WO2021103812A1 - Method for constructing oenococcus oeni engineering bacteria and use thereof - Google Patents

Abstract

Provided are a method for constructing Oenococcus oeni engineering bacteria and the use thereof. The construction method comprises the following steps: (1) preparing a lipase gene lipase; (2) preparing a homologous arm gene having the lipase gene; (3) preparing a plasmid pMG36e-lipase; (4) preparing a nisl fragment; (5) preparing a linear fragment containing no erythromycin sequence; (6) preparing an expression vector pMG36n-lipase; and (7) transforming the expression vector pMG36n-lipase into Oenococcus oeni competent cells to obtain the Oenococcus oeni engineering bacteria. The Oenococcus oeni engineering bacteria with the function of degrading esters obtained by means of the construction method can be applied to the production of wine, such that adverse effects on the taste and aroma of the wine caused by excessive esters in the wine-making process are avoided in order to reduce the preservation time of the wine and shorten the production cycle of the wine while achieving the same taste.

Description

Construction method and application of an oenococcus engineering strain Technical field

The invention relates to a construction method and application of an oenococcus oenococcus engineering bacteria, and belongs to the technical field of microbial engineering.

Background technique

Oenococcus oeni (Oenococcus oeni) is a kind of lactic acid bacteria, which is a very important strain in addition to yeast in the wine production process. Its function is mainly in the post-ripening of wine, that is, in the later stage of winemaking, the special effect of Oenococcus is used to degrade a large amount of malic acid produced during fermentation into lactic acid and CO ₂ , thereby improving the taste of wine and reducing its taste. Acidity, this unique fermentation process is called Malolactic Fermentation (MLF). Therefore, Oenococcus and yeast are the only strains that are allowed to be added in wine production in various countries.

Chinese patent document CN104845811A (application number 201510237503.5) discloses a method for brewing rice wine by using Oenococcus to degrade ethyl carbamate, including soaking rice, steaming rice, sprinkling water, potting, adding koji and adding water, fermentation and post-treatment , On the 5th to 20th days after fermentation, inoculate Oenococcus oeni CICC6066 into the fermentation broth. The present invention degrades the EC produced in the wine making process by adding O. enococcus CICC6066 which can produce EC degrading enzyme. Oenococcus oeni CICC6066 is a lactic acid bacteria. Its addition does not introduce genetically modified strains and does not require subsequent treatment. It can effectively overcome the disadvantages of the two current methods of degrading ethyl carbamate.

In grape winemaking, the presence of esters greatly affects its taste and quality. Among them, if too many esters are produced during the fermentation process, such as ethyl acetate, ethyl butyrate, ethyl caproate, etc., it will have an adverse effect on the taste of the wine, thereby reducing the quality of the wine. However, due to factors such as wine grape quality and winemaking technology, the production of esters in the brewing process cannot be completely avoided. The best way to treat such esters is to use esterase (Easterase) to degrade them. However, the current commercial yeasts suitable for grape winemaking and Oenococcus spp. have weak esterase activity. Therefore, if appropriate methods and means are used to modify yeast or O. enococcus to increase its esterase activity, the above-mentioned problems can be well solved. Considering that as the fermentation progresses, the ethanol content in the fermentation broth gradually increases. When the alcohol concentration reaches 10%, a large number of yeasts will die and cannot exist in the wine for a long time; and O. enococcus has a higher effect on ethanol. Tolerance, it can still survive when the alcohol concentration reaches 16%, and it can survive long-term in wine. Based on the above characteristics, Oenococcus is more suitable for esterase transformation objects.

However, due to the direct application of O. dinococcus to food, the current restrictions on genetic modification of edible fungi for food are strict. In particular, antibiotic resistance genes cannot be used for labeling, and the existing conventional edible fungi source labels cannot be applied to wine. Oenococcus, as a result, there is currently no report on the genetic modification of Oenococcus. In addition, the low transformation rate of competent cells of Oenococcus is also the main reason that restricts its transformation.

Summary of the invention

In view of the shortcomings of the prior art, the present invention provides a construction method and application of Oenococcus oeni engineering bacteria.

This application takes O. enococcus as the research object and adopts molecular biology methods to clone the esterase gene lipase from a strain of Lactobacillus plantarum with high lipase activity, and link it with the expression vector pMG36e of lactic acid bacteria to construct a shaped pMG36e-lipase plasmid. Then, homologous recombination was used to knock out the erythromycin-resistant fragment in the plasmid and replace it with a food-safe lactic acid bacteria-resistant fragment to construct a food-grade (GRAS grade: Generally Recognized As Safe) lactic acid bacteria vector pMG36n- lipase, and then transformed into the host Odonococcus for successful expression. It was verified by wine fermentation experiments that the addition of the modified Oenococcus spp. greatly reduced the content of esters in the fermentation broth.

The technical scheme of the present invention is as follows:

A construction method of Oenococcus oeni engineering bacteria, the steps are as follows:

(1) Extract the genomic DNA of Lactobacillus plantarum and amplify by PCR to obtain the lipase gene lipase;

The specific primers for PCR amplification have nucleotide sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 2;

F:5’-ATGCAAGTTATTAAGCAAAAATTAAC-3’ SEQ ID NO.1

R: 5’-CTAACGATTATCAGCTAGCCATTCAAG-3’ SEQ ID NO.2

(2) Using plasmid pMG36e as a template, the homology arm gene with lipase gene was obtained by PCR amplification;

The specific primers for PCR amplification have nucleotide sequences as shown in SEQ ID NO. 3 and SEQ ID NO. 4;

F:5’-AAGCTTGCAAAGTCTGAAAACGA-3’ SEQ ID NO.3

R: 5’-GAGCTCGAATTACGAATTTTTCTG-3’ SEQ ID NO.4

(3) After linearizing the lipase gene lipase prepared in step (1) and the homology arm gene with lipase gene prepared in step (2), ligating, transforming into E. coli, screening, and obtaining plasmid pMG36e -lipase;

(4) Using the pET30a-nisI plasmid as a template, the nisI fragment was obtained by PCR amplification;

The specific primers for PCR amplification have nucleotide sequences as shown in SEQ ID NO. 5 and SEQ ID NO. 6;

F:5’-CCAAATTAAAGAGGGTTATAATGAGAAGATATTTAATACTTATTGTGGCC-3’ SEQ ID NO.5

R: 5’-CAGTTTATGCATCCCTTAACCTAGTTTCCTACCTTCGTTGCAAG-3’ SEQ ID NO.6

(5) Using the plasmid pMG36e-lipase prepared in step (3) as a template, perform PCR amplification to obtain a linear fragment that does not contain erythromycin sequence;

The specific primers for PCR amplification have nucleotide sequences as shown in SEQ ID NO. 7 and SEQ ID NO. 8;

F:5’-TATAACCCTCTTTAATTTGGTTATATG-3’ SEQ ID NO.7

R: 5’-GTTAAGGGATGCATAAACTGCATC-3’ SEQ ID NO.8

(6) After ligating the nisI fragment prepared in step (4) with the linear fragment not containing erythromycin sequence prepared in step (5), transform Lactococcus lactis (Lactococcus lactis) MG1363 competent cells, screen, and prepare Obtain the expression vector pMG36n-lipase;

(7) The expression vector pMG36n-lipase prepared in step (6) is transformed into competent cells of Oenococcus Oenococcus oeni, and then screened and cultured on a plate containing Nisin to obtain an Oenococcus oeni (Oenococcus oeni) engineered bacteria.

According to the present invention, preferably, in the step (1), the PCR amplification system is as follows, and the total system is 50 μl:

The PCR amplification procedure is as follows:

Pre-denaturation at 98°C for 3min, denaturation at 98°C for 30s, annealing at 55°C for 30s, extension at 68°C for 1min, 35 cycles; final extension at 68°C for 10min.

According to the present invention, preferably, in the step (2), the PCR amplification system is as follows, the total system is 50 μl:

The PCR amplification procedure is as follows:

Pre-denaturation at 98°C for 3min, denaturation at 98°C for 30s, annealing at 50°C for 30s, extension at 68°C for 4min, 35 cycles; final extension at 68°C for 10min.

Preferably according to the present invention, in the step (3), the selection is to spread the transformed Escherichia coli on an LB solid plate medium containing 200 μg/L erythromycin, and cultivate it at 35-37°C After 20-28 hours, select transformants and obtain them after sequencing.

According to the present invention, preferably, in the step (4), the PCR amplification system is as follows, and the total system is 50 μl:

The PCR amplification procedure is as follows:

Pre-denaturation at 98°C for 3min, denaturation at 98°C for 30s, annealing at 55°C for 30s, extension at 68°C for 1min, 35 cycles; final extension at 68°C for 10min.

According to the present invention, preferably, in the step (5), the PCR amplification system is as follows, and the total system is 50 μl:

The PCR amplification procedure is as follows:

Pre-denaturation at 98°C for 3min, denaturation at 98°C for 30s, annealing at 50°C for 30s, extension at 68°C for 4min, 35 cycles; final extension at 68°C for 10min.

Preferably according to the present invention, in the step (6), the screening is to resuscitate the transformed Lactococcus lactis, and then spread it on a plate of M17 medium containing 40 U/mL lactic acid bacteria Nisin at 37°C. Leave it to stand for 48 hours, select positive colonies, and verify by sequencing.

According to the present invention, it is further preferred that the resuscitation culture is static culture in M17 resuscitation medium at 37°C for 2 to 3 hours.

According to the present invention, it is further preferred that the components of the M17 medium are as follows:

Soy peptone 5.0g/L, yeast extract 5.0g/L, animal peptone 5.0g/L, magnesium sulfate 0.25g/L, ascorbic acid 0.5g/L, beef extract 5.0g/L, β-glycerophosphate disodium 19g /L, glucose 5.0g/L, and 15g/L agar should be added to the solid medium.

More preferably, the components of the M17 resuscitation medium are as follows:

Added glucose 0.5g/L, sucrose 17.1g/L, magnesium chloride 1.0mol/L, calcium chloride 1.0mol/L M17 medium.

Preferably, according to the present invention, the competent cell of Oenococcus oeni in the step (7) is prepared by the following steps:

Oenococcus oeni was activated with mFT80 medium supplemented with 2.5 to 3.5% glycine by mass, and cultivated to the logarithmic phase OD ₆₀₀ =0.35. The cells were collected by centrifugation, and then used with 0.5mmoL/L sucrose, 10% (volume percentage) The glycerin solution was washed 3 to 5 times at room temperature to prepare Oenococcus oeni competent cells.

According to the present invention, it is further preferred that the mFT80 medium components are as follows:

Beef powder 5.0g/L, yeast powder 4.0g/L, potassium dihydrogen phosphate 0.6g/L, potassium chloride 0.45g/L, calcium chloride 0.13g/L, magnesium sulfate 0.13g/L, manganese sulfate 0.003 g/L, Tween 80 1mL, L-malic acid 10g/L, fructose 35g/L, glucose 5.0g/L.

According to the present invention, the conversion conditions in the step (7) are preferably: 1.25KV, 200Ω, 25μF electroporation for 1.0 second.

According to the present invention, preferably, in the step (7), the Nisin-containing plate is an mFT80 solid medium containing a concentration of 15-25 U/mL Nisin.

According to the present invention, preferably, in the step (7), the screening and cultivation steps are as follows:

The transformed Oenococcus Oenococcus oeni was added to the mFT80 medium containing 0.5moL/L sucrose, cultured at 27～30℃ for 3～5h, centrifuged to collect the cells, and spread on the mFT80 solid medium containing 20U/mL Nisin , 27～30℃ static culture for 6～8 days.

According to the present invention, it is further preferred that the mFT80 solid medium components are as follows:

MFT80 liquid medium with 15g/L agar added.

A strain of Oenococcus oeni (Oenococcus oeni) engineered bacteria was prepared by the above construction method.

The application of the above-mentioned Oenococcus oeni engineering bacteria as ester-degrading bacteria in the preparation of wine.

According to the present invention, it is further preferred that the wine is fruit wine or grain wine with an alcohol content of less than 16 degrees.

Beneficial effect

1. The present invention uses the lipase gene lipase to transform O. enococcus for the first time, and uses molecular biology to carry out a series of modifications to it to obtain a strain of O. enococcus with the function of degrading esters; this strain is applied to wine During the manufacturing process, it can avoid the adverse effects on the taste and aroma of the wine due to excessive esters in the current wine making process, so as to reduce the preservation time of the wine and shorten the production cycle for the wine to reach the same taste;

2. The present invention uses nisin resistance fragment markers, since it does not contain antibiotic resistance genes, no antibiotics are added for induction during the fermentation process, which fully meets the needs of food safety;

3. The present invention finds for the first time that when Oenococcus Oenococcus oeni is activated with a specific medium, the success rate of transformation of the expression vector pMG36n-lipase can be significantly increased.

Detailed ways

The following examples are to better illustrate the content of the present invention, and those skilled in the art can better understand and master the present invention with the help of the examples. However, the scope of the protection claims of the present invention is not limited to the provided cases.

Materials and reagents

Oenococcus Oenococcus oeni CICC 6057, purchased from China Industrial Microbial Culture Collection and Management Center (CICC), existing known strains, common commercial products;

Lactobacillus plantarum HX1 (Lactobacillus plantarum HX1), deposited at the China Type Culture Collection on September 20, 2017, address: China Type Culture Collection Center, Wuchang Luojia Mountain, Wuhan City, Hubei Province, deposit number: CCTCC No.M 2017522, the existing known strains, please refer to Chinese Patent Document CN108077826A;

Lactococcus lactis MG1363, a common commercially available strain, purchased from Purutin Biotechnology (Beijing) Co., Ltd.;

Plasmid pMG36e and plasmid pET30a-nisI are common commercial products, purchased from Purrutin Biotechnology (Beijing) Co., Ltd.;

Genome extraction, plasmid extraction, gene purification and recombinase reagents were all purchased from Nanjing Novozan Biotechnology Co., Ltd.

Medium composition

M17 broth medium: purchased from Beijing Luqiao Technology Co., Ltd.

mFT80 medium group: beef extract powder 5.0g/L, yeast powder 4.0g/L, potassium dihydrogen phosphate 0.6g/L, potassium chloride 0.45g/L, calcium chloride 0.13g/L, magnesium sulfate 0.13g/ L, manganese sulfate 0.003g/L, Tween 80 1mL, L-malic acid 10g/L, fructose 35g/L, glucose 5.0g/L.

Acid tomato medium (ATB basic medium): peptone 1%, yeast extract 0.5%, glucose 1%, MgSO ₄ ·7H ₂ O 0.02%, MnSO ₄ ·4H ₂ O 0.005%, cysteine hydrochloride 0.5g /L, tomato juice 25%, the liquid medium is adjusted to pH 4.8.

Example 1

A construction method of Oenococcus oeni engineering bacteria, the steps are as follows:

(1) Extract the Lactobacillus plantarum genome according to the method of the bacterial genomic DNA extraction kit, add 50μL ddH ₂ O, and store at -20°C;

(2) The lipase gene of Lactobacillus plantarum on GenBank, design the following primers

F:5’-ATGCAAGTTATTAAGCAAAAATTAAC-3’ SEQ ID NO.1

R: 5’-CTAACGATTATCAGCTAGCCATTCAAG-3’ SEQ ID NO.2

(3) Using step (2) lipase gene primers, using step (1) Lactobacillus plantarum genome as a template, PCR amplifies lipase gene lipase fragments;

The PCR amplification system of the steps is as follows, the total system is 50μl:

The PCR amplification procedure is as follows:

Pre-denaturation at 98°C for 3min, denaturation at 98°C for 30s, annealing at 55°C for 30s, extension at 68°C for 1min, 35 cycles; final extension at 68°C for 10min;

(4) According to the plasmid pMG36e sequence, design the primers of the homology arm with lipase gene. The homology arm sequence is located on both sides of the MCS region of the plasmid multi-cloning site. The primer sequence is as follows:

F:5’-AAGCTTGCAAAGTCTGAAAACGA-3’ SEQ ID NO.3

R: 5’-GAGCTCGAATTACGAATTTTTCTG-3’ SEQ ID NO.4

(5) Extract the pMG36e plasmid with a plasmid extraction cassette, use this as a template, and use step (4) to PCR amplify the linear fragment of pMG36e;

The PCR amplification system of the steps is as follows, the total system is 50μl:

The PCR amplification procedure is as follows:

Pre-denaturation at 98°C for 3 minutes, denaturation at 98°C for 30 seconds, annealing at 50°C for 30 seconds, extension at 68°C for 4 minutes, 35 cycles; final extension at 68°C for 10 minutes;

(6) According to the requirements of the gene recombination ligation kit, the linear pMG36e-fragment purified in steps (3) (5) and the lipase fragment are recombined and ligated.

(7) Transform the ligated product of step (6) into E. coli DH5α competent cells by heat shock, spread it on the LB solid plate medium containing erythromycin (200μg/L), and select the transformants for plasmid extraction Carry out PCR amplification of the lipase gene and sequencing verification, and the transformed transformants after successful verification contain the newly constructed plasmid pMG36e-lipase.

(8) Using the nisI sequence queried on NCBI, design primers with pMG36e homology arms, which are located on both sides of the erythromycin resistance gene in the plasmid;

F:5’-CCAAATTAAAGAGGGTTATAATGAGAAGATATTTAATACTTATTGTGGCC-3’ SEQ ID NO.5

R: 5’-CAGTTTATGCATCCCTTAACCTAGTTTCCTACCTTCGTTGCAAG-3’ SEQ ID NO.6

Using the pET30a-nisI plasmid as a template, the above primers PCR amplify the nisI fragment;

The PCR amplification system of the steps is as follows, the total system is 50μl:

The PCR amplification procedure is as follows:

Pre-denaturation at 98°C for 3min, denaturation at 98°C for 30s, annealing at 55°C for 30s, extension at 68°C for 1min, 35 cycles; final extension at 68°C for 10min;

(9) According to the fragment of plasmid pMG36e-lipase that does not contain erythromycin sequence, the following primers are designed:

F:5’-TATAACCCTCTTTAATTTGGTTATATG-3’ SEQ ID NO.7

R: 5’-GTTAAGGGATGCATAAACTGCATC-3’ SEQ ID NO.8

Use the pMG36e-lipase plasmid extracted in step (7) as a template to perform PCR amplification, and the product is a linear fragment of the plasmid that does not contain erythromycin sequence;

The PCR amplification system of the steps is as follows, the total system is 50μl:

The PCR amplification procedure is as follows:

Pre-denaturation at 98°C for 3 minutes, denaturation at 98°C for 30 seconds, annealing at 50°C for 30 seconds, extension at 68°C for 4 minutes, 35 cycles; final extension at 68°C for 10 minutes;

(10) According to the requirements of the homologous recombinase kit, the fragments purified in steps (8) and (9) are connected by homologous recombination, mixed with 200 μL of Lactococcus lactis (lactis) MG1363 competent cells prepared, and then transferred into In an electro-rotor cup with a spacing of 2mm, let it stand on ice for 5 minutes, and use a high-voltage pulse electro-transmitter for electric shock conversion. The electro-transmission parameters are: voltage 1.5KV, resistance 200. The shock constant is 5ms, the capacitance is 25μF, and the shock is converted.

After the electric shock is finished, quickly add 1800μLMRS resuscitation medium to the electro-rotor cup. Incubate at 37°C for 2-3h. Take 200 μl of the resuscitated bacterial solution, spread it on a plate containing 40 U/mL lactic acid bacteria Nisin in M17 medium, and incubate at 37°C for 48 hours. The positive colonies were selected for expansion and culture, the plasmids were extracted, amplified with nisI primers and lipase primers, and sequenced for verification. If the verification is correct, the construction of the expression vector pMG36n-lipase is successful.

(11) Oenococcus Oenococcus oeni was activated with mFT80 medium supplemented with 3% glycine, cultured to log phase OD600=0.35, collected by centrifugation, and collected with 0.5mmoL/L sucrose, 10%(v/v) glycerol solution at room temperature Rinse 4 times, add a solution containing 5mmoL/L potassium phosphate, 0.2mmoL/L Mgcl2, and 10% (v/v) ethanol in aliquots for storage, and prepare Oenococcus oeni competent.

(12) Mix 500ng of pMG36n-lipase plasmid from step (10) with 100μL of Oenococcus oeni competent from step (11), put it into a 2mm electro-rotor cup, 1.25KV, 200Ω, 25μF electrotransmission, immediately add 1mL containing 0.5moL/L In sucrose-containing mFT80 medium, culture at 28°C for 4h. After centrifugation, the plate was plated in mFT80 solid medium containing 20 U/mL Nisin, and cultured for 7 days.

The positive colonies were selected for expansion and culture, the plasmids were extracted, amplified with nisI primers and lipase primers, and sequenced for verification. If the verification is correct, the transformation is successful, and the new strain is Oenococcus oeni/pMG36n-lipase.

Example 2 Application of the above-mentioned Oenococcus Oenococcus oeni engineering bacteria in the production of wine

The strain constructed by the present invention _{was acclimated for 3-5 days after adding 10mg/L of SO 2} , 10% (volume percentage) absolute ethanol and 20U/mL concentration of Nisin acid tomato medium (ATB medium); 5% by mass is added to the grape juice, cultivated at 20°C for 5 days for expansion; then 1-3% by mass is added to the wine fermentation broth that has been fermented with yeast in the previous stage for post-ripening, and at the same time according to 10-20U /mL concentration of food additive Nisin is added.

Example 3

For the determination of ethyl acetate, butyl acetate, isoamyl acetate and ethyl caproate in wine, refer to the QBT4850-2015 standard.

The content of the above esters was determined by adding different Oenococcus spp. The test results are as follows:

Result analysis

Through the analysis of the above detection results, it is concluded that the Oenococcus oeni engineered bacteria has a certain degree of degradation of the above esters, especially the effect on ethyl acetate is very obvious, and its content is reduced by more than 80%.

Comparative example 1

The method for strain establishment as described in Example 1, the difference is that the method of steps (4) (5) (6) is changed to the traditional method of plasmid digestion and then ligation with the cloned fragment.

By using DNAMAN software to analyze the esterase gene lipase of Lactobacillus plantarum, it was found that there were 21 restriction endonuclease sites in the sequence. Then, based on the multiple cloning site MCS provided in the pMG36e map, two restriction sites were selected. The endonucleases Sac I and Hind III are designed for PCR amplification by designing primers containing restriction sites, then ligated with the cloning vector, transformed, and then subjected to plasmid extraction, restriction digestion, ligation, and transformation again, although pMG36e can also be successfully constructed -lipase, but at least 2-3 days.

Result analysis

Since the lipase gene sequence from Lactobacillus plantarum (Lactobacillus plantarum) cloned in the present invention contains multiple sequences that are the same as restriction enzymes, the PCR product is digested with restriction enzymes and then connected to the vector. The internal sequence of the gene is the same, which will cause the internal breakage of the gene, and the complete cloned fragment cannot be obtained; and the present invention uses the method of homologous recombinase to connect, which solves the above problems, as long as the cloned fragment and the linear fragment of the vector have similar sequences (the same Source arm), it will be recognized and connected by the enzyme. And the connection time is short, less than 1 hour, while the traditional enzyme digestion and connection methods require overnight, which takes a long time.

Comparative example 2

The method for establishing the strain as described in Example 1, the difference is that steps (8), (9), and (10) are omitted, and the plasmid pMG36e-lipase is directly used to transform Oenococcus Oenococcus oeni.

After pMG36e-lipase is successfully transformed into Oenococcus oeni, the Oenococcus oeni/pMG36e-lipase is amplified and cultured, and then added to wine in proportion to post-ripening fermentation. Through the analysis of the fermented wine, the reduction is also achieved. The effect of esters such as ethyl acetate.

Result analysis

Although Oenococcus oeni/pMG36e-lipase also has esterase function, if it is used in wine fermentation, in order to ensure that the plasmid in the cell is not lost, a certain amount of erythromycin must be added during the fermentation process, which is not in line with food safety. Standard production. The pMG36n-lipase in the present invention replaces the erythromycin resistant fragment in the original plasmid with the Nisin resistant fragment. Nisin is a small peptide produced by Lactococcus lactis subsp. , Has a strong inhibitory effect on gram-positive bacteria such as Staphylococcus aureus, and can be used as a selection marker for lactic acid bacteria expression system. Nisin is quickly hydrolyzed into amino acids under the physiological conditions of the human body and the action of α-chymotrypsin after being eaten. It will not change the normal flora in the human intestine, nor will it cause resistance problems caused by other antibiotic resistance genes. Approved by the US FDA and many governments as an additive allowed to be used in food production. Therefore, the strain constructed in Example 1 is used in the production of wine according to the method in Example 2, and it is in full compliance with food safety production specifications.

Comparative example 3

The method for establishing the strain as described in Example 1, except that the addition ratio of glycine in step (11) is 0%, 1%, and 5%.

The Oenococcus oeni competence prepared according to the glycine concentration in Comparative Example 3 is the same as the competence in Example 1. Under the same conditions as other parameters, it is electrotransformed with the plasmid. The number of transformants generated on the plate after resuscitation is shown in the table below Shown.

It can be seen from the above table that the number of transformants produced by the competent cells prepared with glycine concentration in Example 1 is much higher than that of the competent cells prepared with different glycine concentrations in Comparative Example 3, indicating that it has a higher transformation efficiency.

Claims (10)

1. A construction method of Oenococcus oeni engineering bacteria, which is characterized in that the steps are as follows:

   (1) Extract the genomic DNA of Lactobacillus plantarum and amplify by PCR to obtain the lipase gene lipase;

   The specific primers for PCR amplification have nucleotide sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 2;

   (2) Using plasmid pMG36e as a template, the homology arm gene with lipase gene was obtained by PCR amplification;

   The specific primers for PCR amplification have nucleotide sequences as shown in SEQ ID NO. 3 and SEQ ID NO. 4;

   (3) After linearizing the lipase gene lipase prepared in step (1) and the homology arm gene with lipase gene prepared in step (2), ligating, transforming into E. coli, screening, and obtaining plasmid pMG36e -lipase;

   (4) Using the pET30a-nisI plasmid as a template, the nisI fragment was obtained by PCR amplification;

   The specific primers for PCR amplification have nucleotide sequences as shown in SEQ ID NO. 5 and SEQ ID NO. 6;

   (5) Using the plasmid pMG36e-lipase prepared in step (3) as a template, perform PCR amplification to obtain a linear fragment that does not contain erythromycin sequence;

   The specific primers for PCR amplification have nucleotide sequences as shown in SEQ ID NO. 7 and SEQ ID NO. 8;

   (6) After ligating the nisI fragment prepared in step (4) with the linear fragment not containing erythromycin sequence prepared in step (5), transform Lactococcus lactis (Lactococcus lactis) MG1363 competent cells, screen, and prepare Obtain the expression vector pMG36n-lipase;

   (7) The expression vector pMG36n-lipase prepared in step (6) is transformed into competent cells of Oenococcus Oenococcus oeni, and then screened and cultured on a plate containing Nisin to obtain an Oenococcus oeni (Oenococcus oeni) engineered bacteria.
2. The construction method according to claim 1, wherein in the step (1), the PCR amplification system is as follows, the total system is 50 μl:

   

   The PCR amplification procedure is as follows:

   Pre-denaturation at 98°C for 3min, denaturation at 98°C for 30s, annealing at 55°C for 30s, extension at 68°C for 1min, 35 cycles; final extension at 68°C for 10min.
3. The construction method according to claim 1, wherein in the step (2), the PCR amplification system is as follows, the total system is 50 μl:

   

   

   The PCR amplification procedure is as follows:

   Pre-denaturation at 98°C for 3min, denaturation at 98°C for 30s, annealing at 50°C for 30s, extension at 68°C for 4min, 35 cycles; final extension at 68°C for 10min.
4. The construction method according to claim 1, wherein in the step (3), the selection is to spread the transformed E. coli on an LB solid plate medium containing 200 μg/L erythromycin, Cultivate at 35～37℃ for 20～28 hours, select transformants and verify by sequencing, it is obtained;

   Preferably, in the step (4), the PCR amplification system is as follows, the total system is 50 μl:

   

   The PCR amplification procedure is as follows:

   Pre-denaturation at 98°C for 3min, denaturation at 98°C for 30s, annealing at 55°C for 30s, extension at 68°C for 1min, 35 cycles; final extension at 68°C for 10min;

   Preferably, in the step (5), the PCR amplification system is as follows, the total system is 50 μl:

   

   The PCR amplification procedure is as follows:

   Pre-denaturation at 98°C for 3min, denaturation at 98°C for 30s, annealing at 50°C for 30s, extension at 68°C for 4min, 35 cycles; final extension at 68°C for 10min.
5. The construction method according to claim 1, characterized in that, in the step (6), the screening is to resuscitate the transformed Lactococcus lactis and apply it to M17 containing 40 U/mL lactic acid bacteria Nisin. Place the culture medium on the plate at 37°C for 48 hours, select positive colonies, and verify by sequencing to obtain;

   Further preferably, the resuscitation culture is cultured in M17 resuscitation medium at 37°C for 2 to 3 hours;

   Further preferably, the components of the M17 medium are as follows:

   Soy peptone 5.0g/L, yeast extract 5.0g/L, animal peptone 5.0g/L, magnesium sulfate 0.25g/L, ascorbic acid 0.5g/L, beef extract 5.0g/L, β-glycerophosphate disodium 19g /L, glucose 5.0g/L, solid medium should add 15g/L agar;

   More preferably, the components of the M17 resuscitation medium are as follows:

   Added glucose 0.5g/L, sucrose 17.1g/L, magnesium chloride 1.0mol/L, calcium chloride 1.0mol/L M17 medium.
6. The construction method according to claim 1, wherein the competent cell of Oenococcus Oenococcus oeni in step (7) is prepared by the following steps:

   Oenococcus oeni was activated with mFT80 medium supplemented with 2.5 to 3.5% glycine by mass, and cultivated to the logarithmic phase OD ₆₀₀ =0.35. The cells were collected by centrifugation, and then used with 0.5mmoL/L sucrose, 10% (volume percentage) Wash 3 to 5 times with glycerin solution at room temperature to prepare Oenococcus oeni competent cells;

   Further preferably, the mFT80 medium components are as follows:

   Beef powder 5.0g/L, yeast powder 4.0g/L, potassium dihydrogen phosphate 0.6g/L, potassium chloride 0.45g/L, calcium chloride 0.13g/L, magnesium sulfate 0.13g/L, manganese sulfate 0.003 g/L, Tween 801mL, L-malic acid 10g/L, fructose 35g/L, glucose 5.0g/L.
7. The construction method according to claim 1, characterized in that the conversion in step (7), the conditions are: 1.25KV, 200Ω, 25μF electroporation for 1.0 second;

   Preferably, in the step (7), the plate containing Nisin is an mFT80 solid medium containing a concentration of 15-25 U/mL Nisin;

   Preferably, in the step (7), the screening and cultivation are performed as follows:

   The transformed Oenococcus Oenococcus oeni was added to mFT80 medium containing 0.5moL/L sucrose, cultured statically at 27～30℃ for 3～5h, centrifuged to collect cells, and spread on mFT80 solid medium containing 20U/mL Nisin , Static culture at 27～30℃ for 6～8 days;

   Further preferably, the components of the mFT80 solid medium are as follows:

   MFT80 liquid medium with 15g/L agar added.
8. A strain of Oenococcus oeni (Oenococcus oeni) engineered bacteria, prepared by the construction method of claim 1.
9. The use of the Oenococcus oeni engineered bacteria of claim 8 as ester-degrading bacteria in the preparation of wine.
10. The application according to claim 9, wherein the wine is fruit wine or grain wine with an alcohol content of less than 16 degrees.