgtcaat gggaattcat atgttttcat tggctattca 7140
ggaaaaagtt accattgaac gtttagctct actggactat ttctttcttc ctcatttcaa 7200
tcaaccctat aattatatga ccaaagcagc attaaaagct aaatgacaag gagattttag 7260
atggcagaag agaagcgcta tggcgcggat ttgatcgtcg aaagtttaac gaatcacggc 7320
attgactatg tttttgggat tcctggagcc aaaattgacc gggtttttga aagtctggag 7380
catcctaaat cagaaaagag cccgcagcta atcgttgcgc gacatgaaca aaatgcggcg 7440
tttatggccg ctggcatcgg tcgtttgacc ggcaaaccag gggttgtcct gacaacttca 7500
ggaccgggag catcaaactt ggcaaccggc ttggtaaccg caacggctga aggtgatccg 7560
gttttggctt tatccggtca ggtcaaacgc gctgatttgc tgcgaagttc tcatcaaagc 7620
atgcgtaacg ccgatctttt tgccccgatc accaaatatg cagctgaagt tcaggaccct 7680
gataatgtga gtgaaataat cgcgaatgct tatcaagcgg ctgaatctgg caagcaaggc 7740
gcaagttttg tttctatccc gcaagatgtc acagattcgc cagtcaactc agaaccgata 7800
aaaccactgg tcgcaccaaa acttggaccg gctagtccca gtgatatgac gtatctggcg 7860
cacgccatca aggaagcgtc attgccagtg ttgttgttag gaatgcgggc atcgtcaagt 7920
gatgtaacag cagagattcg cgaattactt tcagtaacgg aactacctgt tgtggaaact 7980
ttccaaggtg ctggcatcat ttctcaccgt cagattgaca acttcttcgg ccgggttggc 8040
ctgttccgca atcaaccagg tgacatgttg ttacaacata gtgatttggt cattgctatt 8100
gggtacgatc ccgttgaata tgaaccacgt aactggaatg cggatggcaa ggcgcgcatc 8160
attgtcattg atgatgttcc agctgagatt gatcataact ttcagccgga aactgaactc 8220
attggtgata tttcgcaaac gttagatatt ttggtgccat tgttacgtgg ctaccaagtt 8280
gcacctggca gcaagcgtta tttagaagac cttcaggcaa aactgcagga tagcgatgta 8340
ccgccggcaa ttgctgatca aaaagtgctt catccgctca gcattgttgc tgccttacag 8400
gagcgggtca cggatgaaat gaccgttgct gttgacgttg ggagtcatta tatttggatg 8460
gcccggcact tcagaagtta tgaaccacga catttgctgt tttccaatgg gatgcaaacg 8520
ctcggtgtcg ccttaccatg ggcgattgct gcaacgttgg ttcgcccagg gaaaaaggcg 8580
gtctcagttt ctggcgatgg cggttttctc ttttccggac aggaattaga aacagcagtg 8640
cgcctgcatg cagacttagt gcatattatt tggaatgatg gccattatga tatggtaaaa 8700
tttcaagagg aaatgaagta cggtcgtgca gctggcgttg actttggtcc ggttgacttt 8760
gtgaagtatg cagaagcatt ttgactcgag 8790
<210> 2
<211> 49
<212> DNA
<213> 人工合成
<400> 2
acaaggagat tttagccatg gctatgagta aaatcgttat tgttggagc 49
<210> 3
<211> 25
<212> DNA
<213> 人工合成
<400> 3
ccatctaaaa tctccttgtc attta 25
<210> 4
<211> 38
<212> DNA
<213> 人工合成
<400> 4
gacaaggaga ttttagatgg cagaagagaa gcgctatg 38
<210> 5
<211> 46
<212> DNA
<213> 人工合成
<400> 5
cggggtaccg aattcctcga gtcaaaatgc ttctgcatac ttcaca 46
<210> 6
<211> 22
<212> DNA
<213> 人工合成
<400> 6
gaaatacccg tctaaggaat tg 22
<210> 7
<211> 21
<212> DNA
<213> 人工合成
<400> 7
tggtcatgaa ttagtctcgg a 21
Claims (5)
1.一种提高酸奶中丁二酮和乙偶姻产量的基因工程菌，其特征在于，是一种将pSIP409-nox-alsS质粒导入敲除了alsD基因的干酪乳杆菌中所得的工程菌，所述的工程菌中包含pSIP409-nox-alsS质粒，不包含alsD基因，所述的pSIP409-nox-alsS质粒能够表达NADH氧化酶和α-乙酰乳酸合成酶。
2.如权利要求1所述的一种提高酸奶中丁二酮和乙偶姻产量的基因工程菌，其特征在于，所述的pSIP409-nox-alsS质粒包含串联表达的nox基因和alsS基因。
3.一种细胞，其特征在于，为一种包含pSIP409-nox-alsS质粒的E.coli DH5α感受态细胞，所述的pSIP409-nox-alsS质粒能够表达NADH氧化酶和α-乙酰乳酸合成酶。
4.一种细胞，其特征在于，为一种敲除alsD基因的干酪乳杆菌感受态细胞。
5.权利要求1或2所述的提高酸奶中丁二酮和乙偶姻产量的基因工程菌在酸奶和/或酸奶制品的发酵中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111125496.1A CN113755420A (zh) | 2021-09-24 | 2021-09-24 | 一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111125496.1A CN113755420A (zh) | 2021-09-24 | 2021-09-24 | 一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113755420A true CN113755420A (zh) | 2021-12-07 |
Family
ID=78797412
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111125496.1A Withdrawn CN113755420A (zh) | 2021-09-24 | 2021-09-24 | 一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113755420A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117491526A (zh) * | 2023-11-08 | 2024-02-02 | 山东哲成生物科技有限公司 | 一种果蔬汁乳酸发酵进程的监控方法 |
-
2021
- 2021-09-24 CN CN202111125496.1A patent/CN113755420A/zh not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117491526A (zh) * | 2023-11-08 | 2024-02-02 | 山东哲成生物科技有限公司 | 一种果蔬汁乳酸发酵进程的监控方法 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110241061B (zh) | 提高短乳杆菌γ-氨基丁酸合成能力的方法及其应用 | |
| CN112813085B (zh) | 焦磷酸酶基因的用途 | |
| EP0985043B2 (en) | Lactic acid bacterial starter cultures and compositions thereof | |
| CN108220219B (zh) | 一套植物乳杆菌食品级表达系统及其在异源蛋白表达中的应用 | |
| CN111394288A (zh) | 重组谷氨酸棒杆菌、其构建方法及其生产四氢嘧啶的方法 | |
| CN117645967A (zh) | 一种适用于高密度发酵产酶的枯草芽孢杆菌底盘细胞 | |
| EP1071760A1 (en) | Genetically modified lactic acid bacteria having modified diacetyl reductase activities | |
| JP2000342289A (ja) | ペニシリン生合成遺伝子の単離法 | |
| CN108368523A (zh) | 在代谢工程化的乳酸菌中高水平生产双乙酰 | |
| CN113755420A (zh) | 一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用 | |
| CN111621454A (zh) | 基因工程高产菌株淀粉酶产色链霉菌及ε-聚赖氨酸的生产方法和应用 | |
| CN114874959A (zh) | 一种利用葡萄糖从头发酵生产l-茶氨酸的基因工程菌、方法及应用 | |
| FI111552B (fi) | Menetelmä rekombinanttisen isäntäsolukannan tuottamiseksi ja näin saatu rekombinanttinen isäntäsolu | |
| CN112029701B (zh) | 一种基因工程菌及其在制备22-羟基-23，24-双降胆甾-4-烯-3-酮中的应用 | |
| CN106754555A (zh) | 一株产甘露醇的肠膜明串珠菌突变菌株及其应用方法 | |
| CN112410353B (zh) | 一种fkbS基因、含其的基因工程菌及其制备方法和用途 | |
| CN113462628B (zh) | 一株产血红素的基因工程菌及其构建方法和应用 | |
| EP1642977B1 (en) | Gene involved in growth-promoting function of acetic acid bacteria and uses thereof | |
| CN112410274B (zh) | 一种生产子囊霉素的基因工程菌及其制备方法和用途 | |
| CN114369582B (zh) | 双向伯克霍尔德氏菌来源酯合成酶jg536_25355、编码基因及应用 | |
| CN114107342B (zh) | 一种去除发酵液中乳糖的方法 | |
| CN115125180B (zh) | 一种利用双途径产乙偶姻的重组运动发酵单胞菌及其构建方法与应用 | |
| CN117946984B (zh) | 泛酸合成酶突变体及其制备方法、构建体及其构建方法、泛酸生产菌株和应用及泛酸的制备方法 | |
| CN113789314B (zh) | 一种核糖体σ因子B及其突变体在提升利普司他汀产量中的应用 | |
| CN113980982B (zh) | 增强安丝菌素体内靶标蛋白基因表达的高产安丝菌素方法 |
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 | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20211207 |