Lactococcus lactis recombinant strain and preparation method thereof

Abstract

The application relates to the field of biotechnology, in particular to a recombinant Lactococcus lactis strain and a preparation method thereof. The recombinant Lactococcus lactis strain provided by the application comprises an expression cassette and an expression plasmid, the expression cassette is an expression cassette integrated into the chromosome of the Lactococcus lactis, and comprises a first promoter and a T7 RNA polymerase gene; the expression plasmid comprises a T7 promoter and an exogenous protein gene. The recombinant Lactococcus lactis strain has high transcription intensity, can significantly improve the yield of the exogenous protein, has good genetic stability, does not express an antibiotic-resistant protein, and has high safety.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a recombinant strain of Lactococcus lactis and its preparation method. Background Technology

[0002] Lactococcus lactis is an important model organism for the application and research of heterologous protein expression. Due to its recognized safety, probiotic properties, absence of inclusion bodies and endotoxins, ease of surface display and extracellular secretion, it has become an ideal host for heterologous protein expression.

[0003] Lactococcus lactis expression systems include constitutive expression systems and inducible expression systems. Constitutive expression systems are those where gene expression is unaffected by factors such as timing, location, or environment. Constitutive promoters are key elements regulating normal gene expression, such as P21, P23, P59, P32, and P44. Inducible expression systems are those that can initiate or enhance gene expression under the influence of specific inducers. Promoters that initiate inducible expression systems are called inducible promoters. These promoters require specific stimuli, such as antibiotic infection, changes in temperature or pH, to initiate the transcriptional expression of functional genes.

[0004] The most widely used inducible expression system for *Lactococcus lactis* is the nisin-controlled expression (NICE) system, a self-regulating model of nisin biosynthesis. The NICE system mainly consists of three parts: a host bacterium (containing NisR and NisK genes), the inducible molecule nisin, and a fragment plasmid (containing either a nisA or nisF promoter). Its regulatory principle is as follows: the nisA or nisF promoter controls the target gene, and the NisR / NisK two-component regulatory system senses the nisin signal, thereby initiating the transcription of the exogenous gene. However, the NICE inducible expression system has limited transcriptional intensity and insufficient heterologous expression capacity for exogenous functional enzymes, making it difficult to meet the industrial demands for high-yield synthesis of target products.

[0005] The T7 expression system is a highly efficient recombinant protein expression technology based on the T7 phage transcription mechanism, mainly used in *E. coli*. It leverages the high specificity of T7 RNA polymerase for the T7 promoter to achieve ultra-high-level transcription and translation of the target gene. However, the T7 expression system is rarely used in *Lactococcus lactis*. Furthermore, T7 expression systems often rely on free plasmids for construction, which has drawbacks such as unstable passage, the need for antibiotic screening, and the risk of drug residues in food applications. Summary of the Invention

[0006] In view of this, the present invention provides a recombinant strain of Lactococcus lactis and a method for preparing the same. This recombinant strain exhibits high transcriptional strength, significantly increases the yield of exogenous proteins, demonstrates good genetic stability, shows no expression of antibiotic resistance proteins, and exhibits high safety.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a recombinant strain of *Lactococcus lactis*, the recombinant strain comprising: a) Expression cassette, which is an expression cassette integrated into the chromosome of Lactococcus lactis, including the first promoter and the T7 RNA polymerase gene; b) Expression plasmid, which includes the T7 promoter and the foreign protein gene.

[0008] In this invention, the recombinant lactococcus strain provided by this invention first expresses T7 RNA polymerase through an expression cassette regulated by a first promoter, and then the T7 RNA polymerase specifically recognizes the T7 promoter to mediate efficient transcription of the target gene and increase the yield of exogenous protein.

[0009] In an embodiment of the present invention, the first promoter is an inductive promoter or a constitutive promoter.

[0010] In specific embodiments provided by the present invention, the inducible promoter includes at least one of PnisA, PxylT, PczcD, and PtetO.

[0011] In specific embodiments provided by this invention, the constitutive promoter includes at least one of P32, P45, Pldh, and PpepN. Compared to inducible promoters, constitutive expression mediated by constitutive promoters does not require the addition of an inducer to achieve stable and continuous transcription of the target gene, resulting in better uniformity of expression levels.

[0012] In the embodiments provided by the present invention, the T7 promoter in the expression plasmid can be a T7 promoter or a modified and optimized T7 promoter variant, such as T7PCONSR.

[0013] In a preferred embodiment of the present invention, the expression plasmid further includes a secretion signal peptide coding sequence. The secretion signal peptide can optimize the secretion efficiency of exogenous proteins in recombinant strains and increase the yield of exogenous proteins.

[0014] In specific embodiments provided by this invention, the secretion signal peptide includes, but is not limited to, Usp45 or its variants. Usp45 is a naturally occurring classic secretion signal sequence of Lactococcus lactis, which can significantly enhance the secretion efficiency and total expression level of heterologous proteins through the Sec secretion pathway.

[0015] In the specific embodiments provided by the present invention, the exogenous protein includes, but is not limited to, α-amylase (amyE). Any exogenous protein that is suitable for the expression system of Lactococcus lactis can realize the present invention.

[0016] Secondly, the present invention provides a method for preparing the above-mentioned recombinant strain of Lactococcus lactis, comprising the following steps: The expression cassette was connected to the backbone vector to obtain the suicide plasmid; The suicide plasmid was transformed into competent Lactococcus lactis cells, and after resuscitation culture and screening, an integrated Lactococcus lactis strain was obtained. The expression plasmid was transformed into an integrated strain of Lactococcus lactis to obtain a recombinant strain of Lactococcus lactis.

[0017] In an embodiment of the present invention, the skeleton carrier includes a second promoter, a screening marker, and a homologous fragment (i.e., a homologous arm).

[0018] In this embodiment of the invention, the homologous fragment is a homologous fragment of the target site on the chromosome of Lactococcus lactis. The homologous fragment is highly matched with the upstream and downstream sequences of the target gene, and its main function is to guide the exogenous DNA to undergo homologous recombination with the target region of the genome, much like a navigation system.

[0019] In embodiments of the present invention, homologous segments include upstream homologous segments (up) and downstream homologous segments (down).

[0020] In specific embodiments provided by this invention, the chromosomal target sites of *Lactococcus lactis* include, but are not limited to, those mentioned above. noxD .

[0021] In specific embodiments provided by the present invention, the basic skeleton of the skeleton carrier includes, but is not limited to, at least one of pUC19, pUC18, and pUC57.

[0022] In specific embodiments provided by the present invention, the second promoter includes, but is not limited to, at least one of P32, P45, Pldh, and PpepN.

[0023] In this embodiment of the invention, the filtering markers include positive filtering markers and negative filtering markers.

[0024] In a specific embodiment provided by the present invention, the positive screening marker includes at least one of the following: erythromycin resistance gene EM, chloramphenicol resistance gene Cm, and spectinomycin resistance gene Spc.

[0025] In specific embodiments provided by the present invention, the negative selection markers include at least one of the following: phenylalanine-tRNA synthetase α subunit gene pheS, uracil phosphoribosyltransferase gene upp, and oroate transporter gene oroP.

[0026] In specific embodiments provided by the present invention, the expression plasmid vector includes, but is not limited to, pNZ8148.

[0027] In embodiments of the present invention, the transformation methods include, but are not limited to, electroconversion, chemical transformation, or gene gun.

[0028] In a specific embodiment of the present invention, the conversion method is electrical conversion.

[0029] Preferably, the technical parameters for the electro-conversion include: voltage of 1000~1500 V, capacitance of 20~30 μF, resistance of 150~250 Ω, and shock duration of 0.5~1.5 ms. For example, the voltage is any one of 1000 V, 1050 V, 1100 V, 1150 V, 1200 V, 1250 V, 1300 V, 1350 V, 1400 V, 1450 V, or 1500 V, or falls within any two of these values; the capacitance is any one of 20 μF, 21 μF, 22 μF, 23 μF, 24 μF, 25 μF, 26 μF, 27 μF, 28 μF, 29 μF, or 30 μF, or falls within any two of these values; the resistance is any one of 150 Ω, 160 Ω, 170 Ω, 180 Ω, 190 Ω, 200 Ω, 210 Ω, 220 Ω, 230 Ω, 240 Ω, or 250 Ω, or falls within any two of these values; and the shock duration is 0.5 ms, 0.6 ms, or 0.7 ms. The value can be any one of the following: ms, 0.8 ms, 0.9 ms, 1.0 ms, 1.1 ms, 1.2 ms, 1.3 ms, 1.4 ms, or 1.5 ms, or fall within the range of any two values.

[0030] Preferably, the temperature for resuscitation incubation is 28~32℃, and the time is 1~3 h. For example, the temperature for resuscitation incubation is any one of 28℃, 29℃, 30℃, 31℃, 32℃ or any two of these values, and the time is any one of 1h, 1.5h, 2h, 2.5h, 3h or any two of these values.

[0031] In a specific embodiment of the present invention, the culture medium for resuscitation culture is M17G liquid medium. However, the type of culture medium is not limited to this; any type of culture medium that can achieve the purpose of resuscitation culture is within the scope of protection of the present invention.

[0032] Preferably, the screening method is the positive-negative screening method. The positive-negative screening method selects screening conditions based on the type of screening marker in the backbone vector. For example, if the screening marker is the erythromycin resistance gene EM, then erythromycin is used as the screening condition.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The recombinant *Lactococcus lactis* strain provided by this invention first expresses T7 RNA polymerase via an expression cassette regulated by a first promoter, and then the T7 RNA polymerase specifically recognizes the T7 promoter, mediating efficient transcription of the target gene. The T7 RNA polymerase exhibits high specificity to the T7 promoter and high transcriptional activity, reducing non-specific transcription and optimizing the allocation of host metabolic resources, thus allocating more precursor substances and energy to the synthesis of recombinant proteins. Simultaneously, this transcriptional mechanism is relatively independent of host endogenous transcriptional elements, significantly reducing interference from host endogenous factors. Compared to the *Lactococcus lactis* NICE-induced expression system, the recombinant *Lactococcus lactis* strain provided by this invention can significantly increase the yield of exogenous proteins, making it suitable for low-cost, large-scale fermentation production.

[0034] For inducible expression systems, precise regulation by the NisRK-inducible promoter can achieve moderate expression of T7 RNA polymerase, maintaining high transcription efficiency while reducing the metabolic burden on host cells.

[0035] For constitutive expression systems, constitutive promoter-mediated constitutive expression requires no inducing agent, enabling stable and continuous transcription of the target gene with better uniformity of expression levels. This inducing agent-free constitutive expression system shows promising application prospects in large-scale, low-cost fermentation production.

[0036] 2. The recombinant lactococcus strain provided by this invention, after further introducing a secretory signal peptide, relies on the strong transcriptional ability of the T7 system. The secretory signal peptide can reduce intracellular metabolic pressure and improve protein folding through secretory expression, which can significantly improve the extracellular secretion efficiency and total expression level of exogenous proteins.

[0037] 3. In existing technologies, T7 expression systems constructed using free plasmids not only suffer from unstable passage but also contain genes encoding antibiotic resistance enzymes for ease of screening. This leads to the additional expression of antibiotic resistance enzymes during fermentation, posing a risk of drug residues in food applications. This invention integrates an expression cassette containing the T7 RNA polymerase gene into the chromosome of *Lactococcus lactis*, exhibiting good genetic stability. No antibiotic resistance protein is expressed during T7 RNA polymerase expression, resulting in high safety. Attached Figure Description

[0038] Figure 1 Suicide plasmid map for Lactococcus lactis chromosome integration T7 RNA polymerase and PCR identification results of the integrating strain; (a) Map of the inducible suicide plasmid pUC19-P32-pheS-EM-up-PnisA-T7RNAP-down; this plasmid is used to integrate the T7 RNAP expression cassette regulated by the inducible promoter PnisA into the chromosome of Lactococcus lactis; (b) Map of constitutive suicide plasmid pUC19-P32-pheS-EM-up-P32-T7RNAP-down; this plasmid is used to integrate the T7 RNAP expression cassette regulated by the constitutive promoter P32 into the chromosome of Lactococcus lactis; (c) PCR identification results of T7 RNAP chromosome-integrated strains; verification was performed using two sets of primers, noxJCUF1 / noxJCUR1 and P32F / T7CXR; M: DNA molecular weight standard; lanes 1-10: PCR amplification products of different independent transformants; the upper gel shows the PCR detection results of the PnisA-T7RNAP integrated strain, and the lower gel shows the PCR detection results of the P32-T7RNAP integrated strain; all lanes showed specific bands of the expected size, proving that the T7 RNAP expression cassette was successfully and stably integrated into the chromosomal target site through double crossover recombination. Figure 2 Construction of the T7 expression system in Lactococcus lactis NZ9000 and its effect on α-amylase transcription level and expression activity; (a) Schematic diagrams of the structures by which the inducible promoter PnisA, the constitutive promoter P32, and the T7 promoter drive the expression of the amyE gene, respectively; (b) Schematic diagram of the working principle of the T7 expression system: T7 RNA polymerase is stably expressed on the strain chromosome, and the amyE gene is transcribed under the regulation of the T7 promoter; (c) Comparison of intracellular α-amylase activity between the T7 expression system and the traditional PnisA expression system under induction mode; (d) Comparison of intracellular α-amylase activity between the T7 expression system and the conventional P32 expression system under the compositional pattern; (e) Comparison of amyE gene transcription levels between the T7 expression system and the traditional PnisA expression system under induction mode; (f) Comparison of amyE gene transcription levels between the T7 expression system and the traditional P32 expression system under the compositional pattern; Note: Experimental data are expressed as mean ± standard deviation, and each group has 3 parallel samples; p ≤0.05, p ≤0.01, p ≤0.001.

[0039] Figure 3 Evaluation of expression efficiency of Usp45J signal peptide and T7 expression system; (a) Schematic diagram of the structure driving α-amylase expression by inducible promoter PnisA, constitutive promoter P32, and T7 promoter (T7P); the Usp45J signal peptide sequence is fused to the N-terminus of the amyE gene to achieve protein secretion and transport. (b) Schematic diagram of the T7 secretory expression system: T7 RNA polymerase is stably expressed on chromosomes, and the target gene amyE carrying the Usp45J signal peptide is transcribed under the regulation of the T7P promoter to achieve secretory expression; (c) Comparison of intracellular, extracellular and total α-amylase activities between the T7 expression system and the traditional PnisA expression system under inducible PnisA regulation conditions; (d) Comparison of intracellular, extracellular and total α-amylase activities between the T7 expression system and the traditional P32 expression system under constitutive P32 regulation; Note: Data are expressed as mean ± standard deviation, with 3 biological replicates per group; p ≤ 0.05, p ≤0.01, p ≤ 0.001.

[0040] English translation of the image: Ori: Origin of replication site; AmpR: Ampicillin resistance gene; AmpR promoter: Ampicillin resistance gene promoter; PheS: Gene encoding the alpha subunit of phenylalanyl-tRNA synthetase; RBS: Ribosome Binding Site; EM: Erythromycin resistance gene; up: upstream homologous sequence; T7 RNA polymerase: T7 RNA polymerase, abbreviated as T7RNAP; down: downstream homologous sequence; CAP binding site: the binding site of catabolite activator protein (CAP). T7P: T7 promoter; Enzyme activity; Transcription level; Ext.: extracellular; Int.: intracellular; T.: General. Detailed Implementation

[0041] This invention discloses a recombinant strain of *Lactococcus lactis* and its preparation method. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0042] Terminology Explanation: The term "expression cassette" refers to a single, modular gene expression unit containing the most basic DNA elements necessary for the efficient expression of one or more target genes in a specific host cell. A typical expression cassette contains at least the following elements, which are operatively linked sequentially: (a) a promoter; (b) a target gene coding sequence operatively linked to said promoter; and optionally, (c) a transcription termination signal sequence downstream of the coding sequence that guides transcription termination and polyadenylation. This expression cassette can be integrated into various vectors (such as plasmids, viral vectors, or chromosomes) while maintaining its transcriptional function.

[0043] The term "expression plasmid" refers to an artificially modified vector DNA containing complete gene expression regulatory elements, capable of driving the transcription and translation of exogenous genes in specific host cells.

[0044] The term "promoter" refers to a DNA segment with a specific sequence that is functionally located upstream of the transcription start site of a target gene. This sequence can be specifically recognized and bound by the transcription mechanisms of the host cell (including but not limited to RNA polymerases and their associated transcription factors), thereby initiating and regulating the transcription process of the target gene. Based on their regulatory characteristics, promoters may include, but are not limited to, constitutive promoters, tissue-specific promoters, inducible promoters, or stage-specific promoters.

[0045] The term "inducible promoter" refers to a promoter that selectively expresses coding sequences or functional RNA in response to the presence of endogenous or exogenous stimuli, such as through a chemical compound (chemical inducer), or in response to environmental, hormone, chemical, and / or developmental signals.

[0046] The term "constitutive promoter" refers to a class of regulatory sequences that can drive the continuous expression of genes in all tissues of an organism. Their activity is unaffected by external conditions and lacks spatiotemporal specificity.

[0047] The term "secretion signal peptide" refers to a short peptide that guides the transfer of newly synthesized proteins into the secretory pathway. In mature proteins secreted extracellularly, the secretion signal peptide is cleaved by signal peptidase.

[0048] The term "suicide plasmid" refers to a vector that cannot autonomously replicate and amplify within a specific host. This property means that if a suicide plasmid does not integrate into the host genome after being transferred into a target cell, it will be diluted and eventually lost during cell division. Only during homologous recombination can the genetic information carried by the suicide plasmid be stably integrated into the genome, resulting in functional mutations. The core components of a suicide plasmid structure include: a host-unrecognized / conditional origin of replication (ori), positive selection markers, negative selection markers, promoters and regulatory elements, and conjugate transfer sequences (oriT), etc.

[0049] The term "selection marker" refers to a gene that helps select cells that actively express nucleic acid sequences. It includes positive and negative selection markers.

[0050] The term "positive selection marker" refers to a gene or molecular tag used in genetic engineering or cell selection to endow host cells with the ability to survive under specific selective pressures. These tags are used to directly screen for cells that have successfully incorporated or integrated a target vector.

[0051] The term "negative selection marker" refers to a specific gene sequence located on the outer homologous arm of a gene targeting vector. Its expression product, in the presence of a specific drug, causes cell death. It is primarily used to eliminate non-target cells undergoing random integration, thereby enriching target cells that have undergone homologous recombination.

[0052] The term "homologous recombination" (HR) refers to the process by which two DNA molecules with the same or similar sequences exchange nucleotide sequences through pairing, strand breaking, and rejoining.

[0053] The reagents and biological materials used in this invention are all commercially available.

[0054] The present invention will be further illustrated below with reference to the embodiments: Example 1: Construction of suicide plasmid pUC19- P32 -pheS-EM vector skeleton construction. The specific construction process is as follows: First, the recombinant vector 8148-T7RNAP was constructed, and obtained after double digestion with PstI / SacI. PnisA -T7RNAP fragment, ligating this fragment to pUC19- P32 -pheS-EM-up-down vector, to obtain integration plasmid pUC19- P32 -pheS-EM-up- PnisA -T7RNAP-down (first suicide plasmid); The up- was amplified using primer pairs p32T7RNAPF (5'-CGCGTCGACTTGGAAGCTCCCATG-3') and p32T7RNAPR (5'-ATACCATGGTGAGTGCCTCCTTTTCAAAATTCC TCCGAAT-3'). P32 The fragment, after being double-digested with SalI / NcoI, was ligated into the aforementioned integrative plasmid pUC19- P32 -pheS-EM-up- PnisA -T7RNAP-down, ultimately yielding the integrative plasmid pUC19- P32 -pheS-EM-up- P32 -T7RNAP-down (second suicide plasmid).

[0055] This study selected the chromosomal noxD site, commonly used in Lactococcus lactis genome editing, as the target site for site-specific integration. The upstream homologous fragment (up) is the upstream homologous fragment of the Lactococcus lactis chromosomal noxD site, and the downstream homologous fragment (down) is the downstream homologous fragment of the Lactococcus lactis chromosomal noxD site.

[0056] Table 1 Suicide plasmid of the present invention

[0057] Example 2: Construction of Integrated Strains To obtain an integrated Lactococcus lactis strain capable of stably expressing T7 RNA polymerase (T7RNAP), the two suicide plasmids pUC19- prepared in Example 1 were used. P32 -pheS-EM-up- PnisA -T7RNAP-down, pUC19- P32 -pheS-EM- up- P32 -T7RNAP-down was electroporated into Lactococcus lactis NZ9000 competent cells. In this study, the chromosomal noxD site, commonly used in Lactococcus lactis genome editing, was selected as the site-specific integration target. Electroporation parameters were set as follows: voltage 1250 V, capacitance 25 μF, resistance 200 Ω, and electroporation duration 1 ms. Immediately after transformation, pre-cooled M17G liquid medium was added, and the cells were incubated at 30 ℃ for 2 h. The bacterial culture was then plated on M17G solid plates containing 5 μg / mL erythromycin and incubated at 30 ℃ for 48 h. Single colonies were picked, and single-exchange integrated strains were identified by PCR. Positive strains were diluted and plated on M17G plates supplemented with 20 mM p-chlorophenylalanine (pclphe) and incubated at 30 ℃ for 24 h. Finally, two double-exchange recombinant bacteria with successful T7RNAP gene integration were screened and named NZ01 and NZ02 (see Table 2). Figure 1 ).

[0058] Table 2. Integrative strains of the present invention

[0059] Example 3: Construction of expression plasmids The T7PCONSR promoter was constructed using pNZ8148 plasmid as a backbone (reference: "Improved designs for pET expression plasmids increase protein production yield in..."). Escherichia coli The T7PCONSR promoter is a modified and optimized T7 promoter (with four additional bases added after the T7 promoter to improve expression). This drives the expression vector 8148-T7PCONSR, adaptable to the expression needs of exogenous genes under different regulatory modes. The nucleotide sequence of the T7PCONSR promoter is TAATACGACTCACTATAGGG AGA. Based on this vector, the secretion signal peptide usp45J is fused to further construct the secretory expression vector 8148 T7PCONSRSPJ.

[0060] To evaluate the expression performance of the T7 expression system, the reporter gene amyE was inserted into each vector, resulting in six recombinant expression plasmids (see Table 3). Table 3 Recombinant expression plasmids of the present invention

[0061] Example 4: Construction of Recombinant Strains The expression plasmids constructed in Example 3 were electroporated into strains NZ9000, NZ01, and NZ02, respectively. Transformants were screened using M17G solid plates supplemented with 10 μg / mL chloramphenicol. Single colonies were randomly picked from the chloramphenicol-resistant transformed colonies and further validated by PCR using primer pair cmF(5'-GGACTTCATTTACTGGGTT-3') / 8148R(5'-CCTCTAAAAGGTGAT-3'). Positive clones were collected and named expression strains NZA1 to NZA8 (Table 4).

[0062] Table 4 Recombinant strains of the present invention

[0063] Example 5: Validation of expression and transcription levels 1. Experimental Methods (1) Verification of expression level Constitutive T7 expression system strains (NZA3, NZA4, NZA7, NZA8) were inoculated into their respective culture media and incubated statically at 30 °C for 24 h before cell collection. Inducible T7 expression system strains (NZA1, NZA2, NZA5, NZA6) were cultured under the same conditions until OD500. 600 When the pH was 0.4–0.6, nisin was added for induction, and the cells were collected after culturing for 24 h. The culture supernatant of each group of strains was used for extracellular enzyme activity assay; the cells were collected by centrifugation and then sonicated, and the supernatant after disruption was used as the sample for intracellular enzyme activity assay.

[0064] α-Amylase activity assay: Using 300 g / L sucrose as the reaction substrate, the reaction was carried out at 65 °C for 1 h. The amount of reducing sugar produced by α-amylase was determined by the 3,5-dinitrosalicylic acid (DNS) method, thereby evaluating the intracellular and extracellular enzyme activity levels of α-amylase in constitutive and inducible T7 expression systems.

[0065] Enzyme activity definition: One α-amylase activity unit (U) is defined as the amount of enzyme required to catalyze the hydrolysis of starch to produce 1 μmol of reducing sugar per minute under the above measurement conditions.

[0066] (2) Verification at the transcriptional level To compare the differences in gene transcription levels among different expression systems, the nisin-induced system and the nisin-induced system were measured respectively. P32 Compositional systems and T7 systems T7RNAP and amyE The transcription level was determined. Each strain (NZA1, NZA2, NZA3, NZA4) was cultured under corresponding conditions to a specified time point, after which bacterial cells were collected. Total RNA was extracted using a full-length gold RNA extraction kit. After genomic DNA was removed by DNase treatment, cDNA was synthesized using reverse transcription reagent. The obtained cDNA was used as a template to determine the transcription level using real-time quantitative PCR (qRT-PCR). T7RNAP and amyE The relative amount of transcription, and in terms of 16S rRNA As an internal reference gene.

[0067] Transcription level using 2 - The ΔΔCt method is used for calculation.

[0068] 2. Experimental Results α-Amylase is a simple hydrolytic enzyme that functions without cofactors. Its activity is readily detectable and provides stable and reliable quantitative results, making it an ideal reporter protein for evaluating the performance of heterologous expression systems in *Lactococcus lactis*. This study used *Lactococcus lactis* with chromosome-integrated T7 RNA polymerase as the host and α-amylase as the model protein to systematically evaluate the regulatory effect of the T7 expression system on heterologous proteins and compared it with traditional expression systems.

[0069] In the inducible expression mode, the traditional expression system relies on the NisRK (NisR / NisK)-regulated PnisA promoter to directly drive the transcription of the target gene; while the T7 expression system adopts a cascade regulatory mechanism: first, the NisRK-PnisA regulatory unit expresses the T7 RNA polymerase, and then the polymerase specifically recognizes the T7 promoter, mediating the efficient transcription of the target gene (Figure 2b). As shown in Figure 2c, the intracellular α-amylase activity of the traditional inducible strain NZA1 was 0.386±0.171 U / mL, while the enzyme activity of the engineered strain NZA2 regulated by the T7 cascade reached 2.025±0.028 U / mL, which is about 5.24 times higher than the control group (p<0.05 compared with NZA1), proving that the T7 system can significantly increase the intracellular expression level of α-amylase. The superior expression efficiency of the T7 system stems primarily from two factors: T7 RNA polymerase exhibits high specificity to the T7 promoter and high transcriptional activity, reducing non-specific transcription; simultaneously, this transcriptional mechanism is relatively independent of host endogenous transcriptional elements, significantly minimizing interference from host endogenous factors. Furthermore, precise NisRK regulation enables moderate expression of T7 RNA polymerase, maintaining high transcriptional efficiency while reducing the metabolic burden on host cells.

[0070] In constitutive expression mode, traditional systems directly drive the target gene using the P32 promoter, while the T7 system achieves efficient expression of the target gene by continuously expressing T7 RNA polymerase through P32 (Figure 2b). As shown in Figure 2d, the intracellular α-amylase activity of the traditional constitutive strain NZA3 was 0.208±0.004 U / mL, while that of the T7 system engineered strain NZA4 reached 2.151±0.092 U / mL, a 10.36-fold increase compared to the control group (p<0.01 compared to NZA3). P32-mediated constitutive expression does not require the addition of an inducer, enabling stable and continuous transcription of the target gene with better uniformity of expression levels. The highly specific transcriptional characteristics of the T7 system can optimize the allocation of host metabolic resources, allocating more precursor substances and energy to the synthesis of recombinant proteins. This inducer-free constitutive T7 expression system has promising application prospects in the field of large-scale, low-cost fermentation production.

[0071] To elucidate the mechanism of action of the T7 expression system under different regulatory modes, real-time quantitative PCR was used to detect the transcription level of the amyE gene in each recombinant strain (Figure 2e-f). The results showed that the T7 system could significantly increase the transcription of the target gene in both inducible and constitutive modes, with transcription levels upregulated by 103-fold and 207-fold, respectively, demonstrating that T7 RNA polymerase can strongly drive ultra-high transcription of the target gene.

[0072] Analysis of enzyme activity data revealed that the increase in transcription level was not strictly linearly correlated with the increase in enzyme activity; even with a significant increase in transcription, the increase in amylase activity did not match. This indicates that in the T7 strong expression system, the final yield of the target protein is not only regulated by transcription intensity but also by multiple downstream processes such as translation efficiency and protein folding.

[0073] After confirming that both inducible and constitutive T7 expression systems can significantly enhance α-amylase expression, this study further introduced the usp45J signal peptide to optimize secretion efficiency and increase extracellular amylase production. Usp45 is a classic natural secretion signal sequence of *Lactococcus lactis*, which can significantly enhance the secretion efficiency and total expression of heterologous proteins through the Sec secretion pathway. Existing literature has confirmed its effective promotion of the extracellular functional expression of various exogenous proteins in *Lactococcus lactis*. This study used the modified and optimized usp45J signal peptide (reference: "Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in..."). Lactococcus lactis The specific amino acid sequence is MKKKIISAILMSTVILS AAAPLSGVYALEISSTCDA, which is used for secretory expression.

[0074] As shown in Figure 3, the usp45J signal peptide significantly enhanced the extracellular secretion efficiency and total expression level of α-amylase in the inducible T7 system: the extracellular enzyme activity in the traditional induction system was 0.255±0.019 U / mL, while the extracellular enzyme activity in the T7 system equipped with the usp45J signal peptide reached 1.699±0.042 U / mL, which was 6.65 times higher than that in the control group (p<0.05). Figure 3 c). This indicates that, relying on the strong transcriptional capacity of the T7 system, usp45J can alleviate intracellular metabolic stress, improve protein folding, and significantly enhance extracellular enzyme activity through secretory expression.

[0075] In the constitutive T7 system, USP45J also promoted extracellular amylase secretion, but the increase was weaker than that in the induction system: the extracellular enzyme activity in the traditional constitutive system was 0.250±0.015 U / mL, while that in the T7 system containing USP45J was 0.800±0.071 U / mL, an increase of 3.22 times (p<0.05, Figure 3d). This phenomenon indicates that the sustained high-intensity transcription in constitutive T7 will bring a long-term metabolic and secretory load to the cells. Even with the USP45J signal peptide, the cells will adaptively downregulate protein synthesis, weakening the synergistic effect of the signal peptide. However, overall, the enzyme activity in the T7 system is still better than that in the traditional expression system, proving that the USP45J signal peptide can effectively improve the overall expression level of α-amylase.

[0076] Summarize: This invention utilizes homologous recombination with suicide plasmids to specifically integrate T7 RNAP, regulated by the PnisA inducible promoter and the P32 constitutive promoter, into the noxD chromosomal locus of *Lactococcus lactis*, obtaining two stable engineered hosts. Simultaneously, matching constitutive and T7 promoter-driven expression vectors were constructed, and a secretory vector was constructed by fusing the usp45J signal peptide. Using α-amylase as a reporter protein, the intracellular enzyme activity of the T7 system was increased by 5.24-fold and 10.36-fold in the inducible and constitutive modes, respectively. After introducing the usp45J signal peptide, the extracellular secretory activity was increased by 6.65-fold and 3.22-fold, respectively, significantly superior to traditional expression systems, making it suitable for low-cost, large-scale fermentation production.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant strain of Lactococcus lactis, characterized in that, The recombinant lactococcus strains include: a) An expression cassette, wherein the expression cassette is an expression cassette integrated into the chromosome of Lactococcus lactis, and the expression cassette includes a first promoter and a T7 RNA polymerase gene; b) Expression plasmid, which includes a T7 promoter and a foreign protein gene.

2. The recombinant lactococcus strain according to claim 1, characterized in that, The first promoter is an inductive promoter or a constitutive promoter.

3. The recombinant lactococcus strain according to claim 2, characterized in that, The inducible promoter includes at least one of PnisA, PxylT, PczcD, and PtetO.

4. The recombinant lactococcus strain according to claim 2, characterized in that, The constitutive promoters include at least one of P32, P45, Pldh, and PpepN.

5. The recombinant lactococcus strain according to any one of claims 1 to 4, characterized in that, The expression plasmid also includes a secretion signal peptide coding sequence.

6. A method for preparing the recombinant lactococcus strain according to any one of claims 1 to 5, characterized in that, Includes the following steps: The expression cassette was connected to the backbone vector to obtain the suicide plasmid; The suicide plasmid was transformed into competent Lactococcus lactis cells, and after resuscitation culture and screening, an integrated Lactococcus lactis strain was obtained. The expression plasmid was transformed into the integrated strain of *Lactococcus lactis* to obtain the recombinant strain of *Lactococcus lactis*.

7. The preparation method according to claim 6, characterized in that, The backbone vector includes a second promoter, a selection marker, and a homologous fragment; the homologous fragment is a homologous fragment of the target site on the chromosome of Lactococcus lactis.

8. The preparation method according to claim 7, characterized in that, The basic skeleton of the skeletal carrier is one of pUC19, pUC18, and pUC57.

9. The preparation method according to claim 7, characterized in that, The second promoter includes at least one of P32, P45, Pldh, and PpepN.

10. The preparation method according to any one of claims 7 to 9, characterized in that, The filter markers include positive filter markers and negative filter markers; The positive selection markers include at least one of the following: erythromycin resistance gene EM, chloramphenicol resistance gene Cm, and spectinomycin resistance gene Spc. The negative selection markers include at least one of the following: phenylalanine-tRNA synthetase α subunit gene pheS, uracil phosphoribosyltransferase gene upp, and oroate transporter gene oroP.

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