A method for improving the antioxidant activity of Lactococcus lactis and its application

Abstract

The present invention relates to a method for improving the antioxidant activity of Lactococcus lactis and its application. The method for improving the antioxidant activity of Lactococcus lactis is to construct a recombinant lactic acid bacterium that produces lycopene, thereby improving the antioxidant activity of Lactococcus lactis. The method for constructing the recombinant lactic acid bacterium that produces lycopene is to construct a lycopene heterologous synthesis plasmid, knock out the lactate dehydrogenase (LDH) gene in Lactococcus lactis or overexpress MVA pathway genes in strains of different sources to obtain the recombinant Lactococcus lactis, thereby achieving heterologous synthesis of lycopene by Lactococcus lactis. Compared with the prior art, the present invention constructs a recombinant lactic acid bacterium that produces lycopene, measures its own antioxidant activity and that in skim milk, and finds that the antioxidant activity of the recombinant Lactococcus lactis is more than 1 times that of the original strain, and the DPPH, ABTS and ·OH free radical scavenging abilities are significantly enhanced. The recombinant Lactococcus lactis is expected to be developed into an antioxidant probiotic product or fermentation agent.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for improving the antioxidant activity of Lactococcus lactis and an application thereof. Background Art

[0002] Reactive oxygen species (ROS) are normal products of aerobic metabolism in living cells. When the body is stressed by external environmental factors, excessive ROS, such as superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide, are produced. These ROS can denature intracellular macromolecules such as DNA, proteins, and lipids, leading to cell damage, loss of function, and even apoptosis and necrosis. Therefore, maintaining a balance between oxidation and antioxidant activity is crucial for maintaining normal physiological function and health.

[0003] Lactococcus lactis is one of the important members of lactic acid bacteria and was first isolated from plants. [1] As the most widely studied model strain of lactic acid bacteria, with the completion of the whole genome sequencing of Lactococcus lactis in 2001, it has become the most popular host for exogenous gene expression after Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis. It is currently the most complete lactic acid bacteria gene cloning and expression system. [2] . Lactococcus lactis is widely used as a starter in cheese and other fermented foods, but it is inevitably subjected to ROS oxidative stress in industrial fermentation production, which reduces its fermentation activity. And there is no efficient catalase (CAT) in Lactococcus lactis to scavenge free radicals and hydrogen peroxide, which will cause damage to the bacteria. Common solutions are to express antioxidant enzymes or synthesize active peptides, such as overexpressing SOD or CAT, introducing glutathione (GSH) biosynthesis pathway and other strategies, but such methods have a low overall improvement in the antioxidant activity of Lactococcus lactis. Therefore, improving the antioxidant activity of Lactococcus lactis has important application value.

[0004] Lycopene is a C 40 Isoprenoid compounds have strong antioxidant and immunity-enhancing effects and have been widely used in industries such as food and cosmetics. [3] Lycopene is one of the strongest antioxidants known in nature. Its antioxidant effect is more than twice that of beta-carotene and 100 times that of vitamin E.

[0005] [1] Lv Yichao, Li Xiangao, Wang Kaibo, et al. Research progress on the antibacterial mechanism of lactic acid bacteria as biological protective bacteria and their application in food [J]. Food Science, 2021(19):281-290.

[0006] [2]Xiong ZQ, Wei YY, Kong LH, et al.Short communication: An inducibleCRISPR / dCas9 gene repression system in Lactococcus lactis[J].Journal of DairyScience,2019,103(1):161-165.

[0007] [3]Liang X, Ma C, Yan Summary of the Invention

[0008] The purpose of the present invention is to overcome the defects of the above-mentioned prior art and provide a method for improving the antioxidant activity of Lactococcus lactis and its application. The present invention heterologously expresses a lycopene synthesis gene in Lactococcus lactis, knocks out the lactate dehydrogenase (ldh) gene and optimizes the culture conditions to achieve lycopene synthesis by Lactococcus lactis, and significantly improves the antioxidant activity of the engineered Lactococcus lactis compared with the control strain.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] A method for improving the antioxidant activity of Lactococcus lactis, comprising introducing three heterologous enzyme genes, crtE, crtB, and crtI, into recombinant lactic acid bacteria to achieve heterologous synthesis of lycopene by the recombinant lactic acid bacteria, thereby improving the antioxidant activity of the recombinant lactic acid bacteria;

[0011] The nucleotide sequence of the crtE heterologous enzyme gene is shown in SEQ ID NO.1;

[0012] The nucleotide sequence of the crtB heterologous enzyme gene is shown in SEQ ID NO.2;

[0013] The nucleotide sequence of the crtI heterologous enzyme gene is shown in SEQ ID NO.3.

[0014] Furthermore, the pGZQ01 plasmid was introduced into the recombinant lactic acid bacteria to achieve heterologous synthesis of lycopene by Lactococcus lactis, thereby improving the antioxidant activity of Lactococcus lactis.

[0015] The pGZQ01 plasmid contains three heterologous enzyme genes: crtE, crtB and crtI. The nucleotide sequence of the pGZQ01 plasmid is shown in SEQ ID No.4.

[0016] Lactobacillus uses endogenous IPP and DMAPP to synthesize FPP as a precursor, and then sequentially catalyzes the production of lycopene through three heterologous enzymes: crtE, crtB, and crtI. KEGG metabolic pathway analysis revealed that Lactobacillus possesses a complete MVA pathway but lacks the genes for crtE, crtB, and crtI, which are required for lycopene production. The pGZQ01 plasmid carries these three enzyme genes.

[0017] Furthermore, the recombinant lactic acid bacteria is derived from Lactococcus lactis with the lactate dehydrogenase (ldh) gene knocked out, and the nucleotide sequence of the lactate dehydrogenase (ldh) gene is shown in SEQ ID No.5.

[0018] Furthermore, in the above, the Lactococcus lactis is NZ9000.

[0019] Furthermore, the recombinant lactic acid bacteria are derived from Lactococcus lactis in which the lactate dehydrogenase (ldh) gene is knocked out and the MVA pathway gene is overexpressed.

[0020] Furthermore, the MVA pathway gene is an MVA pathway gene in Lactococcus lactis NZ9000, Streptococcus thermophilus S-3 or Lactobacillus plantarum AR113.

[0021] Furthermore, the gene overexpressing the MVA pathway is the thermophilic Streptococcus 3-hydroxy-3-methylglutaryl-CoA reductase (mvaA) gene, and the nucleotide sequence of the thermophilic Streptococcus 3-hydroxy-3-methylglutaryl-CoA reductase (mvaA) gene is shown in SEQ ID No.6.

[0022] Furthermore, the recombinant lactic acid bacteria are cultured in a culture medium, and acetic acid is added to the culture medium.

[0023] Furthermore, the culture temperature is 30°C, the pH is 7.0, and the fermentation time is 12 hours.

[0024] In addition, the present invention also provides a use of the recombinant lactic acid bacteria obtained based on the above method in the preparation of probiotic products or starter cultures, wherein the recombinant lactic acid bacteria improves the antioxidant function of the probiotic products or starter cultures.

[0025] Compared with the existing technology, the present invention constructs a recombinant lactic acid bacteria that produces lycopene, and measures its own antioxidant activity and that in skim milk. It is found that the antioxidant activity of the recombinant Lactococcus lactis is more than 1 times higher than that of the original strain, and the DPPH, ABTS and ·OH free radical scavenging abilities are significantly enhanced. Therefore, the present invention has the potential to be developed into an antioxidant probiotic product or fermentation agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 For the plasmid construction process;

[0027] Figure 2 PCR verification and double enzyme digestion verification, where A is PCR verification and B is double enzyme digestion verification;

[0028] Figure 3 Colony PCR verification of transformants transformed into lactic acid bacteria, where M is 5000 and 1-3 are all transformants;

[0029] Figure 4 For comparison of bacteria after fermentation;

[0030] Figure 5 The results of HPLC analysis of fermentation broth after extraction, where A is the lycopene standard and B is the fermentation extract of the NZ9000 recombinant strain;

[0031] Figure 6 Lycopene UPC 2 Analyze the results;

[0032] Figure 7 The effect of different acetic acid concentrations on lycopene production;

[0033] Figure 8 The working principle of the L. lactis NZ9000 CRISPR-Cas9 single plasmid system for gene editing;

[0034] Figure 9 The transformant colony PCR verification was performed, where M is 5000, 1 is the control group, and 2-5 are all transformants;

[0035] Figure 10 is the effect of △ldh on lycopene production, where GZQ01 is a lycopene-producing recombinant strain before △ldh, and GZQ02 is a lycopene-producing recombinant strain after △ldh;

[0036] Figure 11 The effect of adding acetic acid and △ldh on lycopene production;

[0037] Figure 12Effects of overexpression of MVA pathway genes from different sources on lycopene biosynthesis, among which GZQ03-GZQ09 overexpressed genes were from NZ9000, GZQ10-GZQ16 overexpressed genes were from S-3, and GZQ017-GZQ23 overexpressed genes were from AR113;

[0038] Figure 13 The effect of culture medium optimization on lycopene production of recombinant strain GZQ12, where A is the amount of glucose added, B is the amount of tryptone added, and C is the amount of yeast powder added;

[0039] Figure 14 The effect of fermentation temperature on lycopene production by recombinant strain GZQ12;

[0040] Figure 15 The effect of fermentation time on lycopene production by recombinant strain GZQ12;

[0041] Figure 16 The effect of initial fermentation pH on lycopene production by recombinant strain GZQ12;

[0042] Figure 17 are the antioxidant test results, where A is the DPPH scavenging ability, B is the ABTS scavenging ability, and C is the ·OH scavenging ability. DETAILED DESCRIPTION

[0043] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0044] In the following examples, the required culture medium is as follows:

[0045] GM17 medium (1 L): 5 g tryptone, 5 g soy peptone, 2.5 g yeast extract powder, 5 g beef extract powder, 20 g lactose, 19 g β-glycerophosphate disodium, MgSO 4. 7 H2O 0.58g, glucose 5g.

[0046] In the following examples, the required reagents are as follows:

[0047] Lycopene (HPLC ≥ 90%, CAS # 502-65-8) standard, Shanghai Yuanye Biotechnology Co., Ltd.;

[0048] Chloramphenicol, Sangon Biotech (Shanghai) Co., Ltd.;

[0049] Dichloromethane and acetone were of analytical grade, methanol was of chromatographic grade, and other reagents were from Sinopharm Group;

[0050] Plasmid extraction kit, Axygen.

[0051] In the following examples, the required strains are as follows:

[0052] The deposit information of Streptococcus thermophilus S-3 is as follows: The deposit number of Streptococcus thermophilus is CGMCC No. 12098, the depositor is China General Microorganism Culture Collection Center, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is January 22, 2016, and it has been disclosed in patent CN108220201A.

[0053] The deposit information of Lactobacillus plantarum AR113 is as follows: The deposit number of Lactobacillus plantarum AR113 is CGMCC No. 13909, the depository is the China General Microorganism Culture Collection Center, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is March 22, 2017, and it has been disclosed in patent CN111304134A.

[0054] Lactococcus lactis NZ9000 was commercially available.

[0055] In the following examples, the required plasmids are as follows:

[0056] The pACLYC plasmid comes from the article Wang JF, Meng HL, Xiong ZQ, Zhang SL, WangY. Identification of novel knockout and up-regulated targets for improving isoprenoid production in E.coli. Biotechnology letters, 2014, 36(5): 1021-1027.

[0057] The pNZ44 plasmid is derived from the article McGrath S, Fitzgerald GF, van Sinderen D. Improvement and optimization of two engineered phage resistance mechanisms in Lactococcus lactis. Applied and environmental microbiology, 2001, 67(2): 608-616.

[0058] The pIB184 plasmid is derived from the article Biswas I, Jha JK, Fromm N. Shuttle expression plasmids for genetic studies in Streptococcus mutans. Microbiology (Reading), 2008, 154(Pt 8): 2275-2282.

[0059] In the following examples, the required instruments are as follows:

[0060] Nanodrop 2000c ultra-micro-quantity nucleic acid and protein quantification instrument, Thermo Fisher Scientific, USA;

[0061] Refrigerated centrifuge, Sigma, Germany;

[0062] Spectrophotometer DU-800, Beckman Company, USA;

[0063] Biochemical incubator, Shanghai Boxun Industrial Co., Ltd.;

[0064] MicroPulse electroporation instrument, Bio-Rad, Germany;

[0065] Waters e2695 high performance liquid chromatography, Waters Technology Co., Ltd. (Shanghai, China).

[0066] In the following examples, the nucleotide sequences are as follows:

[0067] The nucleotide sequence of the crtE heterologous enzyme gene is shown in SEQ ID NO.1:

[0068] The nucleotide sequence of the crtB heterologous enzyme gene is shown in SEQ ID NO.2:

[0069] The nucleotide sequence of the crtI heterologous enzyme gene is shown in SEQ ID NO.3:

[0070] The nucleotide sequence of the pGZQ01 plasmid is shown in SEQ ID No. 4:

[0071] The nucleotide sequence of the lactate dehydrogenase gene is shown in SEQ ID No. 5:

[0072] The nucleotide sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase gene of Streptococcus thermophilus is shown in SEQ ID No. 6:

[0073] The nucleotide sequence of the mvaS heterologous enzyme gene from NZ9000 is shown in SEQ ID NO.7:

[0074] The nucleotide sequence of the mvaA heterologous enzyme gene from NZ9000 is shown in SEQ ID NO.8:

[0075] The nucleotide sequence of the mvaD heterologous enzyme gene from NZ9000 is shown in SEQ ID NO.9:

[0076] The nucleotide sequence of the mvaK1 heterologous enzyme gene from NZ9000 is shown in SEQ ID NO.10:

[0077] The nucleotide sequence of the mvaK2 heterologous enzyme gene from NZ9000 is shown in SEQ ID NO.11:

[0078] The nucleotide sequence of the idi heterologous enzyme gene derived from NZ9000 is shown in SEQ ID NO.12:

[0079] The nucleotide sequence of the IspA heterologous enzyme gene from NZ9000 is shown in SEQ ID NO.13:

[0080] The nucleotide sequence of the mvaS heterologous enzyme gene from S-3 is shown in SEQ ID NO.14:

[0081] The nucleotide sequence of the mvaA heterologous enzyme gene from S-3 is shown in SEQ ID NO.15:

[0082] The nucleotide sequence of the mvaD heterologous enzyme gene from S-3 is shown in SEQ ID NO.16:

[0083] The nucleotide sequence of the mvaK1 heterologous enzyme gene from S-3 is shown in SEQ ID NO.17:

[0084] The nucleotide sequence of the mvaK2 heterologous enzyme gene from S-3 is shown in SEQ ID NO.18:

[0085] The nucleotide sequence of the idi heterologous enzyme gene derived from S-3 is shown in SEQ ID NO.19:

[0086] The nucleotide sequence of the IspA heterologous enzyme gene derived from S-3 is shown in SEQ ID NO.20:

[0087] The nucleotide sequence of the mvaS heterologous enzyme gene derived from AR113 is shown in SEQ ID NO.21:

[0088] The nucleotide sequence of the mvaA heterologous enzyme gene derived from AR113 is shown in SEQ ID NO.22:

[0089] The nucleotide sequence of the mvaD heterologous enzyme gene derived from AR113 is shown in SEQ ID NO.23:

[0090] The nucleotide sequence of the mvaK1 heterologous enzyme gene derived from AR113 is shown in SEQ ID NO.24:

[0091] The nucleotide sequence of the mvaK2 heterologous enzyme gene derived from AR113 is shown in SEQ ID NO.25:

[0092] The nucleotide sequence of the idi heterologous enzyme gene derived from AR113 is shown in SEQ ID NO.26:

[0093] The nucleotide sequence of the IspA heterologous enzyme gene derived from AR113 is shown in SEQ ID NO.27.

[0094] Unless otherwise specified, the rest of the raw materials or processing techniques are conventional commercial products or conventional processing techniques in the art.

[0095] Example

[0096] 1 Construction of lycopene synthesis pathway of recombinant Lactococcus lactis

[0097] (1) Construction of recombinant plasmid

[0098] Using the pACLYC plasmid as a template, the target fragments crtE and crtIB were amplified. Overlap PCR was performed on the two target fragments to amplify the crtEIB fragment. The lactic acid bacteria shuttle plasmid pNZ44 was then digested with the restriction endonucleases XbaI and SpeI and seamlessly cloned with the crtEIB fragment to obtain the recombinant plasmid pGZQ01.

[0099] (2) Verification of recombinant lactic acid bacteria

[0100] The laboratory-collected Lactococcus lactis NZ9000 was selected, and the recombinant plasmid pGZQ01 was electroporated into the competent cells of Lactococcus lactis NZ9000 (L. lactis NZ9000). Transformants were randomly selected and verified by colony PCR using primers pNZ44-YZ-F and pNZ44-YZ-R. After correct verification, the transformants were stored in glycerol to obtain recombinant Lactococcus lactis GZQ01.

[0101] (3) Recombinant bacterial fermentation and fermentation product extraction

[0102] After the recombinant Lactococcus lactis GZQ01 (NZ9000 / pGZQ01) was fermented at 30°C and 200 r / min for 24 h, the cells were collected for lycopene extraction and yield determination. Two equal amounts of fermentation broth were taken, one of which was used to dilute the fermentation broth 5 times and the OD was measured using a spectrophotometer. 600 , total bacterial OD 600 The value is the dilution multiple multiplied by the OD value of the diluted bacterial solution. 600 reading.

[0103] The lycopene extraction method is as follows: lycopene is extracted with 1 mL of acetone, placed in a 55°C water bath for extraction for 15 minutes, vortexed for 20 seconds every 5 minutes, and then centrifuged at 12,000 g for 10 minutes to obtain the supernatant to obtain the lycopene extract.

[0104] (4) Fermentation product detection

[0105] Preparation of lycopene standard curve: Weigh 2 mg of lycopene standard, dissolve in acetone, and dilute to a 10 mL volumetric flask to prepare a 200 μg / mL stock solution. Dilute serially with acetone and filter through a 0.22 μm microporous filter into a sample injection vial. Detect by HPLC. Perform linear regression of the peak area X against the lycopene concentration Y. The regression equation is: Y = 0.0288X - 0.0219, R 2 =0.9998.

[0106] The lycopene extract obtained in step (3) was detected using a C18 column (4.6 mm × 150 mm, Waters). The HPLC conditions were: column temperature, 30°C; mobile phase, methanol:dichloromethane (75:25, v:v); flow rate, 1.0 mL / min; injection volume, 10 μL; DAD lamp detection; and detection wavelength, 472 nm.

[0107] (5) Ultra-high performance convergence chromatography (UPC) 2 ) Identification

[0108] The lycopene extract obtained in step (3) was concentrated using a vacuum concentrator until the organic liquid was completely volatilized, and then redissolved with methyl tert-butyl ether and passed through a 0.22 μm organic membrane and then filtered using a Waters UPC 2 Conduct testing.

[0109] The measurement conditions were: ACQUITY UPC equipped with PDA detector 2 Systems and Specialty Columns HSS C18SB Column 100A, 1.8 μm; mobile phase: 20% methanol and 80% CO2, flow rate: 1 mL / min, column temperature: 40°C, detection wavelength: 450 nm, back pressure: 13.793 MPa (2000 psi), injection volume: 2 μL.

[0110] Example 2 Metabolic Engineering to Improve Heterologous Lycopene Synthesis by Lactococcus Lactis

[0111] 2-1 Strengthening the supply of acetyl-CoA

[0112] (1) Recombinant Lactococcus lactis GZQ01 was supplemented with different concentrations of acetic acid

[0113] The recombinant Lactococcus lactis GZQ01 strain was taken out of a -80°C freezer, thawed on ice, and activated by inoculating the culture into a 5 mL eppendorf tube at a 3% inoculum volume. The activated culture was then transferred to a 250 mL conical flask (containing 50 mL GM17 medium) at a 3% inoculum volume. The culture was shaken at 30°C and 200 rpm / min for 12 h. Acetic acid was added at concentrations of 0, 0.2%, 0.4%, 0.6%, 0.8%, and 1% at the initial fermentation stage.

[0114] (2) Construction of recombinant NZ9000 producing lycopene after LDH knockout

[0115] Using CRISPR-Cas9 technology, an editing system was established in Lactococcus lactis NZ9000 to knock out the ldh gene, resulting in the L. lactis NZ9000-Δldh strain. Competent L. lactis NZ9000-Δldh cells were then transformed with the pGZQ01 plasmid. Electroporation was performed at 12.5 kV / cm, followed by the addition of 900 μL of recovery medium and incubation at 30°C for 2 hours. The recovered bacterial suspension was then spread on GM17 selection plates containing antibiotics, centrifuged at 5000 rpm for 5 minutes, and cells were harvested. 100 μL of the recovery medium was then applied to the plates. After incubation at 30°C for 24–36 hours, individual colonies were selected for PCR verification. If verified, recombinant L. lactis GZQ02 (NZ9000-Δldh / pGZQ01) was obtained.

[0116] (3) Recombinant Lactococcus lactis GZQ02 was supplemented with 0.2% acetic acid

[0117] The recombinant Lactococcus lactis GZQ02 strain was taken out of the -80°C freezer, thawed on ice, and activated by inoculating the culture into a 5 mL eppendorf tube at a 3% inoculum volume. The activated culture was then transferred to a 250 mL conical flask (containing 50 mL GM17 medium) at a 3% inoculum volume and shaken at 30°C and 200 rpm / min for 12 h. Acetic acid was added at a concentration of 0.2% at the initial stage of fermentation.

[0118] 2-2 Enhancement of MVA pathway gene expression levels

[0119] (1) Construction of recombinant plasmid

[0120] The MVA pathway genes from Lactococcus lactis NZ9000, Streptococcus thermophilus S-3 and Lactobacillus plantarum AR113 were overexpressed respectively. The plasmid overexpressing the MVA pathway genes from NZ9000 was constructed as an example. The pIB184 plasmid was used as the expression vector, and the NZ9000 bacterial solution was selected as the template. The target fragments mvaS, mvaA, mvaD, mvaK1, mvaK2, idi and IspA were amplified in sequence, and digested with restriction endonucleases BamHI & EcoRI, and seamlessly cloned and ligated with the target fragments to obtain the recombinant plasmids pGZQ02-pGZQ08.

[0121] The same construction was carried out to obtain recombinant plasmids pGZQ09-pGZQ15 derived from genes of the S-3 pathway;

[0122] The pathway gene derived from AR113 was similarly constructed to obtain the recombinant plasmid pGZQ16-pGZQ21.

[0123] (2) Verification of recombinant bacteria

[0124] The constructed plasmid was transferred into the competent recombinant Lactococcus lactis GZQ02 (NZ9000-△ldh / pGZQ01) and electroporated at 12.5 kV / cm. Then, 900 μL of recovery medium (GM17 medium) was added and the cells were incubated at 30°C for 2 hours. The recovered bacterial suspension was spread on GM17 selection plates with antibiotics. Cells were collected by centrifugation at 5000 rpm for 5 minutes, and 100 μL of the recovery solution was applied to the plates. After incubation at 30°C for 24-36 hours, single colonies were selected for PCR verification. If the results were correct, the GZQ03-GZQ23 strains were obtained.

[0125] (3) Recombinant bacterial fermentation and yield determination

[0126] Refer to the steps (3) and (4) in Example 1.

[0127] Example 3 Optimizing fermentation conditions to improve heterologous lycopene synthesis by Lactococcus lactis

[0128] (1) Effect of culture medium on lycopene production by recombinant bacteria

[0129] Based on GM17 medium, glucose, tryptone and yeast powder were selected as three factors to explore the effects of different addition amounts of glucose (2.5, 5, 7.5 and 10 g / L), different addition amounts of tryptone (2.5, 5, 7.5 and 10 g / L) and different addition amounts of yeast powder (0, 2.5, 5 and 7.5 g / L) on lycopene production. The optimal conditions were determined and the OD of the bacteria was measured after 12 h of fermentation in different culture media. 600 The determination method of lycopene production was similar to steps (3) and (4) in Example 1.

[0130] (2) Effect of fermentation temperature on lycopene production by recombinant bacteria

[0131] The activated recombinant strain GZQ12 was inoculated with 3% of the inoculum into the optimized GM17 medium in step (1) of Example 3 for fermentation. The culture was shaken in a constant temperature shaker at 20°C, 25°C, 30°C and 37°C, and the OD of the bacteria was measured after 12 h of fermentation. 600 The determination method of lycopene production was similar to steps (3) and (4) in Example 1.

[0132] (3) Effect of fermentation time on lycopene production by recombinant bacteria

[0133] The activated GZQ12 was inoculated with 3% of the inoculum into the optimized GM17 medium in step (1) of Example 3 for fermentation. The culture was placed in a constant temperature shaker at 30°C for shaking culture. The shake flasks were fermented for 8 h, 12 h, 16 h, 20 h and 24 h respectively. The OD of the bacteria after fermentation was measured. 600 The determination method of lycopene production was similar to steps (3) and (4) in Example 1.

[0134] (4) Effect of fermentation pH on lycopene production by recombinant bacteria

[0135] The activated GZQ12 was inoculated into the optimized GM17 medium in step (1) of Example 3 at a 3% inoculum for fermentation. The culture was shaken in a constant temperature shaker at 30°C. The initial pH value of the fermentation medium was set to 5.0, 5.5, 6.0, 7.0 and 8.0. The OD value of the bacterial cell was measured after 12 h of fermentation. 600 The determination method of lycopene production was similar to steps (3) and (4) in Example 1.

[0136] Example 4 Determination of Antioxidant Activity of Recombinant Lactococcus lactis

[0137] (1) Sample preparation

[0138] The activated control NZ9000 / pNZ44 strain and recombinant strain GZQ12 were adjusted to the cell density OD 600 =1.0 was inoculated into the optimized GM17 medium, and the recombinant strain GZQ12 was adjusted to a cell density of OD 600 =1.0 was inoculated into skim milk and fermented at 30℃ and 200r / min for 24h. The fermentation liquid was centrifuged at 5000r / min for 10min and the supernatant was collected. The cells were washed 2-3 times with PBS and the cell density was adjusted to OD 600 =1.0, and the cell bacterial suspension was obtained, and the antioxidant activity of the supernatant and cell bacterial suspension was subsequently determined.

[0139] (2) Determination of DPPH scavenging ability

[0140] Take 1 mL of sample and mix it with 2 mL of 0.2 mmol / L DPPH-anhydrous ethanol solution at room temperature in the dark for 30 minutes. Measure the absorbance at 517 nm. Zero the blank by mixing 1 mL of distilled water with 2 mL of anhydrous ethanol. Measure three replicates per group. Calculate the absorbance using the formula (1.1):

[0141] Clearance rate (%) = [A0-(A X -A1)】 / A0×100% (1.1)

[0142] A X : absorbance value of the sample group; A1: absorbance value when an equal volume of anhydrous ethanol replaces the DPPH solution; A0: absorbance value when an equal volume of anhydrous ethanol replaces the sample.

[0143] (3) Determination of ABTS free radical scavenging ability

[0144] Prepare a 7 mmol / L ABTS solution and a 2.45 mmol / L K2S2O8 solution. Mix 10 mL of K2S2O8 solution and 10 mL of ABTS solution evenly. Leave the mixture at room temperature in the dark for at least 12 hours. Dilute the ABTS+ free radical working solution with ethanol to an absorbance of 0.700 ± 0.02 at 734 nm. Mix 200 μL of the ABTS+ free radical working solution and 4 mL of the test solution in a test tube. Place the mixture in a 30°C water bath in the dark for 6 minutes. Measure the absorbance at 734 nm. The scavenging rate is calculated using the following formula (1.2):

[0145] Clearance (%) = (A0-Ax) / A0×100% (1.2)

[0146] A X : absorbance value of the sample group; A0: absorbance value when an equal volume of anhydrous ethanol is used to replace the sample.

[0147] (4) Determination of OH radical scavenging ability

[0148] Using the same sample concentration as above, 3 mL of sample solution was thoroughly mixed with 500 μL of a 6 mmol / L FeSO4·7H2O solution and 500 μL of a 6 mmol / L ethanol-salicylic acid solution. Then, 500 μL of a 6 mmol / L H2O2 solution was added. The mixture was reacted at 37°C for 30 minutes, and the absorbance of the reaction solution was measured at 510 nm. The clearance rate was calculated using the following formula (1.3):

[0149] Clearance rate (%) = [A0-(A X -A1)】 / A0×100% (1.3)

[0150] A X: absorbance value of the sample group; A1: absorbance value when the H2O2 solution is replaced by an equal volume of dd H2O; A0: blank absorbance value, absorbance value when the sample is replaced by an equal volume of dd H2O.

[0151] Example 5 Results

[0152] 5-1 Host screening for heterologous lycopene synthesis by recombinant lactic acid bacteria

[0153] Lactic acid bacteria synthesize lycopene from endogenous IPP and DMAPP using FPP as a precursor, which is then catalyzed sequentially by three heterologous enzymes: crtE, crtB, and crtI. KEGG metabolic pathway analysis revealed that lactic acid bacteria possess a complete MVA pathway, but lack the genes for crtE, crtB, and crtI, thus being unable to synthesize lycopene. The pACLYC plasmid, carrying these three enzyme genes, was used as a template in this study. The target fragments, crtE and crtIB, were amplified using primers pGZQ01-crtE-F, pGZQ01-crtE-R, pGZQ01-SD-crtIB-F, and pGZQ01-SD-crtIB-R. After confirming the correct size of the fragments by agarose gel electrophoresis, the target fragments were recovered using a gel recovery kit. Overlap PCR was then performed to amplify the crtEIB fragment at a pmol ratio of 1:1. After the fragment size was verified to be correct by agarose gel electrophoresis, the target fragment was recovered using a gel tapping recovery kit. The lactic acid bacteria shuttle plasmid pNZ44 was digested with restriction endonucleases XbaI and SpeI, and the digestion product was recovered and seamlessly cloned with the crtEIB fragment, and then transformed into Escherichia coli competent cells, and transformants were screened on chloramphenicol resistance plates ( Figure 1 )

[0154] Transformants were selected for PCR verification and analyzed by agarose gel electrophoresis: an amplified band was found between 3000 bp and 5000 bp, which was consistent with the expected band size (3343 bp). Figure 2 A), then a single colony was inoculated into a 4 mL test tube and cultured at 37°C, 200 rpm for 12 h to extract the plasmid, which was then digested with restriction enzymes XbaI and SpeI to verify the plasmid. Figure 2 B)

[0155] To verify the successful transformation of pGZQ01 into L. lactis NZ9000, randomly selected transformants were verified by colony PCR using primers pNZ44-YZ-F and pNZ44-YZ-R. Both had bands and were consistent with the theoretical value (3343 bp) ( Figure 3 The experimental results showed that the pGZQ01 plasmid was successfully transformed into L. lactis NZ9000.

[0156] Although colony PCR confirmed that the pGZQ01 plasmid had been successfully electroporated into L. lactis NZ9000 ( Figure 3 ), but after centrifuging the fermentation broth and observing the color of the cells, we found a clear difference. We can see that the control strain NZ9000 / pNZ44 cells are white after centrifugation, while the recombinant strain GZQ01 (NZ9000 / pGZQ01) cells appear pink after centrifugation, which is shown as a dark color in the figure ( Figure 4 ), and speculated that the color change of the recombinant strain GZQ01 was caused by the synthesis of lycopene.

[0157] The extract was measured by high performance liquid chromatography, and it can be seen that the lycopene standard showed an absorption peak at around 9 minutes ( Figure 5 A), the extract of recombinant bacteria GZQ01 has the same absorption peak as the standard at the same time ( Figure 5 B) This result indicates that NZ9000 heterologously synthesizes lycopene. Combining the standard curve and HPLC analysis, it was determined that the lycopene production of the recombinant strain GZQ01 was 0.48 mg / L.

[0158] The fermentation extract of recombinant strain GZQ01 was subjected to ultra-high performance chromatography (UPC) using lycopene standard as a control. 2 ) detection. Among them, A and B correspond to the standard and the fermentation extract of the recombinant strain GZQ01, respectively. From the mass spectrometry results, we can find that the NZ9000 fermentation extract and the standard have obvious absorption peaks at the same peak time ( Figure 6 A, B) show that plasmid pGZQ01 was successfully expressed in NZ9000, and the synthesized substance is lycopene.

[0159] 5-2 Study on the characteristics of strains after adding acetic acid

[0160] In microorganisms, acetic acid can be catalyzed by acetyl-CoA synthetase to produce acetyl-CoA. Acetyl-CoA is a precursor for the synthesis of terpenoids such as lycopene. Increasing its supply can further increase lycopene production, which has broad research prospects.

[0161] After adding acetic acid, the production of lycopene synthesized by the recombinant bacteria increased compared with the control, among which the production was the highest at 0.2% addition, reaching 0.73 mg / L, an increase of 58.6% over the control. 600 and lycopene production began to decrease, especially when the acetic acid addition was 1%, the lycopene production and OD 600 Even lower than the control ( Figure 7 ), indicating that an appropriate amount of acetic acid can indeed increase lycopene production, but adding too much acetic acid will be detrimental to bacterial growth and lycopene accumulation.

[0162] 5-3 Study on the knockout of the ldh gene and the characteristics of the recombinant strain

[0163] Lactococcus lactis metabolizes glucose through the glycolysis pathway to produce pyruvate, which serves as an intermediate metabolite for lycopene synthesis. Pyruvate can also be further synthesized into lactic acid through the enzyme lactate dehydrogenase (LDH). Therefore, the lactate synthesis pathway is a competing pathway for lycopene synthesis. To enhance lycopene synthesis, this study blocked lactate synthesis by knocking out the lactate dehydrogenase gene (LDH), thereby reducing pyruvate consumption. Using the single-plasmid pLL gene editing CRISPR-Cas9 system for Lactococcus lactis established in our laboratory, the lactate dehydrogenase gene ldh (945 bp) in the L. lactis NZ9000 genome was selected as the target gene, and sgRNA and homology arms were designed. The construction process is as follows: using plasmid pIB184 as a template and P23-XbaI-up / down as primers, PCR amplification was performed to obtain a 226 bp fragment containing the promoter P23; using pLL24 as a template, XbaI-up, XbaI-P23-down, repA-P23-up, repA-down as primers, PCR amplification of 616 bp and 1174 bp fragments, respectively; then, overlapping PCR amplification of the three obtained fragments was performed using XbaI-up and repA-down as primers to obtain a 1921 bp fragment in length. The fragment was seamlessly cloned into the vector backbone obtained by double-digesting the plasmid pLL24 with Xbal / Spel, and verified using primers XbaI-up and repA-down. The positive transformant obtained was named pLL25.

[0164] pLL25 and the control plasmid pCas9 were electroporated into competent cells of L. lactis NZ9000, and 1 μg of plasmid was added to every 100 μL of competent cells. The whole plasmid was spread on solid medium of GM17 (Em 10 μg / mL) and cultured at 30°C for 24-36 hours. After the knockout plasmid was transformed, the primers Cldh-HA-YZ-F / R were used for verification. The transformants were verified by colony PCR and the fragments recovered after gel tapping by BGI sequencing to confirm the seamless knockout of the gene ldh. The resulting mutant was named L. lactis NZ9000-△ldh( Figure 8 ).

[0165] The L. lactis NZ9000-△ldh strain was prepared into competent cells, and the plasmid pGZQ01 was electroporated and plated on Cm resistance plates. The positive clones were picked out and verified by PCR. The correct strain was named GZQ02 ( Figure 9 ).

[0166] The lycopene production of the recombinant strain GZQ02 after △ldh was determined by HPLC. The lycopene production of the recombinant strain GZQ02 after knockout was 0.92 mg / L, which was 1 times higher than that before knockout ( Figure 10 "*" indicates a difference from the control group when P<0.05; "**" indicates a difference from the control group when P<0.01; "***" indicates a difference from the control group when P<0.001), indicating that the ldh gene does have an effect on lycopene synthesis. Blocking the LDH pathway can allow more pyruvate in the metabolic pathway to flow to acetyl-CoA, increasing its supply and thus increasing lycopene production.

[0167] Adding acetic acid and △ldh can increase the supply of acetyl-CoA and improve the production of lycopene synthesized by the recombinant L. lactis NZ9000 strain. Comparing the yield differences between the two methods, it was found that the recombinant strain GZQ02 had a higher yield after △ldh, which was 25.5% higher than that after adding acetic acid. To further improve the yield, 0.2% acetic acid was added during the fermentation of the recombinant strain GZQ02, and the OD value of the bacteria was significantly increased. 600 and lycopene production decreased sharply, OD 600 is 0.99, and the lycopene yield is 0.35 mg / L ( Figure 11 ).

[0168] 5-4 Overexpression of MVA pathway genes from different sources

[0169] The MVA pathway is the precursor pathway for heterologous lycopene synthesis in Lactococcus lactis. It includes seven pathway genes: mvaE, mvaS, mvaA, mvaK1, mvaK2, mvaD, and idi. Enhancing pathway gene expression can increase IPP and DMAPP accumulation, thereby increasing lycopene production. Overexpressing MVA pathway genes from different sources can enhance lycopene synthesis in recombinant strains.

[0170] The 21 recombinant strains obtained after overexpression of the gene were shake-flask fermented, the pigment was extracted with acetone, and the lycopene production in the fermentation extract was determined by HPLC. We found that the production of lycopene by the recombinant strains after enhanced pathway gene expression was mostly increased. After overexpressing the mvaA gene of the MVA pathway, the lycopene production of the recombinant strains (GZQ05, GZQ12, and GZQ19) was higher than that of the recombinant strains overexpressing other genes ( Figure 12C), mvaA may be a key gene in the MVA pathway. Among them, the recombinant strain GZQ12, which overexpressed the mvaA gene from S-3, produced the highest lycopene production, reaching 1.22 mg / L, an increase of about 30% compared to the control (GZQ02). The yields of genes from other pathways were similar after overexpression. The recombinant strain GZQ13, which overexpressed the mvaD gene from S-3, produced 1.03 mg / L of lycopene, an increase of 12% compared to the control ( Figure 12 D); The recombinant strain GZQ21 overexpressing the mvaK1 gene from AR113 and the recombinant strain GZQ09 overexpressing the idi gene from NZ9000 produced similar lycopene yields, reaching approximately 1.01 mg / L, which was 10.8% higher than the control (GZQ02). Figure 12 G).

[0171] Effects of 5-5 culture medium conditions on lycopene production

[0172] Microbial growth and metabolite accumulation are significantly affected by the culture medium. Culture medium optimization can improve strain growth and affect the synthesis of target metabolites. Glucose is used as the carbon source of GM17 culture medium. By optimizing the addition amount, the production of heterologous lycopene by the recombinant strain GZQ12 can be changed. When the addition amount is from 2.5g / L to 10g / L, the growth of the bacteria shows a trend of increasing first and then gradually leveling off. The same is true for lycopene production. When the addition amount is 7.5g / L, the yield is the highest, reaching 1.17mg / L ( Figure 13 A). Changing the amount of trypsin added from 2.5 g / L to 10 g / L, when the addition amount was 7.5 g / L, OD 600 is 1.95, the lycopene production is the highest, and the lycopene production is 1.21mg / L ( Figure 13 B) Changing the amount of yeast powder added from 0g / L to 7.5g / L, when the addition amount was 5g / L, the bacterial OD 600 and lycopene production were the highest, OD 600 was 2.19, and the lycopene yield was 1.24 mg / L ( Figure 13 C). Therefore, the optimized addition amount of glucose in GM17 was 7.5 g / L, the addition amount of tryptone was 7.5 g / L, and the addition amount of yeast powder was 5 g / L.

[0173] In order to determine the optimal culture temperature for the recombinant strain GZQ12 to synthesize lycopene, this experiment studied the characteristics of the strain at different temperatures. The lycopene production changed significantly when the culture temperature changed. When the culture temperature increased from 20℃ to 30℃, the lycopene production increased. When the culture temperature continued to rise from 30℃ to 37℃, the lycopene production reversed and began to decrease. When the fermentation temperature was 30℃, the bacterial OD 600and lycopene production reached its peak, OD 600 is 1.53, and the yield is 1.21 mg / L ( Figure 14 ), therefore, the optimal culture temperature for the recombinant strain GZQ12 to synthesize lycopene was 30℃.

[0174] With the increase of fermentation time, the production of lycopene and the OD of the bacteria 600 At 12h, the yield and OD 600 When the fermentation time continues to increase, the bacterial concentration and yield reach saturation and no longer increase ( Figure 15 This indicates that after 12 hours of fermentation, there are almost no nutrients left in the culture medium, which inhibits the growth and metabolism of the bacteria and leads to OD 600 Therefore, the optimal fermentation time for the recombinant strain GZQ12 to synthesize lycopene was 12 h, and at the end of the fermentation, the lycopene yield was 1.29 mg / L and the OD 600 Reached 1.52.

[0175] Lycopene's all-trans double bond structure makes its molecular stability sensitive to pH changes. Therefore, to a certain extent, changes in the pH of the culture medium will cause lycopene to isomerize. When the initial pH of the culture medium is 7.0, the yield of synthesized lycopene after fermentation is the highest. Under other pH conditions, the yield of lycopene decreases to varying degrees, especially when the initial pH of the culture medium is 5.0, the yield is the lowest ( Figure 16 Comparing the lycopene production at initial pH of 5.0 and 8.0, it can be seen that lycopene is more sensitive in an acidic environment. In an acidic environment, the lycopene configuration is more unstable, and the external environment is not conducive to bacterial growth, which makes the OD at the end of fermentation 600 Therefore, the optimal fermentation pH for lycopene synthesis by the recombinant strain GZQ12 is 7.0, and the lycopene yield at the end of fermentation is 1.44 mg / L, and the OD 600 Reached 1.66.

[0176] 5-6 Determination of antioxidant activity of recombinant strains

[0177] The L. lactis NZ9000 / pNZ44 strain was selected as a control, and GZQ12 was inoculated into GM17 medium and skim milk, respectively, to determine their DPPH clearance rates. The DPPH clearance efficiency of GZQ12 was much higher than that of the control strain. In the supernatant, the clearance capacity of GZQ12 was 67.58%, while that of L. lactis NZ9000 / pNZ44 was only 31.62%. The clearance rate of GZQ12 in the cell suspension was 62.12%, which was more than double that of the control. At the same time, the clearance rate of GZQ12 inoculated into skim milk was also significantly higher than that of the control empty plasmid strain, with a clearance capacity of 63.72% in the supernatant and 59.04% in the cell suspension. Figure 17 A).

[0178] The ABTS free radical scavenging ability of the supernatant and cell suspension of L. lactis NZ9000 / pNZ44 and GZQ12 after fermentation in GM17 medium and GZQ12 in skim milk was greater than that of the control strain in both the supernatant and cell suspension. The scavenging ability of the supernatant and cell suspension obtained after skim milk fermentation of GZQ12 was also higher than that of the control strain, and the ABTS free radical scavenging ability was increased by 54.5% ( Figure 17 B). The scavenging ability of L. lactis NZ9000 / pNZ44 and GZQ12 in GM17 and skim milk fermentation supernatants and cell suspensions on ·OH free radicals. The scavenging rate of the control strain in the supernatant was 41.69%, while that of GZQ12 reached 67.98%. The scavenging rate of the supernatant after GZQ12 was inoculated into skim milk was significantly higher than that of the control, at 60.29%. The same results were obtained in cell suspensions, with GZQ12 significantly higher than the control strain ( Figure 17 C). Therefore, the antioxidant activity of the recombinant strain GZQ12 was evaluated using DPPH, ABTS, and ·OH. Compared with the control, the clearance rates of the recombinant strain were significantly improved, indicating that GZQ12 had higher antioxidant activity due to the synthesis of lycopene.

[0179] Table 1. Strains and plasmids used in this study

[0180]

[0181]

[0182]

[0183] Table 2. Primer sequences used in this study

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[0185]

[0186]

[0187]

Claims

1. A method for improving the antioxidant activity of Lactococcus lactis, characterized in that: The three heterologous enzyme genes, crtE, crtB and crtI, were introduced into the recombinant lactic acid bacteria to achieve heterologous synthesis of lycopene by the recombinant lactic acid bacteria, thereby improving the antioxidant activity of the recombinant lactic acid bacteria. The nucleotide sequence of the crtE heterologous enzyme gene is shown in SEQ ID NO.1; The nucleotide sequence of the crtB heterologous enzyme gene is shown in SEQ ID NO.2; The nucleotide sequence of the crtI heterologous enzyme gene is shown in SEQ ID NO.3; The recombinant lactic acid bacteria are derived from Lactococcus lactis in which the lactate dehydrogenase gene is knocked out and the MVA pathway gene is overexpressed; The gene overexpressing the MVA pathway is the thermophilic Streptococcus 3-hydroxy-3-methylglutaryl-CoA reductase gene, and the nucleotide sequence of the thermophilic Streptococcus 3-hydroxy-3-methylglutaryl-CoA reductase gene is shown in SEQ ID No.

6.

2. The method for improving the antioxidant activity of Lactococcus lactis according to claim 1, wherein: The pGZQ01 plasmid was introduced into the recombinant lactic acid bacteria to achieve heterologous synthesis of lycopene by Lactococcus lactis, thereby improving the antioxidant activity of Lactococcus lactis. The pGZQ01 plasmid contains three heterologous enzyme genes: crtE, crtB and crtI. The nucleotide sequence of the pGZQ01 plasmid is shown in SEQ ID No.

4.

3. The method for improving the antioxidant activity of Lactococcus lactis according to claim 1, wherein: The recombinant lactic acid bacteria is derived from Lactococcus lactis with the lactate dehydrogenase gene knocked out. The nucleotide sequence of the lactate dehydrogenase gene is shown in SEQ ID No.

5.

4. The method for improving the antioxidant activity of Lactococcus lactis according to claim 3, wherein: The Lactococcus lactis is NZ9000.

5. The method for improving the antioxidant activity of Lactococcus lactis according to claim 1, characterized in that: The MVA pathway gene is an MVA pathway gene in Lactococcus lactis NZ9000, Streptococcus thermophilus S-3 or Lactobacillus plantarum AR113.

6. The method for improving the antioxidant activity of Lactococcus lactis according to claim 1, characterized in that: The culture temperature was 30 °C, the pH was 7.0, and the fermentation time was 12 h.

7. Use of the recombinant lactic acid bacteria obtained by the method according to any one of claims 1 to 6 in the preparation of a probiotic product or a starter, wherein the recombinant lactic acid bacteria improves the antioxidant function of the probiotic product or the starter.

8. A method for improving the antioxidant activity of Lactococcus lactis, characterized in that: The recombinant Lactococcus lactis GZQ01 was cultured in a culture medium to which 0.2% acetic acid was added. The recombinant Lactococcus lactis GZQ01 was obtained by overexpressing three heterologous enzyme genes, crtE, crtB, and crtI, in Lactococcus lactis NZ9000. The nucleotide sequence of the crtE heterologous enzyme gene is shown in SEQ ID NO.1; The nucleotide sequence of the crtB heterologous enzyme gene is shown in SEQ ID NO.2; The nucleotide sequence of the crtI heterologous enzyme gene is shown in SEQ ID NO.3.

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