Screening culture method and application of tolerant streptomyces hygroscopicus

Abstract

The invention belongs to the technical field of epsilon-polylysine production, and particularly relates to a screening culture method and application of tolerant streptomyces hygroscopicus. The screening culture method of the tolerant streptomyces hygroscopicus comprises the following steps: subculturing streptomyces hygroscopicus engineering bacteria in a culture medium taking straw cellulose hydrolysate as a carbon source for at least 30 generations to obtain primary streptomyces hygroscopicus. The primary streptomyces hygroscopicus is subcultured for 10-25 generations in a fermentation culture medium with the epsilon-polylysine concentration being 0.5 g / L-5g / L and straw cellulose hydrolysate as a carbon source, and the tolerant streptomyces hygroscopicus is obtained. Wherein the tolerance means that epsilon-polylysine is produced while an inhibitor is resisted. The straw cellulose hydrolysate contains an inhibitor. According to the screening culture method of the tolerant streptomyces hygroscopicus, disclosed by the invention, the yield of epsilon-polylysine in virus-free cellulose hydrolysate can be up to 1.35 g / L at most, and the yield of epsilon-polylysine can be up to 30.57 g / L at most under the condition of fed-batch fermentation in a 5L fermentation tank.

Description

Technical Field

[0001] This invention belongs to the field of ε-polylysine production technology, specifically relating to a screening and cultivation method for resistant Streptomyces hygroscopicus and its application. Here, resistance refers to tolerance to inhibitors and ε-polylysine production. Background Technology

[0002] ε-Polylysine, abbreviated as ε-PL, is a class of natural antimicrobial peptides formed by the condensation of L-lysine residues through the ε-amino group and the α-carboxyl group. ε-PL possesses broad-spectrum antimicrobial activity, biocompatibility, biodegradability, and thermal stability, and has been widely used in food preservation, biomedical materials, feed mold prevention, and agricultural pathogen control. Current industrial production of ε-PL relies on Streptomyces using fermentable sugars such as glucose and starch syrup as carbon sources for fermentation. However, this method is limited by the high cost and limited availability of carbon sources, as well as the limitation of the metabolic flux of the microorganisms by a single carbon source system.

[0003] Cellulose hydrolysate, derived from biomass such as straw, forestry waste, and agricultural byproducts, is inexpensive, abundant, and has high conversion potential, making it an ideal carbon source to replace glucose. However, various inhibitors are often generated during cellulose extraction and acid or enzymatic hydrolysis, including furfural, acetic acid, and 5-hydroxymethylfurfural. These inhibitors can damage cell membrane structure, inhibit oxidative phosphorylation, and suppress amino acid biosynthesis, significantly reducing the growth rate and secondary metabolic level of Streptomyces, leading to a decrease in biomass and fermentation efficiency.

[0004] The applicant previously obtained an engineered strain capable of synthesizing ε-PL in CN120424840A, "An engineered strain of Streptomyces hygroscopicus and its construction," by heterologously expressing the ε-PL synthase gene into Streptomyces hygroscopicus. However, this strain is inhibited by inhibitors, leading to a decrease in the performance of ε-PL fermentation.

[0005] Therefore, existing ε-PL producing bacteria cannot simultaneously achieve high ε-PL production through fermentation in the presence of inhibitors. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for screening and culturing tolerant hydrophilic Streptomyces and its application.

[0007] To facilitate understanding of this invention, the materials used in this invention and their abbreviations are listed below: ε-Polylysine, abbreviated as ε-PL. Dry weight of the cells, abbreviated as DCW. 5-Hydroxymethylfurfural, abbreviated as 5-HMF.

[0008] The first objective of this invention is to provide a resistant, water-absorbing Streptomyces, the method of which comprises the following steps: First-stage adaptive evolution: The engineered strain of *Streptomyces hygroscopicus* was inoculated into a fermentation medium with straw cellulose hydrolysate as the carbon source and cultured at 28℃~32℃, 180rpm~220rpm for 24h~48h. After at least 30 generations of subculturing, primary *Streptomyces hygroscopicus* was obtained.

[0009] Two-stage adaptive evolution: Primary *Streptomyces hygroscopicus* was inoculated into a fermentation medium with straw cellulose hydrolysate as the carbon source. The concentration of ε-polylysine in the fermentation medium was 0.5 g / L to 5 g / L. The culture was carried out at 28℃ to 32℃ and a rotation speed of 180 rpm to 220 rpm for 24 to 48 hours, followed by 10 to 25 generations of continuous subculturing to obtain tolerant *Streptomyces hygroscopicus* strains. This two-stage adaptive evolution improved the tolerance of the engineered primary *Streptomyces hygroscopicus* strains to the product polylysine and increased the upper limit of the ε-polylysine concentration produced by the strains.

[0010] The tolerance refers to the resistance to the inhibitor while simultaneously producing ε-polylysine.

[0011] The straw cellulose hydrolysate contains inhibitors.

[0012] The inhibitor is one or more of acetic acid, furfural, and 5-hydroxymethylfurfural.

[0013] Preferably, the concentration of acetic acid in the fermentation broth is 1 g / L to 4.00 g / L, the concentration of furfural is 0.1 g / L to 0.5 g / L, and the concentration of 5-hydroxymethylfurfural is 0.03 g / L to 0.15 g / L.

[0014] Preferably, the engineered *Streptomyces hygroscopicus* strain expresses ε-PL synthase.

[0015] The resistant Streptomyces of the present invention can simultaneously tolerate inhibitors and produce high levels of ε-polylysine.

[0016] Preferably, the inoculation amount is 5% to 10% of the fermentation liquid volume.

[0017] Preferably, the culture conditions for the first stage of adaptive evolution are a temperature of 30°C, a rotation speed of 200 rpm, and a time of 36 h.

[0018] Preferably, the concentration of ε-PL is 3 g / L.

[0019] Preferably, the culture conditions for the two-stage adaptive evolution are: temperature 30℃, rotation speed 200rpm, time 36h, and subculture for 18 generations.

[0020] Preferably, the straw cellulose hydrolysate is obtained by mixing straw with sulfuric acid solution for hydrolysis, adjusting the pH, and adding cellulase for further hydrolysis.

[0021] Preferably, the preparation method of the straw cellulose hydrolysate is as follows: Chopped straw is mixed with a 1.0% sulfuric acid solution at a mass ratio of 1:10, and hydrolyzed at 121℃ for 30 min. After cooling to room temperature, the pH is adjusted to 5.0 with Ca(OH)2, and cellulase Cellic® CTec3 is added based on the dextran content in the straw, so that the enzyme protein addition is 40 mg / g dextran. After reacting at 50℃ and 200 rpm for 72 h, the solid matter is removed by centrifugation, and the supernatant is the hydrolysate. The hydrolysate is concentrated to a total sugar concentration of 200 g / L to obtain the straw cellulose hydrolysate.

[0022] A second objective of this invention is to provide the application of a tolerant, hygroscopic *Streptomyces* strain in the fermentation broth for the preparation of ε-polylysine, wherein the method of application comprises the following steps: The inhibitor-resistant and ε-PL-producing *Streptomyces hygroscopicus* were inoculated into a culture medium with straw cellulose hydrolysate as the carbon source and fermented for 48-192 hours at 28-34°C and pH 6.2-7.5. The fermentation broth containing ε-PL was collected.

[0023] Preferably, the straw cellulose hydrolysate is a straw hydrolysate or a corn cob hydrolysate.

[0024] Preferably, the straw is wheat straw, rice straw, or corn straw.

[0025] Preferably, the cellulose hydrolysate has not undergone complete detoxification treatment, or has only undergone pH adjustment, alkali neutralization, and mild activated carbon adsorption treatment.

[0026] Preferably, each liter of the culture medium contains 20g~100g of cellulose hydrolysate, 5g~15g of (NH4)2SO4, 2g~8g of yeast powder, 0.5g~1.0g of K2HPO4, 0.5g~1.5g of KH2PO4, 0.4g~0.8g of MgSO4·7H2O, 0.02g~0.04g of FeSO4·7H2O, and 0.02g~0.06g of ZnSO4·7H2O.

[0027] Compared with the prior art, the present invention has the following beneficial effects: 1. The screening and cultivation method for the tolerant *Streptomyces hygroscopicus* of this invention involves two evolutionary processes of engineered *Streptomyces hygroscopicus* strains on a medium containing straw cellulose hydrolysate with an inhibitor as the carbon source and on a medium containing ε-PL. After two evolutionary processes, the ε-PL concentration of the tolerant *Streptomyces hygroscopicus* strain obtained in this invention significantly increased from 0.31 g / L in undetoxified straw cellulose hydrolysate, reaching a maximum of 1.35 g / L under different fermentation conditions, which is higher than that of the traditional glucose fermentation system. Furthermore, in a 5L fermenter application, the ε-PL concentration reached 30.57 g / L. Therefore, the tolerant *Streptomyces hygroscopicus* strain of this invention can produce high levels of ε-PL in straw cellulose hydrolysate containing an inhibitor.

[0028] The screening and cultivation method for tolerant *Streptomyces hygroscopicus* of the present invention comprises the following steps: First-stage adaptive evolution: Engineered *Streptomyces hygroscopicus* strains are inoculated into a fermentation medium using straw cellulose hydrolysate as the carbon source, and cultured at 28℃~32℃ and 180rpm~220rpm for 24h~48h, followed by at least 30 subcultures to obtain primary *Streptomyces hygroscopicus* strains. This first-stage adaptive evolution allows the engineered *Streptomyces hygroscopicus* strains to adapt to the material environment of the cellulose hydrolysate, including growth and development in an environment containing inhibitors, thus completing fermentation. Second-stage adaptive evolution: Primary *Streptomyces hygroscopicus* strains are inoculated into a fermentation medium using straw cellulose hydrolysate as the carbon source, wherein the concentration of ε-polylysine in the fermentation medium is 0.5g / L~5g / L, and cultured at 28℃~32℃ and 180rpm~220rpm for 24h~48h, followed by 10~25 subcultures to obtain tolerant *Streptomyces hygroscopicus* strains. The straw cellulose hydrolysate contains inhibitors. The inhibitor in the fermentation broth is one or more of the following: acetic acid (1 g / L to 4.00 g / L), furfural (0.1 g / L to 0.5 g / L), and 5-hydroxymethylfurfural (0.03 g / L to 0.15 g / L). Through a two-stage adaptive evolution, *Streptomyces hygroscopicus* has adapted to grow and synthesize ε-PL in the presence of ε-PL. Therefore, the *Streptomyces hygroscopicus* of this invention can ferment and produce ε-PL in cellulose hydrolysate containing the inhibitor.

[0029] This invention achieves, for the first time, the efficient synthesis of ε-PL from incompletely detoxified cellulose hydrolysate. A *Streptomyces hygroscopicus* strain capable of tolerating furfural, acetic acid, and 5-HMF inhibitors in the hydrolysate is obtained, which can stably synthesize ε-PL even under incomplete detoxification conditions. This breakthrough overcomes the technical bottleneck in existing technologies where cellulose hydrolysate is difficult to directly use for ε-PL fermentation.

[0030] 2. The application of the screening and cultivation method for resistant *Streptomyces hygroscopicus* of the present invention in the preparation of fermentation broth for ε-polylysine includes the following steps: inoculating *Streptomyces hygroscopicus* resistant to inhibitors and producing ε-PL into a culture medium using straw cellulose hydrolysate as a carbon source, and fermenting at a temperature of 28℃~34℃ and pH 6.2~7.5 for 48h~192h. The fermentation broth containing ε-PL is then collected.

[0031] The inhibitors and ε-polylysine-producing *Streptomyces hygroscopicus* of this invention can efficiently utilize the mixed sugars in cellulose hydrolysate, improving raw material conversion rate. The evolved strains can simultaneously and efficiently utilize glucose and xylose in the hydrolysate, achieving efficient conversion of mixed sugars and significantly improving the utilization efficiency of agricultural waste. Furthermore, the process of this invention is stable, has good scale-up performance, and is suitable for industrial application. The method of this invention has good scale-up feasibility and significant cost reduction and efficiency improvement advantages, making it suitable for the resource utilization of agricultural waste and the large-scale production of ε-PL. Attached Figure Description

[0032] Figure 1 The diagram shows the fermentation products of Comparative Examples 1 to 3 of this invention.

[0033] Figure 2 The diagram shows the fermentation products of Examples 1, 7, 8, Comparative Example 6, and Comparative Example 7 of the present invention.

[0034] Figure 3 The image shows the results of the fed-batch fermentation of the tolerant hygroscopic Streptomyces obtained in Example 3 of this invention in a 5L fermenter. Detailed Implementation

[0035] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the following detailed description, in conjunction with preferred embodiments and accompanying drawings, provides a clear and complete account of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0036] It should be noted that all technical terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0037] The culture medium used in this invention has the following formulation: TSB liquid culture medium: 30 g / L tryptone soybean broth.

[0038] ISP2 solid medium: 4 g / L yeast extract, 10 g / L malt extract, 4 g / L glucose, 20 g / L agar, pH adjusted to 7.0.

[0039] The preparation method of the straw cellulose hydrolysate of the present invention is as follows: First, chopped straw is mixed with a 1.0% sulfuric acid solution at a mass ratio of 1:10, and hydrolyzed at 121°C for 30 min. After cooling to room temperature, the pH is adjusted to 5.0 with Ca(OH)2, and cellulase Cellic® CTec3 is added according to the dextran content in the straw, so that the enzyme protein added is 40 mg / g dextran. After reacting at 50°C and 200 rpm for 72 h, the solid matter is removed by centrifugation, and the supernatant is the hydrolysate. The hydrolysate is concentrated to a total sugar concentration of 200 g / L to obtain the cellulose hydrolysate. Specifically, when the straw is wheat straw, wheat straw hydrolysate is prepared; when the straw is rice straw, rice straw hydrolysate is prepared; when the straw is corn straw, corn straw hydrolysate is prepared; and when the straw is corn cob, corn cob hydrolysate is prepared.

[0040] The engineered Streptomyces hydrophila used in this invention is the recombinant strain CL-1 / pSET15-SP44-pls disclosed in patent document CN120424840A, "An engineered strain of Streptomyces hydrophila and its construction".

[0041] Example 1 A screening culture method for tolerant hydrophilic Streptomyces includes the following steps: One-stage adaptive evolution: The engineered strain of *Streptomyces hygroscopicus* was inoculated into wheat straw hydrolysate, cultured at 30℃ and 200 rpm for 36 h, and passaged for 36 generations to obtain the primary engineered strain of *Streptomyces hygroscopicus*.

[0042] Two-stage adaptive evolution: The primary engineered Streptomyces hydrophila was inoculated into a medium with 3 g / L ε-PL and cellulose hydrolysate as the carbon source, and cultured at 30℃ and 200 rpm for 36 h. After 18 generations of subculturing, tolerant Streptomyces hydrophila were obtained.

[0043] The application of inhibitor-resistant and ε-PL-producing Streptomyces hydrophila includes the following steps: Tolerant *Streptomyces hygroscopicus* was inoculated into a culture medium using cellulose hydrolysate as the carbon source and fermented at 30°C and pH 6.8 for 72 hours to obtain a fermentation broth containing ε-PL. Each liter of the culture medium contained 50 g of wheat straw hydrolysate (based on total sugars), 10 g of (NH₄)₂SO₄, 6 g of yeast extract, 0.7 g of K₂HPO₄, 1 g of KH₂PO₄, 0.6 g of MgSO₄·7H₂O, 0.03 g of FeSO₄·7H₂O, and 0.04 g of ZnSO₄·7H₂O. The concentration of ε-PL in the fermentation broth was determined to be 1.27 g / L.

[0044] Example 2 A screening culture method for tolerant hydrophilic Streptomyces includes the following steps: One-stage adaptive evolution: The engineered strain of *Streptomyces hygroscopicus* was inoculated into a fermentation medium with wheat straw hydrolysate as the carbon source, and cultured at 28℃ and 180 rpm for 24 h. After 30 generations of subculturing, the primary engineered strain of *Streptomyces hygroscopicus* was obtained.

[0045] Two-stage adaptive evolution: The primary engineered Streptomyces hydrophila was inoculated into a fermentation medium with 1.5 g / L of ε-PL and cellulose hydrolysate as the carbon source. The culture was carried out at 28°C and 180 rpm for 24 h. After 10 generations of subculturing, tolerant Streptomyces hydrophila were obtained.

[0046] The application of inhibitor-resistant and ε-PL-producing Streptomyces hydrophila involves the following steps: Tolerant *Streptomyces hygroscopicus* was inoculated into a culture medium using cellulose hydrolysate as the carbon source and fermented at 28°C and pH 7 for 96 hours to obtain a fermentation broth containing ε-PL. Each liter of the culture medium contained 20 g of wheat straw hydrolysate (based on total sugars), 5 g of (NH4)2SO4, 2 g of yeast extract, 0.5 g of K2HPO4, 0.5 g of KH2PO4, 0.4 g of MgSO4·7H2O, 0.02 g of FeSO4·7H2O, and 0.02 g of ZnSO4·7H2O. The concentration of ε-PL in the fermentation broth was determined to be 1.51 g / L.

[0047] Example 3 A screening culture method for tolerant hydrophilic Streptomyces includes the following steps: One-stage adaptive evolution: The engineered strain of *Streptomyces hygroscopicus* was inoculated into a fermentation medium with wheat straw hydrolysate as the carbon source, cultured at 32℃ and 220 rpm for 48 h, and passaged for 32 generations to obtain the primary engineered strain of *Streptomyces hygroscopicus*.

[0048] Two-stage adaptive evolution: The primary engineered Streptomyces hydrophila was inoculated into cellulose hydrolysate with a concentration of 3 g / L of ε-PL and cultured at 32℃ and 220 rpm for 48 h. After 25 generations of subculturing, tolerant Streptomyces hydrophila were obtained.

[0049] The application of inhibitor-resistant and ε-PL-producing Streptomyces hydrophila involves the following steps: Tolerant *Streptomyces hygroscopicus* was inoculated into a culture medium using cellulose hydrolysate as the carbon source and fermented at 34℃ and pH 6.8 for 72 h to obtain a fermentation broth containing ε-PL. The broth contained, per liter (based on total sugars): 100 g wheat straw hydrolysate, 15 g (NH₄)₂SO₄, 8 g yeast extract, 1.0 g K₂HPO₄, 1.5 g KH₂PO₄, 0.8 g MgSO₄·7H₂O, 0.04 g FeSO₄·7H₂O, and 0.06 g ZnSO₄·7H₂O. The concentration of ε-PL in the fermentation broth was determined to be 1.35 g / L.

[0050] Example 4 A screening culture method for tolerant hydrophilic Streptomyces includes the following steps: The wheat straw hydrolysate in Example 1 was replaced with rice straw hydrolysate, while all other conditions remained the same as in Example 1, resulting in a fermentation broth containing ε-PL. The concentration of ε-PL in the fermentation broth was 1.14 g / L.

[0051] Example 5 A screening culture method for tolerant hydrophilic Streptomyces includes the following steps: The wheat straw hydrolysate in Example 1 was replaced with corn straw hydrolysate, while all other conditions remained the same as in Example 1, resulting in a fermentation broth containing ε-PL. The concentration of ε-PL in the fermentation broth was 1.26 g / L.

[0052] Example 6 A screening culture method for tolerant hydrophilic Streptomyces includes the following steps: The wheat straw hydrolysate in Example 1 was replaced with corn cob hydrolysate, while all other conditions remained the same as in Example 1, resulting in a fermentation broth containing ε-PL. The concentration of ε-PL in the fermentation broth was 0.95 g / L.

[0053] Hydrolysates derived from wheat straw, rice straw, corn straw, and corn cobs were selected as the main carbon source, and ε-PL was produced by fermentation using the evolved strain obtained in Example 2. The types and contents of inhibitors varied in the hydrolysates from different sources, but the evolved strain exhibited stable growth and metabolic performance in all types of hydrolysates. The results indicate that the evolved strain of this invention has good adaptability and stable ε-PL synthesis ability to different agricultural waste hydrolysates, making it suitable for various biomass resource utilization scenarios.

[0054] Table 1. ε-PL production of Examples 1-6 Example 7 A screening and culturing method for tolerant hydrophilic Streptomyces and its application, comprising the following steps: The wheat straw hydrolysate containing 50g of total sugar in Example 1 was replaced with 60g of total sugar, while all other conditions remained the same as in Example 1, resulting in a fermentation broth containing ε-PL. The concentration of ε-PL in the fermentation broth was 1.23g / L.

[0055] Example 8 A screening and culture method for tolerant hydrophilic Streptomyces and its application, comprising the following steps: The wheat straw hydrolysate containing 50g of total sugar in Example 1 was replaced with 70g of total sugar, while all other conditions remained the same as in Example 1, resulting in a fermentation broth containing ε-PL. The concentration of ε-PL in the fermentation broth was 1.12g / L.

[0056] Comparative Example 1 An ε-PL fermentation method for *Streptomyces hygroscopicus* includes the following steps: Engineered *Streptomyces hygroscopicus* strains were inoculated into a culture medium using cellulose hydrolysate as the carbon source. Fermentation was carried out at 34℃ and pH 6.8 for 72 hours to obtain a fermentation broth containing ε-PL. Each liter of the culture medium contained 50 g of wheat straw hydrolysate (based on total sugars), 15 g of (NH₄)₂SO₄, 8 g of yeast extract, 1.0 g of K₂HPO₄, 1.5 g of KH₂PO₄, 0.8 g of MgSO₄·7H₂O, 0.04 g of FeSO₄·7H₂O, and 0.06 g of ZnSO₄·7H₂O. The concentration of ε-PL in the fermentation broth was determined to be 0.31 g / L.

[0057] Comparative Example 2 An ε-PL fermentation method for *Streptomyces hygroscopicus* includes the following steps: In Comparative Example 1, 50g of wheat straw hydrolysate (based on total sugar) was replaced with 50g of glucose, while all other conditions remained the same as in Comparative Example 1, resulting in a fermentation broth containing ε-PL. The concentration of ε-PL in the fermentation broth was 0.44g / L.

[0058] Comparative Example 3 An ε-PL fermentation method for *Streptomyces hygroscopicus* includes the following steps: In Comparative Example 1, 50g of wheat straw hydrolysate (based on total sugar content) was replaced with 50g of xylose, while all other conditions remained the same as in Comparative Example 1, resulting in a fermentation broth containing ε-PL. The concentration of ε-PL in the fermentation broth was 0.67g / L.

[0059] The fermentation results of Comparative Examples 1 to 3 are as follows Figure 1 As shown in the figure, when glucose was used as the carbon source, the ε-PL yield was 0.44 g / L, the cell dry weight was 4.49 g / L, and the total sugar utilization rate was 21.55%. When xylose was used as the sole carbon source, the ε-PL yield increased to 0.67 g / L, the cell dry weight reached 5.83 g / L, and the total sugar utilization rate increased to 39.46%, indicating that this recombinant strain can efficiently utilize xylose to promote ε-PL synthesis. When wheat straw hydrolysate containing inhibitors such as furfural, acetic acid, and 5-HMF was used as the main carbon source, after 72 h of fermentation, the ε-PL yield was 0.31 g / L, the cell dry weight was 3.89 g / L, and the total sugar utilization rate was 12.22%.

[0060] The above results indicate that although the inhibitors in the straw hydrolysate have a certain impact on bacterial growth, the recombinant strain can still utilize the mixed sugars in it for metabolic transformation, demonstrating good substrate adaptability and inhibitor tolerance.

[0061] Comparative Example 4 An ε-PL fermentation method for *Streptomyces hygroscopicus* includes the following steps: Using *Streptomyces hygroscopicus* as the starting strain, and wheat straw hydrolysate with a total sugar concentration of 50 g / L as the sole carbon source, adaptive evolution was carried out. Every 24 hours, 10% (v / v) of the fermentation broth was transferred to fermentation medium containing fresh wheat straw hydrolysate (50 g / L), and the culture was passaged once. This cycle was repeated for 30 days to obtain a recombinant evolved strain after 30 generations of evolution.

[0062] The recombinant evolved strain was inoculated into a culture medium with cellulose hydrolysate as the carbon source and fermented at 30℃ and pH 6.8 for 72 h to obtain a fermentation broth containing ε-PL. Each liter of the culture medium contained 50 g of wheat straw hydrolysate (based on total sugars), 10 g of (NH4)2SO4, 6 g of yeast extract, 0.7 g of K2HPO4, 1 g of KH2PO4, 0.6 g of MgSO4·7H2O, 0.03 g of FeSO4·7H2O, and 0.04 g of ZnSO4·7H2O. As shown in Table 2, the concentration of ε-PL in the fermentation broth was 0.74 g / L, and the biomass was 6.38 g / L. Compared with *Streptomyces hygroscopicus* CL-1, the ε-PL yield and biomass increased by 138.71% and 64.01%, respectively.

[0063] Comparative Example 5 An ε-PL fermentation method for *Streptomyces hygroscopicus* includes the following steps: Using *Streptomyces hygroscopicus* as the starting strain, and wheat straw hydrolysate with a total sugar concentration of 50 g / L as the sole carbon source, adaptive evolution was carried out. Every 24 hours, 10% (v / v) of the fermentation broth was transferred to fermentation medium containing fresh wheat straw hydrolysate (50 g / L), and the culture was passaged once. This cycle was repeated for 30 days to obtain a recombinant evolved strain after 30 generations of evolution.

[0064] The recombinant evolved strain, which had undergone 30 generations of evolution, was subjected to 20 more generations of adaptive evolution by adding 3 g / L ε-PL to 50 g / L wheat straw hydrolysate as a selection pressure.

[0065] The evolved strain was fermented in a fermentation environment with the same culture medium composition and culture conditions as Comparative Example 4 to obtain a fermentation broth containing ε-PL. As shown in Table 2, the ε-PL yield of the evolved strain was further increased to 1.18 g / L, and the biomass reached 6.31 g / L.

[0066] Table 2. Fermentation results of Comparative Examples 4 and 5 Comparative Example 6 A screening and culture method for tolerant hydrophilic Streptomyces and its application, comprising the following steps: The wheat straw hydrolysate containing 50g of total sugar in Example 1 was replaced with 30g of total sugar, while all other conditions remained the same as in Example 1, resulting in a fermentation broth containing ε-PL. The concentration of ε-PL in the fermentation broth was 0.76g / L.

[0067] Comparative Example 7 A screening and culture method for tolerant hydrophilic Streptomyces and its application, comprising the following steps: The wheat straw hydrolysate containing 50g of total sugar in Example 1 was replaced with 40g of total sugar, while all other conditions remained the same as in Example 1, resulting in a fermentation broth containing ε-PL. The concentration of ε-PL in the fermentation broth was 1.02g / L.

[0068] The difference between Examples 1, 7, 8, Comparative Example 6, and Comparative Example 7 lies in the concentration of the wheat straw hydrolysate; the results after fermentation are as follows: Figure 2 As shown in the figure, the unevolved strain represents the fermentation results of directly using the engineered *Streptomyces hygroscopicus* strains corresponding to Examples 1, 7, 8, Comparative Example 6, and Comparative Example 7, with other conditions remaining unchanged. The results show that under different total sugar concentrations in wheat straw hydrolysate, the ε-PL yield of the evolved strain was higher than that of the unevolved strain. When the total sugar concentration was 30 g / L, the ε-PL yields of the unevolved and evolved strains were 0.55 g / L and 0.76 g / L, respectively. With increasing total sugar concentration in the wheat straw hydrolysate, the ε-PL yield of the unevolved strain gradually decreased, while the ε-PL yield of the evolved strain showed a trend of first increasing and then decreasing. Specifically, when the total sugar concentration in the wheat straw hydrolysate was 50 g / L, the ε-PL yield of the evolved strain reached its highest level, 1.27 g / L, indicating that the evolved strain had the best ε-PL production effect under this total sugar concentration condition. 50 g / L is considered the suitable total sugar concentration for wheat straw hydrolysate.

[0069] Application Example 1 Scale-up production validation of recombinant strains in a 5L fermenter To further verify the fermentation performance of the evolved strain of the present invention under scale-up conditions, the *Streptomyces hygroscopicus* obtained in Example 3 was selected and cultured in a 5L fermenter. The results are as follows: Figure 3As shown. The composition of each liter of fermentation medium was: 50g wheat straw hydrolysate, 5g yeast powder, 10g (NH4)2SO4, 1.36g KH2PO4, 0.8g K2HPO4, 0.5g MgSO4·7H2O, 0.04g ZnSO4·7H2O, and 0.03g FeSO4·7H2O. The initial pH was adjusted to 6.8 using ammonia. A 10% (v / v) liquid seed was inoculated into a 3L fermenter, and the fermentation temperature was controlled at 30℃ with an aeration rate of 2 vvm. After 48 hours of fermentation, the pH was controlled at 4.0 to promote the accumulation of ε-PL. When the residual sugar concentration in the fermentation broth was lower than 10g / L, a feed solution consisting of wheat straw hydrolysate and (NH4)2SO4 was added to the tank to maintain the residual sugar concentration at approximately 10g / L. The total fermentation time was 192 hours. The results showed that the maximum concentration of ε-PL reached 30.57 g / L, and the highest cell dry weight reached 39.43 g / L, indicating that the hygroscopic Streptomyces obtained by the screening and culture method of the present invention still have excellent ε-PL production capacity under scale-up fermentation conditions.

[0070] IV. Discussion of Results This invention uses a cellulose hydrolysate containing furfural, acetic acid, and 5-HMF as a selection pressure to induce continuous adaptive evolution of recombinant *Streptomyces hygroscopicus*. The study found that the evolved strains exhibited significantly improved tolerance to the aforementioned inhibitors, and their cell growth performance and metabolic activity were restored and even superior to those of the unevolved strains. In the incompletely detoxified cellulose hydrolysate, the evolved strains were able to fully utilize the mixed sugars of glucose and xylose to achieve stable synthesis of ε-PL, and their yield was higher than that of the control system using glucose as the sole carbon source.

[0071] This technical approach, which enables engineered strains to acquire high tolerance to complex inhibitors through directed adaptive evolution and maintain or even enhance their ε-PL synthesis capacity, overcomes the limitation of existing technologies that "cellulose hydrolysate is not suitable for ε-PL fermentation," and has significant technological advancements and application value.

[0072] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0073] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A method for screening and culturing tolerant hydrophilic Streptomyces, characterized in that, Includes the following steps: First-stage adaptive evolution: The engineered strain of Streptomyces hygroscopicus was inoculated into a fermentation medium with straw cellulose hydrolysate as the carbon source, and cultured at 28℃~32℃, 180rpm~220rpm for 24h~48h, and passaged for at least 30 generations to obtain primary Streptomyces hygroscopicus; Two-stage adaptive evolution: Primary streptomyces hygroscopicus was inoculated into a fermentation medium with straw cellulose hydrolysate as the carbon source. The concentration of ε-polylysine in the fermentation medium was 0.5 g / L to 5 g / L. The culture was carried out at 28℃ to 32℃ and 180 rpm to 220 rpm for 24 h to 48 h. The culture was continuously passaged for 10 to 25 generations to obtain tolerant streptomyces hygroscopicus. The tolerance mentioned refers to the resistance to the inhibitor while simultaneously producing ε-polylysine; The straw cellulose hydrolysate contains inhibitors; The inhibitor is one or more of acetic acid, furfural, and 5-hydroxymethylfurfural.

2. The screening and cultivation method for a tolerant hydrophilic streptomyces according to claim 1, wherein the inoculation amount is 5% to 10% of the fermentation broth volume.

3. The method for screening and culturing tolerant hydrophilic Streptomyces according to claim 1, characterized in that, The culture conditions for the first stage of adaptive evolution were: temperature 30℃, rotation speed 200rpm, and time 36h.

4. The screening and culture method for tolerant hydrophilic Streptomyces according to claim 1, characterized in that, The concentration of the ε-polylysine is 3 g / L.

5. The screening and culture method for tolerant hydrophilic Streptomyces according to claim 1, characterized in that, The culture conditions for the two-stage adaptive evolution are: temperature 30℃, rotation speed 200rpm, time 36h, and 18 generations of subculturing.

6. The screening and culture method for tolerant hydrophilic Streptomyces according to claim 1, characterized in that, The straw cellulose hydrolysate is obtained by mixing straw with sulfuric acid solution for hydrolysis, adjusting the pH, and adding cellulase for further hydrolysis.

7. The application of the screening and cultivation method for tolerant hygroscopic Streptomyces according to claim 1 in the fermentation broth for preparing ε-polylysine, characterized in that, The method of the application includes the following steps: Tolerant Streptomyces hydrophila were inoculated into a culture medium with straw cellulose hydrolysate as the carbon source and fermented at 28℃~34℃ and pH 6.2~7.5 for 48h~192h to obtain the fermentation broth of ε-polylysine.

8. The application of the screening and cultivation method for tolerant hygroscopic Streptomyces according to claim 7 in the fermentation broth for preparing ε-polylysine, characterized in that, The straw is wheat straw, rice straw, or corn straw.

9. The application of the screening and cultivation method for tolerant hygroscopic Streptomyces according to claim 8 in the fermentation broth for preparing ε-polylysine, characterized in that, Each liter of the culture medium contains 20g-100g of straw cellulose hydrolysate (based on total sugars), 5g-15g of (NH4)2SO4, 2g-8g of yeast powder, 0.5g-1.0g of K2HPO4, 0.5g-1.5g of KH2PO4, 0.4g-0.8g of MgSO4·7H2O, 0.02g-0.04g of FeSO4·7H2O, and 0.02g-0.06g of ZnSO4·7H2O.

Images