A method for high efficiency iterative integration of multiple genes in lactococcus lactis

Abstract

The application discloses a method for multi-gene high-efficiency iterative integration of lactococcus lactis, and steps are as follows: a warm-sensitive shuttle plasmid pNZts-Mint-Helper is used to induce expression of TP901-1 or spbc bacteriophage integrase in lactococcus lactis, donor plasmid can be mediated to carry out multi-gene integration at a specific site of a genome, split-Cre expression can be induced to eliminate the plasmid skeleton integrated on the genome, and the warm-sensitive shuttle plasmid pNZts-Mint-Helper in the engineering strain can be removed by increasing the culture temperature after gene integration is completed. The method of the application realizes two rounds of iterative integration of a total of 7 genes in lactococcus lactis. The method disclosed by the application has high gene integration efficiency, continuous experimental operation and few steps, and provides a new genetic engineering tool for realizing stable expression of multiple genes of lactococcus lactis.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, specifically to a method for efficient iterative integration of multiple genes from Lactococcus lactis. Background Technology

[0002] Lactococcus lactis is a Gram-positive, homolactic, facultative anaerobic bacterium with excellent characteristics such as rapid growth, simple metabolism, small genome, few intracellular and extracellular proteins, no production of any endotoxins or toxic substances, separation of decomposition and synthesis metabolism, and no sporulation or extracellular protease production. It has good biosafety and is generally considered a safe microorganism (GRS). It is known as a super "microbial cell processing plant" for producing food-grade metabolites and has broad application prospects in microbial cell factories, food industry, and biomedicine research fields.

[0003] Chromosomal integration of genes / metabolic pathways is a necessary step in developing stable microbial cell factories that meet the requirements of large-scale industrial production and economic efficiency. Current plasmid electroporation methods for *Lactococcus lactis* struggle to transform plasmids larger than 14 kb into *Lactococcus lactis*, and the presence of large plasmids in microbial cell factories can lead to genetic instability and plasmid loss. Another problem is that plasmid expression often results in metabolic burden, and plasmid copy numbers typically change with growth, leading to varying levels of gene expression. The best strategy to avoid these problems is chromosomal integration of genes / metabolic pathways. Many classic tools for gene insertion are available for *Lactococcus lactis*, but these techniques cannot achieve efficient iterative gene integration. Gene integration in *Lactococcus lactis* relying on traditional RecA-mediated plasmid homologous recombination is time-consuming, labor-intensive, and requires a high plasmid transformation efficiency from the host. Although previous studies have explored iterative gene integration in Lactococcus lactis through transient expression of TP901-1 and Cre phage integrase, the entire gene integration process is cumbersome and requires strains with high plasmid transformation efficiency. The relatively backward gene integration technology for lactic acid bacteria and the low transformation efficiency of large plasmids seriously hinder the reconstruction of lactic acid bacteria metabolic pathways. Therefore, developing new, efficient, iterative integration methods for multi-genes in reconstructing genetically engineered strains of Lactococcus lactis is of great significance for the future industrial applications of lactic acid bacteria. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for efficient iterative integration of multiple genes in Lactococcus lactis.

[0005] The technical solution of this invention is summarized as follows:

[0006] A method for efficient iterative integration of multiple genes from Lactococcus lactis includes the following steps:

[0007] 1) The thermosensitive shuttle plasmid pNZts-Mint-Helper, loaded with the TP901-1, spbc, and Split-cre genes, was electroporated into Lactococcus lactis NZ9000 to obtain the engineered strain NZ-A;

[0008] The nucleotide sequence of the thermosensitive shuttle plasmid pNZts-Mint-Helper is shown in SEQ ID NO.1;

[0009] 2) The four gene fragments a1, a2, a3 and a4 were amplified by PCR and purified and recovered. The four purified gene fragments were seamlessly cloned into the Bts I digested pNZint-donor1 plasmid to obtain plasmid pNZint-donor1-4genes.

[0010] The nucleotide sequence of the pNZint-donor1 plasmid is shown in SEQ ID NO.2;

[0011] 3) Electroporate the plasmid pNZint-donor1-4genes into the engineered strain NZ-A obtained in step 1). During resuscitation, use nisin to induce the expression of TP901-1 phage integrase to mediate the integration of plasmid pNZint-donor1-4genes into the genome of engineered strain NZ-A to obtain engineered strain NZ-B.

[0012] 4) The engineered strain NZ-B was plated on a solid plate of SA synthetic medium containing 5-fluoroorotic acid and ZnSO4, and Split-Cre expression was induced and reverse screening was performed to obtain the engineered strain NZ-C, which eliminated the pNZint-donor1-4genes plasmid backbone but integrated 4 genes.

[0013] 5) The three gene fragments b1, b2 and b3 were amplified by PCR and purified and recovered. The three purified gene fragments were seamlessly cloned into the pNZint-donor2 plasmid digested with Bts I to obtain the plasmid pNZint-donor2-3genes.

[0014] The nucleotide sequence of the pNZint-donor2 plasmid is shown in SEQ ID NO.3;

[0015] 6) Electroporate the pNZint-donor2-3genes plasmid into the engineered strain NZ-C obtained in step 4). During resuscitation, use nisin to induce the expression of Spbc phage integrase to mediate the integration of the pNZint-donor2-3genes plasmid into the genome of the engineered strain NZ-C to obtain the engineered strain NZ-D.

[0016] 7) The engineered strain NZ-D was plated on a solid plate of SA synthetic medium containing 5-fluoroorotic acid and ZnSO4, and Split-Cre expression was induced and reverse screening was performed to obtain the engineered strain NZ-E, which eliminated the pNZint-donor2-3 gene plasmid backbone but integrated 7 genes.

[0017] 8) Incubate the engineered strain NZ-E in 1 mL of GM17 broth at 30℃ for 2 hours, then incubate in a 38-42℃ water bath for 4-6 hours, followed by incubation at 30℃ for 2 hours. Pipette 5 μL of the culture solution onto antibiotic-free GM17 plates and streak on designated areas. Incubate at 30℃ for 1-2 days. Transfer colonies using toothpicks to both antibiotic-free and erythromycin-containing GM17 plates.

[0018] The engineered strain NZ-F, which integrates 7 genes and does not contain the pNZts-Mint-Helper plasmid, was obtained through screening.

[0019] Step 4) is preferably: the engineered strain NZ-B is spread onto a solid plate of SA synthetic medium containing 20 μg / mL 5-fluoroorotic acid and 0.7 mM ZnSO4, and Split-Cre expression is induced and reverse screening is performed to obtain the engineered strain NZ-C, which has eliminated the pNZint-donor1-4genes plasmid backbone but integrated 4 genes.

[0020] Step 7) is preferably: the engineered strain NZ-D is plated onto a solid plate of SA synthetic medium containing 20 μg / mL 5-fluoroorotic acid and 0.7 mM ZnSO4, and Split-Cre expression is induced and reverse screening is performed to obtain the engineered strain NZ-E, which has eliminated the pNZint-donor2-3 gene plasmid backbone but integrated 7 genes.

[0021] The a1, a2, a3, a4, b1, b2, or b3 genes are any exogenous genes that are expressed independently or co-expressed in Lactococcus lactis.

[0022] The nucleotide sequence of the a1 gene is shown in SEQ ID NO.4.

[0023] The nucleotide sequence of the a2 gene is shown in SEQ ID NO.5.

[0024] The nucleotide sequence of the a3 gene is shown in SEQ ID NO.6.

[0025] The nucleotide sequence of the a4 gene is shown in SEQ ID NO.7.

[0026] The nucleotide sequence of the b1 gene is shown in SEQ ID NO.8.

[0027] The nucleotide sequence of the b2 gene is shown in SEQ ID NO.9.

[0028] The nucleotide sequence of the b3 gene is shown in SEQ ID NO.10.

[0029] Advantages of this invention:

[0030] This invention uses a single thermosensitive shuttle plasmid, pNZts-Mint-Helper, to mediate multi-gene integration and iterative gene integration at specific sites using a donor plasmid. This results in high gene integration efficiency and a long sequence length for each integration. Controllable expression and reverse selection of the Split-Cre enzyme eliminate the backbone of the donor plasmid on the genome. The entire experimental process is continuous with fewer steps, reducing labor and time costs and accelerating the gene integration process in *Lactococcus lactis*. The thermosensitive shuttle plasmid can be easily and quickly removed from genetically engineered bacteria, leaving room for further modification of the strain. Attached Figure Description

[0031] Figure 1 The pNZts-Mint-Helper plasmid map.

[0032] Figure 2 The plasmid map shows the door plasmids pNZint-donor1, pNZint-donor2, pNZint-donor1-4 genes, and pNZint-donor2-3 genes, which can replicate in Escherichia coli but not in lactic acid bacteria.

[0033] Figure 3 This relates to the principle of efficient iterative integration of multiple genes in Lactococcus lactis.

[0034] Figure 4 This is the result of colony PCR validation for two rounds of multi-gene iterative integration.

[0035] Figure 5 Results of GM17 plates for NZ-F were obtained for resistance screening. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments.

[0037] Lactococcus lactis NZ9000 is the model Lactococcus lactis, purchased from Wuhan Miaoling Biotechnology Co., Ltd. (https: / / miaolingbio.biomart.cn / );

[0038] The culture medium formula used is as follows:

[0039] 1) GM17 broth culture medium: Add 10g / L of glucose to M17 broth (purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.) and autoclave at 121℃ for 15min.

[0040] 2) GM17 plates: Add to M17 agar (purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.)

[0041] 10 g / L glucose, autoclaved at 121°C for 15 min.

[0042] 3) LB medium: Sodium chloride 10g / L, Yeast Extract 5g / L, Tryptone 10g / L. If LB solid medium (LB solid plate) is required, add 15g / L of agar powder. Autoclave at 121℃ for 20min.

[0043] 4) SA synthetic medium: its composition and preparation are referenced in Ref. (Jensen PR, Hammer K. Minimal requirements for exponential growth of Lactococcus lactis[J]. Applied and environmental microbiology, 1993, 59(12): 4363-4366).

[0044] 5) SA synthesis medium solid plate: The SA synthesis medium contains 15 g / L of agar powder.

[0045] Example 1

[0046] Construction of the thermosensitive shuttle plasmid pNZts-Mint-Helper

[0047] 1) a. Temperature-sensitive plasmid backbone: The plasmid backbone mainly consists of two parts, one of which is pWV01 ts-Ery resistance gene, this DNA fragment was obtained by PCR amplification and purification of the target band using primers TSOri-F (SEQ ID NO.11) and TSOri-R (SEQ ID NO.12) with plasmid pNZts-Cre (Ref: Zhu D, Zhao K, Xu H, et al. Construction of thyA deficient Lactococcuslactis using the Cre-loxP recombination system[J]. Annals of Microbiology, 2015, 65(3):1659-1665.) as a template; another part is the UC57ori Escherichia coli high copy replicon, using plasmid pUC57-P purF Using Cre (SEQ ID NO.13) as a template, PCR amplification was performed using primers UCori-F (SEQ ID NO.14) and UCori-R (SEQ ID NO.15), and the DNA fragment UC57ori was purified and recovered.

[0048] b. Site-specific recombinase gene element: Using the TP901-1 gene (SEQ ID NO.16) synthesized by Genewiz as a template, the TP901-1 DNA fragment was obtained by PCR amplification using primers TP901-F (SEQ ID NO.17) and TP901-R (SEQ ID NO.18).

[0049] spbc (SEQ ID NO.19) was obtained by PCR amplification using the genome of Bacillus subtilis 168 strain with accession number ATCC 23857 as a template and primers spbc-F (SEQ ID NO.20) and spbc-R (SEQ ID NO.21).

[0050] c. Promoter amplification and overlap PCR: Using pNZ8148 (purchased from Wuhan Miaoling Biotechnology Co., Ltd. (https: / / miaolingbio.biomart.cn / )) as a template, primer pairs TPPnis-F (SEQ ID NO.22) and TPPnis-R (SEQ ID NO.23), as well as primer pairs SPPnis-F (SEQ ID NO.24) and SPPnis-R (SEQ ID NO.25), were used to amplify, purify, and recover PnisA. TP and PnisA sp DNA fragment; using primers TPPnis-F (SEQ ID NO.22) and TP901-R (SEQ ID NO.18) to synthesize PnisA TPPnisA was obtained by overlapping with TP901-1. TP -TP901-1 and using primers SPPnis-F (SEQ ID NO.24) and spbc-R (SEQ ID NO.21) to synthesize PnisA sp PnisA is obtained by fusing with SPBC. sp -spbc gene fragment;

[0051] The sczA-P fragment was amplified using primers SczA-F (SEQ ID NO.27) and SczA-R (SEQ ID NO.28) with the sczA-P DNA fragment (SEQ ID NO.26) synthesized by Genewiz as a template.

[0052] With plasmid pUC57-P purF Using Cre (SEQ ID NO. 13) as a template, P was amplified using primers CreN-F (SEQ ID NO. 29) and CreN-R (SEQ ID NO. 30). purF -CreN DNA fragment.

[0053] and with plasmid pUC57-P purF Using Cre (SEQ ID NO.13) as a template, CreC was amplified using primers CreC-F (SEQ ID NO.31) and CreC-R (SEQ ID NO.32) to obtain CreC.

[0054] CreC and sczA-P were fused by overlap PCR using primers CreC-R (SEQ ID NO.32) and SczA-R (SEQ ID NO.28) to obtain the DNA fragment sczA-CreC;

[0055] PnisA sp -spbc same as PnisA TP -TP901-1、P purF -CreN and sczA-CreC were respectively subjected to overlap PCR fusion to obtain PnisA sp -spbc-PnisA TP -TP901-1 and sczA-CreC-P purF -CreN gene fragment;

[0056] Finally, UC57ori and pWV01 ts -Ery、PnisA sp -spbc-PnisA TP -TP901-1 and sczA-CreC-P purF-CreN was used to assemble multiple DNA fragments to obtain a thermosensitive shuttle plasmid pNZts-Mint-Helper loaded with the TP901-1, spbc, and Split-cre genes.

[0057] The nucleotide sequence of the thermosensitive shuttle plasmid pNZts-Mint-Helper is shown in SEQ ID NO.1;

[0058] 50 μL PCR reaction mixture: 18 μL ddH2O, 25 μL 2×PCR buffer, 1 μL 2.5 mM dNTPs, 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), 1 μL Phanta Max Super Fidelity DNA Polymerase, and 1 μL template DNA. PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 72℃ annealing for 15 s; 72℃ extension at 1–2 kb / min; 35 cycles; 72℃ extension for 5 min; store at 12℃.

[0059] Purification and recovery of PCR-amplified DNA fragments: The PCR product was mixed with 5 μL of 6× DNA loading buffer and added to the wells of a pre-prepared 1.5% agarose gel for agarose gel electrophoresis. Trans 2K Plus II DNA Marker was added to the wells as a reference. The voltage was 180V, and the electrophoresis time was approximately 25 min. After electrophoresis, the length of the amplified DNA target fragment was determined by referring to the positions of different band lengths on the Trans 2K Plus II DNA Marker under blue light. The gel with the correct band length was excised, and the target gene fragment was purified and recovered using Tiangen's universal DNA purification and recovery kit, following the kit's instructions. The recovered gene fragment was then stored at -20℃ after its concentration was measured using a NanoDrop micro-nucleic acid concentration analyzer.

[0060] Assembly of multiple gene fragments: Seamless cloning and use of multiple gene fragments For detailed steps regarding the reaction system, reaction conditions, etc., please refer to the NEB Seamless Cloning Kit manual on the official website of the HiFi DNAAssembly Master Mix kit.

[0061] Preparation of Escherichia coli TG1 competent cells: Frozen TG1 glycerol bacteria were streaked onto LB agar plates and cultured overnight at 37°C. A single colony was picked and cultured overnight at 37°C with shaking in a shaker. Then, 1% inoculum was transferred to 200 mL of LB medium and cultured until OD500. 600At approximately 0.5 (about 3 hours of culture), the conical flask was placed on ice to cool for 15 minutes, followed by centrifugation at 8000 rpm for 2 minutes to collect the cells, discarding the supernatant. The cells were washed twice with pre-cooled CaCl2-MgCl2 aqueous solution (20 mmol / L CaCl2, 80 mmol / L MgCl2), and finally, CaCl2 (0.1 mmol / L CaCl2)-10% glycerol solution was added to prepare TG1 chemotransformation competent cells. These cells were aliquoted into 100 μL tubes and can be stored at -80°C for 2 months.

[0062] Assembly reaction liquefaction to TG1: Add 10 μL of the assembly reaction solution obtained in step 4) to 50 μL of pre-thawed chemocompetent cells of LE. coli TG1. After incubating on ice for 25 min, heat shock in a water bath at 42 °C for 90 s, then quickly transfer to an ice bath for 2 min. Add 500 μL of LB medium and incubate at 37 °C with shaking at 180 rpm for 45 min. Centrifuge and discard 400 μL of supernatant. Mix the remaining bacterial culture and spread it onto LB agar plates containing appropriate concentrations of antibiotics (final antibiotic concentrations for TG1: 180 μg / mL erythromycin, 25 μg / mL chloramphenicol). Incubate overnight at 37 °C.

[0063] Transformant validation and plasmid sequencing

[0064] Colony PCR reaction system: Pick a single colony and resuspend it in 10 μL ddH2O. Take 3 μL of the resuspended bacterial solution as the PCR template. Add 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM), 1 μL ddH2O, and 5 μL Taq DNA polymerase. PCR reaction conditions: 97℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s; 55℃ for 15 s; 72℃ extension at 1-2 Kb / min; 25 cycles; store at 4℃. DNA band size was checked by gel electrophoresis. The bacterial solution with the correct band size was inoculated into LB liquid medium containing erythromycin resistance (180 μg / ml) and cultured overnight at 37℃ and 180 rpm / min. The next day, plasmid DNA was extracted from *E. coli* using the Tiangen bacterial plasmid miniprep kit and sent to Genewiz for sequencing. The plasmid with the correct sequence was finally identified and named pNZts-Mint-Helper; stored at -20℃. Complete plasmid map as follows Figure 1 As shown, the complete sequence is shown in SEQ ID NO.1.

[0065] Example 2

[0066] Construction of donor plasmid

[0067] 1) Construction of pNZint-donor1 and pNZint-donor2:

[0068] Using the pNZ5319 vector (Ref: Zhu D, Zhao K, Xu H, et al. Construction of thyAdeficient Lactococcus lactis using the Cre-loxP recombination system[J]. Annals of Microbiology, 2015, 65(3): 1659-1665.) as a template, the Cat gene fragment was obtained by PCR amplification using primers Cat-F (SEQ ID NO. 33) and Cat-R (SEQ ID NO. 34). In addition, using the pNZ5319 vector as a template, the P15Aori gene fragment was obtained by PCR amplification using primers P15Aori-F (SEQ ID NO. 35) and P15Aori-R (SEQ ID NO. 36). After purification and recovery, the two fragments were fused by overlap PCR to obtain the P15Aori-Cat fragment.

[0069] Pe-oroP was obtained by PCR amplification using the pCS1966 vector (Ref: Solem C, Defoor E, Jensen PR, et al. Plasmid pCS1966, a new selection / counterselection tool for lactic acid bacterium strain construction based on the oroP gene, encoding an orotate transporter from Lactococcus lactis[J]. Applied and Environmental Microbiology, 2008, 74(15):4772-4775.) as a template and primers oroP-F (SEQ ID NO.37) and oroP-R (SEQ ID NO.38). Subsequently, primers oroP-F (SEQ ID NO.37), T1T2-a (SEQ ID NO.39), T1T2-b (SEQ ID NO.40), and T1T2-R (SEQ ID NO.38) were used. NO.41) A terminator was added downstream of the Pe-oroP fragment by PCR to finally obtain the Pe-oroP-Ter gene fragment.

[0070] Genewiz synthesized the miniGene Dloxp-DBtsI (SEQ ID NO.42) and miniGeneSloxp-DBtsI (SEQ ID NO.43) gene fragments.

[0071] The above P15Aori-Cat, Pe-oroP-Ter, Dloxp-DBtsI or P15Aori-Cat, Pe-oroP-Ter, Sloxp-DBtsI DNA fragments were seamlessly cloned to obtain plasmids pNZint-donor1 and pNZint-donor2.

[0072] Complete plasmid map as follows Figure 2 The complete sequences of pNZint-donor1 and pNZint-donor2 plasmids are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0073] 2) Construction of pNZint-donor1-4 genes and pNZint-donor2-3 genes

[0074] Plasmids pNZint-donor1 and pNZint-donor2 were digested with Bts I. For specific system and procedure, please refer to the NEB BtsI restriction enzyme instruction manual. After purification and recovery, linearized plasmids pNZint-donor1 and pNZint-donor2 were obtained. Using the genome of Bacillus subtilis 168 (ATCC 23857) as a template, the a1-a2-a3 gene fragment was amplified by PCR using primers citA-F (SEQ ID NO.44) and citB-R (SEQ ID NO.45). Using the genome of Bacillus subtilis 168 (ATCC 23857) as a template, the b1-b2 gene fragment was amplified by PCR using primers fmdH-F (SEQ ID NO.46) and fmdH-R (SEQ ID NO.47). Using the genome of Escherichia coli TG1 as a template, the a4 gene fragment was amplified by primers aceA-F (SEQ ID NO.48) and aceA-R (SEQ ID NO.49). Using the genome of Escherichia coli TG1 as a template, the a4 gene fragment was amplified by primers aceB-F (SEQ ID NO.49). The aceB gene fragment was obtained by amplification using primers aceB-F (SEQ ID NO.50) and aceB-R (SEQ ID NO.51); subsequently, the b3 gene fragment was obtained by PCR amplification using primers aceB-F (SEQ ID NO.50) and YQSP7-R1 (SEQ ID NO.52) as a template.

[0075] The artificially synthesized gene fragments P8 promoter (SEQ ID NO.53) and P7 promoter (SEQ ID NO.54) were used to perform PCR overlap of the P8 promoter and the a1-a2-a3 gene fragment using primers P8-F (SEQ ID NO.55) and citB-R to obtain the P8-a1-a2-a3 gene fragment.

[0076] The P7 promoter and the b1-b2 gene fragment were overlapped by PCR using primers P7-F (SEQ ID NO.56) and fmdH-R (SEQ ID NO.47) to obtain the P7-b1-b2 gene fragment.

[0077] Finally, the linearized plasmid pNZint-donor1 was seamlessly cloned with P8-a1-a2-a3, a4, and pNZint-donor2 with P7-b1-b2, b3, respectively, to obtain plasmids pNZint-donor1-4 genes and pNZint-donor2-3 genes. The complete map is shown below. Figure 2 The pNZint-donor1-4 genes and pNZint-donor2-3 genes are shown in the figure.

[0078] The a1, a2, a3, a4, b1, b2, or b3 genes are any exogenous genes that are expressed independently or co-expressed in Lactococcus lactis.

[0079] The nucleotide sequence of the a1 gene is shown in SEQ ID NO.4.

[0080] The nucleotide sequence of the a2 gene is shown in SEQ ID NO.5.

[0081] The nucleotide sequence of the a3 gene is shown in SEQ ID NO.6.

[0082] The nucleotide sequence of the a4 gene is shown in SEQ ID NO.7.

[0083] The nucleotide sequence of the b1 gene is shown in SEQ ID NO.8.

[0084] The nucleotide sequence of the b2 gene is shown in SEQ ID NO.9.

[0085] The nucleotide sequence of the b3 gene is shown in SEQ ID NO.10.

[0086] The PCR reaction system, purification and recovery of amplified DNA fragments, seamless cloning of multiple DNA fragments, preparation of competent E. coli cells and transformation of assembly reaction solution, transformation verification and plasmid extraction and sequencing have been described above and will not be repeated here.

[0087] Example 3

[0088] A method for efficient iterative integration of multiple genes in two rounds of Lactococcus lactis includes the following steps:

[0089] The thermosensitive shuttle plasmid pNZts-Mint-Helper, loaded with the TP901-1, spbc, and Split-cre genes, was electroporated into Lactococcus lactis NZ9000 competent cells to obtain engineered strain NZ-A, and competent cells were prepared.

[0090] The nucleotide sequence of the thermosensitive shuttle plasmid pNZts-Mint-Helper is shown in SEQ ID NO.1;

[0091] The plasmid pNZint-donor1-4 genes were electroporated into the engineered strain NZ-A. During resuscitation, nisin was used to induce the expression of TP901-1 phage integrase, which mediated the integration of the plasmid pNZint-donor1-4 genes into the genome of the engineered strain NZ-A. The results of the verification of the engineered strain NZ-B are as follows: Figure 4 As shown;

[0092] The engineered strain NZ-B was plated onto a solid agar plate containing 20 μg / mL 5-fluoroorotic acid and 0.7 mM ZnSO4. Split-Cre expression was induced, and reverse selection yielded the engineered strain NZ-C, which eliminated the pNZint-donor1-4 genes plasmid backbone but integrated four genes. Colony PCR verification results are shown below. Figure 4 As shown, competent states were prepared;

[0093] The pNZint-donor2-3genes plasmid was electroporated into the engineered strain NZ-C. During resuscitation, nisin was used to induce the expression of Spbc phage integrase, which mediated the integration of the pNZint-donor2-3genes plasmid into the genome of the engineered strain NZ-C, resulting in the engineered strain NZ-D. The colony PCR verification results are as follows: Figure 4 As shown;

[0094] The engineered strain NZ-D was plated onto a solid agar plate containing 20 μg / mL 5-fluoroorotic acid and 0.7 mM ZnSO4. Split-Cre expression was induced, and reverse selection yielded the engineered strain NZ-E, which eliminated the pNZint-donor2-3 gene plasmid backbone but integrated 7 genes. Colony PCR verification results are shown below. Figure 4 As shown;

[0095] The engineered strain NZ-E was cultured at 30℃ in 1 mL of GM17 broth for 2 hours, then placed in a 38℃ (or any value between 38-42℃, such as 39, 40, 41, 42℃, etc.) water bath for 4 hours (or any value between 4-6 hours, such as 4.5 hours, 5 hours, 6 hours), and then cultured at 30℃ for 2 hours. 5 μL of the culture was streaked onto antibiotic-free GM17 plates and incubated at 30℃ for 1.5 days (or any value between 1-2 days, such as 1 day, 2 days, etc.). Colonies were transferred using toothpicks to antibiotic-free GM17 plates and GM17 plates containing erythromycin. The engineered strain NZ-F, which integrates 7 genes and does not contain the pNZts-Mint-Helper plasmid, was obtained through resistance screening on GM17 plates. Figure 5 As shown.

[0096] Preparation of competent cells of Lactococcus lactis NZ9000 and engineered strains NZ-A and NZ-C:

[0097] The preparation of highly competent lactic acid bacteria was carried out according to the method reported in the reference (Ref: Holo H, Nes IF. High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media[J]. Applied and Environmental Microbiology, 1989, 55(12):3119–3123). NZ9000 glycerol bacteria, frozen in a refrigerator, were streaked on antibiotic-free GM17 plates and cultured at 30℃ for 24 h. A single colony was picked and amplified. 2 mL of the activated overnight culture was transferred to 100 mL of GM17 broth and cultured at 30℃ for 3 h. Ampicillin at a final concentration of 20 μg / mL was added to weaken the cell wall of NZ9000. After culturing for another h, the culture was placed on ice for 10 min to allow it to cool completely. The cells were then collected by centrifugation at 8000 rpm for 2 minutes, and washed twice with an equal volume of electroconversion washing buffer (10% glycerol + ultrapure water). Finally, electroconversion washing buffer was added at a ratio of 5 mL of culture medium to prepare 100 μL of electrocompetent cells. After aliquoting into 50 μL tubes, the cells were immediately frozen at -80°C and can be stored for 2 months.

[0098] Plasmid electroconversion:

[0099] Thaw competent *Lactococcus lactis* cells frozen at -80°C on ice for approximately 2 minutes. Then add a certain amount of plasmid (approximately 500 ng for plasmids smaller than 10 kb, and approximately 2 μg for plasmids larger than 10 kb). To ensure transformation efficiency, the plasmid volume should not exceed 5 μL. After thorough mixing, transfer to a 2 mm electroporation cuvette and electroporate using a Bio-Rad electroporator at 2.4 kV for approximately 5 ms. Finally, add 1 mL of GM17 broth and transfer to a 1 mL EP tube, incubating at 30°C for 2 hours. After resuscitation, centrifuge at 8000 rpm for 2 minutes, discard 900 μL of supernatant, mix well, and spread onto GM17 plates containing the appropriate antibiotic resistance (concentrations for *Lactococcus lactis*: 5 μg / ml erythromycin, 5 μg / ml chloramphenicol). Incubate overnight at 30°C.

[0100] The primers and genes used were synthesized by Suzhou Genewise Biotechnology Co., Ltd.

Claims

1. A method for efficient iterative integration of multiple genes from Lactococcus lactis, characterized by: Includes the following steps: 1) The thermosensitive shuttle plasmid pNZts-Mint-Helper, loaded with the TP901-1, spbc, and Split-cre genes, was electroporated into Lactococcus lactis NZ9000 to obtain the engineered strain NZ-A; The nucleotide sequence of the thermosensitive shuttle plasmid pNZts-Mint-Helper is shown in SEQ ID NO.1; 2) The four gene fragments a1, a2, a3 and a4 were amplified by PCR and purified and recovered. The four purified gene fragments were seamlessly cloned into the Bts I digested pNZint-donor1 plasmid to obtain plasmid pNZint-donor1-4genes. The nucleotide sequence of the pNZint-donor1 plasmid is shown in SEQ ID NO.2; 3) Electroporate the plasmid pNZint-donor1-4genes into the engineered strain NZ-A obtained in step 1). During resuscitation, use nisin to induce the expression of TP901-1 phage integrase to mediate the integration of plasmid pNZint-donor1-4genes into the genome of engineered strain NZ-A to obtain engineered strain NZ-B. 4) The engineered strain NZ-B was plated on a solid plate of SA synthetic medium containing 5-fluoroorotic acid and ZnSO4, and Split-Cre expression was induced and reverse screening was performed to obtain the engineered strain NZ-C, which eliminated the pNZint-donor1-4genes plasmid backbone but integrated 4 genes. 5) The three gene fragments b1, b2 and b3 were amplified by PCR and purified and recovered. The three purified gene fragments were seamlessly cloned into the pNZint-donor2 plasmid digested with Bts I to obtain the plasmid pNZint-donor2-3genes. The nucleotide sequence of the pNZint-donor2 plasmid is shown in SEQ ID NO.3; 6) Electroporate the pNZint-donor2-3genes plasmid into the engineered strain NZ-C obtained in step 4). During resuscitation, use nisin to induce the expression of Spbc phage integrase to mediate the integration of the pNZint-donor2-3genes plasmid into the genome of the engineered strain NZ-C to obtain the engineered strain NZ-D. 7) The engineered strain NZ-D was plated on a solid plate of SA synthetic medium containing 5-fluoroorotic acid and ZnSO4, and Split-Cre expression was induced and reverse screening was performed to obtain the engineered strain NZ-E, which eliminated the pNZint-donor2-3 gene plasmid backbone but integrated 7 genes. 8) Incubate the engineered strain NZ-E in 1 mL of GM17 broth at 30℃ for 2 h, then incubate in a water bath at 38-42℃ for 4-6 h, and then incubate at 30℃ for 2 h. Take 5 μL of culture medium and streak it on antibiotic-free GM17 plates. Incubate at 30℃ for 1-2 days. Use a toothpick to transfer the colonies to antibiotic-free GM17 plates and GM17 plates containing erythromycin. Screen to obtain the engineered strain NZ-F, which integrates 7 genes and does not contain the pNZts-Mint-Helper plasmid.

2. The method according to claim 1, characterized in that: Step 4) involves spreading the engineered strain NZ-B onto a solid plate of SA synthetic medium containing 20 μg / mL 5-fluoroorotic acid and 0.7 mM ZnSO4, inducing the expression of Split-Cre, and performing reverse screening to obtain the engineered strain NZ-C, which has eliminated the pNZint-donor1-4genes plasmid backbone but integrated 4 genes.

3. The method according to claim 1, characterized in that: Step 7) involves: spreading the engineered strain NZ-D onto a solid plate of SA synthetic medium containing 20 μg / mL 5-fluoroorotic acid and 0.7 mM ZnSO4, inducing the expression of Split-Cre, and performing reverse screening to obtain the engineered strain NZ-E, which has eliminated the pNZint-donor2-3 gene plasmid backbone but integrated 7 genes.

4. The method according to claim 1, characterized in that: The a1, a2, a3, a4, b1, b2, or b3 genes are any exogenous genes that are expressed independently or co-expressed in Lactococcus lactis.

5. The method according to claim 1 or 4, characterized in that: The nucleotide sequence of the a1 gene is shown in SEQ ID NO.

4.

6. The method according to claim 1 or 4, characterized in that: The nucleotide sequence of the a2 gene is shown in SEQ ID NO.

5.

7. The method according to claim 1 or 4, characterized in that: The nucleotide sequence of the a3 gene is shown in SEQ ID NO.

6.

8. The method according to claim 1 or 4, characterized in that: The nucleotide sequence of the a4 gene is shown in SEQ ID NO.

7.

9. The method according to claim 1 or 4, characterized in that: The nucleotide sequence of the b1 gene is shown in SEQ ID NO.

8.

10. The method according to claim 1 or 4, characterized in that: The nucleotide sequence of the b2 gene is shown in SEQ ID NO.

9.

11. The method according to claim 1 or 4, characterized in that: The nucleotide sequence of the b3 gene is shown in SEQ ID NO. 10.

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