High-yield mutant strain and application thereof in immunosuppressant
By performing ARTP mutagenesis and rifampicin resistance screening on the original strain, a high-yielding mutant strain, Rif-d6, was obtained, which solved the problem of slow antibiotic yield increase in the existing technology and achieved a significant increase in antibiotic yield and optimization of metabolic pathway.
Patent Information
- Application Number
- CN202510848866.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-24
- Publication Date
- 2025-11-04
AI Technical Summary
Existing technologies cannot effectively screen for strains that can promote antibiotic production. Traditional methods, such as ARTP mutagenesis, lack targeting and are inefficient. The transcriptional level of related genes in traditional strains is low, resulting in insufficient precursor supply and affecting yield improvement.
By performing ARTP mutagenesis on the original strain Streptomyces hygroscopicus var. ascomyceticus ATCC 14891, a high-yielding strain AP-270 was obtained, and a high-yielding mutant strain Rif-d6 was obtained through rifampicin resistance screening, thereby increasing antibiotic production.
It significantly increased antibiotic production by 24.69%, optimized nitrogen metabolism and precursor supply, and enhanced the synthesis capacity of secondary metabolites.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biopharmaceuticals, in particular, to a high-yield mutant strain and its application in immunosuppressants. BACKGROUND
[0002] Ascomycin (FK520) is a natural 23-membered macrolide antibiotic, which was first isolated from natural soil components by Fujisawa Pharmaceutical Co., Ltd. of Japan in 1962. Its chemical molecule is C43H69NO12. Ascomycin and its derivatives have various pharmacological activities. Due to its unique chemical structure, ascomycin has various physiological and pharmacological activities and functions such as anti-malaria, immunosuppression, anti-fungal, and nerve function recovery and regeneration. It shows significant clinical research value in immunosuppression, and thus can be used as a potential therapeutic drug for reducing or eliminating organ transplant rejection and autoimmune diseases. FK520 can be used as the starting component for the chemical synthesis of the immunosuppressant pimecrolimus (Pimecrolimus). Pimecrolimus has been approved by the FDA as a first-line treatment drug for mild to moderate atopic dermatitis.
[0003] In the field of antibiotics and biopharmaceuticals, screening and cultivation of high-yield mutant strains is a core technical path to improve the yield of target products (such as FK520 and other important antibiotics). Streptomyces is the main source of microorganisms for antibiotic production, and its metabolic regulation mechanism is complex. Traditional mutagenesis breeding methods have strong randomness and low efficiency, making it difficult to precisely activate the secondary metabolic pathway, resulting in slow improvement of the yield of target products.
[0004] Traditional atmospheric room temperature plasma (ARTP) mutagenesis can improve the mutation rate, but lacks targeting and needs to rely on large-scale screening to obtain performance-optimized strains. For example, the AP-270 strain obtained by ARTP mutagenesis of the starting strain Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 only improves the product synthesis rate through random mutation, without directional optimization of key metabolic nodes (such as glycerol metabolism and nitrogen metabolism).
[0005] Ribosome engineering is a method of screening for resistant mutants by adding antibiotics to the culture medium. The ribosome or RNA polymerase of the screened mutant will undergo some spontaneous mutations, thereby increasing the yield of secondary metabolites or producing some new natural products. In ribosome engineering, streptomycin and rifampicin are commonly used as resistance markers. Streptomycin causes mutations in the rpsL gene of the ribosome, resulting in changes in the conformation of the S12 protein, making it impossible for streptomycin to bind to the ribosome effectively. Thus, the resistant mutant strains screened have more stable translation systems or stronger secondary metabolite synthesis capabilities. The most common mutation induced by rifampicin in actinomycetes is located in the conserved amino acid residues of the RNA polymerase beta-subunit encoded by rpoB, which can also significantly improve the production efficiency of the target product of the strain. The correlation between its resistance mechanism and antibiotic production has not been fully analyzed.
[0006] In addition, the synthesis of FK520 depends on precursor substances such as ethyl malonyl coenzyme A and 4,5-dihydroxy-1-ene-cyclohexanoic acid (DHCHC). The transcription level of related genes (such as fkbO and fkbE) in traditional strains is low, resulting in insufficient supply of precursors, which limits the yield improvement. In the late fermentation stage, Streptomyces often releases intracellular substances due to autolysis, which interferes with the metabolic balance and causes abnormal increase in amino nitrogen level, but the synthesis rate of the product does not increase synchronously.
[0007] Based on this, the present application provides a high-yield mutant strain screening method, which is used to improve the yield of immunosuppressant FK520, and has important practical significance. SUMMARY
[0008] In view of this, the present application provides a high-yield mutant strain and its application in immunosuppressants, aiming to solve the problem that the strains that can promote the yield of antibiotics cannot be effectively screened in the current technology.
[0009] The present application provides a high-yield mutant strain, which is Rif-d6, and the strain was deposited in the China Center for Type Culture Collection on June 2, 2025, with the accession number CCTCC NO: M20251177.
[0010] The present application also provides a method for obtaining the high-yield mutant strain as described in the above technical solution, comprising the following steps:
[0011] The original strain Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 is prepared into a single spore suspension and subjected to ARTP mutagenesis;
[0012] After the mutagenesis is completed, the treated single spore suspension is diluted and screened to obtain a high-yield strain AP-270;
[0013] The high-yield mutant strain AP-270 is subjected to rifampicin resistance screening to obtain the high-yield mutant strain Rif-d6.
[0014] The application further provides application of the high-yield mutant strain in an immunosuppressant FK520, and the high-yield mutant strain is Rif-d6 in the above technical solution.
[0015] Compared with the prior art, the application has the beneficial effects that:
[0016] The high-yield mutant strain is obtained by ARTP mutagenesis of the original strain Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 to obtain AP-270, and then rifampicin resistance screening is performed, and the high-yield mutant strain has rifampicin resistance, can greatly improve the yield of antibiotics, and is further applied to immunosuppressants. BRIEF DESCRIPTION OF DRAWINGS
[0017] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are included to provide a description of preferred embodiments, and are not meant to limit the present application. Furthermore, the same reference numerals are used throughout the several drawings to designate the same or similar parts. In the drawings:
[0018] Figure 1 A yield comparison chart of the high-yield mutant strain Rif-d6 and the starting strain AP-270;
[0019] Figure 2 A comparison chart of changes in amino nitrogen of the high-yield mutant strain Rif-d6 and the starting strain AP-270;
[0020] Figure 3 A comparison chart of changes in ammonium ions of the high-yield mutant strain Rif-d6 and the starting strain AP-270;
[0021] Figure 4 A comparison chart of activity of glutamine synthetase of the high-yield mutant strain Rif-d6 and the starting strain AP-270;
[0022] Figure 5 A comparison chart of transcription levels of the gene fkbO of the high-yield mutant strain Rif-d6 and the starting strain AP-270;
[0023] Figure 6 A comparison chart of transcription levels of the gene fkbN of the high-yield mutant strain Rif-d6 and the starting strain AP-270;
[0024] Figure 7Figure 2 is a graph showing the comparison of the transcription level of gene fkbL between high-yield mutant strain Rif-d6 and starting strain AP-270. DETAILED DESCRIPTION
[0025] Various exemplary embodiments of the present application are described in detail herein, which should not be considered limiting the present application, but rather as being illustrative thereof. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application.
[0026] In addition, for numerical ranges of values recited herein, it is intended that every numerical value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value literally and implicitly falls within the ambit of the application as claimed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.
[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in further detail the methods and / or materials associated with the documents. In the case of conflict between the present specification and any document incorporated by reference, the present specification will control.
[0028] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.
[0029] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms that are intended to mean "including but not limited to".
[0030] The present application provides a high-yield mutant strain, which is Rif-d6, and the strain was deposited in China Center for Type Culture Collection on June 2, 2025, with the accession number CCTCC NO: M20251177.
[0031] The present application also provides a method for obtaining the high-yield mutant strain as described above, comprising the following steps:
[0032] The original strain Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 is prepared into a single spore suspension for ARTP mutagenesis;
[0033] After the mutagenesis, the treated single spore suspension is diluted and screened to obtain a high-yield strain AP-270;
[0034] The high-yield strain AP-270 is subjected to rifampicin resistance screening to obtain the high-yield mutant strain Rif-d6.
[0035] The application also provides a use of the high-yield mutant strain in immunosuppressants, and the high-yield mutant strain is Rif-d6 of claim 1.
[0036] Example 1
[0037] (1) The original strain is prepared into a single spore suspension for ARTP mutagenesis treatment: sterilized round iron pieces are placed in the corresponding grooves in the instrument, 10 μL of the single spore suspension is taken and evenly spread on the round iron pieces, and the mutagenesis operation is performed according to the instrument operation instruction;
[0038] The specific parameters of the ARTP instrument are set as follows: mutagenesis power 120 w, helium flow rate 10 SLM, mutagenesis distance 2 mm, and mutagenesis time preferably 90 s, which can be selected within 30-180 s.
[0039] (2) After the mutagenesis treatment, the treated spore solution is fully diluted and shaken, and then plated on a separation plate, which is incubated at 30°C for about 10 days, and then single colonies are picked on a 24-deep-well plate for high-throughput primary screening, and shake flask re-screening is performed to obtain the high-yield strain AP-270.
[0040] (3) The single spore suspension of the high-yield strain AP-270 is plated on a separation plate containing different rifampicin concentration gradients, and the rifampicin concentration gradient can be selected within 2-10 mg / L, preferably 2-4 mg / L; a separation plate without the addition of a resistance substance is used as a control group, and incubated at 30°C for 10 days, and then single colonies are picked on a 24-deep-well plate for high-throughput primary screening, and shake flask re-screening is performed to obtain the high-yield mutant strain Rif-d6.
[0041] Test Example
[0042] I. The high-yield mutant strain Rif-d6 obtained by the application and the starting strain AP-270 are tested during shake flask fermentation, respectively.
[0043] (1) The yield comparison chart of the high-yield mutant strain Rif-d6 and the starting strain AP-270 is shown in Figure 1 , based on which it can be seen that the yield of the high-yield mutant strain Rif-d6 is higher than that of the starting strain AP-270.Figure 1 It can be seen that, during 24h to 120h, the product synthesis rate of the high-yield mutant strain Rif-d6 and the starting strain has no great difference, and the average yield increase rate is 7.01 and 6.91mg / L / h; after 120h, the two strains show obvious difference, the high-yield mutant strain Rif-d6 maintains higher increase rate to accumulate product, and the average product increase rate is 8.43mg / L / h. At 168h, i.e. the fermentation end time, the average yield of Rif-d6 is 1077.56mg / L, which is increased by 24.69% compared with the starting strain;
[0044] (2) The amino nitrogen process change comparison chart of the high-yield mutant strain Rif-d6 and the starting strain AP-270 is shown in Figure 2 Based on Figure 2 It can be seen that, in the first 24h, the cells release free amino acids and ammonium salt in the process of decomposing organic nitrogen source, resulting in the increase of amino nitrogen concentration, and the amino nitrogen of the starting strain AP-270 increases to the highest point at 24h, about 1000mg / L; the amino nitrogen of the high-yield mutant strain Rif-d6 also increases to the highest point at 24h, 913mg / L, which is about 8.7% lower than that of the starting strain; during 24h to 48h, the two strains continue to consume amino nitrogen, at this time the cell concentration reaches the maximum, and the cells gradually enter secondary metabolism for product biosynthesis. During 24h to 72h, the amino nitrogen consumption rate and the product synthesis rate change trend are consistent. At 72h, the amino nitrogen of the two strains is at the lowest point. Then the amino nitrogen level begins to rise until the fermentation ends, and the change trend is basically consistent, but the amino nitrogen level of the high-yield mutant strain Rif-d6 is almost always lower than that of the starting strain. But the amino nitrogen suddenly rises when the bottle is placed, which may be related to cell lysis;
[0045] (3) The ammonium ion process change comparison chart of the high-yield mutant strain Rif-d6 and the starting strain AP-270 is shown in Figure 3 Based on Figure 3 It can be seen that, during the first 24h, the ammonium ion level of the two strains increases to the maximum, at this time the ammonium ion level of the starting strain AP-270 and the high-yield mutant strain Rif-d6 is 859.3mg / L and 696.6mg / L, respectively. At this stage, the two strains are in lag phase, and the organic nitrogen source in the medium is initially decomposed to release free amino acids and form NH4 +, leading to the accumulation of ammonium ions, and the concentration increased. At 48h to 72h, the ammonium ion levels of the two strains were both in decline, and the change trend was consistent with that of amino nitrogen. In this period, the starting strain consumed ammonium ions at a fast rate in the early stage due to the vigorous growth of the bacterial cells, and the ammonium ions were preferentially used for the synthesis of primary metabolites. In the later stage, the rate slowed down because the bacterial cells entered the stationary phase, the nitrogen demand decreased, or the metabolic efficiency decreased. While the high-yield mutant strain consumed ammonium ions at a slow rate in the early stage, which might be related to the activity of nitrogen metabolism-related enzymes, and the rate increased in the later stage, which might be because the demand for product synthesis increased, driving the intensification of nitrogen metabolism. At 72h to 144h, the ammonium ion level of the starting strain decreased to the lowest point at 96h, and then slowly rose to 144h. While the ammonium ion level of the high-yield mutant strain decreased to nearly zero at 96h, and remained so until 144h. At 144h to 168h, the ammonium ion level began to rise.
[0046] (4) The activity of glutamine synthetase of the starting strain AP-270 and the high-yield mutant strain Rif-d6 was detected, and the results are shown in Figure 4 Based on Figure 4 , it can be known that in the early stage, the ammonium ion concentration was positively correlated with the activity of glutamine synthetase. At 24h, the ammonium ion levels of the starting strain AP-270 and the high-yield mutant strain Rif-d6 were 859.3mg / L and 696.6mg / L, respectively, and the activity of glutamine synthetase of the high-yield mutant strain was higher than that of the starting strain. At 72h, the ammonium ion consumption decreased to a low point, and the activity of glutamine synthetase gradually recovered. From 120h, the activity of glutamine synthetase of the high-yield mutant strain continued to increase, constantly improving the nitrogen precursor for precursor synthesis. At this time, there was a significant difference in product synthesis between the high-yield mutant strain and the starting strain, and the former maintained a high product synthesis rate until the end of fermentation. Due to the different autolysis degrees of the bacterial cells caused by their own characteristics, the enzyme activity of the key enzymes in nitrogen metabolism was different, which further affected the secondary metabolic pathway and product synthesis.
[0047] II. Detection of the transcription level of the genes related to the synthesis of FK520 precursors of the high-yield mutant strain Rif-d6 and the starting strain AP-270 in the early and middle stages of fermentation:
[0048] (1) The transcription level of the gene fkbN belonging to the LAL family transcriptional regulator and positively regulating the biosynthesis of FK520 was detected, and the results are shown in Figure 5 Based on Figure 5 , it can be known that from 48h, the gene fkbN gradually up-regulated, and the transcription level reached the maximum of 11.3 times at 96h. At 48h, the bacterial cells entered the stationary phase from the exponential growth phase, the primary metabolism weakened, the secondary metabolic gene cluster was activated, the secondary metabolism initiation signal triggered, and the gene fkbN began to gradually up-regulate. fkbN activated the downstream target gene fkbO, forming a positive feedback loop;
[0049] (2) Gene fkbO encodes branch hydrolase, under the action of which branch acid is decomposed into 3,4-dihydroxy-1,5-diene-cyclohexanoic acid (DCDC) and pyruvic acid, and DCDC is further oxidized to form the synthesis precursor 4,5-dihydroxy-1-ene-cyclohexanoic acid (DHCHC) of FK520. The transcription level of gene fkbO is detected, and the result is shown in Table 2, based on which it can be known that the transcription level of gene fkbO is significantly down-regulated at 48 h, and is up-regulated by 1.6 times at 96 h. Therefore, when the bacteria enter the secondary metabolism, the up-regulation of the transcription level of gene fkbO provides sufficient precursor DHCHC for the biosynthesis of FK520. Figure 6 Figure 6
[0050] (3) Gene fkbL is an L-lysine cyclization deaminase gene, which catalyzes the cyclization and dehydrogenation of L-lysine to convert it into the precursor L-piperidine acid. The transcription level of gene fkbL is tested, and the result is shown in Table 3, based on which it can be known that gene fkbL is significantly down-regulated at 48 h, and is significantly up-regulated by 7.2 times at 96 h. In the first 48 h, the bacteria consume a large amount of lysine for bacterial growth, reach the maximum bacterial concentration, and the demand for lysine is weakened, so that the expression of gene fkbL is reduced through catabolite repression and feedback inhibition to save energy. When 96 h, the biosynthesis of FK520 enters the peak period, a large amount of L-piperidine acid is needed as a precursor, and the transcription of fkbL is up-regulated due to the increase of lysine concentration to a high point. This is consistent with the gradual increase of the biosynthesis rate of FK520 starting from 96 h; Figure 7 Figure 7
[0051] Based on the above, the high-yield mutant strain Rif-d6 obtained in the application has a general significant up-regulation of the transcription level of the genes related to the synthesis of FK520 precursors in the early and middle stages of fermentation. It can be seen that the strain obtained in the application can be applied to the synthesis of immunosuppressant FK520, and can greatly improve the yield of FK520.
[0052] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it, although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that the specific embodiments of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, any modification or equivalent replacement thereof should be covered within the protection scope of the claims of the present application.
Claims
1. A high-yield mutant strain, characterized in that, The high-yield mutant strain is Rif-d6, which was deposited at the China Center for Type Culture Collection on June 2, 2025, with accession number CCTCC NO: M20251177.
2. A method for obtaining the high-yield mutant strain according to claim 1, characterized in that, Includes the following steps: The original strain Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 was prepared as a single-spore suspension for ARTP mutagenesis. After the mutagenesis was completed, the treated single spore suspension was diluted and screened to obtain the high-yielding strain AP-270. The high-yielding strain AP-270 was screened for rifampicin resistance to obtain the high-yielding mutant strain Rif-d6.
3. The application of a high-yield mutant strain in the immunosuppressant FK520, characterized in that, The high-yield mutant strain is Rif-d6 as described in claim 1.