Streptomyces cinnamomi, microbial inoculum, method for synthesizing monensin and application

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to streptomyces cinnamomi, a fungicide, a method for synthesizing monensin and application. The invention provides streptomyces cinnamomi SDSL6002, the preservation number of which is CGMCC (China General Microbiological Culture Collection Center) No.30409. The streptomyces cinnamomi SDSL6002 provided by the invention has the advantages that the capability of simultaneously metabolizing glucose and xylose is obviously improved, and monensin can be synthesized by simultaneously utilizing the xylose and the glucose. The invention further provides a method for synthesizing the monensin or increasing the yield of the monensin, it is verified that the streptomyces cinnamomi SDSL6002 can be used for synthesizing the monensin through bagasse enzymatic hydrolysate, and the yield of the synthesized monensin is high.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Streptomyces cinnamomi, a bacterial agent, a method for synthesizing monensin and application thereof. Background Art

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

[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 lignocellulose resources. Therefore, it is crucial to achieve 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 in the co-utilization of 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, studies have found that there are more than 200 natural microorganisms with xylose metabolic pathways in nature, mainly including bacteria, yeasts and filamentous fungi. The yeasts that can utilize both xylose and glucose in natural microorganisms are the focus of research, mainly including Pichia stipitis, Kluyveromyces marxianus, Candida utilis and Candida portugalensis, while Streptomyces cinnamomi that can utilize both xylose and glucose have not been reported. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Streptomyces cinnamomi, a bacterial agent, a method for synthesizing monensin and an application thereof. The Streptomyces cinnamomi SDSL6002 provided by the present invention can synthesize monensin by utilizing xylose and glucose simultaneously.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The invention provides a strain of Streptomyces cinnamoneus SDSL6002, whose preservation number is CGMCC No.30409.

[0007] The present invention provides a bacterial agent, comprising the Streptomyces cinnamomi SDSL6002 described in the above technical solution.

[0008] The present invention provides the use of the Streptomyces cinnamomi SDSL6002 described in the above technical solution or the bacterial agent described in the above technical solution in synthesizing monensin and / or improving the yield of monensin.

[0009] Preferably, when the application form of the Streptomyces cinnamomi 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° C.; and the time is 1 to 8 days.

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

[0011] The present invention provides the use of the Streptomyces cinnamomi SDSL6002 described in the above technical solution or the bacterial agent described in the above technical solution in synthesizing monensin from lignocellulose and / or increasing the yield of monensin.

[0012] Preferably, the lignocellulose comprises lignocellulose enzymatic hydrolysate; the lignocellulose enzymatic hydrolysate contains straw sugar; the straw sugar comprises glucose and xylose; the lignocellulose enzymatic hydrolysate comprises bagasse enzymatic hydrolysate.

[0013] The present invention provides a method for synthesizing monensin or improving the yield of monensin, comprising the following steps: mixing the Streptomyces cinnamomi SDSL6002 described in the above technical solution or the bacterial agent described in the above technical solution with a substrate containing sugar, culturing the mixture, and synthesizing monensin; the sugar comprises glucose and xylose.

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

[0015] The invention provides a method for utilizing lignocellulose, comprising the following steps: mixing the cinnamon Streptomyces SDSL6002 described in the above technical solution or the bacterial agent described in the above technical solution with lignocellulose, culturing and synthesizing monensin; the lignocellulose comprises lignocellulose enzymatic hydrolysate; the lignocellulose enzymatic hydrolysate contains straw sugar; the straw sugar comprises glucose and xylose; the lignocellulose enzymatic hydrolysate comprises bagasse enzymatic hydrolysate.

[0016] Beneficial effects of the present invention: The present invention provides a strain of Streptomyces cinnamoneus SDSL6002, with a preservation number of CGMCC No. 30409. The Streptomyces cinnamoneus SDSL6002 provided by the present invention has significantly improved ability to metabolize glucose and xylose simultaneously, and can simultaneously utilize xylose and glucose to synthesize monensin.

[0017] The present invention also provides a method for synthesizing monensin or increasing the yield of monensin. It has been verified that Streptomyces cinnamomi SDSL6002 can synthesize monensin using sugarcane bagasse enzymatic hydrolysate. Monensin is a polyether antibiotic with a pentacyclic monocarboxylic acid polyether structure produced by fermentation of Streptomyces cinnamomi, which has the advantages of high efficiency, low toxicity, low drug resistance, etc., and is widely used in animal husbandry and breeding industry. In recent years, monensin has also been found to have a wide range of anti-tumor and anti-cancer activities, and is expected to become a new type of anti-tumor and anti-cancer drug. In short, the wide application of monensin in animal husbandry and medical treatment has aroused great interest in its research and development, highlighting its broad prospects. At present, microbial fermentation of starch-based glucose is the main source of monensin production, and the production of monensin using lignocellulose has not yet been explored. The Streptomyces cinnamomi SDSL6002 of the present invention can synthesize monensin using lignocellulose enzymatic hydrolysate such as sugarcane bagasse enzymatic hydrolysate, realizes the high-value utilization of lignin, fills the gap in this research field, and has good application prospects.

[0018] The present invention also provides a method for utilizing lignocellulose. Lignocellulose biomass is the most abundant renewable resource. Glucose and xylose are two main sugars in lignocellulose hydrolysate. It has been verified that Streptomyces cinnamomi SDSL6002 can utilize lignocellulose hydrolysate to synthesize monensin, thereby realizing high-value utilization of lignocellulose. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is the result of glucose tolerance experiment, where Glucose is glucose, PMV is bacterial concentration, Monensin is monensin, and Time is culture time, the same below;

[0020] Figure 2 The sugar consumption diagram during the fermentation of mixed sugars with different mass ratios, where Sugar is sugar, Xylose is xylose, Total sugar is total sugar, A is the result of the mass ratio of glucose to xylose of 1:0 and the mass ratio of glucose to xylose of 0:1, B is the result of the mass ratio of glucose to xylose of 3:1; C is the result of the mass ratio of glucose to xylose of 2:1; D is the result of the mass ratio of glucose to xylose of 1:1; E is the result of the mass ratio of glucose to xylose of 1:2; F is the result of the mass ratio of glucose to xylose of 1:3;

[0021] Figure 3 The graphs of pH, PMV and monensin changes during the fermentation of mixed sugars with different mass ratios;

[0022] Figure 4 for Figure 5 A magnified image of Figure 4 The left side in the middle shows the colony morphology after mutagenesis, and the right side shows 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, and C is the mutation rate; D is the monensin yield of the high-yield strain obtained by space mutagenesis; E is the monensin yield result of the subculture stability of the mutant strain F23; F is the bacterial concentration result of the subculture stability of the mutant strain F23; Figure 5 In the table, **** means P < 0.0001, ns means no significant difference;

[0024] Figure 6 The figure is the result of DES mutagenesis, where Fatalityrate in A is the lethality rate, and Volume fraction is the volume fraction; the ordinate of B is the monensin yield after mutagenesis, and Strainnumber represents the strain number; Mutationrate in C is the mutation rate; D is the monensin yield of the high-yield strain obtained by DES mutagenesis; E is the monensin yield result of the subculture stability of the mutant strain F23-65; F is the bacterial concentration result of the subculture stability of the mutant strain F23-65; Figure 6 In the table, **** represents P < 0.0001 ， ns means no significant difference;

[0025] Figure 7 This is the genetic evolution tree of mutant strain F23-65, in which the tested strain represents strain F23-65;

[0026] Figure 8 The figure is a validation diagram of the effects of high-yield mutants F23-65 and M1008, where A represents bacterial concentration and B represents the content of glucose and xylose; Monensin-F23-65 is strain SDSL6002;

[0027] Fig. 9 This is a graph showing the application results of Streptomyces cinnamomi SDSL6002 and M1008 in sugarcane bagasse enzymatic hydrolysate; Monensin-F23-65 or F23-65 are both strains SDSL6002.

[0028] Biological Deposit Description

[0029] Streptomyces cinnamoneus SDSL6002 was deposited in the General Microbiology Center of China Culture Collection Administration on April 22, 2024, with the deposit number CGMCC No. 30409. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

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

[0031] The study found that Streptomyces cinnamomi M1008 has natural glucose and xylose metabolic pathways, and the synthesized product monensin also has high value and has the potential for further development, so its production capacity is improved by means of random mutagenesis. The present invention performs aerospace and diethyl sulfate (DES) composite mutagenesis on Streptomyces cinnamomi M1008 to obtain Streptomyces cinnamomi SDSL6002, which has a high ability to co-ferment glucose and xylose, thereby realizing efficient synthesis of monensin from sugarcane bagasse enzymatic hydrolysate.

[0032] The invention uses the 16s rRNA sequence of Streptomyces cinnamoneus SDSL6002 to perform BLAST homologous sequence search in NCBI, constructs a phylogenetic tree with sequences with high homology, and determines that the strain SDSL6002 is Streptomyces cinnamoneus.

[0033] The present invention provides a bacterial agent, comprising the Streptomyces cinnamomi SDSL6002 described in the above technical solution.

[0034] As an optional embodiment, the application form of the Streptomyces cinnamomi SDSL6002 of the present invention includes culture solution.

[0035] As an optional embodiment, the method for preparing the culture solution of the present invention comprises the following steps: inoculating the cinnamon Streptomyces SDSL6002 in a culture medium for culture to obtain a culture solution. As an optional embodiment, the present invention has no special limitation on the inoculation method, and a conventional method can be used. As an optional embodiment, the culture temperature of the present invention is 30°C to 35°C, or 33 to 34°C; in a specific embodiment of the present invention, the culture temperature is 30, 31, 32, 33, 34 or 35°C. The culture time is 24 to 48 hours. In a specific embodiment of the present invention, the culture time is 24 hours. The culture medium used in the culture of the present invention 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 embodiment, the bacterial concentration of the cinnamon Streptomyces SDSL6002 in the culture solution is 20% to 30%.

[0036] The present invention provides the use of the Streptomyces cinnamomi SDSL6002 described in the above technical solution or the bacterial agent described in the above technical solution in synthesizing monensin and / or improving the yield of monensin.

[0037] As an optional implementation, the application includes: mixing the Streptomyces cinnamomi SDSL6002 described in the above technical solution or the bacterial agent described in the above technical solution with a substrate, and then culturing to synthesize monensin.

[0038] As an optional embodiment, during the culture of the present invention, the bacterial concentration of Streptomyces cinnamomi SDSL6002 in the mixture is 20% to 30%. As an optional embodiment, the culture temperature is 30 to 37° C., or 33 to 34° C. The culture time is 1 to 8 days, or 1 to 5 days. The setting of the synthesis temperature and synthesis time can increase the yield of monensin.

[0039] As an optional embodiment, the substrate for synthesizing monensin of the present invention contains glucose and xylose. As another optional embodiment, the substrate for synthesizing monensin of the present invention contains glucose and xylose. As an optional embodiment, when the substrate for synthesizing monensin contains glucose and xylose, the mass ratio of glucose to xylose is (1-3):(1-3), or 2:(1-3). In a specific embodiment of the present invention, the mass ratio of glucose to xylose is 1:1, 1:2, 1:3, 2:3, 2:1 or 3:1. The setting of the mass ratio of glucose to xylose can increase the yield of monensin.

[0040] The present invention provides the use of the Streptomyces cinnamomi SDSL6002 described in the above technical solution or the bacterial agent described in the above technical solution in synthesizing monensin from lignocellulose and / or increasing the yield of monensin. As an optional embodiment, the lignocellulose described in the present invention includes lignocellulose enzymatic hydrolysate; the lignocellulose enzymatic hydrolysate contains straw sugar; the straw sugar includes glucose and xylose; the lignocellulose enzymatic hydrolysate includes bagasse enzymatic hydrolysate.

[0041] As an optional embodiment, when the bagasse hydrolyzate contains glucose and xylose, the mass ratio of glucose to xylose in the bagasse hydrolyzate of the present invention is (1-3): (1-3), or 2: 1. In a specific embodiment of the present invention, the mass ratio of glucose to 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 sugarcane bagasse enzymatic hydrolysate of the present invention is carried out with reference to a method for pretreating agricultural and forestry biomass using a surfactant-assisted method in Chinese patent CN116426585A. In a specific embodiment of the present invention, the preparation method of the sugarcane bagasse enzymatic hydrolysate of the present invention comprises: mixing sugarcane bagasse, glycerol, an alkaline catalyst and a non-ionic surfactant and heating them to obtain a heating product; filtering the heating product to obtain a solid matrix; enzymolyzing and centrifuging the solid matrix, and the obtained supernatant is the sugarcane bagasse enzymatic hydrolysate. As an optional embodiment, the sugarcane bagasse of the present invention is dried sugarcane bagasse; the sugarcane bagasse is sieved through a 20-mesh sieve. As an optional embodiment, the alkaline catalyst includes sodium hydroxide; the non-ionic surfactant includes PEG; the mass ratio of the sugarcane bagasse, glycerol, NaOH and PEG of the present invention is 10:100:0.5:0.5, the heating of the present invention is carried out under stirring, and the temperature is raised to 202°C during the heating; the temperature of the present invention is raised to 202°C and maintained for 43 minutes. As another optional embodiment, the present invention cools the heated product and then performs a first suction filtration to obtain a solid matrix for the first suction filtration. As an optional embodiment, the present invention cools to a temperature of 100±5°C, and the present invention adds boiling water and stirs during the first suction filtration, and the mass volume ratio of the heated product and boiling water is 1g:1.5mL; the stirring time is 10min. The present invention has no special restrictions on the parameters of the first suction filtration, and conventional methods can be used. As an optional embodiment, the present invention washes and re-filters the solid matrix for the first suction filtration to obtain a solid matrix. The present invention has no special restrictions on the parameters of the second suction filtration, and conventional methods can be used. The washing of the present invention is carried out with tap water, and the number of washings is 2 times. As another optional embodiment, the solid matrix of the present invention is dried and then enzymatically hydrolyzed and centrifuged, and the obtained supernatant is a sugarcane bagasse enzymatic hydrolyzate. The drying method of the present invention includes drying. The drying temperature is 60°C. As an optional embodiment, the enzyme used in the enzymatic hydrolysis of the present invention includes cellulase; a buffer and cellulase are added during the enzymatic hydrolysis of the present invention; the mass volume ratio of the obtained solid matrix to the buffer is 0.5g:25mL. The buffer includes a citric acid buffer. Based on the mass of the solid matrix, the cellulase added in the present invention is 5FPU / g. The temperature of the enzymatic hydrolysis of the present invention is 50°C and the time is 48h. The enzymatic hydrolysis of the present invention is carried out under stirring, and the stirring speed is 180rpm.

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

[0044] As an optional embodiment, the sugar-containing substrate of the present invention includes lignocellulose enzymatic hydrolysate.

[0045] As an optional embodiment, the lignocellulose enzymatic hydrolysate of the present invention contains straw sugar; the straw sugar includes glucose and xylose; the lignocellulose enzymatic hydrolysate includes bagasse enzymatic hydrolysate. The preparation method of the bagasse enzymatic hydrolysate has been discussed above and will not be repeated here.

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

[0047] The function of the nitrogen source and the inorganic salt in the present invention is to promote the growth of Streptomyces cinnamomi SDSL6002. The sources of the nitrogen source and the inorganic salt are not particularly limited in the present invention, and conventional products can be used.

[0048] As an optional embodiment, when the bagasse hydrolyzate of the present invention contains glucose and xylose, the mass ratio of glucose to xylose is (1-3): (1-3), or 2: 1. In a specific embodiment of the present invention, the mass ratio of glucose to xylose is 1: 1, 1: 2, 1: 3, 2: 3, 2: 1 or 3: 1.

[0049] As an optional embodiment, the culture temperature is 30-35°C, or 33-34°C. The culture time is 1-8 days, or 1-5 days. The temperature, time, and mass ratio of glucose to xylose of the present invention are set to increase the yield of monensin in Streptomyces cinnamomi SDSL6002.

[0050] The present invention provides a method for utilizing lignocellulose, comprising the following steps: mixing the cinnamon Streptomyces SDSL6002 described in the above technical solution or the bacterial agent described in the above technical solution with lignocellulose, culturing, and 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 embodiment, the present invention mixes the lignocellulose hydrolysate, a nitrogen source, and an inorganic salt to prepare a culture medium, and cultivates the cinnamon Streptomyces SDSL6002 to synthesize monensin. As an optional embodiment, the nitrogen source described in the present invention is soybean cake powder; based on the volume of the culture medium, the addition amount of the soybean cake powder described in the present invention is 30-40 g / L, or 35 g / L. As an optional embodiment, the inorganic salt is one or more of Na2SO4, K2HPO4, FeSO4·7H2O, Al2(SO4)3·7H2O, and CaCO3. As an optional embodiment, based on the volume of the culture medium, the amount of Na2SO4 added to the culture medium of the present invention is 2.0-2.4 g / L, or 2.2 g / L; based on the volume of the culture medium, the amount of K2HPO4 added to the culture medium of the present invention is 0.06-1.0 g / L, or 0.08 g / L; based on the volume of the culture medium, the amount of FeSO4·7H2O added to the culture medium of the present invention is 0.05-0.15 g / L, or 0.1 g / L; based on the volume of the culture medium, the amount of Al2(SO4)3·7H2O added to the culture medium of the present invention is 0.6-0.8 g / L, or 0.7 g / L; based on the volume of the culture medium, the amount of CaCO3 added to the culture medium of the present invention is 2.0-3.0 g / L, or 2.5 g / L. As an optional embodiment, the culture temperature is 30-35°C, or 33-34°C. The culture time is 1 to 8 days, or 1 to 5 days. The temperature, time, and mass ratio of glucose to xylose in the present invention are set to increase the yield of monensin in Streptomyces cinnamomi SDSL6002.

[0051] The lignocellulose of the present invention includes lignocellulose enzymatic hydrolysate; the lignocellulose enzymatic hydrolysate contains straw sugar; the straw sugar includes glucose and xylose; the lignocellulose enzymatic hydrolysate includes bagasse enzymatic hydrolysate. The preparation method of the bagasse enzymatic hydrolysate has been discussed above and will not be repeated here.

[0052] As an optional implementation manner, the present invention has no special limitation on the mixing, and conventional methods may be used.

[0053] As an optional embodiment, when the bagasse hydrolyzate of the present invention contains glucose and xylose, the mass ratio of glucose to xylose is (1-3): (1-3), or 2: 1. In a specific embodiment of the present invention, the mass ratio of glucose to xylose is 1: 1, 1: 2, 1: 3, 2: 3, 2: 1 or 3: 1.

[0054] The Streptomyces cinnamomi M1008 of the present invention is induced to obtain the Streptomyces cinnamomi SDSL6002, and the Streptomyces cinnamomi SDSL6002 has a high yield of monensin.

[0055] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0056] The method for determining the bacterial concentration used in the following examples is:

[0057] Take 10 mL of fermentation broth, centrifuge at 3000 rpm for 10 min, measure the volume of the supernatant, and calculate the bacterial concentration according to the following formula:

[0058] Bacterial concentration (%) = (fermentation broth volume - obtained supernatant volume) / fermentation broth volume × 100%

[0059] The composition of Gao's medium No. 1 used in the following examples is: 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 used in the following examples is: 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 were performed in the following examples.

[0062] Example 1 Preliminary study on the ability of Streptomyces cinnamomi M1008 to metabolize glucose and xylose

[0063] Streptomyces cinnamoneus is affected by stress conditions such as osmotic stress of substrate sugar during the fermentation of monensin, so it is essential to evaluate the high sugar tolerance of the strain. The present invention uses glucose in the range of 20 to 100 g / L as the carbon source, and uses glucose content, biomass (PMV), pH and monensin production as indicators to detect the growth and metabolism of the strain. The specific experiment is as follows:

[0064] 1. Glucose tolerance test

[0065] Shake flask seed culture: Pick Streptomyces cinnamonii from the fresh agar slant, number it as M1008 for easy identification, and inoculate it into a triangular flask with a volume of 250 mL and 50 mL of seed culture medium.

[0066] After inoculation, the culture was incubated at 33°C with a shaking incubator at 180 rpm for 24 h to obtain seed solution;

[0067] Fermentation culture: The seed liquid was inoculated into a triangular flask at a volume ratio of 10% of the culture medium. The volume of the triangular flask was 500 mL, and each triangular flask contained 50 mL of fermentation medium 1 to 5.

[0068] The composition of fermentation medium 1 was: 20 g / L glucose, 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;

[0069] The composition of fermentation medium 2 was: 40 g / L glucose, 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;

[0070] The composition of fermentation medium 3 was: 60 g / L glucose, 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;

[0071] The composition of fermentation medium 4 was: 80 g / L glucose, 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;

[0072] The composition of fermentation medium 5 was: 100 g / L glucose, 35 g / L soybean cake powder, 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.

[0073] After inoculation, the culture was incubated at 33°C with a shaking speed of 200 rpm for 6 days. The glucose content, bacterial cell concentration (PMV), monensin content and pH of the fermentation broth obtained at different culture times during the culture process were measured: the results are shown in Tables 1 to 4 and Figure 1 . Figure 1 The mean ± standard deviation of three parallel experiments in Tables 1 to 4 was plotted.

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

[0075]

[0076]

[0077] Table 2 Bacterial concentration in fermentation broth at different fermentation cycles (%)

[0078]

[0079] Table 3 pH of fermentation broth at different fermentation cycles

[0080]

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

[0082]

[0083] According to Tables 1 to 4 and Figure 1 It can be seen that the initial concentration of glucose is 60 g / L, the content of monensin in the fermentation broth is high, and the bacterial concentration 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] Shake 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: 60 g / L glucose, 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, wherein the mass ratio of glucose to xylose is 1:0;

[0088] The composition of fermentation medium 7 is: 60 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; wherein the mass ratio of glucose to xylose is 0:1;

[0089] The composition of fermentation medium 8 is: 30 g / L glucose, 30 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; wherein the mass ratio of glucose to xylose is 1:1;

[0090] The composition of fermentation medium 9 is: 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; wherein the mass ratio of glucose to xylose is 2:1;

[0091] The composition of the fermentation medium 10 is: 45 g / L glucose, 15 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; wherein the mass ratio of glucose to xylose is 3:1;

[0092] The composition of the fermentation medium 11 is: 20 g / L glucose, 40 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; wherein the mass ratio of glucose to xylose is 1:2;

[0093] The composition of the fermentation medium 12 is: 15 g / L glucose, 45 g / L xylose, 35 g / L soybean cake powder, 2.2 g / LNa2SO4, 0.08 g / LK2HPO4, 0.1 g / LFeSO4·7H2O, 0.7 g / LAl2(SO4)3·7H2O and 2.5 g / LCaCO3, wherein the mass ratio of glucose to xylose is 1:3.

[0094] After inoculation, the culture was carried out at 33°C with a shaking incubator at 200 rpm for 8 days. The sugar content of the fermentation broth was determined during the culture process. Figure 2 .according to Figure 2 It can be seen that when a single sugar is used as a carbon source, Streptomyces cinnamomi M1008 can not only utilize glucose but also xylose, but the two sugar consumption rates are very different, and the glucose consumption rate is significantly greater than the xylose consumption rate ( Figure 2 A), indicating that the ability of Streptomyces cinnamomi M1008 to utilize glucose is much greater than that of xylose, which is consistent with the metabolism of most microorganisms. Figure 2 B to F), the total sugar consumption trend of all experimental groups is basically the same, and glucose and xylose are consumed at the same time. This metabolic mode is different from the previously reported microorganisms that first use glucose and then use xylose to metabolize carbon sources, overcoming the glucose effect that exists in most microorganisms. The ability of Streptomyces cinnamomi M1008 to simultaneously utilize glucose and xylose is very important for fully utilizing lignocellulose hydrolysate and can realize the resource utilization of lignocellulose. 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. Figure 2 In D, the two curves are close to overlap, and glucose and xylose are consumed almost synchronously. More interestingly, when the mass ratio of glucose to xylose is 3:1 and 2:1, the ratio of their consumption rates is close to the mass ratio of their mixed sugars ( Figure 2 B-C), resulting in the almost simultaneous depletion of both sugars.

[0095] The results of the determination of pH, biomass (PMV) and monensin indexes during the fermentation of mixed sugars with different mass ratios are shown in Figure 2. Figure 3 As shown. Figure 3It can be seen that when the carbon source is a mixed sugar, the fermentation advantages of glucose and xylose are integrated. Glucose ensures the carbon source required for the growth of mycelium in the early stage, and xylose provides a 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, before the 144th hour of fermentation, the pH of the fermentation system is stable below 8.0, the bacterial concentration reaches 40% at 72 hours, and the highest growth reaches 42%. After the 144th hour of fermentation, monensin maintains the growth rate and finally reaches 4.5g / L, which is 309% higher than the monensin production of only glucose fermentation (i.e., glucose group), and 246% higher than the monensin production of only xylose fermentation (i.e., xylose group).

[0096] In summary, when the mass ratio of glucose to xylose was 2:1, the mixed sugars could be metabolized simultaneously, the pH was suitable, and the biomass and monensin yields were the largest. Therefore, the optimal mass ratio of glucose and xylose that Streptomyces cinnamomi M1008 could metabolize simultaneously was 2:1, and it had the potential to produce monensin using mixed sugars.

[0097] Example 2: Spaceflight-DES composite mutagenesis of Streptomyces cinnamomi M1008

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

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

[0100] 1. Spaceflight mutagenesis of Streptomyces cinnamomi M1008

[0101] Scrape the cultured cinnamon Streptomyces M1008 slant spores into a sterile skim milk powder solution to obtain a spore solution, the sterile skim milk powder solution having a milk powder mass concentration of 20%, freeze-dry the obtained spore solution, and prepare it into powder to obtain a freeze-dried powder. The freeze-dried powder was sent into space with the Shenzhou 14 for space mutagenesis. The cabin parameters of the space station and the Shenzhou spacecraft were: orbital altitude 400 kilometers, inclination 41 degrees, radiation dose 0.4 millisievert (mSv), cabin temperature 25°C, and humidity about 30%.

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

[0103] The freeze-dried powder of Streptomyces cinnamomi M1008 induced by space flight was diluted with sterile water and then spread on Gao's No. 1 medium plate for culture. Due to the high lethality of space flight mutation, the number of surviving single bacteria after mutation was small. A total of 479 single colonies were selected from Gao's No. 1 medium plate for microplate culture. The colony morphology of the strain before and after mutation is shown in Figure 5 Middle A.

[0104] Figure 5 A in the middle is the colony map of Streptomyces cinnamomi M1008 before mutagenesis and the colony map of the mutant strain obtained after mutagenesis. Figure 4 for Figure 5 A is an enlarged view of the middle image. Figure 5 The Ground control in A represents before mutagenesis, and the Space mutagenesis represents after mutagenesis.

[0105] The specific process of microplate culture is as follows: Use a sterile toothpick to scrape off half of the spores on each single colony on the culture medium plate, inoculate it in the seed culture medium of the 6-well plate and stir it gently. The seed culture medium in the 6-well plate is filled with 10mL. After the spore inoculation is completed, use another sterile toothpick to spot the remaining half of the spores of the single colony on the Gao's No. 1 medium plate, culture it in a 33℃ constant temperature incubator for 7 days until the colony matures, and then preserve it. If the same strain is inoculated in the 6-well plate and coated on the solid slant, use the same number to facilitate distinction and preservation from other strains.

[0106] After the 6-well plate with seed culture medium was cultured in a constant temperature shaker for 24 hours, the obtained seed solution was inoculated into the fermentation medium of 6 deep-well plates at a volume ratio of 7.5% (v / v), and the volume of the fermentation medium in each well was 10mL. The fermentation culture was carried out for 10 days at 33°C and 300rpm in a constant temperature shaker. The composition of the fermentation medium was: 40g / L glucose, 20g / L xylose, 35g / L bean cake powder, 2.2g / L Na2SO4, 0.08g / L K2HPO4, 0.1g / L FeSO4·7H2O, 0.7g / L Al2(SO4)3·7H2O and 2.5g / L CaCO3; three parallel experiments were set up.

[0107] At the end of fermentation, the content of monensin was detected by ELISA (see Figure 5 (B). Taking the monensin production of the starting strain M1008 ±10% as the standard, the monensin production of the starting strain M1008 +10% was recorded as A1, and the monensin production of the starting strain M1008 -10% was recorded as A2. If the monensin production of the mutant strain was less than A1 and greater than A2, it was defined as an isotropic mutation, if it was greater than A1, it was defined as a positive mutation, and if it was less than A2, it was defined as a negative mutation. Figure 5 FM in C is a positive mutation, EM is an isotropic mutation, and NM is a negative mutation. Figure 5 It can be seen from Figure C that the number of positive mutations, isotropic mutations and negative mutations after mutagenesis were 23, 129 and 322, respectively, and the corresponding positive, isotropic and negative mutation rates were 4.85%, 27.22% and 67.93%, respectively. This also shows that the space mutagenesis method is effective for Streptomyces cinnamomi M1008. Subsequently, five mutant strains of Streptomyces cinnamomi M1008, D16, D42, E38, F23 and H21, which increased the monensin production by 20%, were cultured in shake flasks. Three parallel experiments were set for each strain of shake flask culture. After the fermentation, the monensin production was detected by HPLC. Figure 5 D and Table 5. Figure 5 As shown in Figure D and Table 5, the yield of monensin in mutant F23 increased significantly (5.1 g / L), which was 18.77% higher than that of Streptomyces cinnamomi M1008.

[0108] Then, the mutant strain F23 was subjected to a study on its stability in subsequent generations. Three parallel experiments were set up for each study. Figure 5 The results showed that the main indicators of monensin production and bacterial concentration of mutant strain F23 remained stable within 5 generations. Therefore, mutant strain F23 was selected as the starting strain for the next step of DES mutagenesis. Figure 5 The mean ± standard deviation of three parallel experiments in Tables 5 to 7 were plotted.

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

[0110] Strain No. 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 production results (g / L) of mutant F23 passage stability

[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 The cell concentration results of the 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. Chemical mutagenesis of Streptomyces cinnamomi M1008 with diethyl sulfate

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

[0117] Take 2 mL of spore suspension of the high-yield mutant strain F23 obtained by spaceflight mutagenesis, with a spore concentration of 10 8 , then add 2mL phosphate buffer (pH 7.0), add DES solution, and make the volume fraction of DES in the reaction system 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, and then carry out the reaction in the dark at 33℃ for 20min, and then add 10mL of sodium thiosulfate solution with a mass concentration of 25% to terminate the reaction. After the mutagenesis is completed, the spore suspension is diluted appropriately, spread on Gao's medium No. 1 plate, and cultured at 33℃ for 5d. Observe the number of colonies and calculate the lethality. The formula for calculating the lethality is as follows:

[0118] Lethality rate (%) = (control colony - surviving colony after mutagenesis) / control colony × 100%

[0119] The results are as follows Figure 6 As shown in Tables 8 to 10, Figure 6 Figure A shows that the lethality of mutagenesis increases with the increase of DES addition. When the DES addition is 0.8%, the lethality is 85.6%, and when the DES addition is 1.2%, the lethality reaches 100%. The experiment intends to use 80% to 90% lethality as the mutagenic dose, so the DES addition is selected as 0.8% by volume.

[0120] After that, the F23 spore suspension was treated with 0.8% DES for 20 minutes, and then diluted and spread on a plate containing 25 μg / mL streptomycin resistance medium No. 1 for screening. 240 single bacteria were picked from the resistance plate for 6-well plate culture and preservation. After the fermentation, the monensin production was detected by an ELISA instrument. The results are as follows: Figure 6 As shown in B to C. Taking the monensin production of strain F23 ±10% as the standard, the monensin production of strain F23 +10% was recorded as A3, and the monensin production of strain F23 -10% was recorded as A4. If the monensin production of the mutant strain was less than A3 and greater than A4, it was defined as an isotropic mutation, greater than A3 was defined as a positive mutation, and less than A4 was defined as a negative mutation. Figure 6 FM of C is a positive mutation, EM is an isotropic mutation, and NM is a negative mutation. It can be seen that the positive, isotropic and negative mutation rates are 7.08%, 23.33% and 69.4%, respectively.

[0121] The five mutant strains with the highest monensin production (F23-65, F23-197, F23-229, F23-119, and F23-85) were screened in shake flasks. The monensin production of mutant F23-65 reached 5.65 g / L, which was 10.63% higher than that of the aerospace mutant F23 and 31.4% higher than that of the original strain M1008 ( Figure 6D, Table 8). Genetic stability tests showed that the 5-generation nemonensin production and bacterial concentration of mutant strain F23-65 remained stable ( Figure 6 E-F, Tables 9-10); three parallel experiments were set during the determination process. In addition, the 16SrRNA gene sequence (1390bp) of the first and fifth generations was 100% similar. Therefore, the mutant strain F23-65 was selected for the next study. Figure 6 The mean values ​​± standard deviations of three parallel experiments in Tables 8 to 10 were plotted.

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

[0123] Strain No. 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 passage stability of mutant F23-65

[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 passage stability of mutant strain F23-65 (%)

[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 mutants

[0130] The 16S rRNA of the high-yield mutant strain F23-65 was sequenced, and the result was shown in SEQ ID NO.1: The phylogenetic tree was constructed by comparing with the NCBI database, as shown in Figure 7 As shown, the information of closely related species was obtained, and the mutant strain was in the smallest branch with Streptomyces cinnamoneus DSM1042, which were similar species.

[0131] Combined with the phylogenetic tree, the high-yield mutant strain F23-65 was determined to be Streptomyces cinnamoneus, recorded as Streptomyces cinnamoneus SDSL6002; it was biologically preserved with the accession number CGMCC No.30409.

[0132] SEQ ID NO.1:

[0133]

[0134] 2. Verification of Streptomyces cinnamomi SDSL6002

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

[0136] Treatment Group:

[0137] (1) Shake flask seed culture: the same as the shake flask seed culture step in step 1 of Example 1.

[0138] (2) Mixed sugar fermentation culture: The seed liquid of Streptomyces cinnamomi SDSL6002 was inoculated into a triangular flask at a culture medium volume ratio of 10% for mixed sugar fermentation culture. The mixed sugar fermentation culture temperature was 33°C, the rotation speed was 200 rpm, and the time was 8 days. The volume of the triangular flask was 500 mL, and the triangular flask contained 50 mL of fermentation medium. The composition of the fermentation medium was: 40 g / L glucose, 20 g / L xylose, 35 g / L soybean cake powder, 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, bacterial cell concentration, glucose content and xylose content in the obtained fermentation broth were determined.

[0140] Control group: Same as the treatment group, the only difference is that Streptomyces cinnamomi SDSL6002 is replaced by Streptomyces cinnamomi M1008.

[0141] Results Figure 8 and Tables 11-14.

[0142] from Figure 8 It can be seen that the monensin production and bacterial concentration of the mutant strain SDSL6002 (i.e., F23-65) were significantly higher than those of M1008. The bacterial concentration reached the maximum value at 96h and maintained this concentration to synthesize monensin. The production of monensin gradually increased with the extension of fermentation time. SDSL6002 reached 5.65g / L at 192h of fermentation, which was 30.94% higher than that of M1008 (see Figure 8 A, Tables 11-12). From the perspective of sugar consumption, the presence of glucose does not inhibit the strain's use of xylose. S. cinnamomi SDSL6002 can consume glucose and xylose at the same time, and consumes the mixed sugars 24 h earlier than strain M1008 (see Figure 8 B, Tables 13-14). It can be seen that strain SDSL6002 can utilize glucose and xylose at the same time, and the sugar consumption rate is significantly higher than that of the original strain M1008. Figure 8 The mean values ​​± standard deviations of three parallel experiments in Tables 11 to 14 were plotted.

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

[0144]

[0145]

[0146] Table 12 Bacterial concentration of Streptomyces cinnamomi SDSL6002 and M1008 (%)

[0147]

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

[0149]

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

[0151]

[0152]

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

[0154] 1. Preparation of sugarcane bagasse enzymatic hydrolysate

[0155] Pretreatment of bagasse: crush the bagasse, pass through a 20-mesh sieve, and then dry in an oven at 60°C to constant weight. After drying, accurately weigh 10.0g of bagasse and put it into a 500mL conical flask, add 100.0g of glycerol, 0.50g of NaOH and 0.50g of PEG, mix evenly, place in a constant temperature heating jacket, heat to 202°C under stirring at 180rpm, and maintain for 43min. After the end, remove the heating jacket and continue to stir to dissipate heat and cool to 100°C ± 5°C, add 150mL of boiling water, stir for 10min, and then filter to separate the solid matrix and the pretreatment liquid. The solid matrix is ​​washed twice with 150mL of tap water again. The solid matrix obtained after filtration is placed in an oven and dried at 60°C to obtain a pretreated solid matrix.

[0156] 0.5 g of the pretreated solid matrix was added to 25 mL of citric acid buffer (pH 4.8), and 5 FPU / g of cellulase was added based on the mass of the solid matrix. The mixture was placed in a shaker at a speed of 180 rpm and a temperature of 50°C. After 48 h of enzymolysis, an enzymolysis solution was obtained. 0.4 mL of the enzymolysis solution was taken and centrifuged at 8000 rpm. The supernatant obtained was a bagasse enzymolysis solution, in which the fermentable sugar was 100 g / L and the mass ratio of glucose to xylose was 2:1.

[0157] 2. Fermentation of high-yield mutants in sugarcane bagasse hydrolysate to produce monensin

[0158] The composition of the fermentation medium of bagasse hydrolysate is as follows: the total sugar concentration of bagasse hydrolysate is 60g / L (i.e., the glucose content is 40g / L, the xylose content is 20g / L), soybean cake powder is 35g / L, 2.2g / LNa2SO4, 0.08g / LK2HPO4, 0.1g / LFeSO4·7H2O, 0.7g / LAl2(SO4)3·7H2O and 2.5g / LCaCO3. The preparation method is as follows: dilute the total sugar concentration of bagasse hydrolyzate to 60g / L (i.e., glucose content 40g / L, xylose content 20g / L), then add 35g / L soybean cake powder, 2.2g / L Na2SO4, 0.08g / L K2HPO4, 0.1g / LFeSO4·7H2O, 0.7g / LAl2(SO4)3·7H2O and 2.5g / LCaCO3; adjust the pH of the culture medium to 7.2 with NaOH before sterilizing, and use it after sterilization.

[0159] Treatment group: The mature cinnamon Streptomyces SDSL6002 was cultured on a slant and 1 cm 2 The slant was inoculated into the seed culture medium and cultured at 33°C, 180 rpm shaking for 24 h to obtain seed solution. The seed solution was inoculated into the fermentation medium of sugarcane bagasse enzymatic hydrolysate at an inoculation rate of 10% of the culture medium volume and cultured at 33°C, 200 rpm shaking for 192 h. Three parallel experiments were set up.

[0160] The control group was the same as the treatment group, the only difference being that Streptomyces cinnamomi SDSL6002 was replaced by Streptomyces cinnamomi M1008. Three parallel experiments were set up.

[0161] After the culture was completed, the amount of monensin synthesis, bacterial concentration and sugar content were determined. The results are shown in Fig. 9 and Tables 15-18.

[0162] The monensin synthesis of Streptomyces cinnamomi SDSL6002 was 5.42 g / L, which was 41% higher than that of the original strain Streptomyces cinnamomi M1008 (the increase was 31% in the synthetic medium, which also shows that the mutant strain metabolized more vigorously in the sugarcane bagasse hydrolysate); in terms of bacterial concentration, the mutant strain F23-65 reached a maximum of 44%, which was 10% higher than that of the original strain. (The increase was 5% in the synthetic medium, which again shows that the mutant strain metabolized more vigorously in the sugarcane bagasse hydrolysate); in terms of sugar consumption, the mutant strain F23-65 had a stronger sugar consumption ability than the original strain, and both glucose and glucose were consumed 2 days in advance. It can be seen that the mutant strain F23-65 can make good use of sugarcane bagasse hydrolysate to produce monensin. Fig. 9 The mean ± standard deviation of three parallel experiments in Tables 15 to 18 was plotted.

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

[0164]

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

[0166]

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

[0168]

[0169]

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

[0171]

[0172] In summary, the present invention provides a strain of Streptomyces cinnamomi SDSL6002, which can simultaneously utilize glucose and xylose to synthesize monensin, and has a high yield of monensin produced by fermentation in sugarcane bagasse enzymatic hydrolysate.

[0173] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Streptomyces cinnamoneus SDSL6002, characterized in that: The deposit number is CGMCC No.30409.

2. A bacterial agent, characterized in that It comprises the Streptomyces cinnamomi SDSL6002 described in claim 1.

3. Use of the Streptomyces cinnamomi SDSL6002 according to claim 1 or the bacterial agent according to claim 2 in synthesizing monensin and / or increasing the yield of monensin.

4. The use according to claim 3, characterized in that: When the application form of the Streptomyces cinnamomi SDSL6002 is culture solution, the inoculation amount of the culture solution is 8% to 12%; the temperature for synthesizing monensin is 30 to 35° C.; and the time is 1 to 8 days.

5. The use according to claim 3, characterized in that: The substrates for the synthesis of monensin contain glucose and xylose.

6. Use of the Streptomyces cinnamomi SDSL6002 according to claim 1 or the bacterial agent according to claim 2 in synthesizing monensin from lignocellulose and / or increasing the yield of monensin.

7. The use according to claim 6, characterized in that: The lignocellulose comprises lignocellulose enzymatic hydrolysate; the lignocellulose enzymatic hydrolysate contains straw sugar; the straw sugar comprises glucose and xylose; the lignocellulose enzymatic hydrolysate comprises bagasse enzymatic hydrolysate.

8. A method for synthesizing monensin or increasing the yield of monensin, characterized in that: The method comprises the following steps: mixing the Streptomyces cinnamomi SDSL6002 according to claim 1 or the bacterial agent according to claim 2 and a substrate containing sugar, culturing the mixture and synthesizing monensin; the sugar comprises glucose and xylose.

9. The method according to claim 8, characterized in that The sugar-containing substrate includes a lignocellulose hydrolysate; the lignocellulose hydrolysate contains straw sugar; the straw sugar includes glucose and xylose; the lignocellulose hydrolysate includes a bagasse hydrolysate; when the bagasse hydrolysate contains glucose and xylose, the mass ratio of glucose to xylose is (1-3):(1-3).

10. A method for utilizing lignocellulose, characterized in that: The method comprises the following steps: mixing the cinnamon Streptomyces SDSL6002 according to claim 1 or the bacterial agent according to claim 2 with lignocellulose, culturing and synthesizing monensin; the lignocellulose comprises lignocellulose enzymatic hydrolysate; the lignocellulose enzymatic hydrolysate contains straw sugar; the straw sugar comprises glucose and xylose; the lignocellulose enzymatic hydrolysate comprises bagasse enzymatic hydrolysate.

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