Method for synthesizing strain by streptomyces nosiheptide
By constructing and integrating Streptomyces integrated expression vectors and optimizing the fermentation process, the problems of low translation efficiency, excessive metabolic load and genetic instability in navisex production were solved, and efficient and stable navisex production was achieved.
Patent Information
- Application Number
- CN202510592949.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-09
- Publication Date
- 2025-08-08
AI Technical Summary
There are problems in the existing navisetide production process that have low translation efficiency caused by rare codons, excessive early metabolic load caused by traditional constitutive promoters, lack of dynamic regulation of fermentation processes and genetic instability of engineered strains, which affect yield and process repeatability.
By constructing Streptomyces integrated expression vector pIJ8600-PtipA-tRNA-Asp-AUC-TT, it was integrated into the attB site of the Streptomyces actuosus genome using ΦC31 integrase, combining the thiostreptomyces inducible promoter Ptia and the dynamic feed-soluble oxygen coupling process, the enzyme decoding efficiency and fermentation cycle were optimized.
It significantly improves the yield and production efficiency of navisetide, shortens the fermentation cycle, reduces production costs, and improves the controllability and repeatability of the process, meeting the GMP-grade production requirements.
Smart Images
Figure BDA0005393670810000071 
Figure BDA0005393670810000081
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of Streptomyces nosiheptidylsporin synthesis, and in particular to a method for synthesizing a Streptomyces nosiheptidylsporin strain. Background Art
[0002] Streptomyces nosiheptidinolyticus is a Streptomyces microorganism that produces nosiheptidase, which is used as a feed additive in the livestock industry to promote animal growth and prevent disease. The purpose of this synthetic strain is to optimize nosiheptidase production efficiency through genetic engineering. This approach addresses production bottlenecks faced by natural strains, such as low translation efficiency due to rare codons, excessive metabolic load, extensive fermentation processes, poor genetic stability, and inadequate detection methods. This approach improves nosiheptidase yield, stability, and process reproducibility, reducing production costs and meeting market demand.
[0003] The existing nosiheptide production process relies on Streptomyces strains and faces multiple bottlenecks: the key enzyme gene contains a high-frequency GAT codon, which does not match the host tRNA-Asp, resulting in low translation efficiency; the traditional constitutive promoter causes excessive metabolic load in the early stage, affecting bacterial growth and product synthesis; the fixed feeding strategy lacks dynamic regulation, resulting in long fermentation cycles and fluctuating yields; the engineered strain plasmid is unstable, and the yield decreases significantly after subculture; the existing verification method is limited to phenotypic screening and lacks precise detection at the molecular level, which affects the repeatability of the process. Summary of the Invention
[0004] In view of the shortcomings of the existing technology, the present invention provides a method for synthesizing a strain of Streptomyces nosiheptidylsinicola, which solves the problems of low translation efficiency, unbalanced expression, extensive fermentation and genetic instability in the existing technology.
[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for synthesizing a strain of Streptomyces nosiheptidylsus, specifically comprising the following steps:
[0006] S1. Construction of the Streptomyces integration expression vector pIJ8600-PtipA-tRNA-Asp-AUC-TT, which contains the thiostrepton-inducible promoter Ptia, a synthetic tRNA-Asp gene with an anticodon of AUC, and the strong terminator TT;
[0007] S2. The vector obtained in the above step was integrated into the attB site of the Streptomyces actuosus genome by ΦC31 integrase-mediated site-specific recombination to obtain the engineered strain Nos-S1;
[0008] S3. During the fermentation process containing Gao's medium No. 1, thiostrepton was added after 36-120 hours of culture to induce tRNA-Asp-AUC expression, thereby increasing the recognition efficiency of GAT codon to improve nosiheptide production.
[0009] Preferably, the integration expression vector in step S1 is constructed based on the pIJ8600 backbone and carries the apramycin resistance gene and the ΦC31 phage integrase gene.
[0010] Preferably, in step S1, the anticodon of the tRNA-Asp-AUC gene is transformed from natural GUC to AUC, and the antisense codon sequence is 5'-AUC-3', which is used to recognize the GAT codon encoding aspartic acid.
[0011] Preferably, the step S2 of integrating the vector into the host genome by the Streptomyces protoplast transformation method specifically comprises:
[0012] a) Protoplasts were prepared by treating Streptomyces actuosus cells with 1 mg / mL lysozyme for 30 minutes;
[0013] b) using PEG 6000-mediated transformation to introduce the vector into protoplasts, wherein the final PEG concentration is 25%;
[0014] c). Resistant colonies were screened in R2YE regeneration medium containing 50 μg / mL apramycin, and the integration of the attB site was verified by PCR.
[0015] Preferably, in step S3, thiostrepton is added after 60 hours of culture, and the final concentration is 5 μg / mL.
[0016] Preferably, the composition of Gao's No. 1 medium in step S3 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 pH value is 7.2-7.4.
[0017] Preferably, the total duration of the fermentation process in step S3 is 144 hours, and the control conditions are: temperature 30±2° C., dissolved oxygen content maintained at 30±5%, and stirring speed 300±50 rpm.
[0018] Preferably, step S3 further comprises adding a carbon source and a nitrogen source after 60 hours of fermentation, wherein the carbon source is glucose and the final concentration thereof is 10 g / L; and the nitrogen source is soybean cake powder and the final concentration thereof is 5 g / L.
[0019] The present invention provides a method for synthesizing a strain of Streptomyces nosiheptidylsinicola. It has the following beneficial effects:
[0020] The present invention provides a method for synthesizing a nosiheptide Streptomyces strain. The present invention artificially designs tRNA-Asp-AUC (anticodon AUC) to specifically match the GAT codon, thereby removing the host translation bottleneck, improving the decoding efficiency of key enzyme mRNA, and increasing the rate of the rate-limiting step of nosiheptide synthesis. The thiostrepton-inducible promoter Ptia is used to delay tRNA expression until after the rapid growth period of the bacteria, thereby avoiding early metabolic burden, increasing bacterial biomass, and accurately matching the product synthesis window period. The induction efficiency is high. A dynamic feeding-dissolved oxygen coupling process is used to dynamically adjust the feeding rate based on real-time dissolved oxygen feedback. A carbon source (glucose) is added in a gradient to inhibit acetic acid accumulation. A nitrogen source (soybean cake powder) is supplemented in stages to maintain a stable C / N ratio, thereby shortening the fermentation cycle and reducing the standard deviation of the yield. The vector is site-specifically inserted into the attB site by ΦC31 integrase to avoid the problem of plasmid dependence. After 10 consecutive passages, the engineered strain Nos-S1 has a high exogenous gene retention rate and a low nosiheptide yield attenuation rate, which is significantly superior to the autonomous replication system. The present invention integrates PCR (attB site), Northern Multi-dimensional detection such as blot (tRNA expression) and HPLC (product quantification) ensures the controllability of strain construction and process parameters, with high reproducibility of batch-to-batch production, meeting GMP-level production requirements. Using low-cost Gao's No. 1 culture medium and standard fermentation tank parameters, no special equipment modification is required. The single-tank nosiheptide production is increased, the unit production cost is reduced, and it has the potential for large-scale expansion. DETAILED DESCRIPTION
[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0022] The present invention provides a method for synthesizing a strain of Streptomyces nosiheptidylsus, which specifically includes the following steps:
[0023] (1) Construction of the Streptomyces integration expression vector pIJ8600-PtipA-tRNA-Asp-AUC-TT
[0024] 1. Vector skeleton and element preparation
[0025] Vector selection: The Streptomyces integrative vector pIJ8600 was selected, which carries the apramycin resistance gene (AmR) and the ΦC31 phage integrase gene (int) and can be specifically integrated into the host genome through the attB site.
[0026] Promoter and terminator:
[0027] The inducible promoter Ptia was obtained by PCR amplification from plasmid pTIO1 (primers: Ptia-F: 5'-CCGGAATTCATGACCGCCGACATC-3', Ptia-R: 5'-CCCAAGCTTTTAGCGCCGGTA GAC-3'), with EcoRI / HindIII restriction sites.
[0028] Strong terminator TT: The rrnB terminator sequence (GenBank: X52508) was used and inserted downstream of the vector after synthesis.
[0029] 2. tRNA-Asp-AUC gene design and synthesis
[0030] Genetic modification:
[0031] The anticodon of natural tRNA-Asp is GUC (recognizes GAC codon), and its anticodon sequence was modified to AUC (recognizes GAT codon) through site-directed mutagenesis.
[0032] The complete gene sequence (including the 5' end Ptia promoter binding region and mature tRNA domain) was synthesized. The sequence is as follows:
[0033] 5'-GGTTCGATAGCTCAGTCGGTAGAGCACCAGACTGATTAAGATGTCGGTGTTCGAATCCCGCCGGTTCAAATCCGGATCG-3'
[0034] Cloning verification: The synthetic gene was inserted into the pUC57 vector and the sequence correctness was confirmed by Sanger sequencing.
[0035] 3. Vector Assembly
[0036] Restriction ligation:
[0037] The pIJ8600 vector was double-digested with EcoRI and HindIII to recover the linearized backbone.
[0038] The Ptia promoter, tRNA-Asp-AUC gene and TT terminator fragment (cut by EcoRI / Hind III) were ligated into the backbone to construct pIJ8600-PtipA-tRNA-Asp-AUC-TT.
[0039] Conversion and verification:
[0040] The ligation product was transformed into Escherichia coli DH5α competent cells, coated on LB plates containing 50 μg / mL apramycin, and cultured at 37°C for 16 hours.
[0041] Single colonies were picked to extract plasmids, and the correctness of the vector was confirmed by enzyme digestion (EcoRI / HindIII) and sequencing.
[0042] (II) Construction and verification of engineered strain Nos-S1
[0043] 1. Streptomyces Protoplast Preparation
[0044] Bacterial culture:
[0045] Streptomyces actuosus (Accession No.: CGMCC 4.1235) was inoculated into TSB liquid medium (containing 10.3% sucrose) and cultured at 30°C and 220 rpm with shaking until OD600≈0.6.
[0046] Protoplast isolation:
[0047] The cells were collected by centrifugation (4,000 rpm, 10 min) and washed twice with 10.3% sucrose solution.
[0048] Lysozyme solution (1 mg / mL lysozyme, dissolved in P buffer: 0.3 M sucrose, 25 mM Tris-HCl pH 8.0) was added and gently shaken at 30° C. for 30 min.
[0049] The protoplasts were collected by centrifugation (3,000 rpm, 10 min) and resuspended in P buffer for later use.
[0050] 2. Vector Transformation and Integration
[0051] PEG-mediated transformation:
[0052] 100 μL of protoplast suspension was mixed with 10 μg of linearized vector pIJ8600-PtipA-tRNA-As p-AUC-TT, and 400 μL of 25% PEG 6000 solution (dissolved in P buffer) was added. The mixture was gently mixed and placed on ice for 5 min.
[0053] Incubate in a 30°C water bath for 2 min, and add 1 mL of P buffer to terminate the reaction.
[0054] Regeneration and screening:
[0055] The cells were spread on R2YE regeneration medium (containing 50 μg / mL apramycin) and cultured at 28°C for 5-7 days until single colonies appeared.
[0056] Resistant colonies were picked and inoculated into fresh TSB liquid medium (containing apramycin), cultured at 30°C for 48 hours, and genomic DNA was extracted.
[0057] 3. Integration site verification
[0058] PCR validation:
[0059] Genomic DNA was amplified using primers attB-F (5′-GGCGACCTGCAGGCATGCAAGCT-3′) and attB-R (5′-CGGGATCCGTGTAGGCTGGAGCT-3′), with an expected product size of 1.2 kb.
[0060] The positive strain (Nos-S1) showed a clear band, and the negative control (original strain) had no amplification product.
[0061] 4. tRNA expression detection
[0062] Northern blot analysis:
[0063] The Nos-S1 strain was inoculated into Gao's medium No. 1 containing 5 μg / mL thiostrepton and cultured at 30°C for 48 hours.
[0064] The cells were collected, total RNA was extracted, and tRNA was separated by 10% urea-PAGE electrophoresis.
[0065] After transfer, the cells were hybridized with a digoxigenin-labeled probe (sequence: 5'-CGGAATCCCGCCGGTTC-3') and developed by chemiluminescence to confirm the expression of tRNA-Asp-AUC.
[0066] (III) Fermentation process optimization and nosiheptide production
[0067] 1. Seed Solution Preparation
[0068] Culture medium: Gao's No. 1 seed medium.
[0069] Culture conditions: Inoculate a single colony into 50 mL of seed solution and culture at 30°C, 220 rpm, with shaking for 24 hours until OD600 ≈ 3.0.
[0070] 2. Main fermentation process
[0071] Fermentation tank parameters:
[0072] Filling volume: 60% (volume / volume), temperature 30±2°C, initial pH 7.2, dissolved oxygen (DO) maintained at 30±5% (regulated by stirring rate 300±50 rpm and ventilation volume 1.0 vvm).
[0073] Thiostrepton induction:
[0074] After 60 hours of fermentation, thiostrepton was added to a final concentration of 5 μg / mL.
[0075] Feeding strategy:
[0076] Add twice at 60 and 84 hours of fermentation:
[0077] Carbon source: glucose, each supplementation final concentration 5g / L (total final concentration 10g / L).
[0078] Nitrogen source: soybean cake powder, added at a final concentration of 2.5 g / L each time (total final concentration 5 g / L).
[0079] 3. Product testing and harvesting
[0080] Nosiheptide Quantification:
[0081] Every 12 hours, 5 mL of fermentation broth was sampled, centrifuged (12,000 rpm, 5 min), and the supernatant was collected and filtered through a 0.22 μm filter membrane.
[0082] HPLC was used for detection: a C18 column (4.6×250 mm), a mobile phase of acetonitrile-0.1% phosphoric acid aqueous solution (60:40), a flow rate of 1.0 mL / min, and a detection wavelength of 254 nm.
[0083] The relative yield was calculated using nosiheptide standard (Sigma, N3160) as a control.
[0084] Fermentation termination: The total fermentation time was 144 hours. When the nosiheptide production reached a peak (about 258% relative yield), the fermentation was terminated and the supernatant was collected by centrifugation for downstream purification.
[0085] Table 1
[0086]
[0087]
[0088] Fermentation tests were conducted using Streptomyces actuosus and Nos-S1 strains, and the following fermentation tests were conducted (Table 1). The above results indicate that adding thiostrepton in the middle and late stages of fermentation can effectively promote the biosynthesis of nosiheptide.
[0089] The existing nosiheptide production process mainly relies on natural or traditional mutagenesis-modified Streptomyces strains, and its core bottlenecks are reflected in the following aspects:
[0090] Rare codons limit translation efficiency: The key enzyme genes for nosiheptide synthesis (such as P450 oxidase and non-ribosomal peptide synthetase) often contain high-frequency GAT codons (encoding aspartic acid), while the anticodon of the endogenous tRNA-Asp of the Streptomyces host is GUC, which can only recognize GAC codons, resulting in low decoding efficiency of GAT codons and significant ribosome stalling, which restricts the expression of target proteins and the synthesis of secondary metabolites.
[0091] Mismatch of inducible expression system: Traditional technologies mostly rely on constitutive promoters to continuously express exogenous genes, resulting in excessive metabolic load in the early stages of bacterial growth, excessive energy consumption in non-essential protein synthesis, and reduced bacterial biomass, delayed product synthesis period, and even cell apoptosis.
[0092] Extensive fermentation process: Existing processes often adopt fixed-time feeding or single carbon / nitrogen source supplementation strategies, lacking dynamic regulation, which can easily lead to the accumulation of inhibitory metabolites such as acetic acid due to excess carbon source, or imbalance in amino acid supply due to insufficient nitrogen source, resulting in prolonged fermentation cycle and large fluctuations in yield.
[0093] Defects in genetic stability: Most engineered strains carry exogenous genes through autonomously replicating plasmids, which are prone to plasmid loss or copy number reduction during long-term propagation, resulting in a generational decrease in nosiheptide production, requiring frequent repeated transformations and increasing production costs.
[0094] Insufficient testing and verification: Existing methods for verifying the effects of strain modification are mostly limited to phenotypic screening (such as resistance markers), and lack precise detection at the molecular level (such as tRNA expression levels and integration site specificity). They are prone to introducing off-target mutations or non-functional integrations, affecting process repeatability.
[0095] Advantages of the technology of the present invention
[0096] This technology systematically solves the above problems through molecular design and process synergy optimization. The specific advantages are as follows:
[0097] Precise codon adaptation: By artificially designing tRNA-Asp-AUC (anticodon AUC), the GAT codon is specifically matched, which relieves the host translation bottleneck, improves the decoding efficiency of key enzyme mRNA, and increases the rate of the rate-limiting step in nosiheptide synthesis.
[0098] Controllable induction timing: Using the thiostrepton-inducible promoter Ptia, tRNA expression is delayed until the rapid growth period of the bacteria, avoiding early metabolic burden, increasing bacterial biomass, and accurately matching the product synthesis window period, resulting in high induction efficiency.
[0099] Dynamic feeding-dissolved oxygen coupling process: The feeding rate is dynamically adjusted based on real-time dissolved oxygen feedback, the carbon source (glucose) is added in a gradient to inhibit acetic acid accumulation (concentration <1g / L), and the nitrogen source (soybean cake powder) is supplemented in stages to maintain a stable C / N ratio, shortening the fermentation cycle and reducing the standard deviation of yield.
[0100] Genome integration stability: The vector is inserted into the attB site through ΦC31 integrase to avoid plasmid dependence. After 10 consecutive passages of the engineered strain Nos-S1, the exogenous gene retention rate is greater than 95%, and the nosiheptide production attenuation rate is less than 3%, which is significantly better than the autonomous replication system.
[0101] Multi-level validation system: Integrates PCR (attB site), Northern blot (tRNA expression), and HPLC (product quantification) to ensure the controllability of strain construction and process parameters, with high batch-to-batch yield reproducibility, meeting GMP-grade production requirements.
[0102] Industrial compatibility: Using low-cost Gao's No. 1 culture medium and standard fermentation tank parameters, no special equipment modification is required. The single-tank nosiheptide yield is increased, the unit production cost is reduced, and it has the potential for large-scale expansion.
[0103] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.
Claims
1. A method for synthesizing a strain of Streptomyces nosiheptidylsus, characterized in that: The specific steps include: S1. Construction of the Streptomyces integration expression vector pIJ8600-PtipA-tRNA-Asp-AUC-TT, which contains the thiostrepton-inducible promoter Ptia, a synthetic tRNA-Asp gene with an anticodon of AUC, and the strong terminator TT; S2. The vector obtained in the above step was integrated into the attB site of the Streptomyces actuosus genome by ΦC31 integrase-mediated site-specific recombination, and the engineered strain Nos-S1 was obtained by screening; S3. During the fermentation process containing Gao's medium No. 1, thiostrepton was added after 36-120 hours of culture to induce tRNA-Asp-AUC expression, thereby increasing the recognition efficiency of GAT codon and improving the production of nosiheptide.
2. The method for synthesizing a Streptomyces nosiheptidylsus strain according to claim 1, characterized in that: The integration expression vector in step S1 is constructed based on the pIJ8600 backbone and carries the apramycin resistance gene and the ΦC31 phage integrase gene.
3. The method for synthesizing a Streptomyces nosiheptidylsus strain according to claim 1, characterized in that: In step S1, the anticodon of the tRNA-Asp-AUC gene is transformed from natural GUC to AUC, and its antisense codon sequence is 5'-AUC-3', which is used to recognize the GAT codon encoding aspartic acid.
4. The method for synthesizing a Streptomyces nosiheptidylstrain according to claim 1, characterized in that: In step S2, the vector is integrated into the host genome by the Streptomyces protoplast transformation method, which specifically includes: a) Protoplasts were prepared by treating Streptomyces actuosus cells with 1 mg / mL lysozyme for 30 minutes; b) using PEG 6000-mediated transformation to introduce the vector into protoplasts, wherein the final PEG concentration is 25%; c). Resistant colonies were screened in R2YE regeneration medium containing 50 μg / mL apramycin, and the integration of the attB site was verified by PCR.
5. The method for synthesizing a Streptomyces nosiheptidylsus strain according to claim 1, characterized in that: In step S3, thiostrepton is added after 60 hours of culture, and the final concentration is 5 μg / mL.
6. The method for synthesizing a Streptomyces nosiheptidylstrain according to claim 1, characterized in that: The composition of Gao's No. 1 culture medium in step S3 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 pH value is 7.2-7.
4.
7. The method for synthesizing a Streptomyces nosiheptidylstrain according to claim 1, characterized in that: The total fermentation time in step S3 is 144 hours, and the control conditions are: temperature 30±2° C., dissolved oxygen content maintained at 30±5%, and stirring speed 300±50 rpm.
8. The method for synthesizing a Streptomyces nosiheptidylstrain according to claim 1, characterized in that: The step S3 further includes adding a carbon source and a nitrogen source after 60 hours of fermentation, wherein the carbon source is glucose and the final concentration is 10 g / L; the nitrogen source is soybean meal and the final concentration is 5 g / L.