WO2023098774A1 - 改善食醋风味的醋酸菌及其构建方法 - Google Patents

改善食醋风味的醋酸菌及其构建方法 Download PDF

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WO2023098774A1
WO2023098774A1 PCT/CN2022/135751 CN2022135751W WO2023098774A1 WO 2023098774 A1 WO2023098774 A1 WO 2023098774A1 CN 2022135751 W CN2022135751 W CN 2022135751W WO 2023098774 A1 WO2023098774 A1 WO 2023098774A1
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pacn
peut
acetic acid
sequence
aal
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French (fr)
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郑宇�
王敏
王君
宋佳
夏梦雷
王馨宇
赵航
冯智伟
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天津科技大学
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    • 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
    • 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
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01001—Aspartate ammonia-lyase (4.3.1.1), i.e. aspartase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01007—Ethanolamine ammonia-lyase (4.3.1.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01025—Phenylalanine-tyrosine ammonia-lyase (4.3.1.25)
    • 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

Definitions

  • the invention belongs to the field of bioengineering, and in particular relates to a construction method of acetic acid bacteria for improving the flavor of vinegar and its application.
  • Vinegar the main component of which is acetic acid
  • Acetic acid bacteria are a class of strictly aerobic Gram-negative bacteria, which are chemoheterotrophic.
  • the domestic vinegar industry has developed rapidly, and the acid resistance of bacteria has always been the key factor restricting the production of high-acidity vinegar.
  • the object of the present invention is to provide an acetic acid bacterium for improving the flavor of vinegar and a construction method thereof.
  • Scheme one a kind of acetic acid bacteria that improves the flavor of vinegar with recombinant tyrosine ammonia lyase, with Acetobacter pasteurianus (Acetobacter pasteurianus) as host cell, ethanol aminolysis ammonia with synergistic effect in Acetobacter pasteurianus (Acetobacter pasteurianus)
  • the promoter of the enzyme co-expresses the tyrosine ammonia lyase; the sequence number of the promoter is SEQ ID No.1, and the sequence number of the tyrosine ammonia lyase is SEQ ID No.2.
  • Acetobacter pasteurianus (Acetobacter pasteurianus) as host cell, the promoter of aconitase in the tricarboxylic acid cycle of Acetobacter pasteurianus (Acetobacter pasteurianus) co-expresses aspartate ammonia-lyase to obtain;
  • the sequence number of the enzyme is SEQ ID No.3
  • the sequence number of the aspartate ammonia lyase is SEQ ID No.4.
  • tyrosine ammonia lyase When tyrosine ammonia lyase is used to recombine, tyrosine is converted into p-coumaric acid, which belongs to phenolic compounds, which can promote the improvement of vinegar flavor and efficacy; When ammoniaase is recombined, aspartic acid is converted into fumaric acid, and fumaric acid participates in the TCA cycle, which can enhance energy metabolism to produce ATP and reduce the damage caused by acid to cells.
  • the present invention also includes a method for constructing the acetic acid bacteria for improving the flavor of vinegar.
  • the first scheme includes the steps of: using PCR to obtain the promoter of ethanolamine ammonia-lyase from Acetobacter pasteurianus, and It was connected to the plasmid pBBR1p452 which could be stably replicated in acetic acid bacteria (Verena Kallnik, Maria Meyer, Uwe Deppenmeier, et al. Construction of expression vectors for protein production in Gluconobacter oxydans.
  • 3Plasmid pBBR1p452 and ethanolamine ammonia-lyase promoter sequence Peut were treated with restriction endonuclease at 37°C for 1-2h, purified and recovered, and mixed according to the ratio of plasmid and fragment material 1:0.2-5, using T 4 DNA Ligase 16-22°C for 2-12h ligation reaction, and then transfer the ligated product into Escherichia coli DH5 ⁇ competent to obtain the recombinant plasmid pBBR1-Peut, and use the gene sequencing reaction to verify the sequence of the recombinant plasmid pBBR1-Peut, as shown in Table 2 Composition of pBBR1-Peut enzyme digestion system.
  • the tyrosine ammonia lyase gene tal (GenBank: KF765779.1) derived from Rhodotorula glutinis was obtained by gene synthesis, and linked to a plasmid that can stably replicate in acetic acid bacteria In pBBR1-Peut, the recombinant plasmid pBBR1-Peut-tal was obtained;
  • the present invention also includes another method for constructing acetic acid bacteria that improves the flavor of vinegar, comprising the steps of: using PCR to obtain the promoter of aconitase derived from Acetobacter pasteurianus, and linking it Into the plasmid pBBR1p452 that can be stably replicated in acetic acid bacteria, obtain the recombinant plasmid pBBR1-Pacn; use the PCR method to obtain the aspartate ammonia-lyase gene aal from Escherichia coli, and connect it to the plasmid pBBR1 On -Pacn, obtain the recombinant plasmid pBBR1-Pacn-aal; transfer the recombinant plasmid pBBR1-Pacn-aal into acetic acid bacteria to obtain the improvement of the recombinant expression of aspartate ammonia-lyase controlled by the aconitase
  • 2Acetobacter pasteurianus (Acetobacter pasteurianus) genome was used as a template, using primers Pacn-1 and Pacn-2 to perform PCR to obtain the aconitase promoter sequence Pacn in Acetobacter pasteurianus; Verification of acidase promoter sequence;
  • 3Plasmid pBBR1p452 and aconitase promoter sequence Pacn were treated with restriction endonuclease at 37°C for 1-2h, purified and recovered, and mixed according to the ratio of plasmid and fragment material 1:0.2-5, using T 4 DNA Ligase 16-22°C, carry out ligation reaction for 2-12h, then transfer the ligated product into Escherichia coli DH5 ⁇ competent to obtain recombinant plasmid pBBR1-Pacn, use gene sequencing reaction to verify the sequence of recombinant plasmid pBBR1-Pacn, Table 4 The composition of the pBBR1-Pacn enzyme digestion system is shown.
  • Acetobacter pasteurianus CGMCC 3089 genome is used as a template in step 2 in step (1).
  • the concentration of intracellular ammonium ions in the cells was determined by salicylic acid spectrophotometry.
  • Ammonia nitrogen standard curve drawing prepare 0, 1, 2, 3, 4, 5 ⁇ g/mL standard solution, measure the absorbance according to the above steps, and draw the ammonia nitrogen standard curve.
  • Sample determination After the sample is diluted, measure 0.2ml into a 10ml stoppered colorimetric tube, then follow the standard curve measurement operation, use the standard curve to calculate the total phenol content in the sample, and the result is expressed in mgGAE/ml.
  • Detect the content of p-coumaric acid in the fermentation broth of acetic acid bacteria that expresses recombinant tyrosine ammonia-lyase to improve the flavor of vinegar uses HLB solid-phase extraction column to concentrate and extract the fermentation broth, and uses GC-MS to detect p-coumaric acid in the fermentation broth of recombinant bacteria acid content.
  • Acetobacter of the present invention can be Gluconobacter (Gluconobacter), Acetobacter (Acetobacter) or Gluconacetobacter (Gluconacetobacter), preferred Acetobacter (Acetobacter aceti), Gluconacetobacter (Gluconacetobacter oxydans) ), Acetobacter pasteurianus (Acetobacter pasteurianus), more preferably Acetobacter pasteurianus CGMCC 3089, Acetobacter pasteurianus NBRC 3283.
  • primers involved in the present invention PCR reaction, purification of nucleotide fragments, recovery, digestion, ligation, DNA introduction, artificial synthesis of nucleotide sequences and other operations are well known to those skilled in the art, and can be based on, for example, "Molecular Cloning Experiment Guide” (Science Press, Fourth Edition, 2017), or in accordance with the conditions recommended by the manufacturer.
  • Another object of the present invention is to provide a method for acetic fermentation using the acetic bacteria for improving the flavor of vinegar of the present invention.
  • Using the above-mentioned genetically engineered bacteria to carry out acetic acid fermentation with ethanol as the main raw material has the advantages of short fermentation lag period and high fermentation rate.
  • Described method mainly comprises the following steps:
  • the medium must have the nutrients required for microbial growth, such as carbon sources such as glucose or ethanol, nitrogen sources such as urea, ammonium salt, yeast extract or yeast powder, and phosphate (phosphorus source) and sulfate (sulfur source);
  • carbon sources such as glucose or ethanol
  • nitrogen sources such as urea, ammonium salt, yeast extract or yeast powder, and phosphate (phosphorus source) and sulfate (sulfur source)
  • the concentration of tyrosine or aspartic acid in the medium is 0.2-1g/L; the concentration of ethanol in the medium is 30-200g/L.
  • the acetic acid bacteria for improving the flavor of vinegar of the present invention can be used to ferment and produce vinegar or acetic acid by using ethanol or wine mash containing ethanol as raw material.
  • Liquid acetic acid fermentation Including liquid surface static fermentation and liquid submerged fermentation. After the wine mash fermentation is finished, add activated acetic acid bacteria into the wine mash with an inoculation amount of 10% (v/v), and carry out liquid surface static fermentation or liquid submerged fermentation.
  • the present invention obtains recombinant expression of tyrosine ammonia lyase/aspartate ammonia lyase by constructing a recombinant plasmid containing tyrosine ammonia lyase/aspartate ammonia lyase and transferring it into acetic acid bacteria Acetic acid bacteria that improve the flavor of vinegar, thereby increasing the concentration of intracellular ammonium ions, alleviating pH changes, and improving acid resistance of bacteria.
  • the present invention utilizes the ethanolamine ammonia lyase promoter/aconitase promoter to cooperate with the expression of tyrosine ammonia lyase/aspartate ammonia lyase in the recombinant plasmid, and cooperates with the cell-related metabolism to express ammonia solution Enzyme gene, to achieve the effect of increasing the enzyme activity of ammonia lyase in cells, thus effectively enhancing the level of ammonia metabolism and amino acid metabolism.
  • tyrosine ammonia lyase When recombined with tyrosine ammonia lyase, tyrosine ammonia lyase can convert tyrosine in the medium into p-coumaric acid, which belongs to phenolic compounds, and improves the flavor and efficacy of vinegar There is a promotional effect.
  • aspartate ammonia-lyase When aspartate ammonia-lyase is used to recombine, aspartate ammonia-lyase can convert aspartic acid into fumaric acid, and fumaric acid participates in the TCA cycle, which can enhance energy metabolism to produce ATP and reduce acid damage to cells It can also increase the organic acids such as malic acid and citric acid in the TCA cycle process, and improve the flavor of vinegar.
  • Fig. 1 is the agarose gel electrophoresis of the digested product of plasmid pBBR1-Peut-tal in embodiment 1;
  • Fig. 2 is the vinegar solid-state fermentation process curve in embodiment 1;
  • Escherichia coli DH 5 ⁇ strain The main sources of materials used in the following experiments: Escherichia coli DH 5 ⁇ strain, nucleic acid manipulation tools and enzymes were from Bao Bioengineering (Dalian) Co., Ltd. Primer synthesis, nucleotide sequence artificial synthesis and gene sequencing were entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd.
  • the promoter Peut sequence of ethanolamine ammonia lyase is shown in SEQ ID No.1;
  • the promoter Pacn sequence of aconitase is shown in SEQ ID No.3;
  • Embodiment 1 contains the construction of recombinant plasmid pBBR1-Peut-tal Acetobacter pasteurian genetic engineering bacteria;
  • the PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes, 30 seconds at 94°C, 20 seconds at 56°C, 30 seconds at 72°C, and 10 minutes at 72°C after 26 cycles.
  • the ethanolamine ammonia lyase promoter Peut sequence and plasmid pBBR1p452 (Verena Kallnik, Maria Meyer, Uwe Deppenmeier, et al. Construction of expression vectors for protein production in Gluconobacter oxydans. Journal of Biotechnology 150 (2010) 460–4 65) Use limits separately
  • the endonucleases BamH I and Spe I were treated (37°C, 90 min), and the target fragments were respectively recovered by using the PCR product purification kit (Shanghai Sangon Bioengineering Technology Service Co., Ltd.).
  • the promoter Peut sequence was ligated at a ratio of 1:3 (substance ratio) (22°C, 2 hours); the ligated product was transformed into Escherichia coli DH 5 ⁇ , and the recombinant plasmid pBBR1-Peut was obtained after screening.
  • the tyrosine ammonia lyase gene tal (GenBank: KF765779.1) derived from Rhodotorula glutinis was obtained by gene synthesis, and linked to a plasmid that can stably replicate in acetic acid bacteria In pBBR1-Peut, the recombinant plasmid pBBR1-Peut-tal was obtained;
  • Acetobacter pasteurianus CGMCC 3089 competent cells Take 100 ⁇ L of Acetobacter pasteurianus CGMCC 3089 competent cells in a small centrifuge tube, add 10 ⁇ L of the constructed recombinant plasmid pBBR1-Peut-tal, mix well and add it to a pre-cooled electric pulse cup for ice bath for 3 minutes; turn on the electric pulse instrument, Perform electric shock (2.0kV) according to the set transformation program; quickly add 1mL of pre-cooled sterile YPG medium into the electric pulse cup, mix well, transfer to a test tube, slowly shake and cultivate at 30°C for 2 hours, and then apply Spread it on a selection plate with appropriate antibiotics, and culture it upside down at 30°C for 48 hours to obtain the Acetobacter pasteurianus CGMCC 3089 genetically engineered bacteria containing the recombinant plasmid pBBR1-Peut-tal.
  • Figure 1 is the agarose gel electrophoresis of the digested product of plasmid pBBR1-Peut-tal, M: Marker; 1, 2: The plasmid pBBR1-Peut-tal was double digested with Sal I and Xho I;
  • Medium composition yeast extract 15g/L, glucose 20g/L, tyrosine concentration is 0.5g/L, all the other are water.
  • the bacterial cells sampled under 1% acetic acid and 48 hours were selected, and the intracellular ammonium ion content of the bacterial cells was determined by salicylic acid spectrophotometry. Wash the cells with 1 ⁇ PBS buffer solution, add high-efficiency RIPA tissue/cell fast lysate for ultrasonic disruption, and centrifuge to obtain the supernatant.
  • Ammonia nitrogen standard curve drawing Prepare 0, 1, 2, 3, 4, 5 ⁇ g/mL standard solution, measure the absorbance according to the above steps, and draw the ammonia nitrogen standard curve.
  • Table 8 shows the original strain and the recombinant expression tyrosine ammonia lyase strain Comparison of intracellular ammonium ion content.
  • Acetobacter pasteurianus CGMCC 3089 genetically engineered bacteria and Acetobacter pasteurianus CGMCC 3089 original bacteria containing recombinant plasmid pBBR1-Peut-tal were taken from the slope and inoculated in the seed medium, at 32 ° C, 160 rpm conditions Incubate on a shaker for 24 hours. The 10% (v/v) inoculum was transferred into fresh seed medium for scale-up culture.
  • Seed medium composition yeast extract 15g/L, glucose 20g/L, ethanol 3.5% (v/v), tyrosine concentration is 0.5g/L, all the other are water.
  • the original strain Acetobacter pasteurianus CGMC 3089 and the Acetobacter pasteurianus CGMCC 3089 genetically engineered bacteria containing the recombinant plasmid pBBR1-Peut-tal were used to carry out solid-state fermentation of vinegar at 30°C and 24 hours of fermenting.
  • Concentration is 6.1g/100g vinegar unstrained spirits, then ethanol conversion rate of acid is about 87.1%, average acid production rate is about 0.68g/(100g vinegar unstrained spirits ⁇ d), total phenol content is 1.45mg/ml, wherein p-coumaric acid The content is 0.32mg/L.
  • the final concentration of acetic acid is 6.5g/100g of vinegar fermented grains
  • the acid conversion rate of ethanol is about 92.8%
  • the average acid production rate is about 0.81g/(100gcupei ⁇ d)
  • the total phenol content is 1.73mg/ml
  • the p-coumaric acid content is 0.52mg/L.
  • Fig. 2 is the process curve of producing vinegar by solid-state fermentation using Acetobcter pasteurianus CGMCC 3089 and Acetobcter pasteurianus CGMCC 3089 genetically engineered bacteria containing recombinant plasmid pBBR1-Pacn-tal respectively.
  • the PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes, 30 seconds at 94°C, 20 seconds at 56°C, 30 seconds at 72°C, and 10 minutes at 72°C after 26 cycles.
  • Aconitase promoter Pacn sequence and plasmid pBBR1p452 (Verena Kallnik, Maria Meyer, Uwe Deppenmeier, et al. Construction of expression vectors for protein production in Gluconobacter oxydans.
  • PCR was performed using primers aal-1 and aal-2 to amplify the aal sequence of the aspartate ammonia lyase gene, and the PCR reaction system and reaction conditions were the same as above.
  • the aspartate ammonia-lyase gene aal fragment and the plasmid pBBR1-Pacn were treated with restriction endonucleases EcoR V and Cla I respectively (37°C, 4 hours), and PCR product purification kit (Shanghai Sangon Bioengineering Technology Co., Ltd.
  • Fig. 3 is the agarose gel electrophoresis of plasmid pBBR1-Pacn-aal digestion product; Wherein, in Fig. 3, M: Marker; 1,2: plasmid pBBR1-Pacn-aal is double-digested with EcoR V and Cla I;
  • Acetobacter pasteurianus CGMCC 3089 competent cells Take 100 ⁇ L of Acetobacter pasteurianus CGMCC 3089 competent cells in a small centrifuge tube, add 10 ⁇ L of the constructed recombinant plasmid pBBR1-Pacn-aal, mix well and add it to a pre-cooled electric pulse cup for ice bath for 3 minutes; turn on the electric pulse instrument, Perform electric shock (2.0kV) according to the set transformation program; quickly add 1mL of pre-cooled sterile YPG medium into the electric pulse cup, mix well, transfer to a test tube, slowly shake and cultivate at 30°C for 2 hours, and then apply Spread it on the selection plate with appropriate antibiotics, and culture it upside down at 30°C for 48 hours to obtain the Acetobacter pasteurianus CGMCC 3089 genetically engineered bacteria containing the recombinant plasmid pBBR1-Pacn-aal.
  • Medium composition yeast extract 15g/L, glucose 20g/L, aspartic acid concentration is 0.5g/L, and the rest is water.
  • Add 1% (v/v) acetic acid concentration to the ethanol-free medium inoculate the same acetic acid bacteria and original strains containing the recombinant plasmid pBBR1-Pacn-aal respectively, adjust the initial OD 600 to be consistent, and culture them for 24 hours respectively , 48h sampling to compare the changes in the biomass of the two, Table 10 is the comparison of the acetic acid tolerance of the original strain and the recombinant strain expressing aspartate ammonia-lyase.
  • the bacterial cells were sampled under the condition of 1% acetic acid concentration and fermentation for 48 hours, and the intracellular ammonium ion content of the bacterial cells was determined by salicylic acid spectrophotometry. Wash the cells with 1 ⁇ PBS buffer, add high-efficiency RIPA tissue/cell fast lysate for ultrasonic disruption, and centrifuge to obtain the supernatant.
  • Ammonia nitrogen standard curve drawing prepare 0, 1, 2, 3, 4, 5 ⁇ g/mL standard solution, measure the absorbance according to the above steps, and draw the ammonia nitrogen standard curve.
  • Table 11 is the comparison of the intracellular ammonium ion concentration of the original strain and the recombinant strain expressing aspartate ammonia lyase.
  • Medium composition yeast extract 15g/L, glucose 20g/L, aspartic acid concentration is 0.5g/L, and the rest is water. After culture, take 1mL of the fermentation broth of the original strain and the recombinant strain, collect the bacteria by centrifugation, wash the bacteria with 1 ⁇ PBS buffer, add high-efficiency RIPA tissue/cell rapid lysate for ultrasonic disruption, and centrifuge to take the supernatant as the sample to be tested .
  • the ATP ELISA kit was used to detect the concentration of ATP with a microplate reader (450 nm).
  • HRP horseradish peroxidase
  • Figure 4 is a comparison chart of the ATP content of the Acetobacter pasteurianus CGMCC 3089 genetically engineered bacteria containing the recombinant plasmid pBBR1-Pacn-aal and the original strain Acetobcter pasteurianus CGMCC 3089.
  • Preparation of seed solution Take the Acetobacter pasteurianus CGMCC 3089 genetically engineered bacteria and the original strain containing the recombinant plasmid pBBR1-Pacn-aal from the slant respectively in the seed medium, and culture them on a shaking table at 30°C and 160 rpm for 25 Hour. The 10% (v/v) inoculum was transferred into fresh seed medium for scale-up culture. Seed medium composition: yeast extract 15g/L, glucose 20g/L, ethanol 3.5% (v/v), aspartic acid concentration is 0.5g/L, all the other are water.
  • Alcoholic fermentation Take an appropriate amount of pretreated fruit juice and heat it to 30°C, then sprinkle it with active dry yeast, stir and keep it warm for 30 minutes, then insert it into the fruit juice after the yeast activity is normal, and carry out alcoholic fermentation at 25°C after stirring evenly. Ferment for about 7d.
  • Acetic acid fermentation by the inoculum size of 10% (v/v), the Acetobacter pasteurianus CGMCC 3089 genetically engineered bacterium and the original bacterial strain containing the recombinant plasmid pBBR1-Pacn-aal were respectively inoculated into fermenters containing cider. Fermentation of apple cider vinegar was carried out at 30°C. The ethanol content in cider is 8% (v/v).
  • the original Acetobacter pasteurianus CGMCC 3089 and the Acetobacter pasteurianus CGMCC 3089 genetically engineered bacteria containing the recombinant plasmid pBBR1-Pacn-aal were used to carry out liquid submerged fermentation to produce apple cider vinegar.
  • the concentration of acetic acid is 10g/L, and the final concentration of acetic acid is 85g/L, then the acid conversion rate of ethanol is about 91.1%, and the average acid production rate is about 1.02g/(L ⁇ h).
  • the recombinant strain finished 69h of fermentation the final concentration of acetic acid was 90g/L, the acid conversion rate of ethanol was about 96.0%, and the average acid production rate was about 1.16g/(L ⁇ h).
  • Fig. 5 is the fermentation process curve of producing apple cider vinegar by liquid submerged fermentation of Acetobcter pasteurianus CGMCC 3089 and Acetobcter pasteurianus CGMCC 3089 genetically engineered bacteria containing recombinant plasmid pBBR1-Pacn-aal respectively.

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Abstract

属于基因工程领域，具体提供了一种改善食醋风味的醋酸菌及其构建方法，采用下述方案：方案一、以巴氏醋杆菌为宿主细胞，巴氏醋杆菌中具有协同效果的乙醇胺解氨酶为启动子协同表达酪氨酸解氨酶得到；或方案二、以巴氏醋杆菌为宿主细胞，巴氏醋杆菌三羧酸循环中顺乌头酸酶为启动子协同表达天冬氨酸解氨酶得到；当采用酪氨酸解氨酶重组时，将酪氨酸解氨酶转化为对香豆酸，对香豆酸属于酚类化合物，对于食醋风味的改善和功效有促进作用，当采用天冬氨酸解氨酶重组时，将天冬氨酸解氨酶转化为富马酸，同时富马酸参与TCA循环，能够增强能量代谢产生ATP，减少酸对细胞造成的损伤。

Description

改善食醋风味的醋酸菌及其构建方法
本申请要求2021年12月4日向中国国家知识产权局提交的，专利申请号为202111471319.9，发明名称为“改善食醋风味的醋酸菌及其构建方法”在先申请的优先权。该申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于生物工程领域，具体涉及一种改善食醋风味的醋酸菌构建方法及其应用。
背景技术
食醋，主要成分为醋酸，是世界上消费量最大的调味品之一，也是食品、医药、化工、纺织等领域广泛应用的化合物之一。醋酸菌(Acetic acid bacteria)是一类严格好氧的革兰氏阴性细菌，为化能异养型。最常见的醋杆菌属(Acetobacter)葡萄糖杆菌属(Gluconobacter)以及葡萄糖酸杆菌属(Gluconacetobacter)等，其中醋杆菌属和葡萄糖杆菌属(Gluconobacter)因其强大的氧化乙醇产生乙酸能力而被广泛用于食醋发酵生产。随着社会的不断发展，国内食醋行业发展迅速，而菌体耐酸性一直是制约高酸度醋生产的关键因素，如何提高发酵酸度以及改善菌体耐酸性是提高食醋发酵效率需要解决的关键问题。此外，食醋发酵过程醋酸菌的主要功能是氧化乙醇生成醋酸，对产品其他风味贡献较少，因此在获得较高醋酸浓度的同时，增加其他风味物质的浓度，也有助于食醋产品品质的提升。
发明内容
本发明的目的在于，提供一种改善食醋风味的醋酸菌及其构建方法。
为实现上述目的，本发明采用的技术方案为：
方案一：一种重组酪氨酸解氨酶的改善食醋风味的醋酸菌，以巴氏醋杆菌(Acetobacter pasteurianus)为宿主细胞，巴氏醋杆菌(Acetobacter pasteurianus)中具有协同效果的乙醇胺解氨酶的启动子协同表达酪氨酸解氨酶得到；启动子的序列号为SEQ ID No.1，酪氨酸解氨酶的序列号为SEQ ID No.2。
或者方案二：以巴氏醋杆菌(Acetobacter pasteurianus)为宿主细胞，巴氏醋杆菌(Acetobacter pasteurianus)三羧酸循环中顺乌头酸酶的启动子协同 表达天冬氨酸解氨酶得到；启动子的序列号为SEQ ID No.3，天冬氨酸解氨酶的序列号为SEQ ID No.4。
当采用酪氨酸解氨酶重组时，将酪氨酸转化为对香豆酸，对香豆酸属于酚类化合物，对于食醋风味的改善和功效有促进作用；当采用天冬氨酸解氨酶重组时，将天冬氨酸转化为富马酸，同时富马酸参与TCA循环，能够增强能量代谢产生ATP，减少酸对细胞造成的损伤。
本发明还包括一种所述的改善食醋风味的醋酸菌的构建方法，当采用方案一时，包括步骤：利用PCR的方法获得来源于巴氏醋杆菌中的乙醇胺解氨酶的启动子，并将其连接到可在醋酸菌中稳定复制的质粒pBBR1p452(Verena Kallnik,Maria Meyer,Uwe Deppenmeier,et al.Construction of expression vectors for protein production in Gluconobacter oxydans.Journal of Biotechnology 150(2010)460–465)中，获得重组质粒pBBR1-Peut；利用基因合成的方法获得来源于粘(深)红酵母(Rhodotorula glutinis)中的酪氨酸解氨酶基因tal(GenBank:KF765779.1)，并将其连接到可在醋酸菌中稳定复制的质粒pBBR1-Peut中，获得重组质粒pBBR1-Peut-tal；将重组质粒pBBR1-Peut-tal转入醋酸菌中即可获得利用乙醇胺解氨酶启动子控制的重组表达酪氨酸解氨酶的改善食醋风味的醋酸菌。
具体包括下述步骤：
(1)乙醇胺解氨酶启动子Peut序列的获得和重组质粒pBBR1-Peut的构建:
①设计引物Peut-1和Peut-2，PCR扩增巴氏醋杆菌(Acetobacter pasteurianus)中乙醇胺解氨酶启动子Peut序列，为了便于重组质粒的构建，引物Peut-1上引入BamH I酶切位点，引物Peut-2上引入Spe I酶切位点，如下表1 Peut相关引物序列表所示：
表1
引物名称 引物序列 酶切位点
Peut-1 5'-CGCGGATCCCGTTCAGGTTCAGCATATCTGTG-3' BamH I
Peut-2 5'-AGCACTAGTTGAAGGAACTCCTTAGCTGCG-3' Spe I
②以巴氏醋杆菌(Acetobacter pasteurianus)基因组为模板，利用引物Peut-1和Peut-2进行PCR，获得巴氏醋杆菌中乙醇胺解氨酶启动子序列Peut；利用基因测序反应进行乙醇胺解氨酶启动子序列的验证；
③将质粒pBBR1p452和乙醇胺解氨酶启动子序列Peut分别用限制性内切酶37℃处理1-2h后纯化回收，按质粒和片段物质的量之比1:0.2-5混合,利用T 4 DNA连接酶16-22℃进行2-12h连接反应，之后将连接产物转入大肠杆菌DH5α感受态中，获得重组质粒pBBR1-Peut,利用基因测序反应进行重组质粒pBBR1-Peut的序列验证，表2为pBBR1-Peut酶切体系组成。
表2
Figure PCTCN2022135751-appb-000001
(2)酪氨酸解氨酶tal序列的获得和重组质粒pBBR1-Peut-tal的构建:
利用基因合成的方法获得来源于粘(深)红酵母(Rhodotorula glutinis)中的酪氨酸解氨酶基因tal(GenBank:KF765779.1)，并将其连接到可在醋酸菌中稳定复制的质粒pBBR1-Peut中，获得重组质粒pBBR1-Peut-tal；
(3)重组表达酪氨酸解氨酶改善食醋风味的醋酸菌的获得:
利用电激转化方法，将验证正确的质粒pBBR1-Peut-tal转入醋酸菌中，涂布到加有适当卡那霉素的选择平板上，30℃倒置24-60小时，获得含有重组质粒pBBR1-Peut-tal的改善食醋风味的醋酸菌。
本发明还包括另一种所述的改善食醋风味的醋酸菌的构建方法，包括步骤：利用PCR的方法获得来源于巴氏醋杆菌中的顺乌头酸酶的启动子，并将其连接到可在醋酸菌中稳定复制的质粒pBBR1p452中，获得重组质粒pBBR1-Pacn；利用PCR的方法获得来源于大肠杆菌Escherichia coli中的天冬氨酸解氨酶基因aal，并将其连接到质粒pBBR1-Pacn上，获得重组质粒pBBR1-Pacn-aal；将重组质粒pBBR1-Pacn-aal转入醋酸菌中即可获得利用顺乌头酸酶启动子控制的重组表达天冬氨酸解氨酶的改善食醋风味的醋酸菌。
具体包括下述步骤：
(1)顺乌头酸酶启动子Pacn序列的获得和重组质粒pBBR1-Pacn的构建：
①设计引物Pacn-1和Pacn-2，PCR扩增巴氏醋杆菌(Acetobacter pasteurianus)中顺乌头酸酶启动子Pacn序列，为了便于重组质粒的构建，引物 Pacn-1上引入BamH I酶切位点，引物Pacn-2上引入Spe I酶切位点，如下表3的Pacn相关引物序列表所示：
表3
引物名称 引物序列 酶切位点
Pacn-1 5'-CGCGGATCCAATCGCTCTCTCCCCGATC-3' BamH I
Pacn-1 5'-AGCACTAGTGCTGGTGCTGGATGGTGTC-3' Spe I
②以巴氏醋杆菌(Acetobacter pasteurianus)基因组为模板，利用引物Pacn-1和Pacn-2进行PCR，获得巴氏醋杆菌中顺乌头酸酶启动子序列Pacn；利用基因测序反应进行顺乌头酸酶启动子序列的验证；
③将质粒pBBR1p452和顺乌头酸酶启动子序列Pacn分别用限制性内切酶37℃处理1-2h后纯化回收，按质粒和片段物质的量之比1:0.2-5混合,利用T 4 DNA连接酶16-22℃，进行2-12h连接反应，之后将连接产物转入大肠杆菌DH5α感受态中，获得重组质粒pBBR1-Pacn,利用基因测序反应进行重组质粒pBBR1-Pacn的序列验证，表4示出pBBR1-Pacn酶切体系组成。
表4
Figure PCTCN2022135751-appb-000002
(2)天冬氨酸解氨酶aal序列的获得和重组质粒pBBR1-Pacn-aal的构建：
①设计引物aal-1和aal-2，PCR扩增大肠杆菌中天冬氨酸aal序列，为了便于重组质粒的构建，引物aal-1上引入EcoR V酶切位点，引物aal-2上引入酶切位点Cla I，如下表5示出aal相关引物序列表：
表5
引物名称 引物序列 酶切位点
aal-1 5'-TCGATATCATGTCAAACAACATTCGTATCGAAG-3' EcoR V
aal-2 5'-GTATCGATTTACTGTTCGCTTTCATCAGTATAGCG-3' Cla I
②以大肠杆菌(Escherichia coli)基因组为模板，利用引物aal-1和aal-2进行PCR，获得大肠杆菌中天冬氨酸解氨酶的序列aal；利用基因测序反应进行天冬氨酸序列的验证；
③将重组质粒pBBR1-Pacn和天冬氨酸解氨酶序列aal分别用限制性内切酶处理，37℃，90min后纯化回收，按质粒和片段物质的量之比1:0.2-5混合,利用T 4 DNA连接酶22℃进行2h连接反应，之后将连接产物转入大肠杆菌DH 5α感受态中，获得重组质粒pBBR1-Pacn-aal；利用基因测序反应进行重组质粒pBBR1-Pacn-aal的序列验证；
(3)重组表达天冬氨酸解氨酶改善食醋风味的醋酸菌的获得：利用电激转化方法，将验证正确的质粒pBBR1-Pacn-aal转入醋酸菌中，涂布到加有适当卡那霉素的选择平板上，30℃倒置24-60小时，获得含有重组质粒pBBR1-Pacn-aal的改善食醋风味的醋酸菌。
优选的，步骤(1)中的步骤②中以Acetobacter pasteurianus CGMCC 3089基因组为模板。
改善食醋风味的醋酸菌中胞内铵离子含量测定：
采用水杨酸分光光度法测定菌体内胞内铵离子浓度。
使用1×PBS缓冲液洗涤菌体后添加高效RIPA组织/细胞快速裂解液进行超声破碎，离心取上清液。取8mL上清液样品于10mL比色管中，加入1mL显色剂(水杨酸-酒石酸钾钠溶液)、2滴亚硝基铁氰化钠(0.01g/mL)和2滴次氯酸钠(有效氯3.5g/L、游离碱浓度0.75mol/L)使用液，加水稀释至标线，混匀后显色60min。以水为空白参比，用10mm比色皿检测在697nm处吸光度。
氨氮标准曲线绘制：配制0、1、2、3、4、5μg/mL标准使用液，按照上述步骤测量吸光度，绘制氨氮标准曲线。
改善食醋风味的醋酸菌中总酚含量的测定
标准曲线的绘制：分别吸取不同浓度的没食子酸0.2ml于10ml具塞比色管中，加入0.8ml福林酚试剂，混匀，反应5min，之后加入1.5ml碳酸钠溶液(10％)，加入去离子水至10ml，室温反应2h，测定波长765nm处吸光度值，整个实验避光进行。以没食子酸含量为横坐标，A 765为纵坐标，绘制标准曲线。
样品测定：样品经稀释后量取0.2ml于10ml具塞比色管中，然后按照标曲测定操作，利用标曲计算样品中总酚含量，结果以mgGAE/ml表示。
改善食醋风味的醋酸菌中对香豆酸含量的测定
检测重组表达酪氨酸解氨酶改善食醋风味的醋酸菌发酵液中对香豆酸含量，使用HLB固相萃取柱对发酵液浓缩萃取，采用GC-MS检测重组菌发酵液中对香豆酸含量。
本发明所述的醋酸菌可以为葡糖杆菌(Gluconobacter)、醋杆菌(Acetobacter)或葡糖酸醋杆菌(Gluconacetobacter)，优选的醋化醋杆菌(Acetobacter aceti)、葡糖酸醋杆菌(Gluconacetobacter oxydans)、巴氏醋杆菌(Acetobacter pasteurianus)，更优选的巴氏醋杆菌CGMCC 3089、巴氏醋杆菌NBRC 3283。
本发明涉及的引物的制备、PCR反应、核苷酸片段的纯化、回收、酶切、连接、DNA导入、核苷酸序列人工合成等操作是本领域技术人员所熟知的，可以依据例如《分子克隆实验指南》(科学出版社，第四版，2017年)中记载的方法，或按照制造厂商所建议的条件来实施。
本发明的另一个目的是提供一种利用本发明的改善食醋风味的醋酸菌进行醋酸发酵的方法。利用上述基因工程菌以乙醇为主要原料进行醋酸发酵具有发酵迟缓期短，发酵速率高等优点。
为实现上述目的，本发明技术方案如下。
所述方法主要包括以下步骤：
(1)培养基的制备：
培养基中必须具备微生物生长所需的营养成分，如葡萄糖或乙醇等碳源，尿素、铵盐、酵母浸膏或酵母粉等氮源以及磷酸盐(磷源)和硫酸盐(硫源)；培养基中酪氨酸或天冬氨酸浓度为0.2-1g/L；培养基中乙醇浓度为30-200g/L。
(2)种子培养：
利用250-1000mL的摇瓶，装有如上所述培养基30-100mL，接入含有重组质粒pBBR1-Peut-tal或pBBR1-Pacn-aal的改善食醋风味的醋酸菌进行摇瓶培养，摇床转速为100-300转/分钟，温度为27-30℃，培养时间为20-30小时，制备种子液。
(3)醋酸发酵：
本发明的改善食醋风味的醋酸菌可应用于以乙醇或含有乙醇的酒醪为原料发酵生产食醋或者醋酸。
①固态食醋发酵。醋酸发酵在固态发酵罐中进行。酒醪发酵结束后，在酒醪中加入活化后的醋酸菌，接种量为10％(v/v)，按照酒醪：麸皮：稻壳＝5.5:1.4:0.8的比例在酒醪中加入麸皮和稻壳搅拌均匀，进行醋酸发酵。
②液态醋酸发酵。包括液态表面静置发酵和液态深层发酵方式。酒醪发酵结束后，在酒醪中加入活化后的醋酸菌，接种量为10％(v/v)，进行液态表面静置发酵或者液态深层发酵。
与现有技术相比，本发明的有益效果是：
(1)本发明通过构建含有酪氨酸解氨酶/天冬氨酸解氨酶的重组质粒并转入醋酸菌中，获得重组表达酪氨酸解氨酶/天冬氨酸解氨酶的改善食醋风味的醋酸菌，从而增加胞内铵离子浓度，缓解PH变化，提高菌种耐酸性。
(2)本发明利用乙醇胺解氨酶启动子/顺乌头酸酶启动子协同重组质粒中酪氨酸解氨酶/天冬氨酸解氨酶的表达，与菌体相关代谢协同表达解氨酶基因，达到提高细胞内解氨酶酶活的效果，从而有效增强了氨代谢和氨基酸代谢水平.
(3)利用该改善食醋风味的醋酸菌以乙醇为主要原料进行醋酸发酵具有发酵迟缓期短，发酵速率高等优点，从而减少能源消耗、提高生产效率、提高企业效益。
当采用酪氨酸解氨酶重组时，酪氨酸解氨酶能够将培养基中的酪氨酸转化为对香豆酸，对香豆酸属于酚类化合物，对于食醋风味的改善和功效有促进作用。当采用天冬氨酸解氨酶重组时，天冬氨酸解氨酶能够将天冬氨酸转化为富马酸，富马酸参与TCA循环，能够增强能量代谢产生ATP，减少酸对细胞造成的损伤，同时还可以增加TCA循环过程中苹果酸、柠檬酸等有机酸，改善食醋风味。
附图说明
图1为实施例1中质粒pBBR1-Peut-tal酶切产物琼脂糖凝胶电泳；
图2为实施例1中食醋固态发酵过程曲线；
图3实施例2中质粒pBBR1-Pacn-aal酶切产物琼脂糖凝胶电泳；
图4实施例2中原始菌株与重组菌株ATP含量比较图；
图5实施例2中液态深层发酵苹果醋过程曲线。
具体实施方式
为了使本技术领域的技术人员更好地理解本发明的技术方案，下面结合附图 和最佳实施例对本发明作进一步的详细说明。
以下实验所用主要材料来源：大肠杆菌DH 5α菌株、核酸操作工具酶来自宝生物工程(大连)有限公司，引物合成、核苷酸序列人工合成以及基因测序委托苏州金唯智生物科技有限公司完成。
本发明及实施例涉及的部分基因及引物序列如下：
乙醇胺解氨酶的启动子Peut序列如SEQ ID No.1所示；
酪氨酸解氨酶基因tal的序列如SEQ ID No.2所示；
顺乌头酸酶的启动子Pacn序列如SEQ ID No.3所示；
天冬氨酸解氨酶基因aal的序列如SEQ ID No.4所示：
Figure PCTCN2022135751-appb-000003
实施例1含有重组质粒pBBR1-Peut-tal巴氏醋杆菌基因工程菌的构建；
(1)重组质粒pBBR1-Peut的构建：
以Acetobacter pasteurianus CGMCC 3089(购自中国微生物菌种保藏管理委员会普通微生物中心，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，邮编100101)基因组为模板，利用引物Peut-1和Peut-2进行PCR，扩增获得乙醇胺解氨酶启动子Peut序列，PCR反应体系如下表6：
表6
Figure PCTCN2022135751-appb-000004
Figure PCTCN2022135751-appb-000005
PCR反应条件为：95℃预变性5分钟，94℃30秒，56℃20秒，72℃30秒，26个循环后72℃10分钟。
将乙醇胺解氨酶启动子Peut序列和质粒pBBR1p452(Verena Kallnik,Maria Meyer,Uwe Deppenmeier,et al.Construction of expression vectors for protein production in Gluconobacter oxydans.Journal of Biotechnology 150(2010)460–465)分别用限制性内切酶BamH Ⅰ和Spe Ⅰ处理(37℃，90min)，利用PCR产物纯化试剂盒(上海生工生物工程技术服务有限公司)分别回收目的片段，将酶切后的质粒和乙醇胺解氨酶启动子Peut序列按1:3(物质的量之比)的比例进行连接反应(22℃，2小时)；连接产物转入大肠杆菌DH 5α，经筛选获得重组质粒pBBR1-Peut。
(2)重组质粒pBBR1-Peut-tal的构建：
利用基因合成的方法获得来源于粘(深)红酵母(Rhodotorula glutinis)中的酪氨酸解氨酶基因tal(GenBank:KF765779.1)，并将其连接到可在醋酸菌中稳定复制的质粒pBBR1-Peut中，获得重组质粒pBBR1-Peut-tal；
(3)重组表达酪氨酸解氨酶巴氏醋杆菌基因工程菌的获得：
①Acetobacter pasteurianus CGMCC 3089感受态细胞的制备：
挑取Acetobacter pasteurianus CGMCC 3089接种于YPG培养基中，30℃、190转/分钟预培养12小时，至OD 600约0.6，取1 mL预培养的菌液接入装有100mLYPG培养基的250mL三角瓶中，30℃、220转/分钟培养8h，至OD 600约0.6；将装有菌液的三角瓶置于冰浴上冷却20分钟，4℃下5000转/分钟离心5分钟，弃上清；加80 mL预冷至0℃的10％甘油(质量比)溶液重悬菌体，使菌体充分扩散后于4℃下5000转/分钟离心8分钟，弃上清；加3mL预冷的10％甘油溶液摇匀，置于冰浴中，获得Acetobacter pasteurianus CGMCC 3089感受态细胞。
②质粒pBBR1-Peut-tal的电激转化
取100μL Acetobacter pasteurianus CGMCC 3089感受态细胞于小离心管 中，加入10μL构建好的重组质粒pBBR1-Peut-tal，混合均匀后加入到预冷的电脉冲杯内冰浴3分钟；打开电脉冲仪，按设定的转化程序进行电激(2.0kV)；向电脉冲杯内迅速加入1mL预冷的无菌YPG培养基，混匀后，转入试管中，30℃缓慢振荡培养2小时后，涂布到加有适当抗生素的选择平板上，30℃倒置培养48小时，获得含有重组质粒pBBR1-Peut-tal的Acetobacter pasteurianus CGMCC 3089基因工程菌。
图1为质粒pBBR1-Peut-tal酶切产物琼脂糖凝胶电泳，M：Marker；1、2：质粒pBBR1-Peut-tal用Sal I和Xho I双酶切；
含有重组质粒pBBR1-Peut-tal的改善食醋风味的醋酸菌耐酸性检测：
培养基组成:酵母膏15g/L，葡萄糖20g/L，酪氨酸浓度为0.5g/L，其余为水。
在不含乙醇的培养基中添加1％(v/v)醋酸浓度，接种相同的含有重组质粒pBBR1-Peut-tal的醋酸菌和原始菌株，调整初始OD 600一致后进行培养，分别于24h、48h取样比较两者生物量的变化，表7所示为原始菌株和重组表达酪氨酸解氨酶菌株醋酸耐受性的比较。
表7
Figure PCTCN2022135751-appb-000006
注：表格中的每个数值表示为mean±S.D.(n＝3)。采用邓肯多重方差分析评估显著差异。同列中不同字母表示具有显著性差异(P<0.001)。
同时选取1％的醋酸、48h条件下取样的菌体，采用水杨酸分光光度法对菌体胞内铵离子含量进行测定。使用1×PBS缓冲液洗涤菌体后添加高效RIPA组织/细胞快速裂解液进行超声破碎，离心取上清。取8mL上清液样品于10mL比色管中，加入1mL显色剂(水杨酸-酒石酸钾钠溶液)、2滴亚硝基铁氰化钠(0.01g/mL)和2滴次氯酸钠(有效氯3.5g/L、游离碱浓度0.75mol/L)使用液，加水稀释至标线，混匀后显色60min。以水为空白参比，用10mm比色皿检测在697nm处吸光度。
氨氮标准曲线绘制：配制0、1、2、3、4、5μg/mL标准使用液，按照上述步骤测量吸光度，绘制氨氮标准曲线，表8示出原始菌株和重组表达酪氨酸解氨酶菌株胞内铵离子含量比较。
表8
Figure PCTCN2022135751-appb-000007
注：表格中的每个数值表示为mean±S.D.(n＝3)。采用邓肯多重方差分析评估显著差异。同列中不同字母表示具有显著性差异(P<0.001)。
利用含有重组质粒pBBR1-Peut-tal的醋酸菌固态发酵生产食醋：
(1)制备种子液：分别从斜面取含有重组质粒pBBR1-Peut-tal的Acetobacter pasteurianus CGMCC 3089基因工程菌和Acetobacter pasteurianus CGMCC 3089原始菌接种于种子培养基中，在32℃，160转/分钟条件下摇床培养24小时。按10％(v/v)的接种量转接入新鲜的种子培养基中进行放大培养。
种子培养基组成:酵母膏15g/L，葡萄糖20g/L，乙醇3.5％(v/v)，酪氨酸浓度为0.5g/L，其余为水。
(2)酒精发酵：称取粉碎后的高粱1kg，倒入糊化桶中，加烧开的水至淹没高粱。加入耐高温α-淀粉酶220μL并搅拌均匀。在85-100℃条件下加热30-40min，使高粱糊化。加热结束后自然降温至55℃左右，加入固体糖化酶23g，搅拌均匀后保温1h进行糖化。糖化结束后加入0.65kg大曲，装入发酵用坛子中，加水补重量至5kg。加入3g酵母，并搅拌均匀。液封后放入28℃培养箱中，发酵约7-9d。
(3)醋酸固态发酵：酒醪发酵结束后，在酒醪中加入活化后的醋酸菌，接种量为10％(v/v)，按照酒醪：麸皮：稻壳＝5.5:1.4:0.8的比例在酒醪中加入麸皮和稻壳搅拌均匀，进行醋酸发酵。
分别利用原始菌株Acetobacter pasteurianus CGMC 3089和含有重组质粒pBBR1-Peut-tal的Acetobacter pasteurianus CGMCC 3089基因工程菌，在30℃， 24h翻醅的条件下进行食醋固态发酵，原始菌株9d发酵结束，醋酸终浓度为6.1g/100g醋醅，则乙醇转酸率约为87.1％，平均产酸速率约为0.68g/(100g醋醅·d)，总酚含量为1.45mg/ml，其中对香豆酸含量为0.32mg/L。重组菌株8d发酵结束，醋酸终浓度为6.5g/100g醋醅，则乙醇转酸率约为92.8％，平均产酸速率约为0.81g/(100gcupei·d)，总酚含量为1.73mg/ml，其中对香豆酸含量为0.52mg/L。
图2为分别利用Acetobcter pasteurianus CGMCC 3089和含有重组质粒pBBR1-Pacn-tal的Acetobcter pasteurianus CGMCC 3089基因工程菌固态发酵生产食醋的过程曲线。
实施例2：
含有重组质粒pBBR1-Pacn-aal巴氏醋杆菌基因工程菌的构建：
重组质粒pBBR1-Pacn的构建：以Acetobacter pasteurianus CGMCC 3089(购自中国微生物菌种保藏管理委员会普通微生物中心，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，邮编100101)基因组为模板，利用引物Pacn-1和Pacn-2进行PCR，扩增获得顺乌头酸酶启动子Pacn序列，PCR反应体系如下：
表9
Figure PCTCN2022135751-appb-000008
PCR反应条件为：95℃预变性5分钟，94℃30秒，56℃20秒，72℃30秒，26个循环后72℃10分钟。
将顺乌头酸酶启动子Pacn序列和质粒pBBR1p452(Verena Kallnik,Maria Meyer,Uwe Deppenmeier,et al.Construction of expression vectors for  protein production in Gluconobacter oxydans.Journal of Biotechnology 150(2010)460–465)分别用限制性内切酶BamH Ⅰ和Spe Ⅰ处理(37℃，4小时)，利用PCR产物纯化试剂盒(上海生工生物工程技术服务有限公司)分别回收目的片段，将酶切后的质粒和顺乌头酸酶启动子Pacn序列按1:3(物质的量之比)的比例进行连接反应(16℃，12小时)；连接产物转入大肠杆菌DH 5α，经筛选获得重组质粒pBBR1-Pacn。
(2)重组质粒pBBR1-Pacn-aal的构建
以大肠杆菌BL21(Escherichia coli)基因组为模板，利用引物aal-1和aal-2进行PCR，扩增获得天冬氨酸解氨酶基因aal序列，PCR反应体系与反应条件同上。
将天冬氨酸解氨酶基因aal片段和质粒pBBR1-Pacn分别用限制性内切酶EcoR V和Cla I处理(37℃，4小时)，利用PCR产物纯化试剂盒(上海生工生物工程技术服务有限公司)分别回收目的片段，将酶切后的质粒和天冬氨酸解氨酶aal序列按1:3(物质的量之比)的比例进行连接反应(16℃，12小时)；连接产物转入大肠杆菌DH 5α，经筛选获得重组质粒pBBR1-Pacn-aal。图3为质粒pBBR1-Pacn-aal酶切产物琼脂糖凝胶电泳；其中，图3中，M：Marker；1、2：质粒pBBR1-Pacn-aal用EcoR V和Cla I双酶切；
(3)重组表达天冬氨酸解氨酶巴氏醋杆菌基因工程菌的获得
①Acetobacter pasteurianus CGMCC 3089感受态细胞的制备：
挑取Acetobacter pasteurianus CGMCC 3089接种于YPG培养基中，30℃、190转/分钟预培养12小时，至OD 600约0.6，取1mL预培养的菌液接入装有100mLYPG培养基的250mL三角瓶中，30℃、220转/分钟培养8h，至OD 600约0.6；将装有菌液的三角瓶置于冰浴上冷却20分钟，4℃下5000转/分钟离心5分钟，弃上清；加80mL预冷至0℃的10％甘油(质量比)溶液重悬菌体，使菌体充分扩散后于4℃下5000转/分钟离心8分钟，弃上清；加3mL预冷的10％甘油溶液摇匀，置于冰浴中，获得Acetobacter pasteurianus CGMCC 3089感受态细胞。
②质粒pBBR1-Pacn-aal的电激转化
取100μL Acetobacter pasteurianus CGMCC 3089感受态细胞于小离心管中，加入10μL构建好的重组质粒pBBR1-Pacn-aal，混合均匀后加入到预冷的电 脉冲杯内冰浴3分钟；打开电脉冲仪，按设定的转化程序进行电激(2.0kV)；向电脉冲杯内迅速加入1mL预冷的无菌YPG培养基，混匀后，转入试管中，30℃缓慢振荡培养2小时后，涂布到加有适当抗生素的选择平板上，30℃倒置培养48小时，获得含有重组质粒pBBR1-Pacn-aal的Acetobacter pasteurianus CGMCC 3089基因工程菌。
含有重组质粒pBBR1-Pacn-aal的改善食醋风味的醋酸菌耐酸性检测：
培养基组成:酵母膏15g/L，葡萄糖20g/L，天冬氨酸浓度为0.5g/L，其余为水。在不含乙醇的培养基中添加1％(v/v)醋酸浓度，分别接种相同的含有重组质粒pBBR1-Pacn-aal的醋酸菌和原始菌株，调整初始OD 600一致后进行培养，分别于24h、48h取样比较两者生物量的变化，表10为原始菌株和重组表达天冬氨酸解氨酶菌株醋酸耐受性的比较。
表10
Figure PCTCN2022135751-appb-000009
注：表格中的每个数值表示为mean±S.D.(n＝3)。采用邓肯多重方差分析评估显著差异。同列中不同字母表示具有显著性差异(P<0.001)。
同时选取1％的醋酸浓度、发酵48h条件下取样的菌体，采用水杨酸分光光度法对菌体胞内铵离子含量进行测定。使用1×PBS缓冲液洗涤菌体后添加高效RIPA组织/细胞快速裂解液进行超声破碎，离心取上清液。取8mL上清液样品于10mL比色管中，加入1mL显色剂(水杨酸-酒石酸钾钠溶液)、2滴亚硝基铁氰化钠(0.01g/mL)和2滴次氯酸钠(有效氯3.5g/L、游离碱浓度0.75mol/L)使用液，加水稀释至标线，混匀后显色60min。以水为空白参比，用10mm比色皿检测在697nm处吸光度。
氨氮标准曲线绘制：配制0、1、2、3、4、5μg/mL标准使用液，按照上述步骤测量吸光度，绘制氨氮标准曲线。表11为原始菌株和重组表达天冬氨酸解氨酶菌株胞内铵离子浓度的比较。
表11
Figure PCTCN2022135751-appb-000010
注：表格中的每个数值表示为mean±S.D.(n＝3)。采用邓肯多重方差分析评估显著差异。同列中不同字母表示具有显著性差异(P<0.001)。
含有重组质粒pBBR1-Pacn-aal的改善食醋风味的醋酸菌中ATP含量检测：
培养基组成:酵母膏15g/L，葡萄糖20g/L，天冬氨酸浓度为0.5g/L，其余为水。经培养后分别取1mL原始菌株和重组菌株发酵液，离心收集菌体，使用1×PBS缓冲液洗涤菌体后添加高效RIPA组织/细胞快速裂解液进行超声破碎，离心取上清作为待测样本。采用ATP的ELISA试剂盒，用酶标仪(450nm)检测ATP的浓度。
具体操作步骤如下：
①从冰箱中将试剂盒取出放置在室温下平衡20min。然后从铝箔袋中取出所需板条，剩余板条用自封袋密封放回4℃；
②设置标准品孔和样本孔，选取所需的标准品浓度，然后向标准品孔中分别加入不同浓度的标准品50μL；本实验中ATP标准品浓度选用0、250、500、1000、2000μmol/mL
③取待测样本10μL。
④样本孔中先加待测样本10μL，再加样本稀释液40μL。空白孔不加；
⑤除空白孔外，标准品孔和样本孔中每孔加入辣根过氧化物酶(HRP)标记的检测抗体100μL，用封板膜封住反应孔，37℃水浴锅或恒温箱温育60min；
⑥弃去液体，吸水纸上拍干，每孔加满洗涤液，静置1min，甩去洗涤液，吸水纸上拍干，如此重复洗板5次(也可用洗板机洗板)；
⑦每孔加入底物A、B各50μL，37℃避光孵育15min；
⑧每孔加入终止液50μL，15min内，在450nm波长处测定各孔的OD值。然后绘制标准曲线：以标准品浓度为横坐标，对应OD值为纵坐标，绘制出标准 品线性回归曲线，可得ATP:Y＝0.0005x+0.0697，然后按照曲线方程计算各样本浓度值。
图4为含有重组质粒pBBR1-Pacn-aal的Acetobacter pasteurianus CGMCC 3089基因工程菌和原始菌株Acetobcter pasteurianus CGMCC 3089 ATP含量比较图。
利用含有重组质粒pBBR1-Pacn-aal的醋酸菌液态深层发酵生产苹果醋：
(1)制备种子液：分别从斜面取含有重组质粒pBBR1-Pacn-aal的Acetobacter pasteurianus CGMCC 3089基因工程菌和原始菌株于种子培养基中，在30℃，160转/分钟条件下摇床培养25小时。按10％(v/v)的接种量转接入新鲜的种子培养基中进行放大培养。种子培养基组成:酵母膏15g/L，葡萄糖20g/L，乙醇3.5％(v/v)，天冬氨酸浓度为0.5g/L，其余为水。
(2)酒精发酵：取适量预处理过的果汁加热至30℃后撒入活性干酵母，搅拌并保温30min，待酵母活动正常后接入到果汁中，搅拌均匀后在25℃进行酒精发酵，发酵约7d。
(3)醋酸发酵：按10％(v/v)的接种量，分别将含有重组质粒pBBR1-Pacn-aal的Acetobacter pasteurianus CGMCC 3089基因工程菌和原始菌株接种至含有苹果酒的发酵罐中，在30℃条件下进行苹果醋发酵。苹果酒中乙醇含量为8％(v/v)。
分别利用原始Acetobacter pasteurianus CGMCC 3089和含有重组质粒pBBR1-Pacn-aal的Acetobacter pasteurianus CGMCC 3089基因工程菌进行液态深层发酵产苹果醋，在30℃，通风量0.15vvm条件下，原始菌株73h发酵结束，初始醋酸浓度10g/L，醋酸终浓度为85g/L，则乙醇转酸率约为91.1％，平均产酸速率约为1.02g/(L·h)。而重组菌株69h发酵结束，醋酸终浓度为90g/L，乙醇转酸率约为96.0％，平均产酸速率约为1.16g/(L·h)。
图5为分别利用Acetobcter pasteurianus CGMCC 3089和含有重组质粒pBBR1-Pacn-aal的Acetobcter pasteurianus CGMCC 3089基因工程菌液态深层发酵产苹果醋的发酵过程曲线。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (7)

  1. 一种改善食醋风味的醋酸菌，其特征在于，采用下述方案：
    以巴氏醋杆菌(Acetobacter pasteurianus)为宿主细胞，巴氏醋杆菌(Acetobacter pasteurianus)中具有协同效果的乙醇胺解氨酶的启动子协同表达酪氨酸解氨酶得到；启动子的序列号为SEQ ID No.1，酪氨酸解氨酶的序列号为SEQ ID No.2；或者
    以巴氏醋杆菌(Acetobacter pasteurianus)为宿主细胞，巴氏醋杆菌(Acetobacter pasteurianus)三羧酸循环中顺乌头酸酶的启动子协同表达天冬氨酸解氨酶得到；启动子的序列号为SEQ ID No.3，天冬氨酸解氨酶的序列号为SEQ ID No.4。
  2. 一种权利要求1所述的改善食醋风味的醋酸菌的构建方法，其特征在于，表达酪氨酸解氨酶时，包括步骤：利用PCR的方法获得来源于巴氏醋杆菌中的乙醇胺解氨酶的启动子，并将其连接到可在醋酸菌中稳定复制的质粒pBBR1p452中，获得重组质粒pBBR1-Peut；利用基因合成的方法获得来源于粘(深)红酵母(Rhodotorula glutinis)中的酪氨酸解氨酶基因tal(GenBank:KF765779.1)，并将其连接到可在醋酸菌中稳定复制的质粒pBBR1-Peut中，获得重组质粒pBBR1-Peut-tal；将重组质粒pBBR1-Peut-tal转入醋酸菌中即可获得利用乙醇胺解氨酶启动子控制的重组表达酪氨酸解氨酶的改善食醋风味的醋酸菌。
  3. 根据要求2所述的改善食醋风味的醋酸菌的构建方法，其特征在于，具体包括下述步骤：
    (1)乙醇胺解氨酶启动子Peut序列的获得和重组质粒pBBR1-Peut的构建:
    ①设计引物Peut-1和Peut-2，PCR扩增巴氏醋杆菌中乙醇胺解氨酶启动子Peut序列，为了便于重组质粒的构建，引物Peut-1上引入BamH I酶切位点，引物Peut-2上引入Spe I酶切位点，如下所示：
    引物名称 引物序列 酶切位点 Peut-1 5'-CGCGGATCCCGTTCAGGTTCAGCATATCTGTG-3' BamH I Peut-2 5'-AGCACTAGTTGAAGGAACTCCTTAGCTGCG-3' Spe I
    ②以巴氏醋杆菌(Acetobacter pasteurianus)基因组为模板，利用引物Peut-1和Peut-2进行PCR，获得巴氏醋杆菌中乙醇胺解氨酶启动子序列Peut；利用基因测序反应进行乙醇胺解氨酶启动子序列的验证；
    ③将质粒pBBR1p452和乙醇胺解氨酶启动子序列Peut分别用限制性内切酶 BamH I和Spe I处理,37℃，2-12小时,纯化回收，将酶切纯化后的质粒和乙醇胺解氨酶启动子序列按物质的量之比1:0.2-5混合，利用T 4DNA连接酶进行连接反应,14-16℃，4-12小时,将连接产物转入大肠杆菌DH 5α感受态中，获得重组质粒pBBR1-Peut,利用基因测序反应进行重组质粒pBBR1-Peut的序列验证,
    (2)酪氨酸解氨酶基因tal序列的获得和重组质粒pBBR1-Peut-tal的构建:
    利用基因合成的方法获得来源于粘(深)红酵母(Rhodotorula glutinis)中的酪氨酸解氨酶基因tal(GenBank:KF765779.1)，并将其连接到可在醋酸菌中稳定复制的质粒pBBR1-Peut中，获得重组质粒pBBR1-Peut-tal；
    (3)重组表达酪氨酸解氨酶改善食醋风味的醋酸菌的获得:
    利用电激转化方法，将验证正确的质粒pBBR1-Peut-tal转入醋酸菌中，涂布到加有适当卡那霉素的选择平板上，30℃倒置24-60小时，获得含有重组质粒pBBR1-Peut-tal的改善食醋风味的醋酸菌。
  4. 一种权利要求1所述的改善食醋风味的醋酸菌的构建方法，其特征在于，表达天冬氨酸解氨酶时，包括下述步骤：利用PCR的方法获得来源于巴氏醋杆菌中的顺乌头酸酶的启动子，并将其连接到可在醋酸菌中稳定复制的质粒pBBR1p452中，获得重组质粒pBBR1-Pacn；利用PCR的方法获得来源于大肠杆菌Escherichia coli中的天冬氨酸解氨酶基因aal，并将其连接到质粒pBBR1-Pacn上，获得重组质粒pBBR1-Pacn-aal；将重组质粒pBBR1-Pacn-aal转入醋酸菌中即可获得利用顺乌头酸酶启动子控制的重组表达天冬氨酸解氨酶的改善食醋风味的醋酸菌。
  5. 根据要求4所述的改善食醋风味的醋酸菌的构建方法，其特征在于，具体包括下述步骤：
    (1)顺乌头酸酶启动子Pacn序列的获得和重组质粒pBBR1-Pacn的构建：
    ①设计引物Pacn-1和Pacn-2，PCR扩增巴氏醋杆菌中顺乌头酸酶启动子Pacn序列，为了便于重组质粒的构建，引物Pacn-1上引入BamH I酶切位点，引物Pacn-2上引入Spe I酶切位点，如下所示：
    引物名称 引物序列 酶切位点 Pacn-1 5'-CGCGGATCCAATCGCTCTCTCCCCGATC-3' BamH I Pacn-1 5'-AGCACTAGTGCTGGTGCTGGATGGTGTC-3' Spe I
    ②以巴氏醋杆菌(Acetobacter pasteurianus)基因组为模板，利用引物 Pacn-1和Pacn-2进行PCR，获得巴氏醋杆菌中顺乌头酸酶启动子序列Pacn；利用基因测序反应进行顺乌头酸酶启动子序列的验证；
    ③将质粒pBBR1p452和顺乌头酸酶启动子序列Pacn分别用限制性内切酶BamH I和Spe I处理，37℃，2-12小时，纯化回收，将酶切纯化后的质粒和顺乌头酸酶启动子序列按物质的量之比1:0.2-5混合，利用T 4DNA连接酶进行连接反应，14-16℃，4-12小时，将连接产物转入大肠杆菌DH 5α感受态中，获得重组质粒pBBR1-Pacn；利用基因测序反应进行重组质粒pBBR1-Pacn的序列验证；
    (2)天冬氨酸解氨酶aal序列的获得和重组质粒pBBR1-Pacn-aal的构建：
    ①设计引物aal-1和aal-2，PCR扩增大肠杆菌中天冬氨酸aal序列，为了便于重组质粒的构建，引物aal-1上引入EcoR V酶切位点，引物aal-2上引入酶切位点Cla I，如下所示：
    引物名称 引物序列 酶切位点 aal-1 5'-TCGATATCATGTCAAACAACATTCGTATCGAAG-3' EcoR V aal-2 5'-GTATCGATTTACTGTTCGCTTTCATCAGTATAGCG-3' Cla I
    ②以大肠杆菌(Escherichia coli)基因组为模板，利用引物aal-1和aal-2进行PCR，获得大肠杆菌中天冬氨酸解氨酶的序列aal；利用基因测序反应进行天冬氨酸序列的验证；
    ③将重组质粒pBBR1-Pacn和天冬氨酸解氨酶基因序列aal分别用限制性内切酶EcoR V和Cla I处理，37℃，2-12小时，纯化回收，将酶切纯化后的质粒和天冬氨酸序列按物质的量之比1:0.2-5混合，利用T 4DNA连接酶进行连接反应，14-16℃，4-12小时，将连接产物转入大肠杆菌DH 5α感受态中，获得重组质粒pBBR1-Pacn-aal；利用基因测序反应进行重组质粒pBBR1-Pacn-aal的序列验证；
    (3)重组表达天冬氨酸解氨酶改善食醋风味的醋酸菌的获得：利用电激转化方法，将验证正确的质粒pBBR1-Pacn-aal转入醋酸菌中，涂布到加有适当卡那霉素的选择平板上，30℃倒置24-60小时，获得含有重组质粒pBBR1-Pacn-aal的改善食醋风味的醋酸菌。
  6. 权利要求1所述一种改善食醋风味的醋酸菌在食醋发酵中的应用。
  7. 如权利要求6所述的应用，其特征在于，是在食醋固态发酵或液态发酵中的应用。
PCT/CN2022/135751 2021-12-04 2022-12-01 改善食醋风味的醋酸菌及其构建方法 WO2023098774A1 (zh)

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