An expression vector pHH40 and its application in the construction of engineered *Chlorella vulgaris* strains.
By introducing the heat shock protein gene lphsp into the artificially constructed plasmid pHH40, the problem of low ethanol tolerance of *C. tumefaciens* was solved, achieving a high success rate of gene introduction and rapid MLF fermentation. This improved the ethanol tolerance and fermentation efficiency of *C. tumefaciens*, and shortened the wine production cycle.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- QILU UNIVERSITY OF TECHNOLOGY (SHANDONG ACADEMY OF SCIENCES)
- Filing Date
- 2023-09-13
- Publication Date
- 2026-05-26
AI Technical Summary
Existing technologies make it difficult to efficiently introduce exogenous genes into *Coccus tumefaciens*, resulting in *Coccus tumefaciens* having low tolerance to ethanol and being unable to fully exert its MLF function, especially in high-ethanol environments where it is difficult to grow and ferment.
The artificially constructed plasmid pHH40 was used as an expression vector to introduce the heat shock protein encoding gene lphsp into *Coccus vinifera*. Electroporation technology was used to improve the success rate of gene introduction, and an engineered *Coccus vinifera* strain resistant to 14% ethanol was constructed.
A high success rate of gene introduction was achieved, which improved the tolerance of *C. truncatula* to ethanol, enabling it to introduce foreign genes into the *C. truncatula* host bacteria with a high success rate, shortening the production cycle and accelerating the MLF fermentation process, thus shortening the wine production cycle.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial engineering technology, specifically to an expression vector pHH40 and its application in the construction of engineered *Chlorella vulgaris* strains. Background Technology
[0002] Oenococcus oeni is a common lactic acid bacteria in malolactic fermentation (MLF) of wine. After the ethanol fermentation dominated by Sacchariform yeast is completed, it decarboxylates the excess malic acid in the fermentation broth to produce lactic acid, thereby reducing acidity and increasing pH. In addition, because Oenococcus oeni also plays a positive role in the metabolism of flavor substances in wine, it is one of the few microorganisms allowed to be added to wine production, besides Sacchariform yeast.
[0003] *Coccus spp.*, a common lactic acid bacteria in wine fermentation, exhibits tolerance to pH, sulfur dioxide, and approximately 10% ethanol. The primary mechanism by which *Coccus spp.* tolerates ethanol is the presence of a heat shock protein, Hsp 18. This protein interacts with the cell membrane of *Coccus spp.* under ethanol stress, maintaining cell membrane stability and preventing ethanol damage. However, due to protein activity and other limitations, *Coccus spp.*' tolerance to ethanol concentrations is limited to around 10%. Considering that the ethanol content during wine fermentation often exceeds 12%, many *Coccus spp.* strains struggle to fully utilize their MLF (methyl-lysate fermentation factor) function under such conditions. Therefore, constructing a more ethanol-tolerant *Coccus spp.* strain is essential.
[0004] Heat shock proteins (HSPs), also known as heat stress proteins, are a class of heat stress proteins widely found in organisms. When the body is exposed to high temperatures, the synthesis of these proteins is stimulated to protect the body. Heat shock proteins can enhance the stress response of cells, especially their heat tolerance, and can also regulate intracellular sodium levels. + -K + -ATPase activity promotes intracellular gluconeogenesis and glycogen production, and enhances the cell's resistance to external damage.
[0005] To address the issue of low ethanol tolerance in *C. tumefaciens* during the brewing process, this invention aims to introduce a heat shock protein-encoding gene into *C. tumefaciens* to construct engineered strains that enhance its ethanol tolerance. However, current methods for introducing exogenous genes into *C. tumefaciens* host strains are very immature, with a low success rate, severely hindering the construction of ethanol-resistant engineered *C. tumefaciens* strains. Summary of the Invention
[0006] To address the shortcomings of existing technologies, this invention provides an expression vector pHH40 and its application in the construction of engineered *C. truncatula* strains. This expression vector can introduce exogenous genes into *C. truncatula* host strains with a high success rate, and the constructed engineered *C. truncatula* strains can tolerate ethanol concentrations up to 14%, and can initiate and complete MLF fermentation in wine production more quickly.
[0007] The technical solution of this invention is as follows:
[0008] An expression vector pHH40, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0009] The application of pHH40 in the introduction of exogenous genes into Chlorella vulgaris.
[0010] Preferably, the exogenous gene is the heat shock protein encoding gene lphsp, whose nucleotide sequence is shown in SEQ ID NO.2.
[0011] Preferably, the application method is as follows: using pHH40 as an expression vector, the heat shock protein encoding gene lphsp is introduced into the host bacteria of *Chlorella vulgaris* for expression.
[0012] Preferably, the specific steps include:
[0013] (1) Using Lactobacillus plantarum genomic DNA as a template, PCR amplification was performed using primers lphsp-F and lphsp-R to obtain the heat shock protein gene lphsp; wherein, the nucleotide sequence of primer lphsp-F is shown in SEQ ID NO.3, and the nucleotide sequence of primer lphsp-R is shown in SEQ ID NO.4;
[0014] (2) Using plasmid pHH40 as a template, PCR amplification was performed using primers pHH40-F and pHH40-R to obtain the homologous arm gene of the heat shock protein gene lphsp; wherein, the nucleotide sequence of primer pHH40-F is shown in SEQ ID NO.5, and the nucleotide sequence of primer pHH40-R is shown in SEQ ID NO.6.
[0015] (3) The heat shock protein gene lphsp from step (1) and the homologous arm gene from step (2) were linearized and then ligated to obtain the ligation product; the ligation product was transformed into competent cells of Escherichia coli to obtain transformant 1; after screening, the recombinant expression vector pHH-lphsp was obtained.
[0016] (4) The recombinant expression vector pHH-lphsp from step (3) was transformed into competent cells of the host strain of *Saccharomyces cerevisiae* to obtain transformant 2; after screening, engineered strains of *Saccharomyces cerevisiae* were obtained.
[0017] Preferably, the conversion method in step (3) is as follows: the ligation product is mixed with competent cells of Escherichia coli and heat-shocked in a water bath at 42°C for 30 seconds.
[0018] Preferably, the screening method in step (3) is as follows: transformant 1 is spread on LB solid medium containing 100-500 μg / L erythromycin and cultured at 35-37℃ for 20-28 h. Transformants are selected for sequencing verification, and the correct sequence is the expression vector pHH-lphsp.
[0019] Preferably, the method for preparing competent cells of *Staphylococcus zeolites* host bacteria in step (4) is as follows: activate and culture *Staphylococcus zeolites* host bacteria to the logarithmic growth phase, collect the cells by centrifugation, then wash 3-5 times at room temperature with a 10% (v / v) glycerol solution containing 0.5 mmol / L sucrose, and store in a solution containing 0.5 mmol / L potassium phosphate, 0.2 mmol / L MgCl2, 5% (v / v) ethanol and 5% (v / v) propanol to obtain competent cells of *Staphylococcus zeolites* host bacteria.
[0020] Preferably, the conversion conditions in step (4) are: 2.0KV, 200Ω, 5ms, 25μF electro-conversion for 1.0s.
[0021] Preferably, the screening method in step (4) is as follows: Transformant 2 is added to MRSM medium containing 0.5 mol / L sucrose, and cultured statically at 27-30℃ for 3-5 hours, and the bacterial cells are collected by centrifugation; the bacterial cells are spread on MRSM solid medium containing 25-40 mg / L erythromycin, and cultured statically at 27-30℃ for 8-10 days, and the transformants are selected for sequencing verification. The strain with the correct sequence is the engineered strain of *Chlorella vulgaris*.
[0022] The MRSM medium comprises the following components: yeast extract 5.0 g / L, beef extract 5.0 g / L, peptone 10.0 g / L, sodium acetate 5.0 g / L, triammonium citrate 2.0 g / L, glucose 20.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 mL, potassium dihydrogen phosphate 2.0 g / L, L-malic acid 10.0 g / L, fructose 10.0 g / L, pH 4.5; the MRSM solid medium is MRSM medium with 15 g / L agar added.
[0023] An engineered strain of *C. tumefaciens* was prepared according to the above construction method.
[0024] The application of the engineered strain of *Chlorella vulgaris* in wine production.
[0025] Beneficial effects:
[0026] (1) The present invention uses the artificially constructed plasmid pHH40 as a vector, which can introduce exogenous genes into the host bacteria of Chlorella vulgaris with a high success rate; and the engineered Chlorella vulgaris strain constructed by the present invention can tolerate ethanol concentrations up to 14%, and can start and complete MLF fermentation in wine production more quickly, thus shortening the wine production cycle.
[0027] (2) The present invention uses a solution containing ethanol and propanol to preserve competent cells of *Staphylococcus vinifera*, which can significantly increase the transformation success rate of expression vectors.
[0028] (3) The present invention uses MRSM medium to activate chrysogenum, which can significantly shorten the culture time of chrysogenum. Attached Figure Description
[0029] Figure 1 The growth of engineered *Sacchariformis* strain O.oeni / pHH40-lphsp and the original *Sacchariformis* strain HH1 in a 12% ethanol environment.
[0030] Figure 2 The changes in malic acid content during MLF fermentation of engineered strain O.oeni / pHH40-lphsp and original strain HH1 in 12% ethanol.
[0031] Figure 3 The growth of engineered *Sacchariformis* strain O.oeni / pHH40-lphsp and the original *Sacchariformis* strain HH1 in a 14% ethanol environment.
[0032] Figure 4 The growth of engineered *O. oeni / pHH40-lphsp* strain in MRSM, ATB, and mF80T media was studied. Detailed Implementation
[0033] The following description is based on specific embodiments:
[0034] Source of experimental materials:
[0035] Oenococcus oeni HH1, with accession number CCTCC No.M 2023772, was provided by the Biological Laboratory of Qilu University of Technology.
[0036] Lactobacillus plantarum HX1, with accession number CCTCC No. M 2017522, was provided by the Biological Laboratory of Qilu University of Technology.
[0037] E. coli DH5α / pUC57-gfp: Escherichia coli containing the green fluorescent protein encoding gene (gfp), provided by the Biological Laboratory of Qilu University of Technology; among which E. coli DH5α was purchased from Nanjing Novizan Biotechnology Co., Ltd.
[0038] All molecular biology experimental materials, including genome extraction kits, gene PCR amplification kits, plasmid extraction kits, gene purification kits, and gene recombination ligation kits, were purchased from Nanjing Novizan Biotechnology Co., Ltd.
[0039] Example 1:
[0040] Construction of engineered strain of *O. oeni* / pHH40-lphsp*
[0041] (1) Genomic DNA of Lactobacillus plantarum HX1 was extracted using a genomic DNA extraction kit; the lphsp gene sequence of Lactobacillus plantarum was searched in GenBank and primers lphsp-F (SEQ ID NO.3) and lphsp-R (SEQ ID NO.4) were designed; using the genomic DNA of Lactobacillus plantarum HX1 as a template, PCR amplification was performed using primers lphsp-F and lphsp-R to obtain the heat shock protein gene lphsp (SEQ ID NO.2);
[0042] The PCR amplification system (50 μL) consisted of: 10 μL of 5×PrimeSTAR GXL Buffer; 4 μL of dNTP Mixture (2.5 mM each); 2 μL of lphsp-F; 2 μL of lphsp-R; 1 μL of DNA template; 1 μL of PrimeSTAR GXL DNA Polymerase; and 30 μL of ddH2O.
[0043] The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 68℃ extension for 2 min, 35 cycles; 68℃ final extension for 10 min.
[0044] (2) Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the pHH40 plasmid according to the sequence (SEQ ID NO.1); based on the pHH40 plasmid sequence, primers pHH40-F (SEQ ID NO.5) and pHH40-R (SEQ ID NO.6) carrying the homologous arm of the lphsp gene were designed. The homologous arm sequence is located on both sides of the MCS region of the multiple cloning site of the pHH40 plasmid; using the pHH40 plasmid as a template, PCR amplification was performed using primers pHH40-F and pHH40-R to obtain the homologous arm gene carrying the heat shock protein gene lphsp;
[0045] The PCR amplification system (50 μL) consisted of: 10 μL of 5×PrimeSTAR GXL Buffer; 4 μL of dNTP Mixture (2.5 mM each); 2 μL of pHH40-F; 2 μL of pHH40-R; 1 μL of plasmid pHH40 template; 1 μL of PrimeSTAR GXL DNA Polymerase; and 30 μL of ddH2O.
[0046] The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 68℃ extension for 2 min, 35 cycles; 68℃ final extension for 10 min.
[0047] (3) The heat shock protein gene lphsp from step (1) and the homologous arm gene from step (2) were purified and recovered using a gene purification kit. Then, the purified heat shock protein gene lphsp and the homologous arm gene were recombined and ligated using a gene recombination ligation kit to obtain the ligation product. The ligation product was transformed into competent Escherichia coli DH5α cells under the condition of heat shock at 42℃ for 30s to obtain transformant 1.
[0048] Transformant 1 was spread on LB solid medium containing 400 μg / L erythromycin and cultured at 37°C for 20 h. Transformant 1 was selected for sequencing verification, and the correct sequence was selected as the recombinant expression vector pHH-lphsp.
[0049] (4) Activate and culture *Chlorella vulgaris* HH1 to the logarithmic growth phase (OD). 600 =0.25), centrifuge to collect the bacterial cells, then wash 4 times at room temperature with 10% (v / v) 0℃ glycerol solution containing 0.5 mmol / L sucrose, and store in a solution containing 0.5 mmol / L potassium phosphate, 0.2 mmol / L MgCl2, 5% (v / v) ethanol and 5% (v / v) propanol to obtain competent cells of Chlorella vulgaris HH1;
[0050] The recombinant expression vector pHH-lphsp from step (3) was mixed with competent cells of *Chlorella vulgaris* HH1. After mixing, the mixture was transferred into an electroporation cuvette with a spacing of 2 mm and placed on ice for 10 min. Electroporation was performed using a high-voltage pulse electroporator with the following parameters: voltage 2.0 kV, resistance 200 Ω, electroporation constant 5 ms, capacitance 25 μF, and electroporation for 1.0 s to obtain transformant 2.
[0051] Transformant 2 was added to 1 mL of MRSM medium containing 0.5 mol / L sucrose and incubated at 30°C for 4 h for recovery culture. The bacterial cells were collected by centrifugation and spread on MRSM solid medium containing 40 mg / L erythromycin. The cells were incubated at 30°C for 8 days, and the number of transformants 2 in the medium after recovery culture was recorded. Transformant 2 was selected for sequencing verification. The strain with the correct sequence was identified as the engineered strain of *O. oeni* / pHH40-lphsp.
[0052] The MRSM medium comprises the following components: yeast extract 5.0 g / L, beef extract 5.0 g / L, peptone 10.0 g / L, sodium acetate 5.0 g / L, triammonium citrate 2.0 g / L, glucose 20.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 mL, potassium dihydrogen phosphate 2.0 g / L, L-malic acid 10.0 g / L, fructose 10.0 g / L, pH 4.5; the MRSM solid medium is MRSM medium with 15 g / L agar added.
[0053] Example 2:
[0054] Application of engineered strain O. oeni / pHH40-lphsp in wine production
[0055] (1) Select the engineered strain of *Stellaria media* O.oeni / pHH40-lphsp prepared in Example 1 and incubate it in MRSM medium at 25°C for 48 hours to obtain bacterial solution 1; select the unmodified wild-type strain of *Stellaria media* HH1 and incubate it under the same conditions to obtain bacterial solution 2.
[0056] (2) Select wine with an ethanol concentration of 12% (v / v) and which has completed yeast fermentation, filter and sterilize it for later use; mix bacterial solution 1 and bacterial solution 2 from step (1) with the sterilized wine at a volume ratio of 1:1, and incubate at room temperature for 5 days to obtain a mixture; add the mixture to new wine at 30% of the wine weight, continue to incubate at room temperature for 5 days, and adjust the bacterial solution concentration to 10%. 6 CFU / mL, to obtain culture medium.
[0057] (3) Add 1 mL of the culture medium from step (2) to 100 mL of wine that has completed yeast fermentation to carry out malic-lactic acid (MLF) fermentation; detect the growth of two strains of *Saccharomyces cerevisiae*, *O. oeni / pHH40-lphsp* and *Saccharomyces cerevisiae* HH1, the MLF fermentation activity, and the aroma components such as ethyl acetate in the wine. The detection of MLF fermentation activity is based on the standard SN / T4675.5-2016, and the detection of aroma components is based on the standard QBT4850-2015.
[0058] ① The growth results of the two strains in a 12% ethanol environment are as follows: Figure 1 As shown, it can be seen that the modified *O. oeni* engineered strain O. oeni / pHH40-lphsp* has significantly higher growth rate and growth amount than the original strain *O. oeni* HH1 in a 12% ethanol environment.
[0059] ② Results of MLF fermentation activity tests in wine fermentation without the addition of any strains and with both strains as shown below. Figure 2 As shown, compared with the two groups of wines with added *C. tartrazine*, the malic acid content decreased significantly, indicating that *C. tartrazine* has good MLF fermentation activity. The wine with added engineered *C. tartrazine* strain O.oeni / pHH40-lphsp showed a very low malic acid level on day 5, about 2 days earlier than the original strain *C. tartrazine* HH1 (O.oeni). This indicates that the modified *C. tartrazine* can start and complete MLF fermentation in wine more quickly, thus shortening the wine production cycle and saving production costs.
[0060] ③ The results of the detection of aroma components in wine are shown in Table 1 below:
[0061] Table 1. Detection of ester content in wine
[0062]
[0063]
[0064] As shown in Table 1, the content of aroma components in wines with added *C. oeni* / pHH40-lphsp* strain O.oeni / pHH40-lphsp is not significantly different from that with added original strain *C. oeni* HH1. This indicates that the modified *C. oeni* strain O.oeni / pHH40-lphsp* strain O.oeni / pHH40-lphsp* strain did not have an adverse effect on the aroma of the wine.
[0065] Example 3:
[0066] Application of engineered strain O. oeni / pHH40-lphsp in wine production
[0067] Unlike Example 2, this example uses the bacterial culture 1 from Example 2 to carry out malolactic (MLF) fermentation in wine with an ethanol concentration of 14% (v / v) and which has already undergone yeast fermentation. The remaining steps are the same as in Example 2. The growth of the engineered strain of *O. oeni* / pHH40-lphsp and the MLF fermentation activity were detected.
[0068] The test results are as follows Figure 3As shown, in a 14% ethanol environment, *O. oeni* HH1 essentially stopped growing, while the artificially constructed engineered *O. oeni* / pHH40-lphsp* strain could still grow under these conditions, but its growth quantity and growth rate were slightly lower than in Example 2.
[0069] Example 4:
[0070] Following the construction method described in Example 1, the green fluorescent protein encoding gene gfp (NCBI GI: 690969141) from E. coli DH5α / pUC57-gfp strain was introduced into *E. coli* HH1 using plasmid pHH40. Electroporation was performed under the same conditions as in Example 1, and the number of transformants in the culture medium after resuscitation was recorded, which was 45 ± 2.
[0071] Comparative Example 1:
[0072] Unlike Example 1, in the construction of the recombinant expression vector in step (2), plasmid pHH40 is not used. Instead, the existing lactic acid bacteria universal plasmids pMG36e, pNZ2118, and pLEB590 are selected to construct the recombinant expression vector.
[0073] The recombinant expression vectors constructed using plasmids pMG36e, pNZ2118, and pLEB590 were electroporated with competent cells of *Chlorella vulgaris* HH1 under the same conditions as in Example 1. The number of transformants in the culture medium after resuscitation was detected, and the results are shown in Table 2 below.
[0074] Table 2. Number of transformants obtained using different plasmids
[0075] plasmid Number of transformants in culture medium pHH40 43±3 pMG36e 4±1 pNZ2118 0 pLEB590 0
[0076] As shown in Table 2, the heat shock protein encoding gene could not be transformed into *C. tumefaciens* HH1 using pNZ2118 and pLEB590 as vectors; only about 4 transformants were obtained using pMG36e plasmid as vector; while the number of transformants obtained using the artificially constructed pHH40 plasmid as vector was as high as 43, and the transformation effect was much higher than that of the universal plasmids pMG36e, pNZ2118, and pLEB590.
[0077] Comparative Example 2:
[0078] Unlike Example 1, in step (4) of preparing competent cells of *Chlorella vulgaris* HH1, the "5% (v / v) ethanol and 5% (v / v) propanol" in the preservation solution were replaced with "10% (v / v) ethanol" or "10% (v / v) propanol". Then, electroporation was performed under the same conditions as in Example 1, and the number of transformants in the culture medium after resuscitation was detected. The results are shown in Table 3 below:
[0079] Table 3. Number of transformants in competent *Aeromonas tumefaciens* cells under different preservation solutions
[0080] Ethanol: Propanol Number of transformants in culture medium 5％：5％ 43±3 10％：0％ 23±3 0％：10％ 7±2
[0081] As shown in Table 3, the conversion rate of competent cells of *Chlorella vulgaris* was much higher when both ethanol and propanol were added simultaneously. This was compared to preservation solutions with only ethanol or propanol added.
[0082] Comparative Example 3:
[0083] The engineered strain of *Sacchariformis* O.oeni / pHH40-lphsp obtained in step (4) was placed in 50 mL of MRSM medium, acid tomato medium (ATB medium) and mF80T medium, respectively, and cultured at 30℃ for 4 h. The number of viable bacteria in each medium was then detected.
[0084] The ATB culture medium consists of the following components: 10.0 g / L peptone, 10.0 g / L glucose, 5.0 g / L yeast extract, 2.0 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate tetrahydrate, and 5.0 g / L tomato extract.
[0085] The mF80T medium consists of: 5.0 g / L tryptone, 4.0 g / L yeast extract, 0.6 g / L potassium dihydrogen phosphate, 0.45 g / L potassium chloride, 0.13 g / L calcium chloride, 0.13 g / L magnesium sulfate, 0.003 g / L manganese sulfate, 1.0 mL / L Tween 80, 10.0 g / L L-malic acid, 35.0 g / L fructose, and 5.0 g / L glucose.
[0086] Test results as follows Figure 4 As shown, the viable count of *C. tumefaciens* in MRSM medium was consistently higher than that in ATB and mF80T mediums. In particular, after 120 hours of culture, the growth rate of *C. tumefaciens* in MRSM medium was significantly increased, the logarithmic growth phase was prolonged, and a high cell concentration was eventually reached, which could significantly shorten the culture time of *C. tumefaciens*.
[0087] In summary, this invention uses the artificially constructed plasmid pHH40 as a vector, which can successfully introduce exogenous genes into the host strain of *C. truncatella* with a high success rate. In addition, this invention prepares an engineered strain of *C. truncatella* O.oeni / pHH40-lphsp, which can tolerate ethanol concentrations up to 14% and can initiate and complete MLF fermentation in wine production more quickly, thus shortening the wine production cycle.
Claims
1. The application of expression vector pHH40 in introducing exogenous genes into *Chlorella vulgaris*, characterized in that, The nucleotide sequence of the expression vector pHH40 is shown in SEQ ID NO.1, and the exogenous gene is a heat shock protein encoding gene. lphsp The heat shock protein encoding gene lphsp The nucleotide sequence is shown in SEQ ID NO.
2.
2. The application as described in claim 1, characterized in that, The application method is as follows: using pHH40 as an expression vector, the heat shock protein encoding gene is expressed. lphsp It was obtained by expressing the strain in the host strain of *Chlorella vulgaris*.
3. An engineered strain of *Chlorella vulgaris*, characterized in that, The construction method includes the following steps: (1) Extract genomic DNA from Lactobacillus plantarum as a template and use primers. lphsp -F、 lphsp -R was used for PCR amplification to obtain the heat shock protein gene. lphsp The lphsp The nucleotide sequence is shown in SEQ ID NO.2; wherein, the primer... lphsp The nucleotide sequence of -F is shown in SEQ ID NO.3, primer lphsp The nucleotide sequence of -R is shown in SEQ ID NO.4; (2) Using plasmid pHH40 as a template, PCR amplification was performed using primers pHH40-F and pHH40-R to obtain the heat shock protein gene. lphsp The homologous arm genes; wherein, the nucleotide sequence of pHH40 is shown in SEQ ID NO.1, the nucleotide sequence of primer pHH40-F is shown in SEQ ID NO.5, and the nucleotide sequence of primer pHH40-R is shown in SEQ ID NO.6; (3) The heat shock protein gene from step (1) is respectively lphsp After linearization of the homologous arm gene from step (2), the ligation product was obtained; the ligation product was transformed into competent E. coli cells to obtain transformant 1; after screening, the recombinant expression vector pHH- was obtained. lphsp ; (4) The recombinant expression vector pHH- from step (3) lphsp Transformed into competent cells of the host strain of *Chlorella vulgaris*, transformant 2 was obtained; after screening, engineered *Chlorella vulgaris* strain was obtained.
4. The engineered strain of *Chlorella vulgaris* as described in claim 3, characterized in that, The transformation method in step (3) is as follows: the ligation product is mixed with competent cells of Escherichia coli and heat-shocked in a water bath at 42°C for 30 s.
5. The engineered strain of *Chlorella vulgaris* as described in claim 3, characterized in that, The screening method in step (3) is as follows: Transformant 1 is spread on LB solid medium containing 100~500 µg / L erythromycin and cultured at 35~37℃ for 20~28h. Transformants are selected for sequencing verification. The transformants with the correct sequence are the expression vector pHH- lphsp .
6. The engineered strain of *Chlorella vulgaris* as described in claim 3, characterized in that, The method for preparing competent cells of *Staphylococcus zeolites* host bacteria in step (4) is as follows: activate and culture *Staphylococcus zeolites* host bacteria to the logarithmic growth phase, collect the cells by centrifugation, and then wash 3 to 5 times at room temperature with a 10% (v / v) glycerol solution containing 0.5 mmol / L sucrose. Then, store the cells in a solution containing 0.5 mmol / L potassium phosphate, 0.2 mmol / L MgCl2, 5% (v / v) ethanol and 5% (v / v) propanol to obtain competent cells of *Staphylococcus zeolites* host bacteria.
7. The engineered strain of *Chlorella vulgaris* as described in claim 3, characterized in that, The conversion conditions in step (4) are: 2.0KV, 200Ω, 5ms, 25μF electro-transfer for 1.0s.
8. The engineered strain of *Chlorella vulgaris* as described in claim 3, characterized in that, The screening method in step (4) is as follows: Transformant 2 is added to MRSM medium containing 0.5 mol / L sucrose and cultured statically at 27-30℃ for 3-5 hours. The bacterial cells are collected by centrifugation. The bacterial cells are spread on MRSM solid medium containing 25-40 mg / L erythromycin and cultured statically at 27-30℃ for 8-10 days. Transformants are selected for sequencing verification. The strains with correct sequences are the engineered strains of *Chlorella vulgaris*. The MRSM medium comprises the following components: yeast extract 5.0 g / L, beef extract 5.0 g / L, peptone 10.0 g / L, sodium acetate 5.0 g / L, triammonium citrate 2.0 g / L, glucose 20.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 mL, potassium dihydrogen phosphate 2.0 g / L, L-malic acid 10.0 g / L, fructose 10.0 g / L, pH 4.5; the MRSM solid medium is MRSM medium with 15 g / L agar added.
9. The application of the engineered strain of *Acetobacter tumefaciens* according to any one of claims 3 to 8 in the production of wine.