Protein for improving acetic acid fermentation efficiency and coding gene and application thereof

Abstract

The invention provides a protein capable of improving acetic acid fermentation efficiency and a coding gene and application thereof, and relates to the technical field of fermentation engineering, and the protein composed of an amino acid sequence as shown in SEQ ID No: 2 has a quorum sensing quenching function. The protein obtained by replacing the 67th amino acid residue of the amino acid as shown in SEQ ID No: 2 from S into C through molecular docking and alanine scanning technologies can further improve the quenching effect of quorum quenching enzyme, so that the adverse effect of quorum sensing phenomenon in acetic acid bacteria on acetic acid fermentation can be weakened; acetobacter growth is promoted, and acetic fermentation efficiency and conversion rate are improved. The technical problem that quenching enzyme capable of weakening the quorum sensing phenomenon in acetic fermentation is lacked in the prior art is solved.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, and in particular to a protein for improving acetic acid fermentation efficiency, a coding gene thereof and an application thereof. Background Art

[0002] Vinegar is one of the most consumed condiments in the world. Its main component is acetic acid, which is mainly produced by fermentation. The fermentation of acetic acid is the process of ethanol being oxidized to acetic acid by aerobic respiration under the action of acetic acid bacteria. Among them, ethanol is oxidized to acetaldehyde under the catalysis of alcohol dehydrogenase (ADH). Subsequently, acetaldehyde is oxidized to acetic acid under the action of aldehyde dehydrogenase (ALDH).

[0003] Acetic acid bacteria (AAB) is a type of strictly aerobic Gram-negative bacteria, one of the most important characteristics of which is the ability to oxidize ethanol to acetic acid. The most common acetic acid bacteria include: Acetobacter, Gluconobacter and Gluconacetobacter, among which Acetobacter and Gluconobacter are widely used in the fermentation production of acetic acid due to their high ethanol oxidation and acetic acid tolerance. In the process of acetic acid fermentation, bacterial concentration, acetic acid concentration, ethanol concentration, etc. are all important factors affecting the efficiency of acetic acid fermentation and the rate of acid production. Therefore, improving the fermentation efficiency of acetic acid bacteria is of great significance to the acetic acid fermentation industry.

[0004] Quorum sensing (QS) is a signaling pathway that is closely related to population density and can communicate between cells. Its main function is to regulate important processes in cells, such as the production of virulence factors or the formation of biofilms. With the relevant research on the acid production mechanism of acetic acid bacteria, it was found that quorum sensing exists in a few acetic acid bacteria, such as Gluconacetobacter diazotrophicus, Gluconacctobacter xylinus, and Gluconactobacter sp. QS has a very adverse effect on the growth of acetic acid bacteria and acetic acid fermentation. Mainly, QS signal molecules (N-acylated homoserine lactone, AHL) can be recognized by the QS receptors of bacteria, thereby initiating some regulatory mechanisms, inhibiting the expression of related proteins, resulting in regulated bacterial growth, and no increase in the concentration of related products. Studies have found that quorum quenching enzymes can reduce the concentration of signal molecules by degrading themselves or other bacteria, thereby failing to activate the transcription of receptor proteins, thereby inhibiting adverse factors, such as AiiA in Bacillus spp.240B1 and GqqA in Komagataeibacter europaeus. Therefore, weakening the quorum sensing phenomenon of acetic acid bacteria during acetic acid fermentation may promote the growth of acetic acid bacteria and improve the efficiency of acetic acid fermentation, but there are still few quorum quenching enzymes reported for acetic acid bacteria.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a protein to at least solve one of the technical problems existing in the prior art.

[0007] A second object of the present invention is to provide a nucleic acid molecule encoding the above-mentioned protein.

[0008] A third object of the present invention is to provide a recombinant vector containing the above-mentioned nucleic acid molecule.

[0009] A fourth object of the present invention is to provide a recombinant bacterium.

[0010] A fifth object of the present invention is to provide a method for constructing a recombinant bacterium.

[0011] A sixth object of the present invention is to provide the use of the above-mentioned recombinant bacteria or the recombinant bacteria constructed by the above-mentioned construction method in improving the efficiency of acetic acid fermentation.

[0012] A seventh object of the present invention is to provide a method for improving acetic acid fermentation efficiency.

[0013] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0014] In a first aspect, the present invention provides a protein, wherein the protein is as follows (a1) or (a2):

[0015] (a1) a protein consisting of the amino acid sequence shown in SEQ ID No: 2;

[0016] (a2) A protein obtained by replacing the 67th amino acid residue of the amino acid sequence shown in SEQ ID No: 2 from S to C.

[0017] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned protein.

[0018] Furthermore, the nucleotide sequence of the nucleic acid molecule is as follows (b1) or (b2):

[0019] (b1) a DNA molecule consisting of the nucleotide sequence shown in SEQ ID No: 1;

[0020] (b2) A DNA molecule consisting of the nucleotide sequence shown in SEQ ID No:3.

[0021] In a third aspect, the present invention provides a recombinant vector containing the above-mentioned nucleic acid molecule.

[0022] In a fourth aspect, the present invention provides a recombinant bacterium, wherein the recombinant bacterium comprises the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector, or expresses the above-mentioned protein.

[0023] In a fifth aspect, the present invention provides a method for constructing a recombinant bacterium, comprising integrating the above-mentioned nucleic acid molecule into the genome of a host bacterium;

[0024] or introducing the above-mentioned nucleic acid molecule or the recombinant vector according to claim 4 into a host bacterium;

[0025] Preferably, the host bacteria include any one of Acetobacter, Komabacter, Gluconobacter or Gluconacetobacter;

[0026] Preferably, the Acetobacter includes any one of Acetobacter pasteurianus, Acetobacter occidentalis, Acetobacter aceti or Acetobacter oxydans, preferably Acetobacter pasteurianus.

[0027] In a sixth aspect, the present invention provides the use of the above-mentioned recombinant bacteria or the recombinant bacteria constructed by the above-mentioned construction method in improving the efficiency of acetic acid fermentation;

[0028] Preferably, the acetic acid fermentation includes producing acetic acid by fermenting ethanol as raw material, producing vinegar by fermenting wine mash containing ethanol as raw material, or producing vinegar by solid-state fermentation method and liquid-state fermentation method.

[0029] In a seventh aspect, the present invention provides a method for improving acetic acid fermentation efficiency, comprising subjecting the above-mentioned recombinant bacteria or the recombinant bacteria constructed by the above-mentioned construction method to acetic acid fermentation;

[0030] Preferably, the fermentation ends when the ethanol concentration of the fermentation liquid is less than 10 g / L and the acidity does not change any more. Preferably, the fermentation ends when the ethanol concentration of the fermentation liquid is less than 5 g / L and the acidity does not change any more.

[0031] Furthermore, when liquid fermentation is performed, the inoculation amount of the recombinant bacteria is 5-20% (v / v), preferably 10% (v / v);

[0032] Preferably, the fermentation temperature is 27-35°C, preferably 30°C;

[0033] Preferably, the ventilation rate of the fermentation is 1:0.05-0.25 (v / v) / min, preferably 1:0.1 (v / v) / min;

[0034] Preferably, the liquid fermentation method includes batch fermentation, fed-batch fermentation or split fermentation;

[0035] Preferably, the fermentation time of the batch fermentation is 24 to 160 hours;

[0036] Preferably, ethanol is continuously added during the fed-batch fermentation, the ethanol concentration in the fermentation broth is 10 to 40 g / L, and the fermentation time is 60 to 150 hours;

[0037] Preferably, in the split fermentation, 20-50% (v / v) of the fermentation liquid is taken out each time, and the same volume of fresh culture medium is added, and the fermentation time is 24-100 hours.

[0038] Furthermore, during solid fermentation, the inoculation amount of the recombinant bacteria is 5-20% (v / v), preferably 10% (v / v);

[0039] Preferably, the fermentation temperature is 25-30°C, preferably 30°C;

[0040] Preferably, the fermentation process also includes turning the mash once every 24 hours.

[0041] The protein for improving acetic acid fermentation efficiency provided by the present invention has been experimentally proved to have a quorum sensing quenching function. The protein obtained by replacing the 67th amino acid residue of the amino acid shown in SEQ ID NO: 2 with S to C through molecular docking and alanine scanning technology can further improve the quenching effect of the quorum quenching enzyme, thereby reducing the adverse effect of the quorum sensing phenomenon in acetic acid bacteria on acetic acid fermentation, thereby promoting the growth of acetic acid bacteria and improving the acetic acid fermentation efficiency and conversion rate. The technical problem of the lack of a quenching enzyme that can weaken the quorum sensing phenomenon in acetic acid fermentation in the prior art is solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 The amino acid sequence comparison result of the GqqA protein and the 0046 protein provided in Example 1 of the present invention;

[0044] Figure 2 A phylogenetic tree diagram of the 0046 protein provided in Example 1 of the present invention;

[0045] Figure 3 The 0046 protein Motif analysis diagram provided in Example 1 of the present invention;

[0046] Figure 4 This is a diagram of the quorum sensing phenomenon of acetic acid bacteria verified by the liquid color development method provided in Example 1 of the present invention;

[0047] Figure 5 The positive control plasmid PBBR1-P provided in Example 1 of the present invention 452 - Double-enzyme-digested agarose gel electrophoresis of AiiA and 0046 protein overexpression plasmid construction;

[0048] Figure 6 The functional diagram of protein 0046 verified by the liquid colorimetric method provided in Example 1 of the present invention;

[0049] Figure 7 A three-dimensional structural model of the 0046 protein homology modeling in Example 2 of the present invention;

[0050] Figure 8 This is a docking diagram of the 0046 protein and the ligand molecule in Example 2 of the present invention;

[0051] Fig. 9 Molecular docking of Example 2 of the present invention Schematic diagram of the main amino acids in the range;

[0052] Fig.10 This is a double-enzyme-digested agarose gel electrophoresis diagram of the M0046 protein expression plasmid constructed in Example 2 of the present invention;

[0053] Fig.11 This is an agarose gel electrophoresis diagram of the CRISPR system constructed in Example 4 of the present invention;

[0054] Fig.12 This is the PCR image of the colonies of the positive transformants of the integrated 0046 protein and the M0046 protein in Example 4 of the present invention. DETAILED DESCRIPTION

[0055] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0056] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0057] The present invention provides a protein, wherein the protein is as follows (a1) or (a2):

[0058] (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0059] (a2) A protein obtained by replacing the 67th amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 from S to C.

[0060] Experiments have shown that the protein composed of the amino acid sequence shown in SEQ ID NO:2 has a quorum sensing quenching function, and the protein obtained by replacing the 67th amino acid residue of the amino acid shown in SEQ ID NO:2 from S to C through molecular docking and alanine scanning technology can further improve the quenching effect of the quorum quenching enzyme, thereby weakening the adverse effect of the quorum sensing phenomenon in acetic acid bacteria on acetic acid fermentation, thereby promoting the growth of acetic acid bacteria and improving the efficiency and conversion rate of acetic acid fermentation. The technical problem of the lack of a quenching enzyme that can weaken the quorum sensing phenomenon in acetic acid fermentation in the prior art is solved.

[0061] According to another aspect of the present invention, a nucleic acid molecule encoding the above protein is also provided.

[0062] In some specific embodiments, the nucleotide sequence of the nucleic acid molecule is such as (b1) or (b2):

[0063] (b1) a DNA molecule consisting of the nucleotide sequence shown in SEQ ID No: 1;

[0064] (b2) A DNA molecule consisting of the nucleotide sequence shown in SEQ ID No:3.

[0065] According to another aspect of the present invention, a recombinant vector containing the above nucleic acid molecule is also provided.

[0066] The vector includes but is not limited to pBBR1MCS-2, pBBR1MCS-4, pMV24 or pGRB, and may also be other vectors suitable for expressing 0046 protein or M0046 protein.

[0067] According to another aspect of the present invention, a recombinant bacterium is provided, characterized in that the recombinant bacterium contains the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector, or expresses the above-mentioned protein.

[0068] According to another aspect of the present invention, a method for constructing a recombinant bacterium is also provided, characterized in that it comprises integrating the above-mentioned nucleic acid molecule into the genome of the host bacterium;

[0069] Or the above nucleic acid molecule or the above recombinant vector is introduced into the host bacteria.

[0070] In some specific embodiments, the host bacteria include any one of Acetobacter, Komibacter, Gluconobacter or Gluconacetobacter.

[0071] In some specific embodiments, the Acetobacter comprises any one of Acetobacter pasteurianus, Komagataeibacter europaeus, Acetobacter aceti or Gluconacetobacter oxydans, preferably Acetobacter pasteurianus.

[0072] Specific Acetobacter pasteurianus is preferably Acetobacter pasteurianus CGMCC 3089 or Acetobacter pasteurianus NBRC 3283.

[0073] According to another aspect of the present invention, there is also provided the use of the above-mentioned recombinant bacteria or the recombinant bacteria constructed by the above-mentioned construction method in improving the efficiency of acetic acid fermentation.

[0074] Among them, acetic acid fermentation includes the production of acetic acid by fermentation with ethanol as raw material, the production of vinegar by fermentation of wine mash containing ethanol as raw material, or the production of vinegar by solid fermentation and liquid fermentation.

[0075] According to another aspect of the present invention, a method for improving acetic acid fermentation efficiency is provided, characterized in that it comprises subjecting the above-mentioned recombinant bacteria or the recombinant bacteria constructed by the above-mentioned construction method to acetic acid fermentation.

[0076] Specifically, one situation includes propagating the genetically engineered acetic acid bacteria that overexpresses the M0046 protein or 0046 protein that can improve the acetic acid fermentation efficiency with a plasmid in a culture medium, and then using the propagated acetic acid bacteria for acetic acid fermentation. Another situation includes propagating the genetically engineered acetic acid bacteria that overexpresses the M0046 protein or 0046 protein that can improve the acetic acid fermentation efficiency in the genome through CRISPR-Cas9 technology in a culture medium, and then using the propagated acetic acid bacteria for acetic acid fermentation.

[0077] In some specific embodiments, the fermentation ends when the ethanol concentration of the fermentation liquid is less than 10 g / L and the acidity does not change any more. Preferably, the fermentation ends when the ethanol concentration of the fermentation liquid is less than 5 g / L and the acidity does not change any more.

[0078] When liquid fermentation is performed, the specific fermentation process includes the following steps:

[0079] (1) Preparation of culture medium: The culture medium must contain the nutrients required for microbial growth, such as carbon sources such as glucose or ethanol, nitrogen sources such as urea, ammonium salts, yeast extract or yeast powder, and phosphates (phosphorus sources) and sulfates (sulfur sources). In addition, metal ions such as sodium, potassium, magnesium, calcium, zinc, iron, manganese, copper, cobalt, boron and molybdenum are also required, and the content of each metal ion is approximately 0.001 mg / L to 500 mg / L; the ethanol concentration in the culture medium is 30 to 200 g / L.

[0080] (2) Seed culture: Use a 250-1000 mL shake flask filled with 30-100 mL of the above-mentioned culture medium, inoculate the recombinant bacteria or the control bacteria for shake flask culture at a shaker speed of 100-300 rpm, a temperature of 27-30° C., and a culture time of 20-30 hours to prepare a seed solution.

[0081] (3) Acetic acid fermentation: Acetic acid fermentation can be carried out in a fermenter with an inoculation rate of 5-20% (v / v) and a temperature of 27-35°C. During the fermentation process, air is introduced into the fermenter at a ventilation rate of 1:0.05-0.25 v / v (volume ratio of liquid to gas) per minute. The fermentation method can be batch fermentation, fed-batch fermentation or split fermentation.

[0082] At the end of batch fermentation, the acetic acid concentration can reach 50-150 g / L, and the fermentation time is 24-160 hours.

[0083] During the fed-batch fermentation, ethanol is continuously added to control the ethanol concentration in the fermentation liquid to be 10-40 g / L. The acetic acid concentration can reach 80-200 g / L at the end of the fermentation, and the fermentation time is 60-150 hours.

[0084] In split fermentation, 20-50% (volume ratio) of the fermentation liquid is taken out each time and the same volume of fresh culture medium is added. The acetic acid concentration at the end of each batch of fermentation can reach 50-150 g / L, and the fermentation time is 24-100 hours.

[0085] When solid fermentation is performed, the specific fermentation process includes the following steps:

[0086] (1) Preparation of culture medium: The culture medium must contain the nutrients required for microbial growth, such as carbon sources such as glucose or ethanol, nitrogen sources such as urea, ammonium salts, yeast extract or yeast powder, and phosphates (phosphorus sources) and sulfates (sulfur sources). In addition, metal ions such as sodium, potassium, magnesium, calcium, zinc, iron, manganese, copper, cobalt, boron and molybdenum are also required, and the content of each metal ion is approximately 0.001 mg / L to 500 mg / L; the ethanol concentration in the culture medium is 30 to 200 g / L.

[0087] (2) Seed culture: Use a 250-1000 mL shake flask filled with 30-100 mL of the above-mentioned culture medium, inoculate the recombinant bacteria or the control bacteria for shake flask culture at a shaker speed of 100-300 rpm, a temperature of 27-30° C., and a culture time of 20-30 hours to prepare a seed solution.

[0088] (3) Solid-state acetic acid fermentation: Acetic acid fermentation can be carried out in a solid-state fermentation tank. The inoculation amount of the fermentation tank is 5%-20% (volume ratio). Bran and rice husk are added to the mash according to the ratio of mash: bran: rice husk = 5-5.5:1-1.5:0.5-1 and stirred evenly for acetic acid fermentation. The fermentation temperature is started at 25-30°C, and the mash is turned once a day.

[0089] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0090] Example 1 Acquisition and functional analysis of 0046 protein

[0091] Functional analysis of gene 0046 in Acetobacter pasteurianus CGMCC 3089.

[0092] 1. Compare the CDS sequence of the acetic acid bacteria genome with the typical QS system and key regulatory genes in bacteria, and obtain the amino acid sequence of the 0046 protein gene sequence from the acetic acid bacteria genome. The specific results are as follows Figure 1 shown.

[0093] The 0046 protein gene sequence is shown in SEQ ID No: 1;

[0094] The amino acid sequence of the 0046 protein is shown in SEQ ID No: 2;

[0095] By comparing the amino acid sequence of the 0046 protein in Acetobacter pasteurianus CGMCC 3089, it was found that its amino acid sequence similarity with the GqqA protein (AHL acylase) in Komagataeibacter europaeus was only 62.5%.

[0096] Functional analysis of 2.0046 protein

[0097] (1) Using Mega software, we summarized the phylogenetic diversity of some currently known biochemically characterized quorum quenching enzymes (AttM (Q7D3U0.3), AiiA (P0CJ63), AhlK (A0A098CCK3), AhlS (SCV20999.1), AhlD (AAP57766.1), AidC (I7HB71), GqqA (KOEU_05990), QsdR1 (ACP23138.1), QuiP (Q9I4U2), SisLAc (C4KKZ9), SsoPox (Q97VT7)), and attempted to deduce the evolutionary history of 0046 protein. We found that 0046 protein and GqqA protein have a high degree of genetic relationship and belong to the same PDT type of hydrolase. The specific results are as follows: Figure 2 shown.

[0098] (2) The motif of 0046 protein was analyzed using the MEME (https: / / meme-suite.org / meme / tools / meme) website. It was found that 0046 protein had the same motif as GqqA protein and contained the same PDT and ACT domains as GqqA protein. It was thus inferred that 0046 protein had the same quorum quenching function. The specific results are as follows: Figure 3 shown.

[0099] 3 Verification of quorum sensing of acetic acid bacteria by liquid colorimetric method

[0100] AHLs signal molecules are produced in the quorum sensing phenomenon, while the indicator bacteria (Agrobacterium tumefaciens) do not produce signal molecules themselves, but they contain AHLs inducible promoters. Therefore, when AHLs signal molecules are present, the indicator bacteria can activate the expression of the reporter gene lacZ to produce β-galactosidase, which can then hydrolyze X-Gal, turning it from colorless to blue. Therefore, this method was selected to verify the quorum sensing phenomenon in acetic acid bacteria. The specific steps are as follows:

[0101] (1) Signal molecule extraction: The strains to be tested were cultured to OD 660 When the pH reaches about 1, centrifuge at 5000r / min for 15min to remove the bacteria, extract the supernatant with an equal amount of ethyl acetate (containing 0.5% formic acid), remove the emulsified phase by ultrasound, take the organic phase, and repeat the above operation twice with the remaining aqueous phase, and mix the organic phases extracted three times. Then place the extract in a rotary evaporator and evaporate it to dryness. Then dissolve the evaporated evaporating bottle with anhydrous ethanol, dilute to 2mL to obtain the crude extract of the signal molecule, and store at -20℃ for later use.

[0102] (2) Acetic acid bacteria quorum sensing verification

[0103] Acetobacter pasteurianus CGMCC 3089 was cultured in YPG and GYEA medium, respectively, and Pseudomonas aeruginosa PAO1 (positive control strain) was cultured in LB medium. Then, the crude extract of the signal molecule was extracted according to the above step (1) and added to the liquid test tube culture medium of Agrobacterium tumefaciens A136 (indicator bacteria) containing 40 μg / mL X-Gal. For specific results, see Figure 4 , wherein the concentrations of crude extracts of signal molecules in 1 to 4 are 0 μL / mL, 20 μL / mL, 50 μL / mL and 100 μL / mL, respectively; (A) is crude extracts of signal molecules of Pseudomonas aeruginosa PAO-1 with different doses added; (B) is crude extracts of signal molecules of acetic acid bacteria (YPG medium) with different doses added; (C) is crude extracts of signal molecules of acetic acid bacteria (GYEA medium) with different doses added.

[0104] Depend on Figure 4 It can be seen that the addition of crude extracts of Acetobacter pasteurianus CGMCC3089 signal molecules from YPG and GYEA culture media can cause the indicator bacteria to produce β-galactosidase, which can then hydrolyze X-Gal, changing it from colorless to blue. This result is consistent with the positive control strain. Therefore, the results prove that Acetobacter pasteurianus CGMCC 3089 has quorum sensing.

[0105] 4. Verify the function of 0046 protein by liquid colorimetric method.

[0106] (1) Positive strain (pBBR1-P 452 - Construction of AiiA

[0107] Using the Bacillus thuringiensis genome as a template, primers AiiA-F and AiiA-R were used for PCR reaction to amplify the AiiA gene fragment. The PCR reaction system is shown in Table 1.

[0108] Table 1

[0109] Components Volume (μl) Template DNA 2 AiiA-F (20 μmol / L) 2 AiiA-R (20 μmol / L) 2 Phanta (5U / μl) 25 dd H2O 19

[0110] AiiA-F:5'-CC ATCGAT ATGACAGTAAAGAAGCTTTATTCCATCC-3'; (the underlined part is the Cla I restriction site) (SEQ ID No: 5);

[0111] AiiA-R:5'-CC CTCGAG CTATATATACCCTGGGAACACTCTACAAC-3'; (the underlined part is the Xho I restriction site) (SEQ ID No: 6);

[0112] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 61°C for 20 s, 72°C for 50 s, and 72°C for 10 min after 26 cycles.

[0113] The AiiA gene fragment obtained above and the plasmid pBBR1-P obtained by introducing the constitutive promoter gox0452 from Gluconobacter oxydans into pBBR1MCS-2 were 452 The plasmid was treated with restriction endonucleases Xho I and Cla I (37°C, 2 hours), and the target fragments were recovered using a PCR product purification kit (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.). The plasmid after restriction digestion and the AiiA gene fragment were ligated at a ratio of 1:3 (molar ratio) (16°C, 12 hours); the ligation product pBBR1-P 452 -AiiA recombinant plasmid was transformed into Escherichia coli BL21, and pBBR1-P 452 -AiiA recombinant plasmid strain E.coli BL21 / pBBR1-P 452 -AiiA. Plasmid double restriction enzyme electrophoresis verification Figure 5 -(A) shows plasmid pBBR1-P 452 -Agarose gel electrophoresis of AiiA double digestion products.

[0114] Among them, plasmid pBBR1-P 452The construction method adopts the expression vector construction method disclosed in the publicly published "Construction of expression vectors for protein production in Gluconobacter oxydans" (Kallnik, V., Meyer, M., Deppenmeier, U., Journal of biotechnology & Schweiger, P. (2010). 150 (4), 460-465.).

[0115] (2) Overexpression plasmid pBBR1-P 452 -0046 Construction

[0116] The genome of Acetobacter pasteurianus CGMCC 3089 (purchased from China National Center for Microbiological Culture Collection, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101) was used as a template, and PCR reaction was performed using primers 0046-F and 0046-R to amplify the 0046 gene fragment (shown in SEQ ID No: 1);

[0117] 0046-F:5'-CC CTCGAG ATGACACAAAAGGTTATCGCCTT-3'; (the underlined part is the Xho I restriction site); (SEQ ID No: 7) 0046-R: 5'-GG GGTACC TTATCCTGAAATGGCATGTGCC-3'; (the underlined part is the KpnI restriction site); (SEQ ID No: 8)

[0118] The PCR reaction system is shown in Table 2.

[0119] Table 2

[0120] Components Volume (μl) Template DNA 5 10×Buffer 5 dNTP (25mmol / L) 4 0046-F(20μmol / L) 1 0046-R (20 μmol / L) 1 Pfu DNA polymerase (5U / μl) 1 dd H2O 33

[0121] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 61°C for 20 s, 72°C for 60 s, and 72°C for 10 min after 26 cycles.

[0122] The 0046 gene fragment obtained above and pBBR1-P 452The plasmid was treated with restriction endonucleases Xho I and Kpn I (37°C, 2 hours), and the target fragments were recovered using a PCR product purification kit (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.). The plasmid after restriction digestion and the 0046 gene fragment were ligated at a ratio of 1:3 (molar ratio) (16°C, 12 hours); the ligation product pBBR1-P 452 -0046 recombinant plasmid was transformed into E. coli BL21, and the plasmid containing pBBR1-P 452 -0046 recombinant plasmid strain E.coli BL21 / pBBR1-P 452 -0046. Plasmid double enzyme digestion electrophoresis verification see Figure 5 -(B) shows plasmid pBBR1-P 452 -0046 double enzyme digestion product agarose gel electrophoresis.

[0123] (3) Acetobacter pasteurianus CGMCC 3089 (pBBR1-P 452 -0046), Acetobacter pasteurianus CGMCC 3089(pBBR1-P 452 -AiiA), Acetobacterpasteurianus CGMCC 3089(pBBR1-P 452 )Genetically engineered bacteria.

[0124] Take 100 μL of Acetobacter pasteurianus CGMCC 3089 competent cells in a small centrifuge tube and add 10 μL of the constructed recombinant plasmid pBBR1-P 452 -0046, mix well and add to the pre-cooled electric pulse cup and ice bath for 3 minutes; turn on the electric pulse instrument and perform electric stimulation (2.0 kV) according to the set transformation program; quickly add 1 mL of pre-cooled sterile YPG medium to the electric pulse cup, mix well, transfer to a test tube, and culture slowly with shaking at 30°C for 2 hours, then spread on a selection plate with kanamycin, and culture inverted at 30°C for 48 hours, randomly pick positive transformants with good growth, and use the primers used in plasmid construction for colony PCR verification. The correct strain is the one containing the recombinant plasmid pBBR1-P 452 -0046 of Acetobacter pasteurianus CGMCC 3089 genetically engineered bacteria.

[0125] At the same time, the above method was used to construct the recombinant plasmid pBBR1-P 452 , pBBR1-P 452-AiiA-derived Acetobacter pasteurianus CGMCC 3089 genetically engineered bacteria.

[0126] (4) 0046 protein function verification

[0127] The Acetobacter pasteurianus CGMCC 3089 (pBBR1-P 452 ), Acetobacter pasteurianus CGMCC 3089(pBBR1-P 452 -0046), Acetobacter pasteurianusCGMCC 3089(pBBR1-P 452 -AiiA) were cultured in YPG medium, and then the crude extract of signal molecules was extracted according to the above 3 steps and added to the liquid test tube culture medium of Agrobacterium tumefaciens A136 (indicator bacteria) containing 40μg / mL X-Gal, and anhydrous ethanol was added as a negative control. The crude extract of signal molecules and anhydrous ethanol were added to a concentration of 50μL / mL and 100μL / mL, respectively. The specific results are shown in Figure 6 As shown, 1 to 4 are respectively added with Acetobacter pasteurianus CGMCC 3089 (pBBR1-P452) signal molecule crude extract, Acetobacter pasteurianus CGMCC 3089 (PBBR1-P 452 -AiiA) signal molecule crude extract, Acetobacter pasteurianus CGMCC 3089 (PBBR1-P 452 -0046) signal molecule crude extract and anhydrous ethanol. (A) and (C) are crude extracts with a dosage of 50 μL / mL; (B) and (D) are crude extracts with a dosage of 100 μL / mL.

[0128] Depend on Figure 6 It can be seen that the addition of the positive control strain Acetobacter pasteurianus CGMCC 3089 (pBBR1-P 452 -AiiA) signal molecule crude extract was added to the test tube compared with Acetobacter pasteurianus CGMCC 3089 (pBBR1-P 452) The color of the test tube containing the crude extract of the signal molecule is lighter, but darker than that of the test tube containing anhydrous ethanol, indicating that the strain expressing the quorum quenching enzyme AiiA has the effect of quenching the signal molecule. The color trend of the test tube containing the crude extract of the signal molecule of the strain overexpressing the 0046 protein is consistent with that of the positive control group, which verifies that the 0046 protein has the function of degrading the quorum sensing signal molecule, thereby reducing the negative impact of quorum sensing on the growth of acetic acid bacteria.

[0129] Therefore, through the above functional analysis and color development verification, 0046 protein has the function of quorum sensing quenching. The quorum sensing phenomenon in acetic acid bacteria has an adverse effect on acetic acid fermentation, and the quorum quenching enzyme can reduce this adverse effect.

[0130] Example 2 Positive breeding of mutated 0046 protein (M0046)

[0131] 1. 0046 protein homology modeling

[0132] The amino acid sequence of the protein was input into the Swiss-Model (https: / / swissmodel.expasy.org / ) platform, and homology modeling of the 0046 protein was performed with reference to the GQQA protein in Komagataeibacter europaeus. After the platform was automatically operated, the PDB file of the three-dimensional structure model of the homology modeling of the required protein was obtained, such as Figure 7 shown.

[0133] 2. Obtaining a 3D model of a small molecule ligand

[0134] (1) Two signal molecules from Acetobacter pasteurianus, octanoyl-DL-homoserine lactone and butyryl-DL-homoserine lactone, were used as ligands, and the three-dimensional model was downloaded in mol2 format using the PubChem website (https: / / pubchem.ncbi.nlm.nih.gov / ).

[0135] (2) Processing of small molecule ligands: Energy minimization of small molecule ligands was performed on the AlphaFold Protein Structure Database (alphaFold: http: / / alphaford.com).

[0136] 3. Molecular docking

[0137] (1) The protein body and two ligands, N-butanoyl-L-homoserine lactone and N-octanoyl-L-homoserine lactone, were simulated and docked on the alphaFold platform, and the appropriate conformation was found. Figure 8As shown, (A) is the docking diagram of 0046 protein and N-butanoyl-L-homoserine lactone, and (B) is the docking diagram of 0046 protein and N-octanoyl-L-homoserine lactone.

[0138] (2) Docking with ligand small molecule The amino acids in the range were used as the main mutation sites and alanine scanning was performed. Fig. 9 shown.

[0139] 4. Alanine scanning

[0140] (1) Modify the protein mut.pdb file at the position where alanine is needed to be replaced. Only the CACBN atom of a certain residue needs to be retained, the others are deleted, and the residue name is changed to ALA. After leap, the original residue is replaced by alanine, and the top information after mutation is obtained.

[0141] (2) Ligand small molecule After alanine scanning of the amino acids in the range, molecular docking with the ligand was performed again to compare the changes in binding energy (Mutation Energy) and molecular force (VDW Term). If the binding energy decreases and the molecular force increases, the mutation may be a positive mutation; if the binding energy increases and the molecular force decreases, the mutation may be a negative mutation. According to the above rules, positive mutation screening was performed, and the results are shown in Table 3.

[0142] Table 3

[0143] Mutation site Binding Energy Molecular forces Mutation Effect M0046:SER67>CYS -0.56 0.43 Stablize

[0144] Example 3 Effect of Plasmid Expression of 0046 Protein and M0046 on Acetic Acid Fermentation of Production Strain

[0145] 1. Overexpression plasmid pBBR1-P 452 - Construction of M0046

[0146] Gene fragments of the screened positive mutation results are synthesized by GENEWIZ Biotech.

[0147] The gene sequence of M0046 protein is shown in SEQ ID No: 3;

[0148] The amino acid sequence of M0046 is shown in SEQ ID No: 4;

[0149] Using the synthesized M0046 gene fragment as a template, primers 0046-F and 0046-R were used for PCR reaction to amplify the M0046 gene fragment with Xho I and Kpn I restriction sites;

[0150] 0046-F:5'-CC CTCGAG ATGACACAAAAGGTTATCGCCTT-3'; (the underlined part is the Xho I restriction site); (SEQ ID No: 7) 0046-R: 5'-GG GGTACC TTATCCTGAAATGGCATGTGCC-3'; (the underlined part is the KpnI restriction site); (SEQ ID No: 8)

[0151] The PCR reaction system is shown in Table 2. The PCR reaction conditions were: 95°C pre-denaturation for 5 min, 94°C for 30 s, 61°C for 20 s, 72°C for 60 s, and 72°C for 10 min after 26 cycles.

[0152] The M0046 gene fragment with Xho I and Kpn I restriction sites obtained above was combined with pBBR1-P 452 The restriction enzymes Xho I and Kpn I were used to treat the fragments (37°C, 2 hours), and the target fragments were recovered using a PCR product purification kit (Shanghai Shenggong Biotechnology Technology Service Co., Ltd.). The plasmid after enzyme digestion and the M0046 gene fragment were ligated at a ratio of 1:3 (substance ratio) (16°C, 12 hours); the ligation product was transformed into Escherichia coli BL21, and the strain E. coli BL21 / pBBR1-P containing the recombinant plasmid was obtained after screening. 452 -M0046. Plasmid single enzyme electrophoresis verification Fig.10 shown.

[0153] 2. Acetobacter pasteurianus CGMCC 3089 (pBBR1-P 452 -M0046) genetically engineered bacteria.

[0154] Take 100 μL of Acetobacter pasteurianus CGMCC 3089 competent cells in a small centrifuge tube and add 10 μL of the constructed recombinant plasmid pBBR1-P 452-M0046, mix well and add to the pre-cooled electric pulse cup and ice bath for 3 minutes; turn on the electric pulse instrument and perform electric stimulation (2.0kV) according to the set transformation program; quickly add 1mL of pre-cooled sterile YPG medium to the electric pulse cup, mix well, transfer to a test tube, and culture slowly with shaking at 30℃ for 2 hours, then spread on a selection plate with kanamycin, and culture inverted at 30℃ for 48 hours, randomly pick positive transformants with good growth, and use the primers used in plasmid construction for colony PCR verification. The verified correct strain is the one containing the recombinant plasmid pBBR1-P 452 -M0046 Acetobacter pasteurianus CGMCC 3089 genetically engineered bacteria.

[0155] 3. Acetic acid fermentation verification

[0156] Acetobacter pasteurianus CGMCC 3089 (pBBR1-P 452 -M0046) and Acetobacter pasteurianus CGMCC 3089 (pBBR1-P 452 -0046), Acetobacter pasteurianus CGMCC 3089(pBBR1-P 452 ) for apple cider vinegar fermentation.

[0157] (1) Preparation of seed solution: Acetobacter pasteurianus CGMCC 3089 (pBBR1-P 452 -0046), Acetobacter pasteurianus CGMCC 3089(pBBR1-P 452 -M0046) and Acetobacterpasteurianus CGMCC 3089 (pBBR1-P 452 ) The genetically engineered bacteria were inoculated into the seed culture medium and cultured in a shaking incubator at 30°C and 160 rpm for 25 hours. The inoculation volume was transferred to a fresh seed culture medium at 10% (v / v) for amplification culture.

[0158] The composition of the seed culture medium: glucose 20 g / L, yeast extract 15 g / L, ethanol 3.5% (v / v), and the rest water.

[0159] (2) Cider fermentation: Take an appropriate amount of pretreated juice, heat it to 30°C, sprinkle in active dry yeast, stir and keep warm for 30 minutes, and add it to the juice after the yeast activity becomes normal. Stir evenly and then carry out alcohol fermentation at 25°C. The fermentation is completed after about 7 days. The ethanol content in the cider is 8% (v / v).

[0160] (3) Apple cider vinegar fermentation

[0161] According to the inoculum amount of 10% (v / v), it was inoculated into the fermentation tank containing apple cider, and apple cider vinegar fermentation was carried out at 30°C and ventilation rate of 1:0.1 (v / v) / min. When the ethanol concentration of the fermentation liquid was less than 5g / L and the acidity did not change, the fermentation was completed. The specific fermentation results are shown in Table 4.

[0162] Table 4

[0163]

[0164] Each value in the table is expressed as mean±SD (n=3). Duncan's multiple variance analysis was used to evaluate significant differences. Different letters in the same column indicate significant differences (P<0.001).

[0165] As shown in Table 4, compared with Acetobacter pasteurianus CGMCC 3089 (pBBR1-P 452 ), after overexpression of 0046 protein, the biomass of acetic acid bacteria increased by 11.21%, the fermentation efficiency increased by 18.33%, and the raw material utilization rate increased by 4.6%, indicating that 0046 protein weakened the quorum sensing phenomenon in acetic acid bacteria, thereby improving the acetic acid fermentation efficiency; after overexpression of M0046 protein, the biomass of acetic acid bacteria increased by 17.36%, the fermentation efficiency increased by 24.50%, and the raw material utilization rate increased by 7.1%, indicating that M0046 protein can better weaken the quorum sensing phenomenon in acetic acid bacteria, increase the biomass of the bacteria and thus improve the acetic acid fermentation efficiency, and the effect is more significant than that of 0046 protein.

[0166] Example 4 Effect of Genome-Integrated Expression of 0046 Protein and M0046 Protein on Acetic Acid Fermentation

[0167] The CRISPR-Cas9 editing technology was used to integrate the 0046 protein and M0046 protein genes into the Acetobacterpasteurianus CGMCC 3089 genome, and the 0046 protein and M0046 protein were overexpressed, followed by acetic acid fermentation.

[0168] 1. Construction of pCas9 plasmid in Acetobacter pasteurianus

[0169] (1)pBBR1-Ptrc -Construction of Cas9 plasmid

[0170] ① Using pTrc99a as the starting plasmid as the gene template, reverse PCR was performed using primers Trc-F and Trc-R to amplify and obtain a linear Ptrc plasmid without a replicon. The PCR reaction system is shown in Table 5.

[0171] Table 5

[0172] Components Volume (μl) Template DNA 2 Forward primer (20 μmol / L) 2 Reverse primer (20 μmol / L) 2 Phanta (5U / μl) 25 dd H2O 19

[0173] Trc-F: 5'-GCTGCCGCGCCGGTAGAACGCCAGCAACGCGG-3'; (SEQ ID No: 9)

[0174] Trc-R: 5'-GAGCCAGCCGGTGGCCGCGAAGATCCTTTG-3'; (SEQ ID No: 10)

[0175] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 58°C for 20 s, 72°C for 3 min 40 s, and 72°C for 10 min after 26 cycles.

[0176] ② Using pBBR1-P 452 The primers ori-F and ori-R were used as gene templates for PCR reaction to amplify the replicon pBBR1 fragment. The PCR reaction system is shown in Table 5.

[0177] ori-F: 5'-AAGATCAAAGGATCTTCGCGGCCACCGG-3'; (SEQ ID No: 11)

[0178] ori-R: 5'-GGCCGCGTTGCTGGCGTTCTACCGGCGCGGC-3'; (SEQ ID No: 12)

[0179] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 58°C for 20 s, 72°C for 3 min 40 s, and 72°C for 10 min after 26 cycles.

[0180] The linear Ptrc plasmid without replicon and the replicon pBBR1 fragment were respectively recovered using a PCR product purification kit (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.), and homologous ligation reactions were performed (50°C, 30 min) according to the ratios shown in Table 6. The ligation products were transformed into Escherichia coli DH5α, and the recombinant plasmid pBBR1-P was obtained after screening. trc strains.

[0181] Table 6

[0182] Ptrc plasmid sequence 2μl Replicon pBBR1 fragment 2μl C115 recombinase 5μl dd H2O 1μl

[0183] ③ Using pBBR1-P trc The plasmid was used as a template and the primers TF and TR were used for inverse PCR reaction to amplify the linear pBBR1-P trc The plasmid and PCR reaction system are shown in Table 5.

[0184] TF:5'-GTCAGCTAGGAGGTGACTGATCTAGAGTCGACCTG-3'; (SEQ ID No: 13)

[0185] TR:5'-TATTGAGTATTTCTTATCCATCATGGTCTGTTTCCTG-3'; (SEQ ID No:14)

[0186] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 61°C for 20 s, 72°C for 5 min 20 s, and 72°C for 10 min after 26 cycles.

[0187] ④ Using the Predcas9 plasmid as a template, the primers Cas9-F and Cas9-R were used for PCR reaction to amplify the Cas9 fragment. The PCR reaction system is shown in Table 5.

[0188] Cas9-F:5'-GGAAACAGACCATGATGGATAAGAAATACYCAAT-3'; (SEQ ID No:15)

[0189] Cas9-R:5'-CCTGCAGGTCGACTCTAGATCAGTCACCTCCTAG-3'; (SEQ ID No:16)

[0190] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 62°C for 20 s, 72°C for 4 min 30 s, and 72°C for 10 min after 26 cycles.

[0191] Linearize pBBR1-P trc The plasmid and Cas9 fragment were respectively recovered using a PCR product purification kit (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.), and homologous ligation reaction (50°C, 30 min) was performed according to the ratio shown in Table 7. The ligation product was transformed into Escherichia coli DH5α, and the recombinant plasmid pBBR1-P was obtained after screening. trc -Cas9 strains.

[0192] Table 7

[0193] Linearized pBBR1-Ptrc plasmid 2μl Cas9 fragment 2μl C115 recombinase 5μl dd H2O 1μl

[0194] (2)pBBR1-P 452 -Construction of Cas9 plasmid

[0195] ① Using the Predcas9 plasmid as a template, the primers Cas9-F1 and Cas9-R1 were used for PCR reaction to amplify the Cas9 fragment. The PCR reaction system is shown in Table 5.

[0196] Cas9-F1:5'-CC ATCGAT ATGGATAAGAAATACTCAATAGGCTTAG-3'; (the underlined part is the Cla I restriction site), (SEQ ID No: 17);

[0197] Cas9-R1:5'-CC CTCGAG TCAGTCACCTCCTAGC-3'; (the underlined part is the Xho I restriction site), (SEQ ID No: 18);

[0198] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 55°C for 20 s, 72°C for 4 min 20 s, and 72°C for 10 min after 26 cycles.

[0199] ② Plasmid pBBR-P 452 The Cas9 fragment was treated with restriction endonucleases Xho I and Cla I, respectively, for 2 h at 37 °C. The plasmid and Cas9 fragment were purified and recovered, and the purified plasmid and Cas9 fragment were mixed in a molar ratio of 1:3. Then, the ligation reaction was carried out using T4 DNA ligase at 22 °C for 2 h. The ligation product was then transferred into Escherichia coli DH 5α competent cells to obtain the recombinant plasmid pBBR-P 452 -Cas9 strain;

[0200] (3)pBBR1-P BAD -Construction of Cas9 plasmid

[0201] ①Using Predcas9 plasmid as template, PCR reaction was performed using primers BAD-F and BAD-R to amplify promoter P BAD Fragment;

[0202] BAD-F:5'-AT CGATCG TTATGACAACTTGACGGCTACATCA-3'; (the underlined part is the Pvu I restriction site); (SEQ ID No: 19);

[0203] BAD-R:5'-CG GAATTCATGGAGAAACAGTAGAGAGTTGCGA-3'; (the underlined part is the EcoR I restriction enzyme cleavage site); (SEQ ID No: 20).

[0204] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 55°C for 20 s, 72°C for 1 min 20 s, and 72°C for 10 min after 26 cycles.

[0205] ② Plasmid pBBR-P 452 and promoter P BAD The fragment sequence was treated with restriction endonucleases EcoR I and Pvu I, respectively, for 2 h at an ambient temperature of 37 ° C; purified and recovered, the purified plasmid and the promoter PBAD fragment sequence were mixed at a molar ratio of 1:3, and then ligated using T4 DNA ligase at a reaction temperature of 22 ° C for 2 h; then the ligation product was transferred into Escherichia coli DH 5α competent cells to obtain the recombinant plasmid pBBR1-P BAD strains;

[0206] ③Transfer plasmid pBBR1-P BAD The Cas9 fragment obtained in step (2) was treated with restriction endonucleases Xho I and ClaI, respectively, for 2 h at an ambient temperature of 37 ° C; purified and recovered, the plasmid purified by enzyme digestion and the Cas9 fragment were mixed in a molar ratio of 1:3, and then ligated using T4 DNA ligase at a reaction temperature of 22 ° C for 2 hours; the ligated product was then transferred into Escherichia coli DH 5α competent cells to obtain a recombinant plasmid pBBR1-P BAD -Cas9 strain;

[0207] The three plasmids pBBR1-P trc -Cas9, pBBR1-P 452 -Cas9, pBBR-P BAD -Cas9 were transformed into Acetobacter pasteurianus CGMCC 3089 by electroporation to obtain Acetobacter pasteurianus CGMCC 3089 containing pCas9 plasmid (pBBR1-P 452 -Cas9), CGMCC 3089 (pBBR1-P trc -Cas9), CGMCC3089 (pBBR1-P BAD -Cas9).

[0208] (4) Screening the most suitable promoter for expressing Cas9 protein in Acetobacter pasteurianus CGMCC 3089.

[0209] CGMCC 3089, CGMCC 3089(pBBR1-P 452 -Cas9), CGMCC 3089

[0210] (pBBR1-P trc -Cas9), CGMCC 3089 (pBBR1-P BAD -Cas9) was inoculated into YPG test tube culture medium and pre-cultured at 30°C and 220 rpm for 12 hours until OD600 was about 0.6. 1 mL of pre-cultured bacterial solution was inoculated into a 250 mL Erlenmeyer flask containing 100 mL YPG culture medium and cultured at 30°C and 220 rpm for 8 hours until OD600 was about 0.6; then, CGMCC 3089 (pBBR1-P trc -Cas9), CGMCC 3089 (pBBR1-P BAD -Cas9) culture medium, IPTG with a final concentration of 0.5 mM and L-arabinose with a final concentration of 1% (W / V) were added, and the culture was carried out at 30°C and 220 rpm for 12 h (CGMCC 3089, CGMCC 3089 (pBBR1-P 452 -Cas9) without any other manipulations). Then, IPTG-induced CGMCC 3089 (pBBR1-P trc -Cas9), CGMCC 3089 (pBBR1-P BAD -Cas9), CGMCC 3089 (pBBR1-P 452 -Cas9) and CGMCC3089 culture medium were each taken 2 ml of bacterial solution and centrifuged at 5000 rpm for 5 minutes to collect the bacteria. Then 100 μl of physiological saline was pipetted and mixed, and CGMCC 3089 was spread on a non-antibiotic YPG plate. CGMCC 3089 (pBBR1-P 452 -Cas9) and CGMCC 3089 (pBBR1-P BAD -Cas9) were coated on kanamycin-resistant YPG plates, CGMCC 3089 (pBBR1-P trc -Cas9) was coated on an ampicillin-resistant YPG plate, and statically cultured in a 30°C incubator for 12 hours, and the number of transformants on the plate was compared. The promoter used by the strain with a smaller number of transformants on the plate was the promoter most suitable for Cas9 protein expression in acetic acid bacteria. The specific results are shown in Table 8.

[0211] Table 8

[0212] strain Fatality rate% CGMCC 3089 / CGMCC 3089 (pBBR-P452-Cas9) 5.5％ CGMCC 3089 (pBBR-PBAD-Cas9) 11.0％ CGMCC 3089(pBBR-Ptrc-Cas9) 98.0％

[0213] As shown in Table 8, compared with the constitutive promoter P 452 and arabinose-inducible P BAD Promoter, IPTG-inducible P trc The promoter has the highest lethality rate to the host bacteria and is most suitable for expressing Cas9 protein. Therefore, the CRISPR-Cas9 editing system in Acetobacter CGMCC 3089 uses pBBR-P trc -Cas9 plasmid expresses Cas9 protein.

[0214] 2. Construction of sgRNA plasmid (pTarget-1)

[0215] The sgRNA: 5′-ACGAAGCCTTTGCTGGCGAA-3′ (SEQ ID No: 21) was obtained through the website http: / / crispor.tefor.net / .

[0216] Using the pTarget plasmid as a template, perform an inverse PCR reaction using primers GF and GR with gRNA as homology arms to amplify and obtain a linear PTarget plasmid with homology arms:

[0217] GF: 5'-GTACGAAGCCTTTCTGGCGAAGTTTTAG-3'; (SEQ ID No: 22)

[0218] GR: 5'-CTTCGCCAGCAAAGGCTTCGTACTAGTATTATAC-3'; (SEQ ID No: 23)

[0219] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 60°C for 20 s, 72°C for 2 min 20 s, and 72°C for 10 min after 26 cycles.

[0220] The linear PTarget plasmid with homology arms was used to recover the target fragment using a PCR product purification kit (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.), and a single fragment homology ligation reaction (50°C, 30 min) was performed with a ratio of 3 μl of PTarget fragment sequence, 5 μl of C115 recombinase, and 2 μl of ddH2O. The ligation product was transferred into E. coli DH5α, and the E. coli DH5α containing the pTarget-1 plasmid containing sgRNA was obtained after screening. The specific results are shown in the figure. Fig.11 As shown, (A) is the plasmid pBBR1-P trc-Cas9 double digestion product agarose gel electrophoresis; (B) is the sgRNA plasmid agarose gel electrophoresis; (C) Lane 1 is the DonorDNA (0046) agarose gel electrophoresis, and Lane 2 is the DonorDNA (M0046) agarose gel electrophoresis.

[0221] 3. Construction of Donor DNA

[0222] (1) Construction of Donor DNA integrating 0046 protein

[0223] Using the CGMCC 3089 genome as a template, PCR reactions were performed using primers 0046T-F1 and 0046T-R2, 0046T-F3 and 0046T-R4, 0046T-F5 and 0046T-R6, respectively, to obtain the 0046 gene upstream homology arm fragment, 0046 gene fragment, and 0046 gene downstream homology arm fragment;

[0224] 0046T-F1:5'-CTGGCTGCAGGTGGGTGATGGC-3' (SEQ ID No: 24);

[0225] 0046T-R2:5'-CCTTTTGTGGGAGCTCATCAGTTCA-3' (SEQ ID No: 25);

[0226] 0046T-F3:5'-GAACTGATGAGCTCCAATGACACAAAAGG-3' (SEQ ID No: 26);

[0227] 0046T-R4:5'-CATCAGCAGCTTGCGCGGTTATCCTG-3' (SEQ ID No: 27);

[0228] 0046T-F5:5'-CAGGATAACCGCGCAAGCTGCTGATG-3' (SEQ ID No: 28);

[0229] 0046T-R6:5'-GCTTGGCTGATTAAATATTTAAACAGGCAAACGG-3' (SEQ ID No: 29);

[0230] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 60°C for 20 s, 72°C for 30 s, and 72°C for 10 min after 26 cycles.

[0231] The upstream homology arm fragment, 0046 fragment, and downstream homology arm fragment were respectively recovered using a PCR product purification kit (Shanghai Sangon Bioengineering Technology Service Co., Ltd.), and fusion PCR reactions were performed according to the ratios in Table 9.

[0232] Table 9

[0233] Components Volume (μl) Upstream homology arm fragment DNA 1 Downstream homology arm fragment DNA 1 0046 Clip 1 0046T-F1(20μmol / L) 2 0046T-R6(20μmol / L) 2 Phanta (5U / μl) 25 <![CDATA[dd H2O]]> 1

[0234] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 60°C for 20 s, 72°C for 1 min 45 s, and 72°C for 10 min after 26 cycles.

[0235] The fused PCR fragment was purified using a PCR product purification kit (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.) to recover the target fragment, namely Donor DNA (0046). Fig.11 As shown in (C).

[0236] (2) Construction of Donor DNA integrating M0046 protein

[0237] Using the CGMCC 3089 genome as a template, PCR reactions were carried out with primers 0046T-F1 and 0046T-R2, 0046T-F5 and 0046T-R6, respectively, to obtain the upstream homology arm fragment of the M0046 gene and the downstream homology arm fragment of the M0046 gene; using the synthetic M0046 protein gene fragment as a template, PCR reactions were carried out with primers 0046T-F3 and 0046T-R4 to obtain the M0046 gene fragment with homology arms.

[0238] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 60°C for 20 s, 72°C for 30 s, and 72°C for 10 min after 26 cycles.

[0239] The upstream homology arm fragment, 0046 fragment, and downstream homology arm fragment were respectively recovered using a PCR product purification kit (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.), and fusion PCR reactions were performed according to the ratios in Table 9.

[0240] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 60°C for 20 s, 72°C for 1 min 45 s, and 72°C for 10 min after 26 cycles.

[0241] The fused PCR fragment was purified using a PCR product purification kit (Shanghai Shenggong Biotechnology Service Co., Ltd.) to recover the target fragment, namely Donor DNA (M0046). Fig.11 As shown in lane 2 in (C).

[0242] 4. Obtaining CGMCC 3089 strain overexpressing 0046 protein and M0046 protein in the genome.

[0243] (1) Obtaining the CGMCC 3089 strain that overexpresses the 0046 protein in the genome.

[0244] ① The plasmid pTarget-1 and Donor DNA (0046) were transformed into CGMCC 3089 (pBBR1-P trc -Cas9) competent cells, spread them in a culture medium containing corresponding antibiotics, and culture them upside down at 30°C for 1-2 days.

[0245] ②Verification of positive transformants

[0246] Use the transformants in the plate as templates, use primers YF and YR to perform PCR reaction, perform colony PCR, and verify the positive transformants:

[0247] YF: 5'-CTGGCTGCAGGTGGGTGATGGC-3' (SEQ ID No: 30);

[0248] YR: 5'-GACACACGTGGTACCGTAAAATGGTACAAC-3' (SEQ ID No: 31);

[0249] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 60°C for 20 s, 72°C for 120 s, and 72°C for 10 min after 26 cycles.

[0250] Through colony PCR verification, the strain with a PCR product length of 2000bp is the CGMCC 3089 strain that overexpresses the 0046 protein in the genome, that is, the CGMCC 3089 (0046) strain. The strain screening results are shown in Fig.12 As shown in lane 1.

[0251] (2) Obtaining the CGMCC 3089 strain that overexpresses the M0046 protein in the genome.

[0252] ① The plasmid pTarget-1 and Donor DNA (M0046) fragment were transformed into CGMCC 3089 (pBBR1-P trc -Cas9) competent cells, spread them in a culture medium containing corresponding antibiotics, and culture them upside down at 30°C for 1-2 days.

[0253] ②Verification of positive transformants

[0254] Use the transformants in the plate as templates, use primers YF and YR to perform PCR reaction, perform colony PCR, and verify the positive transformants:

[0255] AA-F: 5'-CTGGCTGCAGGTGGGTGATGGC-3' (SEQ ID No: 32);

[0256] AB-R: 5'-GACACACGTGGTACCGTAAAATGGTACAAC-3' (SEQ ID No: 33);

[0257] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 94°C for 30 s, 60°C for 20 s, 72°C for 120 s, and 72°C for 10 min after 26 cycles.

[0258] Through colony PCR verification, the strain with a PCR product length of 2000bp is the CGMCC 3089 strain that overexpresses the M0046 protein in the genome, namely the CGMCC 3089 (M0046) strain. The strain screening results are shown in Fig.12 Shown in lane 2.

[0259] 5. Acetic acid fermentation was carried out using the CGMCC 3089 strain that overexpressed 0046 protein and M0046 in the genome.

[0260] (1) Preparation of seed solution: CGMCC 3089 (0046), CGMCC 3089 (M0046) genetically engineered bacteria and CGMCC 3089 original bacteria were taken from the slant and inoculated into the seed culture medium, and cultured in a shaking incubator at 30°C and 160 rpm for 24 hours. 10% (v / v) inoculum was transferred into fresh seed culture medium for amplification culture. The seed culture medium composition: 20 g / L glucose, 15 g / L yeast extract, 3.5% (v / v) ethanol, and the rest water.

[0261] (2) Acetic acid fermentation: The seed solution was inoculated into a fermentation tank containing a fermentation medium at an inoculum of 10%, and acetic acid fermentation was carried out at 30°C and a ventilation rate of 1:0.15 (v / v) / min. The fermentation was terminated when the ethanol concentration in the fermentation liquid was lower than 5 g / L and the acidity did not change. The specific fermentation results are shown in Table 10.

[0262] Table 10

[0263]

[0264] Each value in the table is expressed as mean±SD (n=3). Duncan's multiple variance analysis was used to evaluate significant differences. Different letters in the same column indicate significant differences (P<0.001).

[0265] As shown in Table 10, compared with CGMCC 3089, after overexpressing 0046 protein in the genome, the biomass of acetic acid bacteria increased by 12.99% and the fermentation efficiency increased by 13.84%. After overexpressing M0046 protein in the genome, the biomass of acetic acid bacteria increased by 17.73% and the fermentation efficiency increased by 19.74%, indicating that M0046 protein can better alleviate the impact of quorum sensing than the original 0046 protein, promote bacterial growth, and improve fermentation efficiency.

[0266] The fermentation medium comprises 20 g / L glucose, 15 g / L yeast extract, 8% (v / v) ethanol, 10 g / L acetic acid, 2 g / L MgSO4, 3 g / L CaCl2, and the rest is water. Acetic acid is then produced by fermentation at 30°C and a ventilation rate of 1:0.15 (v / v) / min.

[0267] Example 5 Effect of Genome-Integrated Expression of 0046 Protein and M0046 Protein on Acetic Acid Solid-State Fermentation

[0268] The CGMCC 3089 strain with the 0046 protein integrated into its genome and the CGMCC 3089 strain with the M0046 protein integrated into its genome in Example 4 were used to produce vinegar by solid-state acetic acid fermentation.

[0269] 1. Prepare seed solution: Take genetically engineered bacteria Acetobacter pasteurianus CGMCC 3089 (0046), genetically engineered bacteria Acetobacter pasteurianus CGMCC 3089 (M0046) and original bacteria Acetobacter pasteurianus CGMCC 3089 from the slant and inoculate them into seed culture medium, and culture them in a shaking incubator at 30°C and 160 rpm for 24 hours. Transfer the inoculated amount of 10% (v / v) into fresh seed culture medium for amplification culture.

[0270] The composition of the seed culture medium: yeast extract 15 g / L, glucose 20 g / L, ethanol 3.5% (v / v), and the rest water.

[0271] 2. Alcohol fermentation: Weigh 1kg of crushed sorghum, pour it into a gelatinization barrel, add boiling water to cover the sorghum. Add 220μl of high-temperature resistant α-amylase and stir evenly. Heat at 85-100℃ for 30-40min to gelatinize the sorghum. After heating, naturally cool to about 55℃, add 23g of solid saccharifying enzyme, stir evenly and keep warm for 1h for saccharification. After saccharification, add 0.65kg of Daqu, put it into the fermentation jar, and add water to make up the weight to 5kg. Add 3g of yeast and stir evenly. After liquid sealing, put it in a 28℃ incubator and ferment for about 7d. The ethanol content at the end of alcohol fermentation is 6.0-7.0% (w / w).

[0272] 3. Acetic acid solid fermentation: After the fermentation of the mash is completed, activated acetic acid bacteria are added to the mash, with an inoculation amount of 10%. Bran and rice husk are added to the mash in a ratio of mash: bran: rice husk = 5.5:1.4:0.8 and stirred evenly for acetic acid fermentation. The initial ethanol content is about 5.5% (w / w). Fermentation is started at 25-30℃, and the mash is turned over once a day. Fermentation is completed when the ethanol concentration in the fermentation liquid is lower than 5g / L and the acidity no longer changes. See Table 11 for specific fermentation results.

[0273] Table 11

[0274]

[0275] Each value in the table is expressed as mean±SD (n=3). Duncan's multiple variance analysis was used to evaluate significant differences. Different letters in the same column indicate significant differences (P<0.001).

[0276] From the comparison results in Table 11, it can be seen that compared with CGMCC 3089, after overexpressing 0046 protein in the genome and performing solid-state fermentation, the biomass of acetic acid bacteria increased by 8.63%, and the fermentation efficiency increased by 7.87%; and after overexpressing M0046 protein in the genome and performing solid-state fermentation, the biomass of acetic acid bacteria increased by 11.44%, and the fermentation efficiency increased by 11.24%, indicating that M0046 protein can better alleviate the impact of quorum sensing than the original 0046 protein, promote the growth of bacteria, and improve the fermentation efficiency.

[0277] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A protein, wherein the protein is (a1) or (a2): (a1) a protein consisting of the amino acid sequence shown in SEQ ID No: 2; (a2) A protein obtained by replacing the 67th amino acid residue of the amino acid sequence shown in SEQ ID No: 2 from S to C.

2. A nucleic acid molecule encoding the protein according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of the nucleic acid molecule is as follows (b1) or (b2): (b1) a DNA molecule consisting of the nucleotide sequence shown in SEQ ID No: 1; (b2) A DNA molecule consisting of the nucleotide sequence shown in SEQ ID No:

3.

4. A recombinant vector containing the nucleic acid molecule according to claim 2 or 3.

5. A recombinant bacterium, characterized in that: The recombinant bacterium comprises the nucleic acid molecule according to claim 2 or 3 or the recombinant vector according to claim 4, or expresses the protein according to claim 1.

6. A method for constructing a recombinant bacterium, characterized in that: The method comprises integrating the nucleic acid molecule of claim 2 or 3 into the genome of a host bacterium; or introducing the nucleic acid molecule according to claim 2 or 3 or the recombinant vector according to claim 4 into a host bacterium; Preferably, the host bacteria include any one of Acetobacter, Komabacter, Gluconobacter or Gluconacetobacter; Preferably, the Acetobacter includes any one of Acetobacter pasteurianus, Acetobacter occidentalis, Acetobacter aceti or Acetobacter oxydans, preferably Acetobacter pasteurianus.

7. Use of the recombinant bacteria according to claim 5 or the recombinant bacteria constructed by the construction method according to claim 6 in improving the efficiency of acetic acid fermentation; Preferably, the acetic acid fermentation includes producing acetic acid by fermenting ethanol as raw material, producing vinegar by fermenting wine mash containing ethanol as raw material, or producing vinegar by solid-state fermentation method and liquid-state fermentation method.

8. A method for improving acetic acid fermentation efficiency, characterized in that: The method comprises subjecting the recombinant bacteria according to claim 5 or the recombinant bacteria constructed by the construction method according to claim 6 to acetic acid fermentation; Preferably, the fermentation ends when the ethanol concentration of the fermentation liquid is less than 10 g / L and the acidity does not change any more. Preferably, the fermentation ends when the ethanol concentration of the fermentation liquid is less than 5 g / L and the acidity does not change any more.

9. The method according to claim 8, characterized in that When liquid fermentation is performed, the inoculation amount of the recombinant bacteria is 5-20% (v / v), preferably 10% (v / v); Preferably, the fermentation temperature is 27-35°C, preferably 30°C; Preferably, the ventilation rate of the fermentation is 1:0.05-0.25 (v / v) / min, preferably 1:0.1 (v / v) / min; Preferably, the liquid fermentation method includes batch fermentation, fed-batch fermentation or split fermentation; Preferably, the fermentation time of the batch fermentation is 24 to 160 hours; Preferably, ethanol is continuously added during the fed-batch fermentation, the ethanol concentration in the fermentation broth is 10 to 40 g / L, and the fermentation time is 60 to 150 hours; Preferably, in the split fermentation, 20-50% (v / v) of the fermentation liquid is taken out each time, and the same volume of fresh culture medium is added, and the fermentation time is 24-100 hours.

10. The method according to claim 8, characterized in that When solid fermentation is performed, the inoculation amount of the recombinant bacteria is 5-20% (v / v), preferably 10% (v / v); Preferably, the fermentation temperature is 25-30°C, preferably 30°C; Preferably, the fermentation process also includes turning the mash once every 24 hours.

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