A high-yield strain of ε-polylysine and its application, and methods to improve ε-polylysine production by *Streptomyces simulans*.

Abstract

This invention belongs to the field of food fermentation technology, specifically relating to a high-yield strain of ε-polylysine, its application, and a method for improving the ε-polylysine production of *Streptomyces albulus*. The strain, PL-711, was deposited on July 2, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.35082, classified as *Streptomyces albulus*, and located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This strain produced 98 g / L of ε-polylysine through fermentation. Furthermore, transcriptome analysis revealed that the inactivation of the NRPS-PKS-NRPS heterozygous gene cluster was key to high ε-polylysine production, confirming that this gene cluster is crucial for high ε-polylysine yield.

Description

Technical Field

[0001] This invention belongs to the field of food fermentation technology, specifically relating to a strain that produces high levels of ε-polylysine, its application, and a method for improving the production of ε-polylysine by Streptomyces leucovorum. Background Technology

[0002] ε-Polylysine is a polymer composed of 25-35 L-lysine residues with α-amino and ε-carboxyl groups linked by amide bonds. As a natural microbial metabolite, ε-polylysine possesses broad-spectrum antibacterial properties, exhibiting excellent inhibitory effects against Gram-positive bacteria, Gram-negative bacteria, yeasts, molds, and bacteriophages. Compared to traditional chemical food preservatives such as benzoic acid and sorbic acid, and other biological food preservatives (such as Nisin), ε-polylysine offers advantages including a broad antibacterial spectrum, strong antibacterial ability, high temperature resistance (120℃), good water solubility, no impact on food flavor, low dosage, and high safety. It has obtained certifications from the US FDA and the EU, making it safe for use in food and humans. It is currently considered a microbial food preservative with excellent preservative properties and significant commercial potential among natural preservative products. Currently, microbial fermentation is the most commonly used method for synthesizing ε-polylysine. Although several microorganisms have been reported to have the ability to synthesize ε-polylysine, *Streptomyces leucovora* is the most frequently used microorganism for fermentation. Japan's Chisun Corporation has achieved industrial-scale production of ε-polylysine on an annual scale of thousands of tons using a mutant strain of *Streptomyces leucovora*. Domestic efforts to breed high-yield ε-polylysine-producing *Streptomyces leucovora* strains are also ongoing, but yields still need to be increased. Summary of the Invention

[0003] To address the problem of the lack of high-yielding ε-polylysine strains, the present invention aims to provide a high-yielding ε-polylysine strain, its application, and a method for improving the ε-polylysine production of Streptomyces leucovora.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] A strain producing high levels of ε-polylysine, strain PL-711, was deposited on July 2, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.35082 and classified as Streptomyces albulus. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0006] Application of the strain that produces high levels of ε-polylysine, and its application in the production of ε-polylysine.

[0007] Furthermore, the activated strain is inoculated into a solid culture medium containing 1.5-2.0 g / 100 mL agar and cultured at 28℃-32℃ for 5-7 days. After culture, the culture solution is inoculated into a seed culture medium at an inoculation rate of 1%-5% (v / v) and cultured at 28℃-32℃ with shaking for 24-48 hours to obtain a seed culture. After culture, the seed culture is inoculated into a fermentation culture medium at a volume ratio of 10% and cultured at 28℃-32℃ with an air flow rate of 1-3 vvm (the ratio of the air flow rate per minute to the actual volume of the liquid in the tank). The dissolved oxygen is controlled at 20%-40% by adjusting the stirring speed. When the pH of the fermentation broth naturally drops to 4.0, the pH is controlled at 4.0, and the glucose concentration is controlled at 1-10 g / L. Fermentation is carried out for 7-10 days to obtain ε-polylysine fermentation broth.

[0008] A method to improve the production of ε-polylysine by Streptomyces simulans involves regulating the heterozygous gene clusters in Streptomyces simulans and then fermenting to obtain ε-polylysine.

[0009] Furthermore, by regulating the heterozygous gene cluster of NRPS-PKS-NRPS in Streptomyces leucovorum, followed by fermentation culture, ε-polylysine can be obtained.

[0010] Furthermore, ε-polylysine can be obtained by inactivating the heterozygous gene cluster NRPS-PKS-NRPS in Streptomyces leucovorum and then fermenting it.

[0011] The NRPS-PKS-NRPS heterozygous gene cluster has a total size of approximately 13.4 kb and contains 8 genes, including 4 PKS genes (gene0450, gene0448, gene0445, and gene0444), 3 NRPS genes (gene0449, gene0447, and gene0446), and 1 coenzyme F420 hydrogenase (gene0451).

[0012] The fermentation culture involves inoculating the regulated strain into a solid culture medium containing 1.5-2.0 g / 100 mL agar and culturing at 28℃-32℃ for 5-7 days. After culturing, the culture solution is inoculated into a seed culture medium at an inoculation rate of 1%-5% (v / v) and cultured at 28℃-32℃ with shaking for 24-48 hours to obtain a seed culture. After culturing, the seed culture is inoculated into the fermentation culture medium at a volume ratio of 10% and cultured at 28℃-32℃ with an air flow rate of 1-3 vvm (the ratio of the aeration rate per minute to the actual volume of the liquid in the tank). The dissolved oxygen is controlled at 20%-40% by adjusting the stirring speed. When the pH of the fermentation broth naturally drops to 4.0, the pH is controlled at 4.0, and the glucose concentration is controlled at 1-10 g / L. Fermentation is carried out for 7-10 days to obtain ε-polylysine fermentation broth.

[0013] Advantages of this invention:

[0014] This invention screened ε-polylysine-producing strains from soil and, through ARTP mutagenesis, obtained a high-yield ε-polylysine strain, PL-711, which produced 98 g / L of ε-polylysine through fermentation. Simultaneously, transcriptome analysis revealed that the inactivation of the NRPS-PKS-NRPS heterozygous gene cluster was key to high ε-polylysine production, confirming that this gene cluster is crucial for high ε-polylysine yield. Attached Figure Description

[0015] Figure 1 The fermentation curve of ε-polylysine obtained by the high-yield strain in this invention is shown. Detailed Implementation

[0016] The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings and examples, so as to better understand the solutions and advantages of the present invention. However, the specific embodiments and examples described below are merely explanations of the present invention, and the present invention is not limited to the following embodiments.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0018] The quantitative data used in the following examples are the average of the results from at least three repeated experiments.

[0019] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0020] The method for synthesizing ε-polylysine by fermentation of Streptomyces simulans in the following embodiments includes the following steps:

[0021] Activated *Streptomyces simulans* was inoculated into a solid culture medium containing 1.5–2.0 g / 100 mL agar and cultured at 28–32°C for 5–7 days. After culturing, the culture was transferred to a seed culture medium and cultured at 28–32°C with shaking for 24–48 hours to obtain a seed culture. The seed culture was then inoculated into a fermentation medium at a volume ratio of 10% and cultured at 28–32°C with an air flow rate of 1–3 vvm (the ratio of air flow rate per minute to the actual volume of the liquid in the tank). The dissolved oxygen was controlled to be 20%–40% by adjusting the stirring speed. When the pH of the fermentation broth naturally dropped to 4.0, the pH was controlled to 4.0, and the glucose concentration was controlled to be 1–10 g / L. Fermentation was carried out for 7–10 days to obtain ε-polylysine fermentation broth.

[0022] The culture medium involved is:

[0023] The solid culture medium contains 20 grams of mannitol, 20 grams of soybean flour, and 20 grams of agar powder per liter of water, with a pH of 7.0-7.5;

[0024] The seed culture medium contains 50 g glucose, 5 g yeast powder, 10 g ammonium sulfate, 0.8 g K2HPO4, 1.36 g KH2PO4, 0.04 g ZnSO4·7H2O, 0.5 g MgSO4·7H2O, and 0.03 g FeSO4·7H2O per liter of water, with a pH of 7.0~7.5.

[0025] The fermentation medium contains 10-100 g of glucose, 2-20 g of yeast powder, 2-10 g of ammonium sulfate, 0.2-1 g of K2HPO4, 1-2 g of KH2PO4, 0.01-1 g of ZnSO4·7H2O, 0.1-1 g of MgSO4·7H2O, and 0.01-0.1 g of FeSO4·7H2O per liter of water, with a pH of 7.0-7.5. Example 1

[0026] Screening of ε-polylysine-producing Streptomyces albopictus

[0027] Soil samples were obtained from a farm in Pinggu District, Beijing. Each gram of soil sample was added to 50 ml of seed culture medium (containing 50 g glucose, 5 g yeast powder, 10 g ammonium sulfate, 0.8 g K₂HPO₄, 1.36 g KH₂PO₄, 0.04 g ZnSO₄·7H₂O, 0.5 g MgSO₄·7H₂O, and 0.03 g FeSO₄·7H₂O per liter of water, pH 7.0–7.5) and enriched at 30°C for 48 hours. The resulting solution was then diluted and spread onto solid culture medium (containing 20 g mannitol, 20 g soybean flour, and 20 g agar powder per liter of water, pH 7.0–7.5) and incubated at 30°C and 200 rpm for 7 days. After single colonies have grown, select Streptomyces colonies and inoculate them into fermentation medium (containing 10-100 g glucose, 2-20 g yeast extract, 2-10 g ammonium sulfate, 0.2-1 g K₂HPO₄, 1-2 g KH₂PO₄, 0.01-1 g ZnSO₄·7H₂O, 0.1-1 g MgSO₄·7H₂O, 0.01-0.1 g FeSO₄·7H₂O per liter of water, pH 7.0-7.5). Incubate at 30℃ and 200 rpm for 3 days, measure the yield of ε-polylysine, and select the strain with the highest yield, naming it PL-1.

[0028] The genome of strain PL-1 was extracted, and its 16S rDNA sequence was amplified using primers 27F and 1492R. The sequence of primer 27F was 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1), and the sequence of primer 1492R was 5'-TACGGTTACCTTGTTACGACTT-3' (SEQ ID NO.2). Sequencing results of the PCR amplification products showed that the 16S rDNA sequence of strain PL-1 (SEQ ID NO.3) was identical to the 16S rDNA sequence of *Streptomyces albulus*. Strain PL-1 was named *Streptomyces albulus* PL-1.

[0029] SEQ ID NO.3

[0030] >16S rDNA sequence

[0031]

[0032] The obtained Streptomyces albulus PL-1 was then cultured on seed culture medium at 30°C and 200 rpm until the logarithmic growth phase. The culture was then streaked onto solid medium, and single colonies were selected based on colony size and the time it took for the bacteria to form pods. An OD suspension was prepared using sterile 0.9% (w / v) physiological saline. 600 =1), 10 μL of bacterial suspension was evenly spread onto an irradiated metal sheet for ARTP mutagenesis. Helium was used as the working gas, with a working power of 140 W and a gas flow rate of 10 slpm. Treatment times were 0 s, 50 s, 100 s, 150 s, 200 s, and 250 s. After mutagenesis, the different mutagenesis times were serially diluted with sterile physiological saline and spread onto solid culture medium. The plates were inverted and incubated at 30℃. Single colonies were selected and inoculated into fermentation medium. Fermentation was carried out at 30℃ and 200 rpm for 4 days (see Table 1). The ε-polylysine yield of each strain was determined, and the strains with high yields were preserved.

[0033] The above method for testing ε-polylysine uses the Itzhaki methyl orange colorimetric method: The fermentation supernatant is appropriately diluted with 0.1 mmol / L phosphate buffer, and 2 mL of the diluted solution is mixed with 2 mL of 1 mmol / L methyl orange solution. The mixture is shaken at 30°C for 30 minutes, centrifuged at 5000 rpm for 15 minutes, and the precipitate is removed. 0.5 mL of the supernatant is diluted to 10 mL, and the absorbance is measured at 465 nm. The content of ε-polylysine is calculated based on the standard curve. ε-polylysine standard solutions: Concentration gradients of 0, 0.02, 0.04, 0.06, 0.08, and 0.1 g / L are prepared using 0.1 mmol / L phosphate buffer.

[0034] The strain with the highest ε-polylysine production was named Streptomyces albulus PL-711, and was deposited on July 2, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.35082.

[0035] Table 1. Results of ARTP mutagenesis

[0036] . Example 2

[0037] Key sites for high ε-polylysine production in Streptomyces albulus PL-711 identified

[0038] Streptomyces albulus PL-711, a high-yield ε-polylysine-producing strain, and Streptomyces albulus PL-1 (wild-type strain) were inoculated into the fermentation medium at an inoculation rate of 10%. The fermentation broth obtained by fermenting Streptomyces albulus PL-711 in the fermentation medium for 24 hours was used as sample one, and the fermentation broth obtained by fermenting wild-type strain in the fermentation medium for 24 hours was used as sample two.

[0039] The two samples obtained above were used by Shanghai Meiji Biomedical Technology Co., Ltd. to construct RNA libraries and perform differential gene expression analysis, resulting in the NRPS-PKS-NRPS heterozygous gene cluster.

[0040] In *Streptomyces albulus* PL-711, a heterozygous NRPS-PKS-NRPS gene cluster is inactivated. This cluster, approximately 13.4 kb in size, contains eight genes: four PKS genes (gene0450, gene0448, gene0445, and gene0444), three NRPS genes (gene0449, gene0447, and gene0446), and one coenzyme F420 hydrogenase (gene0451). The NRPS-PKS-NRPS heterozygous gene cluster is crucial for high-yield production of ε-polylysine. Example 3

[0041] Production of ε-polylysine by fermentation with Streptomyces albulus PL-711

[0042] Streptomyces Streaking albulus PL-711 onto solid medium (mannitol 20 g / L, soybean flour 20 g / L, agar powder 20 g / L, pH 7.2) and incubating at 30°C for 6 days. Once the medium surface is covered with black spores, collect the spores from the plate and inoculate them into a conical flask containing 50 ml of seed culture medium. Incubate at 30°C and 220 rpm on a shaker for 24 hours to obtain the seed culture. Inoculate the prepared seed culture medium at a rate of 10% (v / v) into a fermenter containing fresh fermentation medium. Control the fermentation temperature at 30°C and the air flow rate at 3 vvm (the ratio of aeration rate per minute to the actual volume of the liquid in the tank). Control the dissolved oxygen to 30% by adjusting the stirring speed. Do not control the pH in the early stage of fermentation. When the pH of the fermentation broth naturally drops to 4.0, turn on the ammonia water to control the pH at 4.0. When the glucose concentration is below 5 g / L, start adding glucose and control the glucose concentration in the fermentation broth to 5-10 g / L. The levels of ε-polylysine and glucose in the fermentation broth were measured every 24 hours (see [reference]). Figure 1 After 10 days of fermentation, the yield of ε-polylysine was 98.5 g / L. Example 4

[0043] Validation of key sites for high-yield production of ε-polylysine

[0044] Streptomyces albulus CICC11022 was purchased from the China Industrial Microbial Culture Collection Center. The NRPS-PKS-NRPS heterozygous gene cluster in this strain was inactivated using conventional genetic engineering methods, resulting in the strain Streptomyces albulus CICC11022-∆PKS-NRPS.

[0045] Streptomyces albulus CICC11022 and CICC11022-∆PKS-NRPS were streaked onto solid medium (mannitol 20 g / L, soybean meal 20 g / L, agar powder 20 g / L, pH 7.2) and incubated at 30°C for 6 days. Once the medium surface was covered with black spores, the spores were collected from the plates and inoculated into Erlenmeyer flasks containing 50 mL of seed culture medium. The flasks were then incubated at 30°C and 220 rpm for 24 hours to obtain the seed culture. The culture medium was inoculated into a fermenter containing fresh fermentation medium at a 10% (volume) inoculation rate. The fermentation temperature was controlled at 30℃, and the air flow rate was 3 vvm (the ratio of aeration rate per minute to the actual volume of the liquid in the tank). The dissolved oxygen was controlled at 30% by adjusting the stirring speed. pH was not controlled in the early stages of fermentation. When the pH of the fermentation broth naturally dropped to 4.0, ammonia was added to maintain the pH at 4.0. When the glucose concentration fell below 5 g / L, glucose was added to maintain a glucose concentration of 5-10 g / L in the fermentation broth. The content of ε-polylysine and glucose in the fermentation broth was measured every 24 hours. After 10 days of fermentation, the yield of ε-polylysine from *Streptomyces albulus* CICC11022 was 1.7 g / L, and the yield of ε-polylysine from *Streptomyces albulus* CICC11022-∆PKS-NRPS was 15.5 g / L.

Claims

1. A strain that produces high levels of ε-polylysine, characterized by: The strain is PL-711, which was deposited on July 2, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.35082 and classified as Streptomyces albulus. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The application of the strain with high ε-polylysine production as described in claim 1, characterized in that: Application of the strain in the production of ε-polylysine.

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