Streptomyces cinnamoneus, microbial inoculum, method for synthesizing monensin and application thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a Streptomyces cinnamoneus, a microbial agent, a method for synthesizing monensin and application. The present application provides a Streptomyces cinnamoneus SDSL6002 with a preservation number of CGMCC No. 30409. The ability of the Streptomyces cinnamoneus SDSL6002 provided by the present application to simultaneously metabolize glucose and xylose is significantly improved, and the Streptomyces cinnamoneus SDSL6002 can simultaneously utilize xylose and glucose to synthesize monensin. The present application further provides a method for synthesizing monensin or improving the yield of monensin. It has been verified that the Streptomyces cinnamoneus SDSL6002 can utilize bagasse enzymolysis liquid to synthesize monensin, and the yield of the synthesized monensin is high.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a Streptomyces cinnamoneus, a microbial agent, a method for synthesizing monensin and application. BACKGROUND

[0002] Lignocellulosic biomass is the most abundant renewable resource, and is a very promising bio-production feedstock because it does not compete with the global food supply chain. Glucose and xylose are two major sugars in lignocellulosic hydrolysate, which can be used as carbon sources for microbial fermentation. Therefore, efficient production of high-value chemicals from mixed carbon sources is an important step to realize an economic and sustainable lignocellulosic biomass bioconversion process.

[0003] Lignocellulose is mainly composed of cellulose, hemicellulose and lignin. Compared with glucose produced by cellulose hydrolysis, xylose produced by hemicellulose hydrolysis is difficult to be effectively utilized by microorganisms, which greatly limits the application potential of lignocellulosic resources. Therefore, it is crucial to realize efficient co-utilization of glucose and xylose by microorganisms. However, so far, only a few natural microorganisms have xylose metabolic pathways, and these microorganisms generally have strong substrate preference, carbon catabolite repression and low yield when co-utilizing xylose and glucose. Therefore, screening or constructing strains that can efficiently co-utilize mixed sugars, especially xylose and glucose, is crucial for the large-scale industrial utilization of lignocellulose. So far, more than 200 natural microorganisms with xylose metabolic pathways have been found in nature, mainly including bacteria, yeast and filamentous fungi. Yeast that can utilize both xylose and glucose is the focus of research, mainly including Pichia stipitis, Kluyveromyces marxianus, Candida utilis and Candida lusitaniae, etc., and Streptomyces cinnamoneus that can utilize both xylose and glucose has not been reported. SUMMARY

[0004] The application provides a Streptomyces cinnamoneus, a microbial agent, a method for synthesizing monensin and application, and the Streptomyces cinnamoneus SDSL6002 provided by the application can synthesize monensin while utilizing both xylose and glucose.

[0005] In order to solve the above technical problems, the application provides the following technical solutions.

[0006] The application provides a Streptomyces cinnamoneus SDSL6002, and the preservation number is CGMCC No.30409.

[0007] The application provides a microbial agent, which comprises the Streptomyces cinnamoneus SDSL6002 described in the above technical solution.

[0008] The application provides application of the Streptomyces cinnamonensis SDSL6002 or the bacterial agent in synthesis of monensin and / or improvement of monensin yield.

[0009] Preferably, when the application form of the Streptomyces cinnamonensis SDSL6002 is a culture solution, the inoculation amount of the culture solution is 8% to 12%; the temperature for synthesizing monensin is 30 to 35 DEG C; and the time is 1 to 8 days.

[0010] Preferably, the substrate for synthesizing monensin contains glucose and xylose.

[0011] The application provides application of the Streptomyces cinnamonensis SDSL6002 or the bacterial agent in synthesis of monensin from lignocellulose and / or improvement of monensin yield.

[0012] Preferably, the lignocellulose contains lignocellulose enzymolysis solution; the lignocellulose enzymolysis solution contains straw sugar; the straw sugar contains glucose and xylose; and the lignocellulose enzymolysis solution contains bagasse enzymolysis solution.

[0013] The application provides a method for synthesizing monensin or improving monensin yield, which comprises the following steps: mixing the Streptomyces cinnamonensis SDSL6002 or the bacterial agent and a substrate containing sugar, and culturing to synthesize monensin; and the sugar contains glucose and xylose.

[0014] Preferably, the substrate containing sugar contains lignocellulose enzymolysis solution; the lignocellulose enzymolysis solution contains straw sugar; the straw sugar contains glucose and xylose; the lignocellulose enzymolysis solution contains bagasse enzymolysis solution; and when the bagasse enzymolysis solution contains glucose and xylose, the mass ratio of the glucose to the xylose is (1 to 3):(1 to 3).

[0015] The application provides a method for utilizing lignocellulose, which comprises the following steps: mixing the Streptomyces cinnamonensis SDSL6002 or the bacterial agent and lignocellulose, and culturing to synthesize monensin; the lignocellulose contains lignocellulose enzymolysis solution; the lignocellulose enzymolysis solution contains straw sugar; the straw sugar contains glucose and xylose; and the lignocellulose enzymolysis solution contains bagasse enzymolysis solution.

[0016] The application provides a Streptomyces cinnamoneus SDSL6002, and the preservation number is CGMCC No.30409.

[0017] The application also provides a method for synthesizing monensin or improving the yield of monensin. Monensin is a polyether antibiotic with a five-ring monocarboxylic acid polyether structure, which is produced by fermentation of Streptomyces cinnamoneus, and has the advantages of high efficiency, low toxicity and small drug resistance, and is widely used in the livestock breeding industry. In recent years, monensin has also been found to have extensive antitumor and anticancer activity, and is expected to become a new drug for antitumor and anticancer. At present, microbial fermentation of starch-based glucose is the main source for producing monensin, and the production of monensin by using lignocellulose has not been explored. The application of the Streptomyces cinnamoneus SDSL6002 to lignocellulose enzymolysis liquid such as bagasse enzymolysis liquid for synthesizing monensin realizes high-value utilization of lignin, fills the gap in this research field, and has good application prospect.

[0018] The application also provides a utilization method of lignocellulose. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a glucose tolerance experiment result graph, wherein Glucose is glucose, PMV is the concentration of bacteria, Monensin is monensin, and Time is the culture time, and the same below;

[0020] Figure 2 is a sugar consumption graph in the fermentation process of mixed sugars with different mass ratios, wherein Sugar is sugar, Xylose is xylose, Total sugar is total sugar, A is the result of the glucose and xylose mass ratio being 1:0 and the glucose and xylose mass ratio being 0:1, B is the result of the glucose and xylose mass ratio being 3:1, C is the result of the glucose and xylose mass ratio being 2:1, D is the result of the glucose and xylose mass ratio being 1:1, E is the result of the glucose and xylose mass ratio being 1:2, and F is the result of the glucose and xylose mass ratio being 1:3;

[0021] Figure 3 The pH, PMV and monensin changes of mixed sugar fermentation process with different mass ratios;

[0022] Figure 4 For Figure 5 Enlarged view of A, Figure 4 The left side is the colony morphology after mutagenesis, and the right side is the colony morphology before mutagenesis;

[0023] Figure 5 The results of space mutagenesis, where A is the colony morphology before and after mutagenesis, B is the monensin yield after mutagenesis, C is the mutation rate; D is the monensin yield of high-yield strains obtained by space mutagenesis; E is the monensin yield results of the passage stability of mutant strain F23; F is the cell concentration results of the passage stability of mutant strain F23; Figure 5 In the middle, **** represents P<0.0001, and ns represents no significant difference;

[0024] Figure 6 The results of DES mutagenesis, where Fatality rate in A is the mortality rate, and Volume fraction is the volume fraction; the vertical coordinate of B is the monensin yield after mutagenesis, and Strain number represents the strain number; Mutation rate in C is the mutation rate; D is the monensin yield of high-yield strains obtained by DES mutagenesis; E is the monensin yield results of the passage stability of mutant strain F23-65; F is the cell concentration results of the passage stability of mutant strain F23-65; Figure 6 In the middle, **** represents P<0.0001 ， ns represents no significant difference;

[0025] Figure 7 The gene phylogenetic tree of mutant strain F23-65, where the submitted strain represents strain F23-65;

[0026] Figure 8 The effect verification diagram of high-yield mutant strain F23-65 and M1008, where A represents cell concentration, and B represents the content of glucose and xylose; Monensin-F23-65 is strain SDSL6002.

[0027] Figure 9 The application results of Streptomyces cinnamoneus SDSL6002 and M1008 in sugarcane bagasse enzymatic hydrolysate; where Monensin-F23-65 or F23-65 is strain SDSL6002.

[0028] Biological preservation instructions

[0029] Streptomyces cinnamoneus SDSL6002, deposited on April 22, 2024 in the China General Microbiological Culture Collection Center, with the accession number of CGMCC No.30409, and the address of the deposit unit is No.3, Beichen West Road, Beijing City, Chaoyang District. DETAILED DESCRIPTION

[0030] The application provides a Streptomyces cinnamoneus SDSL6002, with the accession number of CGMCC No.30409. The 16s rRNA gene sequence of the Streptomyces cinnamoneus SDSL6002 is shown in SEQ ID NO.1.

[0031] It is found in the research that the Streptomyces cinnamoneus M1008 has a natural glucose and xylose metabolic pathway, and the product synthesized by the Streptomyces cinnamoneus M1008, i.e., monensin, also has a high value and has a potential for further excavation, so that the production capacity of the Streptomyces cinnamoneus M1008 is improved by means of random mutagenesis. The Streptomyces cinnamoneus SDSL6002 is obtained by spaceflight and diethyl sulfate (DES) compound mutagenesis on the Streptomyces cinnamoneus M1008, and the Streptomyces cinnamoneus SDSL6002 has a high glucose and xylose co-fermentation capacity, so that the efficient synthesis of monensin from bagasse enzymolysis liquid is realized.

[0032] The 16s rRNA sequence of the Streptomyces cinnamoneus SDSL6002 is used for BLAST homologous sequence retrieval in NCBI, the sequences with high homology are used for construction of an evolutionary tree, and it is determined that the strain SDSL6002 is Streptomyces cinnamoneus.

[0033] The application provides a microbial agent, which comprises the Streptomyces cinnamoneus SDSL6002.

[0034] As an optional implementation form, the application provides the application form of the Streptomyces cinnamoneus SDSL6002, which comprises a culture solution.

[0035] As an optional implementation, the method for preparing the culture solution comprises the following steps: inoculating the Streptomyces cinnamonensis SDSL6002 into a culture medium for culture to obtain the culture solution. As an optional implementation, the method for inoculating is not particularly limited in the present application, and a conventional method can be used. As an optional implementation, the culture temperature is 30-35℃, or 33-34℃. In specific embodiments of the present application, the culture temperature is 30, 31, 32, 33, 34 or 35℃. The culture time is 24-48h. In specific embodiments of the present application, the culture time is 24h. The culture medium used in the present application comprises a seed culture medium, and the composition of the seed culture medium comprises 20g / L dextrin, 15g / L soybean cake powder, 5g / L glucose, 2.5g / L yeast powder and 1g / L CaCO3. As an optional implementation, the concentration of the Streptomyces cinnamonensis SDSL6002 in the culture solution is 20-30%.

[0036] The present application provides the use of the Streptomyces cinnamonensis SDSL6002 or the microbial agent in the synthesis of monensin and / or the improvement of the yield of monensin.

[0037] As an optional implementation, the use comprises mixing the Streptomyces cinnamonensis SDSL6002 or the microbial agent with a substrate, and then culturing to synthesize monensin.

[0038] As an optional implementation, the concentration of the Streptomyces cinnamonensis SDSL6002 in the mixture is 20-30% during the culture. As an optional implementation, the culture temperature is 30-37℃, or 33-34℃. The culture time is 1-8d, or 1-5d. The setting of the synthesis temperature and the synthesis time can improve the yield of monensin.

[0039] As an optional implementation, the substrate for synthesizing monensin comprises glucose and xylose. As another optional implementation, the substrate for synthesizing monensin comprises glucose and xylose. As an optional implementation, when the substrate for synthesizing monensin comprises glucose and xylose, the mass ratio of the glucose to the xylose is (1-3):(1-3), or 2:(1-3). In specific embodiments of the present application, the mass ratio of the glucose to the xylose is 1:1, 1:2, 1:3, 2:3, 2:1 or 3:1. The setting of the mass ratio of the glucose to the xylose can improve the yield of monensin.

[0040] The application provides application of the Streptomyces cinnamonensis SDSL6002 or the microbial agent in synthesis of monensin from lignocellulose and / or improvement of monensin yield.

[0041] As an optional implementation form, when the bagasse enzymolysis liquid contains glucose and xylose, the mass ratio of the glucose and the xylose in the bagasse enzymolysis liquid is (1-3):(1-3), and can also be 2:1. In specific embodiments of the application, the mass ratio of the glucose and the xylose is 1:1, 1:2, 1:3, 2:3, 2:1 or 3:1.

[0042] As an optional embodiment, the preparation method of the bagasse enzymatic hydrolysate provided by the present application is carried out with reference to the method for pretreating agricultural and forestry biomass with the assistance of surfactants in Chinese patent CN116426585A. In a specific embodiment of the present application, the preparation method of the bagasse enzymatic hydrolysate provided by the present application comprises the following steps: mixing bagasse, glycerol, an alkaline catalyst and a non-ionic surfactant and then heating to obtain a heated product; performing suction filtration on the heated product to obtain a solid substrate; performing enzymatic hydrolysis and centrifugation on the solid substrate, and the obtained supernatant is the bagasse enzymatic hydrolysate. As an optional embodiment, the bagasse provided by the present application is dried bagasse; the bagasse is passed through a 20-mesh sieve. As an optional embodiment, the alkaline catalyst comprises sodium hydroxide; the non-ionic surfactant comprises PEG; the mass ratio of the bagasse, glycerol, NaOH and PEG provided by the present application is 10:100:0.5:0.5, the heating provided by the present application is carried out under stirring, the temperature of the heating provided by the present application is raised to 202℃, and the temperature of 202℃ provided by the present application is maintained for 43 min. As another optional embodiment, the heated product is cooled and then subjected to first suction filtration to obtain a first suction-filtered solid substrate. As an optional embodiment, the temperature of the cooled product provided by the present application is 100±5℃, the first suction filtration provided by the present application is carried out by adding boiling water and then stirring, the mass-to-volume ratio of the heated product and the boiling water is 1g:1.5mL, and the stirring time is 10 min. The parameters of the first suction filtration provided by the present application are not particularly limited and can be determined by using a conventional method. As an optional embodiment, the first suction-filtered solid substrate is washed and then subjected to second suction filtration to obtain a solid substrate. The parameters of the second suction filtration provided by the present application are not particularly limited and can be determined by using a conventional method. The washing provided by the present application is carried out by using tap water, and the washing is performed twice. As another optional embodiment, the solid substrate is dried and then subjected to enzymatic hydrolysis and centrifugation, and the obtained supernatant is the bagasse enzymatic hydrolysate. The drying method provided by the present application comprises oven drying. The temperature of the oven drying is 60℃. As an optional embodiment, the enzyme used in the enzymatic hydrolysis provided by the present application comprises cellulase; a buffer solution and cellulase are added during the enzymatic hydrolysis provided by the present application; and the mass-to-volume ratio of the solid substrate and the buffer solution is 0.5g:25mL. The buffer solution comprises a citric acid buffer solution. The cellulase added in the present application is 5FPU / g based on the mass of the solid substrate. The temperature of the enzymatic hydrolysis provided by the present application is 50℃, and the time is 48h. The enzymatic hydrolysis provided by the present application is carried out under stirring, and the stirring speed is 180rpm.

[0043] The present application provides a method for synthesizing monensin or increasing the yield of monensin, comprising the following steps: mixing the Streptomyces cinnamoneus SDSL6002 or the bacterial agent of the above technical solution and a substrate containing sugar, culturing, and synthesizing monensin; the sugar includes glucose and xylose.

[0044] As an optional embodiment, the sugar-containing substrate of the present application comprises lignocellulose enzymolysis solution.

[0045] As an optional embodiment, the lignocellulose enzymolysis solution of the present application comprises straw sugar; the straw sugar comprises glucose and xylose; and the lignocellulose enzymolysis solution comprises bagasse enzymolysis solution. The preparation method of the bagasse enzymolysis solution has been discussed above, and will not be repeated here.

[0046] As an optional embodiment, the sugar-containing substrate, nitrogen source and inorganic salt of the present application are mixed to prepare a culture medium, and the Streptomyces cinnamoneus SDSL6002 is cultured to synthesize monensin. As an optional embodiment, the nitrogen source of the present application is soybean cake powder; and the addition amount of the soybean cake powder of the present application is 30-40 g / L, or 35 g / L, based on the volume of the culture medium. As an optional embodiment, the inorganic salt of the present application is one or more of Na2SO4, K2HPO4, FeSO4·7H2O, Al2(SO4)3·7H2O and CaCO3. As an optional embodiment, the addition amount of Na2SO4 in the culture medium of the present application is 2.0-2.4 g / L, or 2.2 g / L, based on the volume of the culture medium; the addition amount of K2HPO4 in the culture medium of the present application is 0.06-1.0 g / L, or 0.08 g / L, based on the volume of the culture medium; the addition amount of FeSO4·7H2O in the culture medium of the present application is 0.05-0.15 g / L, or 0.1 g / L, based on the volume of the culture medium; the addition amount of Al2(SO4)3·7H2O in the culture medium of the present application is 0.6-0.8 g / L, or 0.7 g / L, based on the volume of the culture medium; and the addition amount of CaCO3 in the culture medium of the present application is 2.0-3.0 g / L, or 2.5 g / L, based on the volume of the culture medium.

[0047] The nitrogen source and inorganic salt of the present application promote the growth of the Streptomyces cinnamoneus SDSL6002, and the source of the nitrogen source and inorganic salt of the present application is not particularly limited, and a conventional product can be used.

[0048] As an optional embodiment, when the bagasse enzymolysis solution of the present application comprises glucose and xylose, the mass ratio of the glucose and xylose is (1-3):(1-3), or 2:1. In the specific embodiments of the present application, the mass ratio of the glucose and xylose is 1:1, 1:2, 1:3, 2:3, 2:1 or 3:1.

[0049] As an optional implementation, the temperature of the culture is 30-35℃, or 33-34℃. The culture time is 1-8d, or 1-5d. The temperature, time, glucose and xylose mass ratio of the present application is set to improve the production of monensin of Streptomyces cinnamonensis SDSL6002.

[0050] The present application provides a method for utilizing lignocellulose, comprising the following steps: mixing the above technical solution described Streptomyces cinnamonensis SDSL6002 or the above technical solution described bacterial agent and lignocellulose, culturing, synthesizing monensin; the lignocellulose includes lignocellulose hydrolysate; the lignocellulose hydrolysate contains straw sugar; the straw sugar includes glucose and xylose; the lignocellulose hydrolysate includes bagasse hydrolysate. As an optional implementation, the present application mixes the lignocellulose hydrolysate, nitrogen source and inorganic salt to prepare a culture medium, and the Streptomyces cinnamonensis SDSL6002 is cultured to synthesize monensin. As an optional implementation, the nitrogen source of the present application is soybean meal; the addition amount of the soybean meal of the present application is 30-40g / L, or 35g / L, based on the volume of the culture medium. As an optional implementation, the inorganic salt is one or more of Na2SO4, K2HPO4, FeSO4·7H2O, Al2(SO4)3·7H2O and CaCO3. As an optional implementation, the addition amount of Na2SO4 in the culture medium of the present application is 2.0-2.4g / L, or 2.2g / L, based on the volume of the culture medium; the addition amount of K2HPO4 in the culture medium of the present application is 0.06-1.0g / L, or 0.08g / L, based on the volume of the culture medium; the addition amount of FeSO4·7H2O in the culture medium of the present application is 0.05-0.15g / L, or 0.1g / L, based on the volume of the culture medium; the addition amount of Al2(SO4)3·7H2O in the culture medium of the present application is 0.6-0.8g / L, or 0.7g / L, based on the volume of the culture medium; the addition amount of CaCO3 in the culture medium of the present application is 2.0-3.0g / L, or 2.5g / L, based on the volume of the culture medium. As an optional implementation, the temperature of the culture is 30-35℃, or 33-34℃. The culture time is 1-8d, or 1-5d. The temperature, time, glucose and xylose mass ratio of the present application is set to improve the production of monensin of Streptomyces cinnamonensis SDSL6002.

[0051] The lignocellulose of the present application comprises lignocellulose enzymolysis liquid; the lignocellulose enzymolysis liquid contains straw sugar; the straw sugar comprises glucose and xylose; and the lignocellulose enzymolysis liquid comprises bagasse enzymolysis liquid. The preparation method of the bagasse enzymolysis liquid has been discussed above, and will not be repeated here.

[0052] As an optional embodiment, the mixing has no special limitation, and a conventional method can be used.

[0053] As an optional embodiment, when the bagasse enzymolysis liquid of the present application contains glucose and xylose, the mass ratio of the glucose and the xylose is (1-3):(1-3), and can also be 2:1. In the specific embodiments of the present application, the mass ratio of the glucose and the xylose is 1:1, 1:2, 1:3, 2:3, 2:1 or 3:1.

[0054] The cinnamon Streptomyces M1008 is mutagenized to screen out the cinnamon Streptomyces SDSL6002, and the production of monensin of the cinnamon Streptomyces SDSL6002 is high.

[0055] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0056] The determination method of the cell concentration applied in the following examples is as follows:

[0057] 10 mL of fermentation liquid is centrifuged at 3000 rpm for 10 min, the volume of the supernatant is measured, and the cell concentration is calculated according to the following formula:

[0058] Cell concentration (%) = (fermentation liquid volume - supernatant volume) / fermentation liquid volume x 100%

[0059] The composition of the Gause No. 1 medium applied in the following examples is as follows: soluble starch 20 g / L, KNO3 1 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 g / L, FeSO4·7H2O 0.01 g / L and agar 20 g / L.

[0060] The composition of the seed culture medium applied in the following examples is as follows: 20 g / L dextrin, 15 g / L soybean cake powder, 5 g / L glucose, 2.5 g / L yeast powder and 1 g / L CaCO3.

[0061] Three parallel experiments are set in the following examples.

[0062] Example 1 Preliminary study on the glucose and xylose metabolic capacity of cinnamon Streptomyces M1008

[0063] Streptomyces cinnamoneus is affected by stress conditions such as osmotic stress of substrate sugar during the fermentation of monensin, therefore it is necessary to evaluate the high sugar tolerance performance of the strain. In the present application, glucose in the range of 20-100 g / L is used as carbon source, and glucose content, biomass (PMV), pH and monensin yield are used as indexes to detect the growth and metabolism of the strain. The specific experiment is as follows:

[0064] 1. Glucose tolerance experiment

[0065] Seed culture in a flask: Streptomyces cinnamoneus is picked from a fresh agar slant and numbered as M1008 for easy identification, inoculated into a flask, the volume of the flask is 250 mL, and the flask contains 50 mL of seed culture medium.

[0066] After inoculation, the seed liquid is obtained by incubation at 33°C with a shaking speed of 180 rpm for 24 h;

[0067] Fermentation culture: the seed liquid is inoculated into a flask according to the medium volume ratio of 10%, the volume of the flask is 500 mL, and the flask contains 50 mL of fermentation medium 1-5, respectively,

[0068] The composition of fermentation medium 1 is: glucose 20 g / L, soybean meal 35 g / L, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4·7H2O, 0.7 g / L Al2(SO4)3·7H2O and 2.5 g / L CaCO3; three parallel experiments are set;

[0069] The composition of fermentation medium 2 is: glucose 40 g / L, soybean meal 35 g / L, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4·7H2O, 0.7 g / L Al2(SO4)3·7H2O and 2.5 g / L CaCO3; three parallel experiments are set;

[0070] The composition of fermentation medium 3 is: glucose 60 g / L, soybean meal 35 g / L, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4·7H2O, 0.7 g / L Al2(SO4)3·7H2O and 2.5 g / L CaCO3; three parallel experiments are set;

[0071] The fermentation medium 4 was composed of glucose 80 g / L, soybean cake powder 35 g / L, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4-7H2O, 0.7 g / L Al2(SO4)3-7H2O and 2.5 g / L CaCO3; three parallel experiments were set;

[0072] The fermentation medium 5 was composed of glucose 100 g / L, soybean cake powder 35 g / L, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4-7H2O, 0.7 g / L Al2(SO4)3-7H2O and 2.5 g / L CaCO3; three parallel experiments were set.

[0073] After inoculation, the fermentation was carried out at 33°C for 6 days at a rotation speed of 200 rpm. The glucose content, PMV, Monensin content and pH of the fermentation broth obtained at different fermentation time were determined: the results are shown in Tables 1-4 and Figure 1 . Figure 1 The average value ± standard deviation of the three parallel experiments of Tables 1-4 was plotted.

[0074] Table 1 Glucose content (g / L) in the fermentation broth at different fermentation periods

[0075]

[0076]

[0077] Table 2 PMV in the fermentation broth at different fermentation periods (%)

[0078]

[0079] Table 3 pH of the fermentation broth at different fermentation periods

[0080]

[0081] Table 4 Monensin content (g / L) in the fermentation broth obtained at the 144th hour of fermentation in different media

[0082]

[0083] According to Tables 1-4 and Figure 1 It can be seen that when the initial concentration of glucose is 60 g / L, the Monensin content in the fermentation broth is high, and the PMV is high, therefore, the optimal initial concentration of glucose in the fermentation medium is 60 g / L.

[0084] 2. Determination of the mass ratio of mixed sugars

[0085] Shaking flask seed culture: same as step 1.

[0086] Fermentation culture: same as step 1, the only difference is the change of fermentation medium, the composition of fermentation medium is as follows:

[0087] The composition of fermentation medium 6 is: glucose 60 g / L, bean cake powder 35 g / L, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4·7H2O, 0.7 g / L Al2(SO4)3·7H2O and 2.5 g / L CaCO3, wherein the mass ratio of glucose to xylose is 1:0;

[0088] The composition of fermentation medium 7 is: xylose 60 g / L, bean cake powder 35 g / L, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4·7H2O, 0.7 g / L Al2(SO4)3·7H2O and 2.5 g / L CaCO3; wherein the mass ratio of glucose to xylose is 0:1;

[0089] The composition of fermentation medium 8 is: glucose 30 g / L, xylose 30 g / L, bean cake powder 35 g / L, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4·7H2O, 0.7 g / L Al2(SO4)3·7H2O and 2.5 g / L CaCO3; wherein the mass ratio of glucose to xylose is 1:1;

[0090] The composition of fermentation medium 9 is: glucose 40 g / L, xylose 20 g / L, bean cake powder 35 g / L, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4·7H2O, 0.7 g / L Al2(SO4)3·7H2O and 2.5 g / L CaCO3; wherein the mass ratio of glucose to xylose is 2:1;

[0091] The composition of fermentation medium 10 is: glucose 45 g / L, xylose 15 g / L, bean cake powder 35 g / L, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4·7H2O, 0.7 g / L Al2(SO4)3·7H2O and 2.5 g / L CaCO3; wherein the mass ratio of glucose to xylose is 3:1;

[0092] The fermentation medium 11 is composed of glucose 20 g / L, xylose 40 g / L, soybean cake powder 35 g / L, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4·7H2O, 0.7 g / L Al2(SO4)3·7H2O and 2.5 g / L CaCO3; wherein the mass ratio of glucose to xylose is 1:2;

[0093] The fermentation medium 12 is composed of glucose 15 g / L, xylose 45 g / L, soybean cake powder 35 g / L, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4·7H2O, 0.7 g / L Al2(SO4)3·7H2O and 2.5 g / L CaCO3; wherein the mass ratio of glucose to xylose is 1:3.

[0094] After inoculation, the culture was incubated at 33°C with a shaking speed of 200 rpm for 8 days. The sugar content of the fermentation broth during the culture was determined, and the results are shown in Table 1. Figure 2 According to the results in Table 1, when a single sugar is used as the carbon source, Streptomyces cinnamoneus M1008 can not only utilize glucose but also utilize xylose, but the sugar consumption rates are quite different, the glucose consumption rate is significantly greater than the xylose consumption rate (Table 1, A), indicating that the ability of Streptomyces cinnamoneus M1008 to utilize glucose is much greater than that of xylose, which is consistent with the metabolism of most microorganisms. Figure 2 When the carbon source is mixed sugar (Table 1, B-F), the total sugar consumption trends of all experimental groups are basically the same, and both glucose and xylose are consumed simultaneously. This metabolic mode is different from the sequential metabolism of carbon sources reported previously, in which glucose is utilized first and then xylose is utilized, and overcomes the glucose effect existing in most microorganisms. The ability of Streptomyces cinnamoneus M1008 to utilize glucose and xylose simultaneously is very important for the full utilization of lignocellulose hydrolysate, and can realize the utilization of lignocellulosic resources. It is worth noting that when the mass ratio of glucose to xylose is 1:1, the consumption rates of the two are basically the same, as shown in Table 1, D, where the two curves are nearly coincident, and glucose and xylose are almost consumed synchronously. Figure 2 More interestingly, when the mass ratio of glucose to xylose is 3:1, 2:1, the ratio of the consumption rates of the two is close to the mass ratio of the mixed sugar (Table 1, B-C), resulting in almost simultaneous depletion of the two sugars. Figure 2 Figure 2 The determination results of pH, biomass (PMV) and monensin indicators during the fermentation of mixed sugars with different mass ratios are shown in Table 2. Figure 2 According to the results in Table 2, when the mass ratio of glucose to xylose is 1:1, the pH value is the lowest, and the biomass is the highest, which is consistent with the results in Table 1, D.

[0095] The determination results of pH, biomass (PMV) and monensin indicators during the fermentation of mixed sugars with different mass ratios are shown in Table 2. Figure 3 According to the results in Table 2, when the mass ratio of glucose to xylose is 1:1, the pH value is the lowest, and the biomass is the highest, which is consistent with the results in Table 1, D. Figure 3 ​It can be seen that when the carbon source is mixed sugar, the fermentation advantages of glucose and xylose are integrated. Glucose ensures the carbon source needed for the growth of mycelium in the early stage, and xylose provides guarantee for the stable pH environment and monensin synthesis in the later stage. It is worth noting that when the mass ratio of glucose to xylose is 2:1, the pH of the fermentation system is stable below 8.0 before the 144th hour of fermentation, and the cell concentration reaches 40% in 72h, with the highest growth of 42%. After the 144th hour of fermentation, monensin maintains the growth rate, and finally reaches 4.5g / L, which is 309% higher than that of glucose fermentation (i.e. glucose group) and 246% higher than that of xylose fermentation (i.e. xylose group).

[0096] In summary, when the mass ratio of glucose to xylose is 2:1, mixed sugar can be metabolized simultaneously, the pH is suitable, and the biomass and monensin yield are the largest. Therefore, Streptomyces cinnamoneus M1008 can synchronously metabolize glucose and xylose with an optimal mass ratio of 2:1, and has the potential to produce monensin using mixed sugar.

[0097] Example 2 Spaceflight-DES Compound Mutagenesis of Streptomyces cinnamoneus M1008

[0098] In order to further improve the ability of Streptomyces cinnamoneus M1008 to utilize mixed sugar efficiently and increase the yield of monensin, spaceflight-DES compound mutagenesis was used.

[0099] The mutation of Streptomyces cinnamoneus M1008 was carried out in two steps. The first step was spaceflight mutagenesis on Shenzhou 14 spacecraft, and the second step was chemical mutagenesis using diethyl sulfate (DES). The specific process is as follows:

[0100] 1. Spaceflight mutagenesis of Streptomyces cinnamoneus M1008

[0101] The cultured slant spores of Streptomyces cinnamoneus M1008 were scraped into a sterile skim milk solution to obtain a spore solution. The skim milk solution had a milk powder mass concentration of 20%. The obtained spore solution was freeze-dried to prepare a powder, obtaining a freeze-dried powder. The freeze-dried powder was taken into space on Shenzhou 14 for spaceflight mutagenesis. The space station and Shenzhou spacecraft cabin parameters: orbital height 400 kilometers, inclination 41 degrees, radiation dose 0.4 millisiemens (mSv), cabin temperature 25°C, humidity about 30%.

[0102] The space physical environment is very different from that on the ground. The main characteristics of the space physical environment are strong radiation, microgravity, high vacuum, and weak geomagnetic field, etc. Under the synergistic effect of multiple factors or the effect of a single factor in the space physical environment, microorganisms can produce physiological damage and genetic variation, achieving spaceflight mutagenesis.

[0103] The lyophilized powder of *Streptomyces cinnamon* M1008, after space-induced mutagenesis, was diluted with sterile water and spread onto Gao's No. 1 agar plates for cultivation. Due to the high lethality rate of space-induced mutagenesis, the number of surviving single colonies was relatively small. A total of 479 single colonies were selected from the Gao's No. 1 agar plates for microplate cultivation. The colony morphology of the strain before and after mutagenesis is shown in the figure. Figure 5 A.

[0104] Figure 5 Image A shows the colony diagrams of Streptomyces cinnamon M1008 before and after mutagenesis. Figure 4 for Figure 5 A magnified view of A in the middle. Figure 5 In the equation A, Ground control represents pre-mutation and Space mutagenesis represents post-mutation.

[0105] The specific procedure for microplate culture is as follows: Using a sterile toothpick, scrape half of the spores from each single colony on the culture medium plate and inoculate them into a 10 mL seed culture medium in a 6-well plate. After spore inoculation, use another sterile toothpick to spot the remaining half of the spores from each colony onto a Gao's No. 1 medium plate. Incubate at 33°C for 7 days until colonies mature, then store. If the same strain is inoculated in the 6-well plate and spread on the solid slant, use the same numbering system for easy differentiation and preservation from other strains.

[0106] After culturing the seed culture medium in a constant temperature shaker for 24 h, the resulting seed culture was inoculated into the fermentation medium in a 6-well plate at a volume ratio of 7.5% (v / v), with 10 mL of fermentation medium in each well. Fermentation was carried out at 33℃ and 300 rpm for 10 days in a constant temperature shaker. The fermentation medium consisted of: 40 g / L glucose, 20 g / L xylose, 35 g / L soybean meal, 2.2 g / L Na₂SO₄, 0.08 g / L K₂HPO₄, 0.1 g / L FeSO₄·7H₂O, 0.7 g / L Al₂(SO₄)₃·7H₂O, and 2.5 g / L CaCO₃. Three parallel experiments were set up.

[0107] At the end of fermentation, the monensin content was detected using an enzyme-linked immunosorbent assay (ELISA) reader (see [link to ELISA reader]). Figure 5 (B) Using the monensin yield of the starting strain M1008 ±10% as the standard, the monensin yield of the starting strain M1008 +10% is recorded as A1, and the monensin yield of the starting strain M1008 -10% is recorded as A2. If the monensin yield of the mutant strain is less than A1 but greater than A2, it is defined as an isotropic mutation; if it is greater than A1, it is defined as a positive mutation; and if it is less than A2, it is defined as a negative mutation. Figure 5 In C, FM is a positive mutation, EM is an isotropic mutation, and NM is a negative mutation.Figure 5 From Table D and Table 5, it can be seen that the number of positive mutation, isogenic mutation and negative mutation strains after mutagenesis are 23, 129 and 322 respectively, and the corresponding positive mutation rate, isogenic mutation rate and negative mutation rate are 4.85%, 27.22% and 67.93% respectively. This also shows that the spaceflight mutagenesis method is effective for Streptomyces cinnamonensis M1008. Subsequently, the five mutant strains D16, D42, E38, F23 and H21 of Streptomyces cinnamonensis M1008 with 20% higher monensin yield were subjected to shake flask culture, and each strain of shake flask culture was set up three parallel experiments. After fermentation, the monensin yield of each strain was detected by HPLC, and the results are shown in Table D and Table 5. Figure 5 Figure 5 From Table D and Table 5, it can be seen that the number of positive mutation, isogenic mutation and negative mutation strains after mutagenesis are 23, 129 and 322 respectively, and the corresponding positive mutation rate, isogenic mutation rate and negative mutation rate are 4.85%, 27.22% and 67.93% respectively. This also shows that the spaceflight mutagenesis method is effective for Streptomyces cinnamonensis M1008. Subsequently, the five mutant strains D16, D42, E38, F23 and H21 of Streptomyces cinnamonensis M1008 with 20% higher monensin yield were subjected to shake flask culture, and each strain of shake flask culture was set up three parallel experiments. After fermentation, the monensin yield of each strain was detected by HPLC, and the results are shown in Table D and Table 5.

[0108] Then the passage stability of mutant strain F23 was studied, and three parallel experiments were set up. The results are shown in Table E-F and Table 6-7. Figure 5 Figure 5 From Table D and Table 5, it can be seen that the number of positive mutation, isogenic mutation and negative mutation strains after mutagenesis are 23, 129 and 322 respectively, and the corresponding positive mutation rate, isogenic mutation rate and negative mutation rate are 4.85%, 27.22% and 67.93% respectively. This also shows that the spaceflight mutagenesis method is effective for Streptomyces cinnamonensis M1008. Subsequently, the five mutant strains D16, D42, E38, F23 and H21 of Streptomyces cinnamonensis M1008 with 20% higher monensin yield were subjected to shake flask culture, and each strain of shake flask culture was set up three parallel experiments. After fermentation, the monensin yield of each strain was detected by HPLC, and the results are shown in Table D and Table 5.

[0109] Table 5 Monensin yield (g / L) of high-yield strains obtained by spaceflight mutagenesis

[0110] Strain number M1008 D16 D42 E38 F23 H21 Parallel 1 4.37 4.76 4.28 4.94 5.03 4.52 Parallel 2 4.25 4.81 4.42 4.82 5.16 4.55 Parallel 3 4.29 4.94 4.46 4.99 5.13 4.65

[0111] Table 6 Monensin yield results (g / L) of passage stability of mutant strain F23

[0112] Number of passages F1 F2 F3 F4 F5 Parallel 1 5.03 5.13 5.04 4.92 4.98 Parallel 2 5.16 5.15 5.07 4.95 5.14 Parallel 3 5.13 5.35 5.34 4.90 5.14

[0113] Table 7 Bacterial concentration results (%) of passage stability of mutant strain F23

[0114] Number of passages F1 F2 F3 F4 F5 Parallel 1 38 42 41 40 41 Parallel 2 41 41 41 39 39 Parallel 3 41 43 38 38 40

[0115] 2. Diethyl sulfate chemical mutagenesis of Streptomyces cinnamonensis M1008

[0116] Compared with single mutagenesis method, physical and chemical compound mutagenesis has better mutation frequency. Diethyl sulfate (DES) is an alkylating agent that can alkylate part of the bases in the gene and is widely used in microbial mutagenesis breeding. The experiment explored the relationship between the amount of DES added and the lethality of mutant S. cinnamonensis F23.

[0117] ​​The spore suspension 2 mL of high yield mutant strain F23 obtained by spaceflight mutagenesis was taken, and the spore concentration was 10 8 Then 2 mL of phosphate buffer (pH 7.0) was added, and a DES solution was added to make the volume fraction of DES in the reaction system 0.2%, 0.4%, 0.6%, 0.8%, and 1.0%, respectively. Then the reaction was carried out in the dark at 33°C for 20 min, and then 10 mL of sodium thiosulfate solution with a mass concentration of 25% was added to terminate the reaction. After the mutagenesis, the spore suspension was diluted by an appropriate multiple and plated on Gao's No. 1 medium plate, which was incubated at 33°C for 5 d. The colony number was observed and the mortality rate was calculated. The calculation formula of the mortality rate was as follows:

[0118] Mortality rate (%) = (control colonies - survival colonies after mutagenesis) / control colonies x 100%

[0119] The results are shown in Table 8-10, Figure 6 and Table 8-10, Figure 6 A in Table 8 shows that the mutagenesis mortality rate gradually increases with the increase of the DES addition amount, and when the DES addition amount is 0.8%, the mortality rate is 85.6%, and when the DES addition amount is 1.2%, the mortality rate reaches 100%. Therefore, the DES addition amount is selected as 0.8% as the mutagenesis dose.

[0120] Then, the F23 spore suspension was treated with DES with a volume fraction of 0.8% for 20 min, and then diluted and plated on Gao's No. 1 medium plate containing 25 μg / mL of streptomycin resistance for screening. 240 single bacteria were picked from the resistant plate, cultured in a 6-well plate, and preserved. The monensin yield was detected by an enzyme-labeled instrument at the end of fermentation, and the results are shown in Table 8-10. Figure 6 B-C in Table 8. Taking the monensin yield of strain F23 ± 10% as the standard, the monensin yield of strain F23 + 10% is recorded as A3, the monensin yield of strain F23 - 10% is recorded as A4, the monensin yield of the mutant strain is less than A3 and greater than A4, which is defined as equal mutation, greater than A3 is defined as positive mutation, and less than A4 is defined as negative mutation. Figure 6 C in Table 8. The FM is positive mutation, the EM is equal mutation, and the NM is negative mutation. It can be seen that the positive, equal and negative mutation rates are 7.08%, 23.33% and 69.4%, respectively.

[0121] The 5 mutant strains with the highest monensin yield (F23-65, F23-197, F23-229, F23-119, and F23-85) were re-screened in a shake flask. The monensin yield of mutant strain F23-65 reached 5.65 g / L, which increased by 10.63% compared with the spaceflight mutant strain F23 and increased by 31.4% compared with the original strain M1008. Figure 6Table 8). Genetic stability test showed that the mutant strain F23-65 was stable in monensin production and cell concentration in 5 generations Figure 6 Table 9-10); three parallel experiments were set during the determination process. In addition, the 16S rRNA gene sequences (1390bp) of the 1st and 5th generations were 100% similar. Therefore, the mutant strain F23-65 was selected for further study. Figure 6 The average value ± standard deviation of the three parallel experiments of Tables 8-10 was plotted.

[0122] Table 8 Monensin production (g / L) of six high-yield strains obtained by DES mutagenesis

[0123] Strain number M1008 F23 F23-65 F23-197 F23-229 F23-119 F23-85 Parallel 1 4.37 5.03 5.63 5.47 5.56 5.47 5.24 Parallel 2 4.25 5.16 5.60 5.68 5.43 5.27 5.49 Parallel 3 4.28 5.13 5.72 5.57 5.43 5.42 5.42

[0124] Table 9 Monensin production results (g / L) of the mutant strain F23-65 for passage stability

[0125] Number of passages F1 F2 F3 F4 F5 Parallel 1 5.63 5.61 5.57 5.72 5.54 Parallel 2 5.60 5.74 5.61 5.61 5.63 Parallel 3 5.72 5.92 5.59 5.91 5.69

[0126] Table 10 Cell concentration results (%) of the mutant strain F23-65 for passage stability

[0127] Number of passages F1 F2 F3 F4 F5 Parallel 1 39 41 42 40 41 Parallel 2 40 42 41 39 42 Parallel 3 41 43 40 41 41

[0128] Example 3

[0129] 1. Identification of high-yield mutant strains

[0130] By sequencing the 16S rRNA of the high-yield mutant strain F23-65, the determination result is shown in SEQ ID NO. 1: by comparing with the NCBI database, a phylogenetic tree is constructed, as shown in Figure 7 As shown, the information of the proximal species is obtained, and the mutant strain is in the smallest branch with Streptomyces cinnamoneus DSM1042, which is a proximal species.

[0131] Combined with the phylogenetic tree, it is determined that the high-yield mutant strain F23-65 is Streptomyces cinnamoneus, which is recorded as Streptomyces cinnamoneus SDSL6002; biological preservation is carried out, and the preservation number is CGMCC No. 30409.

[0132] SEQ ID NO. 1:

[0133]

[0134] 2. Validation of Streptomyces cinnamonensis SDSL6002

[0135] To further evaluate the performance of the mutant, mixed sugar fermentation analysis was performed on Streptomyces cinnamonensis SDSL6002.

[0136] Treatment group:

[0137] (1) Seed culture in shake flask: The seed culture step in shake flask was the same as step 1 in Example 1.

[0138] (2) Mixed sugar fermentation culture: Streptomyces cinnamonensis SDSL6002 seed liquid was inoculated into a flask at a medium volume ratio of 10% for mixed sugar fermentation culture. The temperature of the mixed sugar fermentation culture was 33°C, the rotation speed was 200 rpm, and the time was 8 days. The volume of the flask was 500 mL, and the flask contained 50 mL of fermentation medium. The composition of the fermentation medium was: glucose 40 g / L, xylose 20 g / L, soybean cake powder 35 g / L, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4·7H2O, 0.7 g / L Al2(SO4)3·7H2O, and 2.5 g / L CaCO3.

[0139] During the culture process, the monensin yield, cell concentration, glucose content, and xylose content in the obtained fermentation broth were determined.

[0140] Control group: The same as the treatment group, the only difference was that Streptomyces cinnamonensis SDSL6002 was replaced by Streptomyces cinnamonensis M1008.

[0141] The results are shown in Figure 8 and Tables 11-14.

[0142] As can be seen from Figure 8 , the monensin yield and cell concentration of the mutant Streptomyces cinnamonensis SDSL6002 (i.e., F23-65) were significantly higher than those of Streptomyces cinnamonensis M1008. The cell concentration reached a maximum at 96 h and maintained that concentration for monensin synthesis. The monensin yield gradually increased with the extension of the fermentation time, and Streptomyces cinnamonensis SDSL6002 reached 5.65 g / L at 192 h of fermentation, which was 30.94% higher than M1008 (see Figure 8 A, Tables 11-12). In terms of sugar consumption, the presence of glucose did not inhibit the use of xylose by the strain. Streptomyces cinnamonensis SDSL6002 could consume both glucose and xylose, and it consumed the mixed sugar 24 h earlier than strain M1008 (see Figure 8 B, Tables 13-14). It can be seen that strain SDSL6002 can utilize both glucose and xylose, and the sugar consumption rate is significantly higher than that of the original strain M1008.Figure 8 Plotted with the average value ± standard deviation of three parallel experiments of Tables 11-14.

[0143] Table 11 Monensin production (g / L) of Streptomyces cinnamoneus SDSL6002 and M1008

[0144]

[0145]

[0146] Table 12 Cell concentration (%) of Streptomyces cinnamoneus SDSL6002 and M1008

[0147]

[0148] Table 13 Glucose content (g / L) in fermentation broth of Streptomyces cinnamoneus SDSL6002 and M1008

[0149]

[0150] Table 14 Xylose content (g / L) in fermentation broth of Streptomyces cinnamoneus SDSL6002 and M1008

[0151]

[0152]

[0153] Example 4 Application of Streptomyces cinnamoneus SDSL6002 in bagasse enzymatic hydrolysate

[0154] 1. Preparation of bagasse enzymatic hydrolysate

[0155] Pretreatment of bagasse: The bagasse was crushed and passed through a 20 mesh sieve, and then dried to constant weight in an oven at 60°C. After drying, 10.0 g of bagasse was accurately weighed into a 500 mL Erlenmeyer flask, 100.0 g of glycerol, 0.50 g of NaOH and 0.50 g of PEG were added and mixed evenly, and placed in a constant temperature heating jacket, heated to 202°C under stirring at 180 rpm, and maintained for 43 min. After completion, the heating jacket was removed and the temperature was allowed to drop to 100°C ± 5°C under continuous stirring, 150 mL of boiling water was added, and after stirring for 10 min, the solid substrate and pretreatment liquid were separated by suction filtration, and the solid substrate was washed twice with 150 mL of tap water. The solid substrate obtained after suction filtration was placed in an oven and dried at 60°C to obtain the pretreated solid substrate.

[0156] Take 0.5 g of the pretreated solid matrix and add it to 25 mL of citrate buffer (pH 4.8). Add 5 FPU / g cellulase based on the mass of the solid matrix. Place the mixture in a shaker at 180 rpm and 50 °C for 48 h to obtain the enzymatic hydrolysate. Take 0.4 mL of the hydrolysate and centrifuge at 8000 rpm. The resulting supernatant is the sugarcane bagasse hydrolysate, containing 100 g / L of fermentable sugars and a glucose to xylose mass ratio of 2:1.

[0157] 2. Production of monensin by fermentation of high-yield mutant strains in sugarcane bagasse enzymatic hydrolysate.

[0158] The fermentation medium for sugarcane bagasse enzymatic hydrolysate consists of: a total sugar concentration of 60 g / L (i.e., glucose content 40 g / L and xylose content 20 g / L), 35 g / L soybean meal, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4·7H2O, 0.7 g / L Al2(SO4)3·7H2O, and 2.5 g / L CaCO3. The preparation method is as follows: dilute the total sugar concentration of the sugarcane bagasse enzymatic hydrolysate to 60 g / L (i.e., glucose content 40 g / L, xylose content 20 g / L), then add 35 g / L soybean meal, 2.2 g / L Na2SO4, 0.08 g / L K2HPO4, 0.1 g / L FeSO4·7H2O, 0.7 g / L Al2(SO4)3·7H2O and 2.5 g / L CaCO3; adjust the pH of the culture medium to 7.2 with NaOH before sterilization, and apply after sterilization.

[0159] Treatment group: Mature Streptomyces cinnamon SDSL6002 slant culture was used to prepare 1cm... 2 The slant culture was inoculated into the seed culture medium and cultured at 33℃ and 180 rpm for 24 h to obtain the seed liquid. The seed liquid was then inoculated into sugarcane bagasse enzymatic hydrolysate fermentation medium at an inoculation rate of 10% of the culture medium volume and cultured at 33℃ and 200 rpm for 192 h. Three parallel experiments were set up.

[0160] The control group was the same as the treatment group, except that *Streptomyces cinnamon* SDSL6002 was replaced with *Streptomyces cinnamon* M1008. Three parallel experiments were set up.

[0161] After cultivation, the amount of monensin synthesized, the cell concentration, and the sugar content were measured. The results are shown in [Table / Reference]. Figure 9 See Tables 15-18.

[0162] The monensin synthesis amount of Streptomyces cinnamoneus SDSL6002 is 5.42 g / L, which is increased by 41% (increased by 31% in the synthetic culture medium, which also shows that the mutant strain metabolizes more vigorously in the bagasse enzymatic hydrolysate) than the original strain Streptomyces cinnamoneus M1008; in terms of cell concentration, the mutant strain F23-65 reaches a maximum of 44%, which is increased by 10% than the original strain (increased by 5% in the synthetic culture medium, which again shows that the mutant strain metabolizes more vigorously in the bagasse enzymatic hydrolysate); in terms of sugar consumption, the sugar consumption ability of the mutant strain F23-65 is stronger than that of the original strain, and the glucose and glucose are consumed 2 days in advance. It can be seen that the mutant strain F23-65 can well utilize the bagasse enzymatic hydrolysate to produce monensin. Figure 9 The average values ± standard deviations of three parallel experiments in Tables 15-18 are plotted.

[0163] Table 15 Monensin yield (g / L) of two strains

[0164]

[0165] Table 16 PMV cell concentration (%) of two strains

[0166]

[0167] Table 17 Glucose content (g / L) in the fermentation broth of two strains

[0168]

[0169]

[0170] Table 18 Xylose content (g / L) in the fermentation broth of two strains

[0171]

[0172] In summary, the application provides a Streptomyces cinnamoneus SDSL6002 which can synthesize monensin by simultaneously utilizing glucose and xylose, and the yield of monensin produced by fermentation in the bagasse enzymatic hydrolysate is high.

[0173] Although the above embodiment has made a detailed description of the application, it is only a part of the embodiments of the application, but not all the embodiments, and other embodiments can be obtained according to the embodiment without creativity, which all belong to the protection scope of the application.

Claims

1. A strain of Streptomyces cinnamoneus (S. cinnamoneus) D- SDSL6002, characterized in that, Streptomyces cinnamoneus ) SDSL6002, characterized in that, The preservation number is CGMCC No. 30409.

2. An inoculant characterized in that, The cinnamoneus Streptomyces SDSL6002 in claim 1.

3. The cinnamoneus Streptomyces SDSL6002 in claim 1 or the microbial agent in claim 2 is applied in synthesis of monensin and / or improvement of monensin yield.

4. Use according to claim 3, characterized in that, When the application form of the cinnamoneus Streptomyces SDSL6002 is culture solution, the inoculation amount of the culture solution is 8%~12%; the temperature for synthesis of monensin is 30~35℃; and the time is 1~8d.

5. Use according to claim 3, characterized in that, The substrate for synthesis of monensin contains glucose and xylose.

6. The cinnamoneus Streptomyces SDSL6002 in claim 1 or the microbial agent in claim 2 is applied in synthesis of monensin from lignocellulose and / or improvement of monensin yield. The lignocellulose is lignocellulose enzymolysis solution.

7. Use according to claim 6, characterized in that, The lignocellulose enzymolysis solution contains straw sugar; the straw sugar includes glucose and xylose; and the lignocellulose enzymolysis solution includes bagasse enzymolysis solution.

8. A method of synthesizing monensin or increasing the production of monensin, characterized by, The method comprises the following steps: mixing the cinnamoneus Streptomyces SDSL6002 in claim 1 or the microbial agent in claim 2 and substrate containing sugar, culturing, and synthesizing monensin; the sugar includes glucose and xylose.

9. The method of claim 8, wherein, The substrate containing sugar includes lignocellulose enzymolysis solution; the lignocellulose enzymolysis solution contains straw sugar; the straw sugar includes glucose and xylose; the lignocellulose enzymolysis solution includes bagasse enzymolysis solution; when the bagasse enzymolysis solution contains glucose and xylose, the mass ratio of the glucose and the xylose is (1~3):(1~3).

10. A method of utilizing lignocellulose, characterized by, The method comprises the following steps: mixing the cinnamoneus Streptomyces SDSL6002 in claim 1 or the microbial agent in claim 2 and lignocellulose, culturing, and synthesizing monensin; the lignocellulose is lignocellulose enzymolysis solution; the lignocellulose enzymolysis solution contains straw sugar; the straw sugar includes glucose and xylose; and the lignocellulose enzymolysis solution includes bagasse enzymolysis solution.

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