A strain producing coronatine based on a new mechanism, an efficient breeding method for coronatine high-yield strains and fermentation application

Abstract

The present invention discloses a strain that produces coronatine based on a new mechanism, an efficient breeding method for a high-yield coronatine strain, and fermentation application, and relates to the technical field of bioengineering. The strain that produces coronatine based on a new mechanism has a deposit number of CGMCC NO.33248, and is named Pseudomonas syringae ( Pseudomonas syringae )XZY; after mutagenesis, XZY was efficiently bred to obtain a gene carrying α‑AB R The phenotype of the coronatine high-producing strain, whose deposit number is CGMCC NO.30035, is named Pseudomonas syringae ( Pseudomonas syringae )XZY02. The fermentation yield of coronatine by the strain is greatly improved compared with the fermentation yield before mutation. In addition, the present invention also provides a new method for direct breeding of high-yield coronatine strains based on improving the downstream metabolic flow of threonine.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a strain for producing coronatine based on a new mechanism, and an efficient breeding method and fermentation application of a coronatine high-yield strain. Background Art

[0002] Coronatine (COR) is a structural analogue of jasmonic acid (JA), which is formed by connecting coronatine and coronanoid acid through an amide bond. Its molecular formula is C 18 H 25 NO 4 , with a molecular weight of 319 and the following structural formula:

[0003] .

[0004] Ichihara et al. discovered a plant effector factor produced by microorganisms and named it coronatine. Coronatine can regulate multiple physiological processes such as plant growth and development, especially inducing plants to produce defense responses to adverse environments such as pathogens, herbivores and abiotic stresses, and has great application value in the agricultural field.

[0005] According to reports, the following five microorganisms can produce coronatine, namely, Pseudomonas syringae pv. crimsonii, Pseudomonas syringae pv. sojae, Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. mulberry, and Burkholderia cepacia. Initially, Japanese scholar Ichihara Ogimita (1998) obtained 60 mg of coronatine (0.24 mg / L) from 250 L of fermentation broth. He believed that the low fermentation yield of coronatine led to high fermentation costs, which became an important factor limiting the large-scale industrial application of coronatine. Therefore, discovering high-yield strains of coronatine and improving the fermentation yield of coronatine have become research hotspots in recent years.

[0006] Wang Xiaofei et al. mentioned in the article "Preliminary Study on the Selection of Coronatine High-yielding Strains": Using pingyangmycin as a mutagen to isolate Burkholderia cepacia ( Burkholderia cepacian )189-20 (this strain carries kanamycin resistance, but its deposit information is not disclosed.) Three rounds of mutagenesis were performed and finally the cepacia Burkholderia ( Burkholderia cepacian) PWLII-12 mutant strain, the fermentation yield of coronatine of this mutant strain reached 333.4 mg / L, but the peak area calculation of the HPLC detection graph used in the article to prove the fermentation content of coronatine was wrong: the response peak height of 50 ug / mL coronatine standard was 0.06, and the peak width was 0.5 min; the response peak height of coronatine in the fermentation broth of 189-20 strain was 0.03, and the peak width was 0.5 min; the response peak height of coronatine in the fermentation broth of PWLII-12 strain was 0.05, and the peak width was 0.5 min; the response peak height of coronatine in the fermentation broth of PWLII-24 strain was 0.05, and the peak width was 0.5 min; thus, it can be concluded that the order of the response peak areas of coronatine from small to large is 189-20, PWLII-12, PWLII-24, and 50ug / mL standard, that is, the coronatine concentrations in the fermentation broths of strains 189-20, PWLII-24, and PWLII-12 are all less than 50 mg / L coronatine standard solution, and do not reach the claimed 333.4 mg / L.

[0007] Yu Sha et al. revealed in the paper "Optimization of Coronatine Fermentation Process and Study on the Mechanism of Action of Key Environmental Factors" that in strain Z2-6, the synthetic precursor of coronatine is isoleucine (Ile), and the addition of isoleucine can increase the fermentation yield of coronatine in strain Z2-6; the strain used for fermentation of coronatine is Pseudomonas syringae pv. sojae ( Pseudomonas syringae pv. glycinea ) Z2-6; the GC optimized culture medium (hereinafter referred to as GC) was obtained by optimizing the carbon source, nitrogen source and inorganic salt culture medium in the GC fermentation culture medium. 于优 ), strain Z2-6 was inoculated to GC 于优 Under shake flask conditions (250 rpm, 18°C ​​for 14 days), the fermentation concentration of coronatine in the culture medium was increased to 214.7 mg / L. 于优 The fermentation concentration of the culture medium in a fermenter (100L) was 187.1 mg / L. If 0.7 g / L isoleucine (Ile) was added to the fermenter, the concentration of the crown bacteria fermented by strain Z2-6 could be increased to 192.7 mg / L.

[0008] Using traditional random mutagenesis technology to screen mutant strains that meet the requirements is a classic method, but screening mutant strains that meet the requirements has the disadvantages of being time-consuming and labor-intensive. The reason is that mutagens are lethal to strains, and 95%-99% of strains will die after mutagenesis; for those surviving strains (accounting for 1%-5%), 50% may mutate, and 50% may not mutate; further, mutagens are random in their mutation of strain genes, that is, there is no guarantee that mutagens only mutate the target gene; for example, after the coronatine-producing strain XZY is mutated, it may be that any one of the genes 1, 2, 3...n in its body mutates, or it may be that any of the genes 1 to n mutate at the same time; in addition, for any single gene, 33% may mutate positively, 33% may mutate negatively, and 33% may not mutate; Gemperlein et al. in Synthetic biology approaches to establish a heterologous production system for The article "The Study on the Production of Coronatines" reported that there are dozens of genes related to coronatine synthesis. Therefore, it is extremely difficult to make all genes that may be related to coronatine synthesis mutate in the direction of increasing coronatine production. Therefore, it requires a huge screening base and accumulation of workload, and the process is time-consuming and laborious. If those possible high-yield mutant strains can be marked by specific substances, it will greatly shorten the breeding time and help improve the efficiency of breeding.

[0009] Currently, there is a lack of breeding methods that can efficiently obtain high-yield coronatine mutant strains. In the actual production process, due to the lack of higher-yield coronatine-producing strains, the fermentation production efficiency of coronatine cannot be further improved.

[0010] In view of this, the present invention is proposed. Summary of the invention

[0011] The object of the present invention is to provide a high-yield strain for producing coronatine based on a new mechanism, a high-yield fermentation medium for coronatine, and a method for efficiently breeding a high-yield strain for coronatine, thereby increasing the fermentation yield of coronatine.

[0012] The present invention is achieved in that:

[0013] In the first aspect, the present invention provides a novel mechanism of coronatine production strain, which has a deposit number of CGMCC No. 33248 and is named Pseudomonas syringae ( Pseudomonas syringae )XZY.

[0014] In a second aspect, the present invention also provides a method for breeding a high-producing strain of coronatine based on a new mechanism, which comprises the following steps: mutagenizing the basic bacteria, and then using α-aminobutyric acid (α-AB) as a screening marker to screen strains with α-AB resistance as high-producing coronatine strains, wherein the basic bacteria are selected from the genus Pseudomonas, and the deposit number of the basic bacteria is: CGMCC No. 33248, which is named Pseudomonas syringae ( Pseudomonas syringae )XZY.

[0015] In a third aspect, the present invention provides a coronatine high-yielding strain XZY02 obtained by mutation using the above strain and breeding method, and its deposit number is CGMCC NO.30035, which is named Pseudomonas syringae ( Pseudomonas Syringae )XZY02.

[0016] In a fourth aspect, the present invention also provides a coronatine fermentation medium (GC 2 Thr), each liter or kg of culture medium includes: ammonium chloride 1.0-1.2 grams, potassium dihydrogen phosphate 1.1-4.1 grams, potassium hydrogen phosphate trihydrate 3.6-8.6 grams, magnesium sulfate heptahydrate 0.2 grams, potassium nitrate 0.6-1.8 grams, glycerol 20 grams, ferric chloride hexahydrate 0.0054 grams and threonine 0.5-1.5 grams, pH value is 7.0-7.8.

[0017] In a fifth aspect, the present invention further provides a method for producing coronatine, comprising the following steps: culturing the above-mentioned coronatine high-yield strain or culturing the above-mentioned coronatine production strain based on the new mechanism.

[0018] The present invention has the following beneficial effects:

[0019] The present invention is effective against coronatine-producing Pseudomonas syringae ( Pseudomonas syringae ) XZY was studied and it was found that although strain XZY and Z2-6 of Yu Sha et al. are of the same genus, they are not the same subspecies; and strain XZY does not use isoleucine as a precursor to synthesize coronatine, but uses threonine downstream metabolites (ɑ-ketobutyric acid, ɑ-acetyl-ɑ-hydroxybutyric acid, ɑ, β-dihydroxy-β-methylvaleric acid, ɑ-keto-β-methylvaleric acid) as precursors to synthesize coronatine, which indicates that the coronatine synthesis mechanism in strain XZY and strain Z2-6 is essentially different; Based on this discovery, the present invention also provides a new strategy method for efficiently breeding high-yield coronatine strains based on improving the downstream metabolic flow of threonine, and successfully obtained a high-yield coronatine strain CGMCC NO.30035, whose coronatine fermentation yield is greatly improved compared with the fermentation yield before mutation.

[0020] The mechanism of the method for breeding a high-yield coronatine strain provided by the present invention is:

[0021] Because the strain XZY of the present invention synthesizes coronatine using the downstream metabolites of threonine as precursors, after the strain XZY is mutated, only those mutant strains that survive and can tolerate high concentrations of α-aminobutyric acid have a greater chance of positive mutations in threonine dehydratase (TD) and acetohydroxyacid synthase (AS), which enhances the activity of the two enzymes, thereby increasing the downstream metabolic flow of threonine, including the accumulation of α-ketobutyric acid, α-acetyl-α-hydroxybutyric acid, α, β-dihydroxy-β-methylvaleric acid, and α-keto-β-methylvaleric acid, thereby increasing the coronatine yield in the mutant strain XZY02. Therefore, the present invention provides a method for efficiently breeding a high-yield coronatine strain by selectively breeding a mutant strain with α-aminobutyric acid resistance after mutagenesis of a specific microorganism.

[0022] The high-yield coronatine strain CGMCC NO.30035 can further increase the fermentation yield of coronatine in the strain by fermenting coronatine in the fermentation medium containing threonine provided by the present invention, and the fermentation content can reach about 400 mg / L.

[0023] In addition, the present invention also analyzed the amino acid-related transcriptional differential genes of the coronatine high-yield strain (CGMCC No. 30035) and the basic strain XZY (CGMCC No. 33248), and found that the threonine metabolic pathway was significantly enhanced. This further confirms that α-aminobutyric acid, as a breeding screening marker, enhances the metabolic flow of threonine to produce ɑ-ketobutyric acid, ɑ-acetyl-ɑ-hydroxybutyric acid, ɑ, β-dihydroxy-β-methylvaleric acid, and ɑ-keto-β-methylvaleric acid, and this metabolic flow can be used as a precursor to promote the production of coronatine. These transcriptional differential genes are expected to become targets for the next step of genetic modification or editing, which is of great significance for improving the fermentation production efficiency of coronatine. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is the experimental result of the effect of different C / N ratios on the shake flask yield of coronatine;

[0026] Figure 2 For GC 2 Effects of adding different composite organic nitrogen sources to the culture medium on the yield of coronatine in shake flasks;

[0027] Figure 3 For GC 2 Effects of adding different amino acids to the culture medium on the yield of coronatine in shake flasks;

[0028] Figure 4 These are five amino acid metabolic flows that have significant inhibitory or promoting effects on coronatine;

[0029] Figure 5 MA plots for visualization of differentially transcribed genes comparing XZY and XZY02;

[0030] Figure 6 This is a diagram showing the transcriptional differences of genes in the amino acid metabolism pathway of the mutant strain XZY02 compared to the original strain XZY;

[0031] Figure 7 This is a graph showing the experimental results of the effect of different initial pH values ​​on the shake flask yield of coronatine in mutant strain XZY02. DETAILED DESCRIPTION

[0032] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0033] In the first aspect, the present invention provides a novel coronatine metabolic mechanism strain (i.e., it can use threonine metabolic flow as a precursor to synthesize coronatine, but cannot use isoleucine to synthesize coronatine), which has a deposit number of CGMCC No. 33248 and is named Pseudomonas syringae ( Pseudomonas syringae )XZY; also provided a strain based on XZY, which was selected from a number of mutant strains after mutagenesis and had a high coronatine production rate. Its deposit number is CGMCCNO.30035, and it is named Pseudomonas syringae ( Pseudomonas syringae )XZY02.

[0034] The biological deposit information of strain XZY is as follows: the strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on December 30, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 33248. The classification name is Pseudomonas syringae Pseudomonas syringae , the identification result is survival.

[0035] The biological deposit information of strain XZY02 is as follows: The strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on March 15, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 30035. It is classified and named Pseudomonas syringae Pseudomonas syringae , the identification result is survival.

[0036] By random chemical mutagenesis (specifically NTG) of the basic strain XZY (CGMCC No. 33248) that produces coronatine with a new mechanism, we obtained a strain carrying α-AB by breeding. R The XZY02 strain, which has an α-aminobutyric acid-resistant phenotype and high coronatine production, has the following colony characteristics on the LB plate: light yellow, round, slightly convex in the center, sticky in texture, and neat at the edges.

[0037] In a second aspect, the present invention also provides the use of a coronatine high-yielding strain in producing coronatine.

[0038] The coronatine high-yield strain provided by the present invention can efficiently accumulate coronatine and can be used for industrial production of coronatine.

[0039] Coronatine has at least one of the following uses: plant stress resistance, disease and pest resistance, growth regulation, and improving the quality of agricultural products; improving the quality of agricultural products includes but is not limited to fruit coloring and sugar enhancement.

[0040] In a third aspect, the present invention further provides a fermentation medium for producing coronatine, wherein per liter or per kg of the medium comprises: 1.0-1.2 g of ammonium chloride, 1.1-4.1 g of potassium dihydrogen phosphate, 3.6-8.6 g of dipotassium hydrogen phosphate trihydrate, 0.2 g of magnesium sulfate heptahydrate, 0.6-1.8 g of potassium nitrate, 20 g of glycerol, 0.0054 g of ferric chloride hexahydrate and 0.5-1.5 g of threonine, and the pH value of the fermentation medium is 7.0-7.8.

[0041] In a preferred embodiment of the present invention, the pH value of the fermentation medium is 7.4-8.0.

[0042] In a fourth aspect, the present invention further provides a method for producing coronatine, comprising the following steps: culturing the coronatine high-yield strain XZY02 or culturing the coronatine basic strain XZY.

[0043] The coronatine high-producing strain is inoculated and cultured in a fermentation medium. The fermentation medium contains 1.0-1.2 grams of ammonium chloride, 1.1-4.1 grams of potassium dihydrogen phosphate, 3.6-8.6 grams of potassium dihydrogen phosphate trihydrate, 0.2 grams of magnesium sulfate heptahydrate, 0.6-1.8 grams of potassium nitrate, 20 grams of glycerol, 0.0054 grams of ferric chloride hexahydrate and 0.5-1.5 grams of threonine per liter or per kg. The pH value of the fermentation medium is 7.0-8.0. Under the above formula, the strain is more conducive to producing coronatine.

[0044] The C / N ratio is a key parameter for culturing bacteria. A suitable C / N ratio is conducive to promoting microbial growth and improving metabolic efficiency. For example, the C / N ratio can affect the arginine production in Escherichia coli and the lipid production in oleaginous yeast. The specific method for adjusting the C / N ratio of the present invention is to optimize the amount of glycerol, potassium nitrate and ammonium chloride in the GC culture medium: under the condition of the fermentation medium with a C / N ratio of 15:1, it is more conducive to the production of coronatine by the strain, and NO 3 - Than NH 4+ More conducive to the production of coronatine; but NO 3 - Too much will result in a too low C / N ratio, which is not conducive to the production of coronatine.

[0045] In order to increase the growth rate of the bacteria and shorten the fermentation cycle of coronatine, the inventors tried to add a variety of composite organic nitrogen sources commonly used in industrial fermentation (N%<10%) to the culture medium with the best fermentation effect, and tested the effects of different composite nitrogen sources on the shake flask yield of coronatine. Through the test, 6 composite organic nitrogen sources with greater impact on industrial fermentation were screened out. Adding any of the above composite nitrogen sources to the culture medium can shorten the fermentation cycle of coronatine, but it has a very significant negative effect on the fermentation concentration of coronatine. The negative effects are ranked from large to small: peptone> yeast powder> fish meal> peanut cake powder> soybean cake powder> cottonseed cake powder. Therefore, the inventors speculated that the different types and contents of amino acids contained in the composite nitrogen source are the main reasons for this negative difference; this also suggests that some amino acids are not conducive to the accumulation of coronatine, while some amino acids are conducive to the accumulation of coronatine. Therefore, the inventors further investigated the effects of different amino acids on the fermentation concentration of coronatine.

[0046] By testing the effects of 18 kinds of amino acids on the yield of coronatine in shake flasks, it was found that when isoleucine (Ile), valine (Val), leucine (Leu), methionine (Met) or lysine (Lys) were added to the culture medium, the production of coronatine was significantly inhibited, while the addition of threonine (Thr) to the culture medium significantly promoted the production of coronatine. Therefore, the inventors chose to add threonine to the fermentation medium to promote the production of coronatine. Therefore, the inventors inferred that in the basic strain XZY, the downstream metabolites of threonine (Thr) (ɑ-ketobutyric acid, ɑ-acetyl-ɑ-hydroxybutyric acid, ɑ, β-dihydroxy-β-methylvaleric acid, ɑ-keto-β-methylvaleric acid) are the precursors for the synthesis of coronatine. Based on this inference, the inventors also randomly induced mutagenesis on the basic strain XZY, and then used α-AB to selectively breed mutant strains with enhanced downstream metabolic flow of threonine, and obtained the coronatine high-producing strain XZY02. In addition, this inference was proven to be correct after the subsequent analysis of differentially transcribed genes.

[0047] By exploring the effect of the initial pH of the culture solution on the yield of coronatine, it was found that an alkaline environment is conducive to the fermentation of coronatine by strain XZY02, while an acidic environment is not conducive to its fermentation to produce coronatine. Therefore, the initial pH value of the fermentation culture is set to 7.0-8.0. For example, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0048] In a preferred embodiment of the present invention, the culture is performed at 18°C ​​± 0.5°C with shaking at 225 rpm ± 25 rpm.

[0049] In a preferred embodiment of the present invention, shaking culture is performed for 10-14 days.

[0050] In a preferred embodiment of the present invention, the pH value of the fermentation medium is 7.4-8.0. Under such culture conditions, the strain XZY02 is conducive to fermenting and producing more coronatine.

[0051] The fermentation medium can be a solid medium, a liquid medium or a semi-solid medium.

[0052] In a fifth aspect, the present invention also provides use of a fermentation medium for producing coronatine in fermenting coronatine.

[0053] In a sixth aspect, the present invention further provides a method for breeding a high-producing strain of coronatine, wherein the basic bacteria are mutated by a mutagenesis technique, α-aminobutyric acid (α-AB) is used as a screening marker, and strains having α-AB resistance are screened as strains with high coronatine production, the basic bacteria are selected from the genus Pseudomonas, and the deposit number of the basic bacteria is: CGMCC No. 33248, which is named Pseudomonas syringae ( Pseudomonas syringae )XZY.

[0054] The method for breeding a high-yield coronatine strain has the following technical advantages:

[0055] Compared with the previous blind screening after random mutagenesis, the screening method provided by the present invention can more efficiently and quickly screen out coronatine high-producing strains through α-aminobutyric acid labeling.

[0056] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0057] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0058] Example 1

[0059] Strain XZY in GC medium and GC 于优 Fermentation yield in culture broth.

[0060] Strain XZY was isolated from the rhizosphere soil of kiwi fruit trees in Pujiang County, Chengdu City, Sichuan Province, and has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.33248.

[0061] Strain XZY was inoculated into GC (according to Table 1) and GC 于优 (The culture medium was prepared according to the optimized formula published by Yu Sha, see Table 2) The culture medium was placed at 18°C ​​with continuous shaking (250 rpm) for 14 days, and the fermentation yield of coronatine was measured.

[0062] Table 1 GC culture medium

[0063]

[0064] Table 2 GC 于优 Culture medium

[0065]

[0066] The results showed that under shake flask conditions, the fermentation yield of XZY in GC culture medium was ~87 mg / L, while in GC 于优 The fermentation yield in GC was ~81 mg / L. 于优 The cost of ammonium chloride, phosphate and glycerol in the culture medium is higher than that of GC culture medium, but GC 于优 The culture solution did not significantly improve the fermentation yield of coronatine in strain XZY, so strain XZY is not suitable for GC 于优 The culture broth is used for coronatine fermentation; therefore, in order to further increase the coronatine fermentation yield of strain XZY, the present invention will re-optimize the GC culture broth in subsequent examples.

[0067] Example 2

[0068] Optimize the carbon-nitrogen ratio (C / N ratio) in GC culture medium.

[0069] The C / N ratio is a key parameter for culturing bacteria. A suitable C / N ratio is conducive to promoting microbial growth and improving metabolic efficiency. For example, the C / N ratio can affect the arginine production in Escherichia coli and the lipid production in oleaginous yeast. The specific method for adjusting the C / N ratio is to adjust the amount of glycerol, potassium nitrate and ammonium chloride in the GC culture medium so that the C / N element mass ratio is adjusted from 25:1 to 20:1, 15:1 and 10:1 respectively (Table 3).

[0070] Table 3 C / N element mass ratio adjustment scheme

[0071]

[0072] After adjusting the C / N ratio of the GC culture medium, XZY was inoculated into a shake flask for fermentation (fermented at 18°C ​​and 250 rpm for 14 days, during which the coronatine content was tested every day. The test results showed that the coronatine fermentation concentration in the GC culture medium was 88 mg / L ( Figure 1 A in ); while GC 1 and GC 2 The fermentation concentrations of coronatine in the culture medium reached 121 mg / L and 161 mg / L, respectively, which were 38.5% and 82.9% higher than that of GC (88 mg / L), indicating that with the increase of nitrate ions (NO 3 - ) concentration increases, the fermentation level of coronatine gradually increases ( Figure 1 B and Figure 1 C in ); but when GC 3 NO 3 - When the concentration increased to 3.8 g / L, the peak fermentation concentration of coronatine decreased to 77 mg / L ( Figure 1 D in the figure indicates that a low C / N ratio is not conducive to the formation of coronatine. 4 + ) concentration, although the bacterial growth rate accelerated and the coronatine concentration reached its peak on the 10th to 12th day (2 to 4 days earlier), all the 4 + Concentration treatment group (GC 4 , GC 5 , GC 6 ) were only 61.3%, 45%, and 38.9% of those in GC culture medium, respectively. Figure 1 E in Figure 1 F and Figure 1 G in the figure), indicating that excess NH 4 + Therefore, the culture medium suitable for XZY strain fermentation of coronatine is GC with a C / N ratio of 15:1. 2 culture medium; and NO 3 - It is more conducive to the production of coronatine by strain XZY.

[0073] GC 2 The culture medium is as follows:

[0074]

[0075] Example 3

[0076] Test on the effect of different composite nitrogen sources on the yield of coronatine in shake flasks.

[0077] The test results of Example 2 show that NH 4 + Although it is not suitable for the accumulation of coronatine, it can shorten the fermentation cycle. Therefore, in order to increase the growth rate of the bacteria and shorten the fermentation cycle of coronatine, we tried to obtain the GC with the best fermentation effect in Example 2. 2 Six kinds of composite organic nitrogen sources commonly used in industrial fermentation (N%<10%) were added to the culture medium. In order to minimize the impact of the composite nitrogen source on the C / N ratio, the addition amount was set at 1 g / L (due to the small amount of nitrogen added, its impact on the C / N ratio is negligible). The addition scheme is shown in Table 4.

[0078] Table 4 Addition scheme of composite nitrogen source

[0079]

[0080] After the addition, strain XZY was inoculated into the shake flasks containing the above-mentioned culture solutions, and the culture solutions were shaken and fermented at 18°C ​​and 250 rpm for 14 days. The coronatine fermentation concentration in each culture solution was then tested. The test results showed that: GC 2 Adding any of the above composite nitrogen sources to the culture medium can shorten the fermentation period of coronatine, but it has a very significant negative effect on the fermentation concentration of coronatine. The negative effects are ranked from largest to smallest: peptone>yeast powder>fish meal>peanut cake powder>soybean cake powder>cottonseed cake powder ( Figure 2 ). Therefore, it is speculated that the difference in the types and contents of amino acids contained in the composite nitrogen source is the main reason for this negative difference; this also suggests that some amino acids are not conducive to the accumulation of coronatine, while some amino acids are conducive to the accumulation of coronatine. Therefore, the effects of different amino acids on the fermentation concentration of coronatine will be investigated in subsequent examples.

[0081] Example 4

[0082] Test on the effect of different types of amino acids on the yield of coronatine in shake flasks.

[0083] In order to further clarify the effects of different types of amino acids on the fermentation yield of coronatine, in this example, 1 g / L of different amino acids were added to the GC-containing 2 In the shake flask of culture medium; 1 GC is formed 2 The control group and 18 treatment groups supplemented with 1 g / L amino acids (Table 5).

[0084] Table 5 Amino acid addition scheme

[0085]

[0086] After the addition, strain XZY was inoculated into each group of shake flasks, and the fermentation was carried out at 18°C ​​and 250 rpm for 14 days. Then the coronatine fermentation concentration in each group was measured. 2 After threonine was added to the Thr culture medium, the concentration of coronatine produced by fermentation increased to 196 mg / L, which was higher than that of GC 2 The fermentation concentration of coronatine (158.6 mg / L) increased by 23% ( Figure 3 ); when the GC 2 After adding isoleucine (Ile), valine (Val), leucine (Leu), methionine (Met) or lysine (Lys) to the culture medium, all of them had a very significant negative effect on the fermentation concentration of coronatine. 2 Ile, GC 2 Val, GC 2 Leu, G.C. 2 Met, GC2 Lys) were compared with GC 2 decreased by 145.9%, 174.9%, 240.3%, 210.4% and 135.0% ( Figure 3 ).according to Figure 3 Result: To GC 2 The addition of threonine (Thr) to the culture medium significantly promoted the production of coronatine, while the addition of any of the five amino acids, isoleucine (Ile), valine (Val), leucine (Leu), methionine (Met) and lysine (Lys), significantly inhibited the production of coronatine.

[0087] Therefore, in strain XZY, threonine (Thr) downstream metabolites (ɑ-ketobutyric acid, ɑ-acetyl-ɑ-hydroxybutyric acid, ɑ, β-dihydroxy-β-methylvaleric acid, ɑ-keto-β-methylvaleric acid) are precursors for coronatine synthesis.

[0088] Example 5

[0089] Breeding of high-producing coronatine strains based on the strategy of improving the downstream metabolic flow of threonine.

[0090] In the bacterial amino acid metabolic pathway, leucine (Leu), valine (Val) and isoleucine (Ile) can be converted into each other under the action of branched-chain aminotransferase (BCAT); excessive accumulation of leucine (Leu), valine (Val) and isoleucine (Ile) will inhibit the activity of threonine dehydratase (TD), thereby cutting off the pathway for threonine (Thr) to be metabolized downstream to form α-ketobutyrate; excessive accumulation of lysine (Lys) will inhibit the activity of aspartate kinase (AK), thereby cutting off the metabolic pathway for the formation of threonine (Thr); excessive accumulation of methionine (Met) will inhibit the activity of homoserine kinase / dehydrogenase (HK / HD), thereby cutting off the metabolic pathway for the formation of threonine (Thr).

[0091] In strain XZY, the precursor of coronatine is one or more of the downstream metabolites of threonine (Thr), namely, ɑ-ketobutyric acid, ɑ-acetyl-ɑ-hydroxybutyric acid, ɑ, β-dihydroxy-β-methylvaleric acid, and ɑ-keto-β-methylvaleric acid. This also explains why the fermentation concentration of coronatine decreased significantly after adding isoleucine (Ile), valine (Val), leucine (Leu), methionine (Met), or lysine (Lys) in Example 4, and the fermentation concentration of coronatine was increased only after adding threonine (Thr). Figure 4 ).

[0092] Therefore, in this embodiment, α-AB resistant mutant strains are selected to release the repression of the three amino acids, isoleucine (Ile), valine (Val), and leucine (Leu), on the activities of threonine dehydratase (TD) and acetohydroxyacid synthase (AS), thereby enhancing the metabolic flow of threonine (Thr).

[0093] The strain XZY was chemically mutagenized by using NTG at a concentration of 0.5 g / L for 20 min, and then using α-AB at a concentration of 0.16-0.35 g / L as a resistance screening marker. R The XZY02 strain with high coronatine production has the following colony characteristics on the LB plate: light yellow, round, slightly convex in the center, sticky, and neat edges. It has been submitted to the General Microbiology Center of the China Microbiological Culture Collection Administration, and the deposit number is CGMCC NO.30035. 2 Thr culture medium was used for shake flask fermentation, and the fermentation concentration of coronatine in mutant strain XZY02 reached ~250 mg / L, which was 25.0% higher than that of the original strain XZY (~200 mg / L).

[0094] GC 2 The formula of Thr culture solution (i.e., the fermentation medium provided by the present invention) is as follows:

[0095]

[0096] Example 6

[0097] Transcriptome differences between strains XZY and XZY02.

[0098] The original strain XZY and mutant strain XZY02 were inoculated into GC 2 Thr culture medium was fermented in shake flasks at 18°C ​​and 250 rpm for 14 days, and then the two strains were collected to determine the transcriptome differences between the two. It was found that under the same fermentation conditions, compared with the original strain XZY, the transcription levels of 1074 genes (20%) in the mutant strain XZY02 were significantly upregulated, and the transcription levels of 195 genes (3.6%) were significantly downregulated ( Figure 5 ). Up-regulated genes indicates the number of genes with upregulated transcriptional levels between XZY and XZY02; down-regulated genes indicates the number of genes with downregulated transcriptional levels between XZY and XZY02; notdifferentially expressed indicates the number of genes with no significant difference in transcriptional levels between XZY and XZY02.

[0099] KEGG pathway analysis of amino acid metabolism-related genes in the differentially expressed genes of XZY02 vs XZY revealed that threonine metabolic flux was significantly enhanced ( Figure 6 ). Among them, red indicates up-regulation and green indicates down-regulation. This fully demonstrates that α-AB R As a breeding selection marker, it enhances the metabolic flow of threonine to the downstream production of ɑ-ketobutyric acid, ɑ-acetyl-ɑ-hydroxybutyric acid, ɑ, β-dihydroxy-β-methylvaleric acid, and ɑ-keto-β-methylvaleric acid, and this metabolic flow can serve as a precursor to promote the production of coronatine.

[0100] Example 7

[0101] GC 2 Effect of initial pH of Thr culture medium on coronatine production.

[0102] Since the transcription levels of most genes in strain XZY02 have changed, and pH is an important indicator affecting strain culture, it is crucial to find the initial pH of the culture medium suitable for the growth of this mutant strain. The appropriate initial pH may increase the final fermentation concentration of coronatine. Therefore, GC with different initial pH values ​​was prepared. 2 Thr culture medium (Table 6).

[0103] Table 6 GC at different initial pH values 2 Thr culture medium grouping scheme

[0104]

[0105] After the preparation, strain XZY02 was inoculated into each culture medium, and the fermentation was carried out at 18°C ​​and 250 rpm for 14 days. The fermentation concentration of coronatine in each culture medium was measured. After the test, the results showed that the GC at initial pH of 6.2, 6.5, 7.1, 7.4, 7.7 and 8.0 2 In Thr culture medium, the coronatine fermentation concentrations of strain XZY02 reached 25 mg / L, 49 mg / L, 306 mg / L, 378 mg / L, 399 mg / L and 353 mg / L, respectively; the GC with an initial pH of 6.8 was reached 2 10%, 19%, 123%, 152%, 161% and 142% of the Thr concentration in the culture medium (248 mg / L) Figure 7 ), indicating that an alkaline environment is conducive to the fermentation of coronatine by strain XZY02, while an acidic environment is not conducive to its fermentation to produce coronatine.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain for producing coronatine, characterized in that: Its deposit number is CGMCC NO.30035, and it is named Pseudomonas syringae ( Pseudomonas syringae )XZY02.

2. Use of the strain producing coronatine as claimed in claim 1 in producing coronatine.

3. A method for producing coronatine, characterized in that: The method comprises the following steps: culturing the coronatine-producing strain according to claim 1.

4. The method for producing coronatine according to claim 3, characterized in that: The coronatine-producing strain is inoculated in a fermentation medium for cultivation. The fermentation medium contains, per liter or per kg, 1.0-1.2 g of ammonium chloride, 1.1-4.1 g of potassium dihydrogen phosphate, 3.6-8.6 g of dipotassium hydrogen phosphate trihydrate, 0.2 g of magnesium sulfate heptahydrate, 0.6-1.8 g of potassium nitrate, 20 g of glycerol, 0.0054 g of ferric chloride hexahydrate and 0.5-1.5 g of threonine. The pH value of the fermentation medium is 7.0-8.

0.

5. The method for producing coronatine according to claim 4, characterized in that: The pH value of the fermentation medium is 7.4-8.

0.

6. The method for producing coronatine according to claim 4, characterized in that: The culture was incubated at 18°C ​​± 0.5°C with shaking at 225 rpm ± 25 rpm.

7. The method for producing coronatine according to claim 6, characterized in that: The culture was shaken for 10-14 days.

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