Tandem expression recombinant industrial strain for efficiently producing epsilon-polylysine and application of tandem expression recombinant industrial strain

Abstract

The invention discloses a tandem expression recombinant industrial strain for efficiently producing epsilon-polylysine and application of the tandem expression recombinant industrial strain, the strain TG1003-ddcpgkakpls is obtained by integrating a tandem expression cassette on a genome attB site of a wild strain TG1003 of streptomyces albidus, the strain TG1003-ddcpgkakpls is preserved in China Center for Type Culture Collection on June 16, 2025, and the preservation number of the strain TG1003-ddcpgkakpls is CCTCC M 20251400; the tandem expression cassette sequentially comprises a pls gene driven by a kasOP promoter, and a ddc gene, a pgk gene and an ak gene which are respectively driven by a permEP promoter. Related genes produced by epsilon-PL are expressed in series on a genome of a wild type strain, and different promoters are combined with the genes, so that the stable expression quantity of the related genes is improved, and the yield of epsilon-PL is further improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, relates to a tandem expression recombinant industrial strain for the efficient production of ε-polylysine and its application. Background Technology

[0002] ε-polylysine (ε-PL), a biopolymer composed of L-lysine residues linked by amide bonds between α-carboxyl and ε-amino groups, possesses excellent properties such as edibility, water solubility, thermal stability, broad-spectrum antibacterial activity, and biodegradability, making it promising for applications in numerous fields including food preservation, pharmaceuticals, and materials. In food preservation, ε-PL effectively inhibits the growth of various bacteria, molds, and yeasts, significantly extending shelf life. In pharmaceuticals, its antibacterial properties can be used to develop novel antibacterial drugs and formulations. In materials science, it can be applied as a biocompatible material in tissue engineering and other fields.

[0003] Currently, microbial fermentation is the main method for producing ε-PL, with *Streptomyces albulus* being one of the commonly used production strains. To increase ε-PL yield, existing technologies typically involve optimizing the fermentation process and modifying the production strains using genetic engineering techniques. The literature "Zhang Chongyang. Identification of key genes in ε-polylysine biosynthesis and research on improving ε-polylysine yield through combined overexpression [D]. Jiangnan University. 2022." reports that tandem or tritandem overexpression of ppc, pyc, and pls promotes ε-PL synthesis. However, strains with multi-gene tandem overexpression exhibit problems such as inter-gene interference and genetic instability, showing decreased cell mass and increased glucose consumption rate.

[0004] Therefore, developing a recombinant industrial strain and its construction method that can efficiently and stably express multiple key genes and significantly increase ε-PL production is of great significance for promoting the industrialization of ε-PL. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient tandem expression recombinant industrial strain for producing ε-polylysine and its application, in order to solve the technical problems of low production efficiency and unstable strain expression in the production of ε-PL by Streptomyces leucovorum.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a highly efficient tandem expression recombinant industrial strain TG1003-ddcpgkakpls for the production of ε-polylysine. The strain is obtained by integrating a tandem expression cassette at the attB site of the genome of wild-type Streptomyces albulus strain TG1003. It was deposited at the China Center for Type Culture Collection on June 16, 2025, with the accession number CCTCC M 20251400.

[0008] The wild-type strain TG1003 was screened from soil in the suburbs of Xiangtan City and deposited at the China Center for Type Culture Collection on June 16, 2025, with accession number CCTCC M 20251399.

[0009] The expression cassette sequentially contains: — ε-polylysine synthase gene pls driven by the kasOP promoter; — diaminopimelic acid decarboxylase gene ddc driven by the permEP promoter; — pyruvate kinase gene pgk driven by the permEP promoter; — aspartate kinase gene ak driven by the permEP promoter.

[0010] Preferably, the 16S rDNA sequence of the wild-type strain TG1003 is shown in SEQ ID NO:1.

[0011] Preferably, the nucleotide sequence of the expression cassette is shown in SEQ ID NO:2.

[0012] Preferably, the expression cassette is integrated into the attB site of the Streptomyces TG1003 genome via the pSET152 vector.

[0013] A second aspect of the present invention provides a method for constructing the recombinant industrial strain, comprising:

[0014] The ddc, pgk, ak, and pls genes driven by the kasOP promoter, permEP promoter, and permEP promoter, respectively, were sequentially inserted into the pSET152 vector using In-Fusion one-step cloning technology to obtain the recombinant plasmid pSET152-ddcpgkakpls. This recombinant plasmid was transformed into *E. coli* ET12567 / pUZ8002, and then conjugated with *Streptomyces leucocephala* strain TG1003 for transfer. After screening for apramycin resistance, PCR verification, and sequencing confirmation, TG1003-ddcpgkakpls was obtained.

[0015] Preferably, the conditions for conjugation transfer are as follows: Escherichia coli and strain TG1003 are mixed at a 1:1 ratio, spread on MS plates, co-cultured at 28°C for 18–20 h, and then screened using MS plates containing 80–100 µg / mL Apr and 50 µg / mL Na1.

[0016] A third aspect of the present invention provides a method for producing ε-PL using the recombinant industrial strain, comprising:

[0017] Inoculate the strain into seed culture medium and incubate at 28-32 ℃ and 200-220 rpm for 16-18 h;

[0018] Transfer the inoculum to the fermentation medium at a rate of 2-5% (v / v), and ferment at 28-32 ℃, 1-2 vvm aeration, 15-30% dissolved oxygen, and pH controlled at 3.0-3.2 for 180 h.

[0019] The fermentation broth was centrifuged, purified with resin, eluted with hydrochloric acid, nanofiltered, and dried to obtain the ε-PL product.

[0020] Preferably, the fermentation medium is formulated as follows: glucose 50-60 g / L, yeast extract 4-8 g / L, (NH4)2SO4 22.5-25.5 g / L, K2HPO4·3H2O 0.56-1.36 g / L, KH2PO4 0.68-0.78 g / L, MgSO4·7H2O 0.7-0.8 g / L, FeSO4·7H2O 0.057-0.068 g / L, ZnSO4·7H2O 0.04-0.06 g / L, pH 6.8; glucose is sterilized separately.

[0021] Preferably, the pH control specifically includes: maintaining the pH at 6.8 for 0–48 h, and then reducing the pH to 3.0–3.5 after 48 h.

[0022] In a fourth aspect, the present invention provides an ε-polylysine product prepared by the method thereof, wherein the ε-PL content is ≥95% (w / w), the molecular weight distribution is 3.5–4.5 kDa, and the yield is ≥6.5 g / L.

[0023] A fifth aspect of the invention provides the use of the recombinant industrial strain as an ε-PL production strain in the food, pharmaceutical, materials, or agricultural fields.

[0024] Biological Preservation Information

[0025] The wild-type strain of Streptomyces albulus TG1003 was deposited at the China Center for Type Culture Collection (CCTCC) on June 16, 2025, with accession number CCTCC M 20251399, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0026] Streptomyces albulus TG1003-ddcpgkakpls was deposited at the China Center for Type Culture Collection on June 16, 2025, with accession number CCTCC M 20251400, located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0027] In *Streptomyces albopictus*, ε-PL synthesis occurs via the diaminopimelic acid pathway. Under the mediation of key enzymes such as aspartate kinase and diaminopimelic acid decarboxylase, L-lysine is first synthesized, followed by ε-PL synthesis via α-polylysine synthase (pls) located on the cell membrane, and then secreted extracellularly. However, wild-type strains exhibit low ε-PL production, making it difficult to meet the growing market demand.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] The recombinant industrial strain (Streptomyces albulus) TG1003-ddcpgkakpls provided by this invention is obtained by integrating a tandem expression cassette at the attB site of the genome of the wild-type Streptomyces albulus strain TG1003. This strain can produce high levels of ε-polylysine, with a yield of 59.97 g / L in a 5L fermenter (180 h), which is 2.66 times that of the initial strain (22.5 g / L). This invention achieves high ε-polylysine production by linking promoters to the 5' end of the genes related to ε-polylysine production, enabling stable expression of these genes. Even after multiple passages, the strain maintains the same superior traits, and the overexpression strain consistently maintains a high expression intensity. Attached Figure Description

[0030] Figure 1 Microscopic morphology of wild-type Streptococcus thuringiensis strain TG1003.

[0031] Figure 2 Flowchart for constructing the recombinant plasmid pSET152-ddcpgkakpls.

[0032] Figure 3 Comparison of ε-PL production between industrial recombinant strain TG1003-ddcpgkakpls and other process strains (180h, 5 L tank).

[0033] Figure 4 Biomass comparison of industrial recombinant strain TG1003-ddcpgkakpls with other process strains (180 h, 5L tank).

[0034] Figure 5 Comparison of key gene expression levels between industrial recombinant strain TG1003-ddcpgkakpls and wild-type Streptococcus leuciscus strain TG1003 (180 h, 5 L tank). Detailed Implementation

[0035] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways than those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. It should be noted that the reagents and other materials used in these embodiments are all commercially available products.

[0037] Example 1: Isolation and Screening of Strains

[0038] A wild-type Streptomyces albulus strain, TG1003, was screened from soil in the suburbs of Xiangtan City. This strain can produce high levels of ε-polylysine, reaching a yield of 22.5 g / L in a 5L fermenter. Soil samples for screening high-yield ε-polylysine strains were obtained from the suburbs of Xiangtan City. The soil samples were air-dried at room temperature for 10-15 days, crushed, and passed through a 100-mesh sieve, then dried in an oven at 40°C for 1 hour. 10-11 g of the dried soil sample was added to 50-100 mL of sterile 5-6 mM phosphate buffer (pH=7.2) and shaken for 10-15 min to obtain the screening solution. 10 mL of this solution was added to a 500 mL Erlenmeyer flask containing 200 mL of LB liquid medium and mixed thoroughly. The flask was then incubated on a shaker for 24 hours at 30°C and 200 rpm. This step was to enrich the strain contained in the sample.

[0039] The screening process for high-yield ε-polylysine strains consists of two steps: primary screening and secondary screening. First, a compound antibiotic is added to a sterile ISP2 plate, and the enriched strains are then spread on the plate. The compound antibiotic antibacterial agent is prepared as follows: Appropriate amounts of actinomycin, nystatin, and sodium naphthaleneate are weighed and spread evenly at the bottom of an Erlenmeyer flask. The flask is then covered with ether until the antibiotic powder is submerged. The flask is sealed with plastic wrap and placed in a fume hood. After the ether evaporates, a measured amount of sterile water is added, and the mixture is dissolved at 40°C to achieve final concentrations of 10-15 mg / L, 10-15 mg / L, and 20-25 mg / L, respectively. The solution is then filtered through a 0.22 μm sterile filter membrane before use. Note that nystatin cannot be completely dissolved at pH 7.2; the pH must be adjusted to 10-11 with 1 M NaOH. After complete dissolution, the solution is neutralized and titrated with 1 M HCl to restore the pH to neutral.

[0040] Using an inoculation spatula, scrape the bacterial strains grown on the screening medium plate, dilute them appropriately with sterile water, and spread them onto isolation medium plates containing 2-5 g / L ε-polylysine to screen for ε-polylysine-tolerant strains. Pick colonies from the ε-polylysine isolation medium plate and transfer them using a sterile toothpick to SG plates containing 0.003-0.005% methylene blue, and incubate for 7 days. Screen single colonies based on the size of the clear zone, selecting those showing a clear zone for further amplification, in preparation for secondary screening.

[0041] The single colonies obtained from the initial screening were inoculated into 15-20 mL / 250 mL of M3G liquid shake flask medium and fermented for 96 h. The fermentation broth was collected, centrifuged at 7000 rpm for 10 min, and 1-2 mL of the fermentation supernatant was placed in a test tube. DR reagent was added in an equal proportion, and the mixture was allowed to stand for 20-30 min before observation.

[0042] The selected ε-polylysine-producing single colonies were inoculated into 15-20 mL / 250 mL M3G liquid shake flask medium for further activation and expansion. The culture conditions were 28-30°C for 24-32 h with a constant temperature shaker speed of 200 rpm. The activated bacterial solution was streaked onto BTN test tubes for preservation and incubated at 30°C for 3-4 days, then placed at room temperature for subsequent experiments. Simultaneously, the activated bacterial solution was mixed thoroughly with sterilized 30% glycerol at a 1:1 ratio and added to preservation tubes for storage at -80°C.

[0043] The culture medium formulations used for screening strains are all standard culture media:

[0044] LB medium (%): yeast extract 5%, peptone 10%, NaCl 10%, agar powder 15%, pH 7.0

[0045] ISP2 medium (%): yeast extract 4%, malt extract 1%, glucose 4%, agar powder 15%, trace salt solution 10mL, pH 7.2

[0046] SG medium (%): Glycerol 10, yeast extract 0.1, KH₂PO₄ 0.25, NaH₂PO₄·2H₂O 0.88, MgSO₄·7H₂O 0.25, (NH₄)₂SO₄ 0.66, ZnSO₄·7H₂O 0.05, FeSO₄·7H₂O 0.01, agar powder 15, pH 6.8

[0047] SGB ​​medium (%): Methylene blue 0.03, glycerol 10, yeast extract 0.2, KH₂PO₄ 0.25, NaH₂PO₄·2H₂O 0.88, MgSO₄·7H₂O 0.25, (NH₄)₂SO₄ 0.86, ZnSO₄·7H₂O 0.05, FeSO₄·7H₂O 0.01, agar powder 15, pH 6.8

[0048] M3G medium (%): glucose 5%, yeast extract 0.5%, (NH4)2SO4 1%, K2HPO4·3H2O 1.048%, KH2PO4 1.36%, concentrated salt solution 100mL, pH 6.8

[0049] BTN medium (%): glucose 10, beef extract 1, peptone 2, yeast extract 1, agar powder 20, pH 7.5

[0050] Of the above-mentioned culture media, those containing glucose were sterilized at 115°C for 15 min before use; those without glucose were sterilized at 121°C for 20 min and then cooled to room temperature before use. In M3G culture medium, the glucose solution was prepared and sterilized separately.

[0051] Single colonies were collected from screening plates and transferred to 15-20 mL / 250 mL M3G liquid shake flask medium. Incubation was carried out at 30°C for 24 h for enrichment. 1-2 mL of the bacterial culture was centrifuged, and bacterial genomic DNA was extracted using a rapid bacterial genomic DNA extraction kit (see kit instructions). The 16S rDNA of strain TG1003 was amplified using universal primers 27F and 1492R. PCR was performed using PrimerSTAR DNA polymerase in a 50 μL reaction volume. The PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The 16S rDNA sequence of strain TG1003 is shown in SEQ ID No. 1. The sequencing results were compared with sequences of related species in the GenBank database. A phylogenetic tree was constructed using MATLAB software, as shown below. Figure 2 As shown, it was identified as Streptomyces albopictus.

[0052] The seed culture medium (g / L) for the *Streptomyces leucocephala* strain TG1003 comprises: glucose 40–60 g / L, yeast extract 4–6 g / L, (NH4)2SO4 8–12 g / L, K2HPO4·3H2O 0.8–1.2 g / L, KH2PO4 1.0–1.5 g / L, MgSO4·7H2O 0.5–0.8 g / L, FeSO4·7H2O 0.04–0.06 g / L, ZnSO4·7H2O 0.03–0.05 g / L, pH 6.8–7.0.

[0053] Due to its excellent shape, it was selected as the starting strain for genetic engineering modification.

[0054] Example 2 Construction of recombinant industrial strain TG1003-ddcpgkakpls

[0055] The wild-type Streptococcus leucis wild-type strain TG1003 from Example 1 was used as the initial strain.

[0056] 1. Serial expression box structure

[0057] The following elements are included sequentially from 5' to 3':

[0058] kasOP promoter → ε-polylysine synthase gene pls;

[0059] permEP promoter → diaminopimelic acid decarboxylase gene ddc;

[0060] permEP promoter → pyruvate kinase gene pgk;

[0061] permEP promoter → aspartate kinase gene ak.

[0062] The nucleotide sequence of the tandem expression cassette is shown in SEQ ID No. 2.

[0063] 2. Construction of recombinant plasmid pSET152-ddcpgkakpls

[0064] (1) Gene amplification

[0065] Using the genomic DNA of wild-type Streptococcus tumefaciens strain TG1003 as a template, high-fidelity DNA polymerase was used to amplify the following:

[0066] permEP promoter (314 bp)

[0067] kasOP promoter (96 bp)

[0068] pls gene (XbaI–EcoRI site, 2223 bp in length)

[0069] ddc gene (EcoRI–BamHI site, 1257 bp in length)

[0070] pgk gene (BamHI–HindIII site, 1506 bp in length)

[0071] ak gene (HindIII–SpeI site, 1401 bp in length)

[0072] PCR reaction system (50 μL): 100 ng template, 0.4 μM each of forward and reverse primers, 0.2 mM dNTPs, 25 μL of 2× high-fidelity enzyme mixture, and ddH2O to a final volume of 50 μL. Program: 98 ℃ for 3 min; 98 ℃ for 15 s, 60 ℃ for 15 s, 72 ℃ for 60 s, 35 cycles; 72 ℃ for 5 min.

[0073] (2) Carrier linearization

[0074] Take 2 μg of pSET152 vector, digest it with SpeI / SphI for 2 h, and recover the 5.9 kb backbone fragment by electrophoresis to obtain the linearized vector.

[0075] (3) In-Fusion one-step cloning

[0076] The four gene fragments were mixed with the linearized vector at a molar ratio of 3:1, and 2 μL of In-Fusion HD enzyme was added. The mixture was incubated at 50 °C for 15 min, followed by an ice bath for 5 min, and then transformed into E. coli DH5α. Antibiotic selection was performed at Apr (50 μg / mL), single clones were picked, plasmids were extracted, and sequencing was performed to obtain the recombinant plasmid pSET152-ddcpgkakpls.

[0077] (4) Joining transfer

[0078] Donor bacteria: pSET152-ddcpgkakpls was transformed into E. coli ET12567 / pUZ8002;

[0079] Recipient bacteria: TG1003 spore suspension (10^8 CFU / mL);

[0080] Conjugation: Pre-spread 200 μL of donor bacteria + 200 μL of recipient bacteria on MS plates and co-incubate at 28 ℃ for 18–20 h.

[0081] Screening: Cover MS plates containing Apr (80–100 μg / mL) and Nal (50 μg / mL) and incubate at 30 °C for 3–5 days;

[0082] Verification: Select resistant single colonies, perform colony PCR, and send the products to Shanghai Sangon Biotech for sequencing.

[0083] Example 3 Production of ε-polylysine by recombinant industrial strain TG1003-ddcpgkakpls

[0084] The recombinant industrial strain TG1003-ddcpgkakpls obtained in Example 2 was cultured. The strain was inoculated into seed culture medium and cultured at 30℃ and 200 rpm for 16 h. It was then transferred to 5 L of fermentation medium at an inoculum of 5% (v / v). The liquid volume was 2.5 L, the inoculum volume was 5% (v / v), the temperature was 30℃, the aeration was 1 vvm, the stirring rate was 200 rpm, and the dissolved oxygen was ≥30%. The pH was maintained at 3.0–3.5 throughout the process (with automatic ammonia addition), and fermentation was carried out for 180 h. The fermentation broth was separated and purified to obtain the ε-PL product.

[0085] Seed culture medium (g / L): glucose 50, yeast extract 5, (NH4)2SO4 22.5, K2HPO4·3H2O 0.56, KH2PO4 0.68, MgSO4·7H2O 0.7, FeSO4·7H2O 0.057, ZnSO4·7H2O 0.04; pH 6.8 (25 ℃).

[0086] The fermentation medium (g / L) formula is as follows: glucose 50-60, yeast extract 4-85, (NH4)2SO4 22.5-25.5, K2HPO4·3H2O 0.56-1.36, KH2PO4 0.68-0.78, MgSO4·7H2O 0.7-0.8, FeSO4·7H2O 0.057-0.068, ZnSO4·7H2O 0.04-0.06, pH 6.8; glucose is sterilized separately.

[0087] The specific fermentation process for the 5 L tank is as follows: liquid volume 2.5 L; inoculum 5% (v / v); temperature 30℃; aeration 1 vvm; stirring 200 rpm; dissolved oxygen ≥30%; pH maintained at 3.0–3.5 throughout (with automatic ammonia addition).

[0088] Fermentation parameters were measured and recorded, including cell mass, ε-polylysine yield in the fermentation broth, and gene expression levels of the overexpressing strain during fermentation. qRT-PCR results are shown below. Figure 5As shown. Collect 10 mL of bacterial culture in a 15 mL EP tube, centrifuge at 8000 rpm for 15 min, pour off the supernatant, measure the volume of the supernatant, and subtract the volume of the supernatant from the volume of the supernatant to obtain the bacterial volume. The unit of bacterial volume is %.

[0089] Weigh 1.2 g of ε-polylysine standard, dissolve it completely in deionized water, and bring the volume to 1 L. Dilute this solution to obtain eight concentration gradients of ε-polylysine: 1.2, 1.0, 0.8, 0.6, 0.5, 0.4, 0.2, and 0 g / L. Establish a standard curve for ε-polylysine determination by measuring the peak areas of ε-polylysine at different concentration gradients using HPLC. The chromatographic column used for ε-polylysine concentration determination was a C18 column, with a mobile phase of 12% acetonitrile, a flow rate of 0.5 mL / min, a detection wavelength of 215 nm, an injection volume of 20 μL, an oven temperature of 40℃, a sample rack temperature of 4℃, and a peak elution time of 4.2–5.0 min. Fermentation broth from ε-polylysine-producing strains was collected, centrifuged at 12000 rpm for 10 min, and diluted with deionized water to ensure the final concentration was within the standard curve range. Samples were prepared by HPLC analysis of the peak area corresponding to ε-polylysine, and the concentration of ε-polylysine in the fermentation broth was calculated from the plotted standard curve. Figure 3 As shown, the yield of the recombinant industrial strain TG1003-ddcpgkakpls after 180 h was 59.97 g / L. Figure 4 The biomass of the industrial recombinant strain TG1003-ddcpgkakpls and other process strains were compared. Due to the threshold of biomass carrying capacity in the fermenter, the difference in biomass was not significant, but it also showed a trend of gradual accumulation and increase with modification.

[0090] The specific process of ε-PL separation and purification is as follows:

[0091] a) Centrifuge the fermentation broth at 8000 rpm and 4 ℃ for 30 min, and collect the supernatant;

[0092] b) Adjust the pH of the supernatant to 8.5 with 2 N NaOH, let it stand for 30 min, and then filter to remove impurities;

[0093] c) The filtrate was passed through a D155 resin column (3 L) at a rate of 0.8 BV / h, first washed with 4 BV 0.2 N acetic acid, and then eluted with 12 BV 0.1 N HCl;

[0094] d) Add 1% (w / v) activated carbon to the eluent, stir at 25°C for 30 min to decolorize, and filter;

[0095] e) Nanofiltration (1 kDa molecular weight cutoff) concentrates the molecules 5 times;

[0096] f) Freeze-dry to obtain white ε-PL powder.

[0097] The powder contains ≥95% (w / w) ε-PL, has a purity of 96.3% (HPLC), a molecular weight of 3967 Da (MALDI-TOF-MS) as the main peak, 3.8% moisture, <10 ppm heavy metals (as Pb), and pH (1% aqueous solution): 6.5–7.5.

[0098] Total bacterial count: ≤100 CFU / g; Molds and yeasts: ≤10 CFU / g; Pathogenic bacteria: not detected; Microbial limits comply with the 2020 edition of the Chinese Pharmacopoeia.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A highly efficient tandem expression recombinant industrial strain TG1003-ddcpgkakpls for producing ε-polylysine, wherein the strain was obtained by integrating a tandem expression cassette at the attB site of the genome of wild-type Streptomyces albulus strain TG1003, and was deposited at the China Center for Type Culture Collection on June 16, 2025, with accession number CCTCCM20251400; The wild-type strain TG1003 was deposited at the China Center for Type Culture Collection on June 16, 2025, with accession number CCTCC M 20251399. The expression box sequentially includes: —The ε-polylysine synthase gene pls driven by the kasOP promoter; —The diaminopimelic acid decarboxylase gene ddc driven by the permEP promoter; —The pyruvate kinase gene pgk driven by the permEP promoter; —The aspartate kinase gene ak driven by the permEP promoter.

2. The recombinant industrial strain according to claim 1, characterized in that, The nucleotide sequence of the expression cassette is shown in SEQ ID NO:

2.

3. The recombinant industrial strain according to claim 1, characterized in that, The expression cassette was integrated into the attB site of the Streptomyces TG1003 genome via the pSET152 vector.

4. A method for constructing the recombinant industrial strain according to any one of claims 1-3, characterized in that, include: The ddc gene driven by the kasOP promoter, the pgk gene driven by the permEP promoter, the ak gene driven by the permEP promoter, and the pls gene driven by the permEP promoter were sequentially inserted into the pSET152 vector using the In-Fusion one-step cloning technology to obtain the recombinant plasmid pSET152-ddcpgkakpls. The recombinant plasmid was transformed into Escherichia coli ET12567 / pUZ8002, and then conjugated with Streptomyces thuringiensis strain TG1003 for transfer. After screening for resistance to apramycin, PCR verification and sequencing confirmation, the recombinant industrial strain TG1003-ddcpgkakpls was obtained.

5. The method according to claim 4, characterized in that, The conjugation transfer conditions were as follows: Escherichia coli and strain TG1003 were mixed at a 1:1 ratio, spread on MS plates, co-cultured at 28°C for 18–20 h, and then screened using MS plates containing 80–100 μg / mL Apr and 50 μg / mL Na1.

6. A method for producing ε-PL using the recombinant industrial strain according to any one of claims 1-3, characterized in that, include: Inoculate the strain into seed culture medium and incubate at 28-32℃ and 200-220rpm for 16-18h. Transfer the inoculum to the fermentation medium at a rate of 2-5%, and ferment at 28-32℃, 1-2 vvm aeration, 15-30% dissolved oxygen, and pH controlled at 3.0-3.2 for 180 hours. The fermentation broth was centrifuged, purified with resin, eluted with hydrochloric acid, nanofiltered, and dried to obtain the ε-PL product.

7. The method according to claim 6, characterized in that, The fermentation medium formula is as follows: glucose 50-60 g / L, yeast extract 5-8 g / L, (NH4)2SO4 22.5-25 g / L, K2HPO4·3H2O 0.56-1.08 g / L, KH2PO4 0.68-1.08 g / L, MgSO4·7H2O 0.7-0.9 g / L, FeSO4·7H2O 0.057-0.068 g / L, ZnSO4·7H2O 0.04-0.05 g / L, pH 6.8-7.2; glucose is sterilized separately.

8. The method according to claim 6 or 7, characterized in that, The pH control specifically includes maintaining the pH at 6.8 for 0–48 hours, and then reducing the pH to 5.0–5.5 after 48 hours.

9. An ε-polylysine product, characterized in that, The product is prepared by the method according to any one of claims 6–8, and the product has an ε-polylysine content ≥95% (w / w), a molecular weight distribution of 3.5–4.5 kDa, and a yield ≥6.5 g / L.

10. Use of the recombinant industrial strain according to any one of claims 1-3 as an ε-polylysine producing strain in the food, pharmaceutical, materials or agricultural fields.

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