Method for increasing yield of dsRNA

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a method for increasing the yield of dsRNA. The method is suitable for an escherichia coli expression system carrying a dsRNA expression vector, and the dsRNA yield is remarkably increased to 728 mg / L by performing system optimization on various components such as carbon and nitrogen sources, phosphate, metal ion inorganic salts, vitamins and effectors in a fermentation culture medium and combining regulation and control of fermentation parameters (including dissolved oxygen, a feeding strategy and induction opportunity). The dsRNA fermentation process has excellent amplification and industrial application potential, and a reliable solution is provided for large-scale and low-cost production of the dsRNA.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically to a method for increasing dsRNA production. Background Technology

[0002] Double-stranded RNA (dsRNA), as an important biomolecule, has shown great potential in fields such as agricultural biopesticides and gene function research. For example, dsRNA-based RNA interference (RNAi) technology has become a core method for developing next-generation targeted insecticides and novel therapeutics. However, to achieve large-scale deployment of these applications, the high cost of dsRNA production must be addressed. Currently, using recombinant E. coli fermentation to produce dsRNA is one of the most cost-effective solutions.

[0003] Although *E. coli* expression systems offer advantages such as ease of operation, rapid growth, and high yield, their inherent metabolic networks and physiological characteristics are not always compatible with the efficient synthesis of exogenous dsRNA. Traditional basal media such as LB are designed to promote rapid bacterial growth, and their nutrient composition is not optimized for the accumulation of specific products like dsRNA. Therefore, deficiencies or imbalances in the types and ratios of carbon and nitrogen sources, phosphate concentration, metal ions, vitamins, and effectors (such as nucleosides and amino acids) in the culture medium can limit the production potential of strains, resulting in low yields, high costs, and difficulty in meeting the needs of industrial production. In large-scale fermentation, the synergistic optimization of culture medium composition and fermentation process is even more critical, but systematic research on dsRNA culture media is relatively scarce. The ability to achieve a balance between high-density culture and efficient expression at the fermenter level has become a bottleneck hindering the scale-up of dsRNA production lines. For example, the highest yield of dsRNA produced by bacterial fermentation was reported in 2024 as 182 mg / L (GUAN R, MIAO X, et al. Bacteria-based double-stranded RNA production to develop cost-effective RNA interference application for insect pest management [M]. Methods Mol Biol, 2024, 2771: 73-81). This result was obtained on a fermenter scale, but it is still far from meeting the economic requirements for industrial production.

[0004] Currently, while some studies have attempted to improve dsRNA stability by modifying host strains through genetic engineering (such as knocking out nuclease genes), research on optimizing culture medium components and directly enhancing the supply of synthetic substrates and energy levels from a metabolic engineering perspective remains relatively scarce and unsystematic. An optimized culture medium that can provide the necessary "building materials" and "catalytic tools" for dsRNA synthesis is crucial for releasing the production capacity of engineered bacteria. More importantly, precisely controlled fermentation processes (such as dynamic feeding based on metabolic feedback, dissolved oxygen stepwise control, and growth-coupled induction strategies) are also key pathways to further overcome yield bottlenecks and achieve efficient, large-scale production.

[0005] Therefore, this invention aims to provide a method for increasing dsRNA production. It focuses on optimizing fermentation media with well-defined and efficient components for dsRNA production and large-scale fermentation processes. Through systematic research and optimization of the types and concentrations of carbon and nitrogen sources, phosphates, metal ions, vitamins, and specific effectors in the culture medium, combined with process control at the fermenter scale, a balance is established between the cell growth of recombinant *E. coli* and product synthesis, significantly improving dsRNA yield and productivity. This lays a solid foundation for the large-scale, low-cost industrial production of dsRNA. Summary of the Invention

[0006] The technical problem this invention aims to solve is to address the shortcomings of existing technologies in terms of low dsRNA yield and low production efficiency, and to provide a method for increasing dsRNA production. Specifically, by systematically optimizing and proportioning the carbon and nitrogen sources, inorganic salts, vitamins, and effector components in the fermentation medium, and utilizing the synergistic effects between components, an extracellular metabolic environment conducive to efficient dsRNA synthesis is created. This promotes the growth of the host bacteria and enhances the synthesis of its metabolically directed product, dsRNA. Simultaneously, carbon sources are dynamically supplemented during fermentation to maintain a low sugar concentration, and an inducer is added during the logarithmic growth phase of the cells to induce efficient dsRNA expression, ultimately achieving high and stable dsRNA production.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for increasing dsRNA production involves inoculating the seed culture of a recombinant strain carrying a dsRNA expression vector into an optimized fermentation medium for fermentation. When the dissolved oxygen level first rises to above 80% and the residual sugar concentration is below 4 g / L, a carbon source is added to maintain the residual sugar concentration within the range of 0-5 g / L. An inducer is added to induce dsRNA expression when the bacterial cell ferments to the logarithmic growth phase.

[0009] The extraction method of the dsRNA is disclosed in Chinese patent CN118240729A.

[0010] The recombinant strain carrying the dsRNA expression vector is a recombinant strain carrying the dsRNA expression vector constructed using Escherichia coli as the expression system.

[0011] In some embodiments of the present invention, the recombinant strain carrying the dsRNA expression vector is obtained by overexpressing the BcSAS1 gene fusion fragment, or the DCL1+2 gene fusion fragment, or the ERG gene fusion fragment as the chassis strain of Escherichia coli BL21(DE3)-ΔC-Δrnc.

[0012] The Escherichia coli BL21(DE3)-ΔC-Δrnc mentioned therein is the host B-Δr-ΔC disclosed in Chinese patent CN118291510A.

[0013] The recombinant strain that uses *Escherichia coli* BL21(DE3)-ΔC-Δrnc as the chassis strain and overexpresses the BcSAS1 gene fusion fragment is the recombinant strain B-Δr-ΔC-BcSAS1-CAG disclosed in Chinese patent CN118291510A. 40 -BcSAS1.

[0014] In some embodiments of the present invention, the DCL1+2 gene fusion fragment is obtained using CAG. 40 The tag is obtained by linking the DCL1+2 gene and the DCL1+2 reverse complement gene; the ERG gene fusion fragment is obtained using CAG. 40 The tag is obtained by linking the ERG gene and the ERG reverse complement gene.

[0015] In some embodiments of the present invention, the DCL1+2 gene has the nucleotide sequence shown in SEQ ID NO.1; the CAG 40 The tag, the nucleotide sequence of which is shown in SEQ ID NO.2; the DCL1+2 reverse complement gene, the nucleotide sequence of which is shown in SEQ ID NO.3; the ERG gene, the nucleotide sequence of which is shown in SEQ ID NO.5; the ERG reverse complement gene, the nucleotide sequence of which is shown in SEQ ID NO.6.

[0016] In some embodiments of the present invention, the optimized fermentation medium is obtained by adding metal ion inorganic salts, vitamins, and effectors to LB medium as the base medium.

[0017] Specifically, the LB medium is a commercially available medium composed of peptone, yeast extract, and sodium chloride (NaCl).

[0018] Specifically, the inorganic salt containing metal ions includes inorganic salts that provide divalent metal ions, wherein the divalent metal ions include Mg.2+ Ca 2+ Zn 2+ Fe 2+ Mn 2+ Co 2+ Cu 2+ Any one or more of the following combinations.

[0019] Specifically, the inorganic metal ion salt includes any one or a combination of several of the following: 10-30 mg / L calcium chloride, 0.15-1.5 mg / L cobalt chloride, 0.15-1.5 mg / L copper chloride, 7-21 mg / L ferrous sulfate, 3-9 g / L magnesium sulfate, 0.5-1.5 mg / L manganese sulfate, and 5-15 mg / L zinc sulfate.

[0020] In some embodiments of the present invention, the metal ion inorganic salt is composed of 10-30 mg / L calcium chloride, 0.15-1.5 mg / L cobalt chloride, 0.15-1.5 mg / L copper chloride, 7-21 mg / L ferrous sulfate, 3-9 g / L magnesium sulfate, 0.5-1.5 mg / L manganese sulfate, and 5-15 mg / L zinc sulfate.

[0021] Specifically, the vitamins include any one or a combination of several of the following: 0.1-5 mg / L VB1, 0.1-1 mg / L VB2, 0.1-1 mg / L VB3, 0.1-1 mg / L nicotinamide, 0.1-1 mg / L VB6, 0.03-3 mg / L VB7, and 0.1-1 mg / L VB12.

[0022] In some embodiments of the present invention, the vitamins are composed of 0.1-5 mg / L VB1, 0.1-1 mg / L VB2, 0.1-1 mg / L VB3, 0.1-1 mg / L nicotinamide, 0.1-1 mg / L VB6, 0.03-3 mg / L VB7, and 0.1-1 mg / L VB12.

[0023] Specifically, the effector is selected from any one or a combination of several groups of amino acids, nucleotides / nucleotide precursors.

[0024] The amino acids include any one or a combination of several of the following: 0.05-1 g / L L-tryptophan, 0.05-1 g / L aspartic acid, 0.03-1 g / L threonine, 0.03-1 g / L methionine, and 0.03-1 g / L isoleucine; the nucleotides / nucleotide precursors include 1.5-5 g / L hypoxanthine and / or 0.05-1 g / L uracil.

[0025] In some embodiments of the present invention, the amino acids are composed of 0.05-1 g / L L-tryptophan, 0.05-1 g / L aspartic acid, 0.03-1 g / L threonine, 0.03-1 g / L methionine, and 0.03-1 g / L isoleucine; the nucleotides / nucleotide precursors are composed of 1.5-5 g / L hypoxanthine and 0.05-1 g / L uracil.

[0026] The initial fermentation parameters for the fermentation culture are set as follows: temperature 35~40°C, stirring speed 100~800 rpm, aeration rate 0.5~4 vvm, tank pressure 0.02~0.1 MPa, dissolved oxygen maintained at 20~60% throughout the process, and fermentation pH maintained at 6.0~8.0; the carbon source is 100~1000 g / L glucose; the inducer is IPTG with a final concentration of 0.01~10 mM; the induction parameters are set as follows: temperature 20~37°C, stirring speed 100~800 rpm, aeration rate 0.5~4 vvm, tank pressure 0.02~0.1 MPa, dissolved oxygen maintained at 20~60% throughout the process, and fermentation pH maintained at 6.0~8.0.

[0027] In some embodiments of the present invention, the initial fermentation parameters for the fermentation culture are set as follows: temperature 35-40°C, stirring speed 500 rpm, aeration rate 2 vvm, tank pressure 0.05 MPa, dissolved oxygen maintained at 30-40% throughout the process, and fermentation pH maintained at 7.0; the carbon source is 600 g / L glucose; the inducer is IPTG with a final concentration of 0.04 mM; the induction parameters are set as follows: temperature 20-37°C, stirring speed 500 rpm, aeration rate 2 vvm, tank pressure 0.05 MPa, dissolved oxygen maintained at 20-30% throughout the process, and fermentation pH maintained at 7.0.

[0028] In some embodiments of the present invention, when the dissolved oxygen value first rises to above 80% and the residual sugar concentration is close to 0 g / L, a carbon source is added to maintain the residual sugar concentration in the range of 0~2 g / L.

[0029] In some embodiments of the present invention, an inducer is added to induce dsRNA expression when the bacterial cells have fermented to the logarithmic growth phase, at a time when the culture period is 8-14 hours after inoculation or at OD600. 600 Ferment to 20°C.

[0030] Beneficial effects:

[0031] (1) This invention provides a method for increasing dsRNA yield, which is applicable to E. coli expression systems carrying dsRNA expression vectors. By systematically optimizing various components such as carbon and nitrogen sources, phosphates, metal ions, inorganic salts, vitamins, and effectors on the basis of LB medium, the synergistic effect between the components is fully utilized. After verification by shake-flask fermentation, the optimized dsRNA fermentation medium significantly increased dsRNA yield by about 29%, with the highest yield reaching about 151.122 mg / L.

[0032] (2) This invention further achieves process scale-up and optimization from laboratory shake flasks to 6 L fermenters by precisely controlling fermentation parameters (including dissolved oxygen, feeding strategy, and induction timing). Combined with dsRNA fermentation medium, a yield of 728 mg / L was achieved in LB medium-based medium, significantly superior to the 182 mg / L reported in existing bacterial expression systems. This result fully demonstrates that the method of synergistically improving dsRNA yield through the medium formulation and fermentation process described in this invention possesses excellent scalability and industrial application potential, providing a reliable solution for the large-scale, low-cost production of dsRNA. Attached Figure Description

[0033] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0034] Figure 1 This is a gel electrophoresis image of recombinant Escherichia coli.

[0035] Figure 2 The results of optimizing the addition of different concentrations of carbon / nitrogen sources to LB medium.

[0036] Figure 3 Optimization results for adding different phosphates to LB medium.

[0037] Figure 4 The results of optimizing the addition of different concentrations of inorganic salts containing metal ions to LB medium.

[0038] Figure 5 dsRNA yield and OD of LB medium supplemented with different concentrations of vitamins 600 Value relationship diagram.

[0039] Figure 6 The results of optimizing the addition of different concentrations of effector (nucleotide) to LB medium.

[0040] Figure 7 Experimental results of adding the composition to LB medium.

[0041] Figure 8dsRNA was produced in a 6 L fermenter in LB medium. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0043] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0044] Example 1: Screening of recombinant Escherichia coli expressing exogenous RNA

[0045] I. Recombinant Escherichia coli pET28a-ERG-CAG 40 -ERG-BL21(DE3)-ΔC-Δrnc, pET28a-DCL1+2-CAG 40 Construction of -DCL1+2-BL21(DE3)-ΔC-Δrnc

[0046] 1. Fusion gene fragment DCL1+2-CAG 40 -DCL1+2, ERG-CAG 40 Construction of -ERG and corresponding recombinant plasmid

[0047] DCL1+2 gene fragment, CAG 40 Tags, DCL1+2 reverse complementary gene fragments according to DCL1+2, CAG 40 The nucleotide sequences of DCL1+2 were ligated in reverse complementary order to obtain the fusion gene fragment DCL1+2-CAG, as shown in SEQ ID NO.4. 40 -DCL1+2. The nucleotide sequence of the DCL1+2 gene is shown in SEQ ID NO.1; the CAG... 40 The tag, the nucleotide sequence of which is shown in SEQ ID NO.2; the DCL1+2 reverse complementary gene, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0048] EGR gene fragment, CAG 40 Tags, EGR reverse complementary gene fragments according to ERG, CAG 40 The nucleotide sequences of the reverse complementary ERG fragments were ligated to obtain the fusion gene fragment ERG-CAG, as shown in SEQ ID NO.7. 40 -ERG. The EGR gene, whose nucleotide sequence is shown in SEQ ID NO.5; the CAG... 40The tag, the nucleotide sequence of which is shown in SEQ ID NO.2; the ERG reverse complementary gene, the nucleotide sequence of which is shown in SEQ ID NO.6.

[0049] The fusion gene fragment ERG-CAG 40 -ERG, DCL1+2-CAG 40 -DCL1+2 was ligated to plasmid pET28a after double digestion with XbaI and EcoRI, respectively, and the recombinant plasmids pET28a-ERG-CAG were obtained. 40 -ERG, pET28a-DCL1+2-CAG 40 -DCL1+2.

[0050] The aforementioned fusion gene fragment and recombinant plasmid were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0051] 2. Construction of recombinant Escherichia coli

[0052] The synthesized recombinant plasmid was introduced into the BL21(DE3)-ΔC-Δrnc recombinant strain via heat shock transformation. The results were verified to be correct, thus yielding a small cell system containing CAG. 40 Recombinant Escherichia coli pET28a-ERG-CAG tagged 40 -ERG-BL21(DE3)-ΔC-Δrnc, pET28a-DCL1+2-CAG 40 -DCL1+2-BL21(DE3)-ΔC-Δrnc. The BL21(DE3)-ΔC-Δrnc recombinant strain is either the strain B-Δr-ΔC disclosed in Example 17 of Chinese Patent CN118291510A, or the strain BL21(DE3)ΔrncΔminC disclosed in Example 1 of Chinese Patent CN118240729.

[0053] II. Construction of recombinant Escherichia coli pET28a-DCL1+2-DCL1+2-BL21(DE3)-Δrnc and pET28a-ERG-ERG-BL21(DE3)-Δrnc

[0054] 1. Construction of fusion gene fragments DCL1+2-DCL1+2 and ERG-ERG and their corresponding recombinant plasmids

[0055] CAG 40The tag was replaced with a meaningless, equal-length sequence. The DCL1+2 gene fragment and the DCL1+2 reverse complementary gene fragment were linked in the order of DCL1+2, equal-length sequence, and DCL1+2 complement, to obtain the nucleotide sequence shown in SEQ ID NO.8, which is the fusion gene fragment DCL1+2-DCL1+2.

[0056] CAG 40 The tag was replaced with a meaningless, equal-length sequence. The ERG gene fragment and the ERG reverse complement gene fragment were then linked in the order of ERG, equal-length sequence, and ERG complement to obtain the nucleotide sequence shown in SEQ ID NO.9, which is the fusion gene fragment ERG-ERG.

[0057] The fusion gene fragments DCL1+2-DCL1+2 and ERG-ERG were ligated to plasmid pET28a after double digestion with Xba I and EcoR I, respectively. The recombinant plasmids pET28a-DCL1+2-DCL1+2 and pET28a-ERG-ERG were obtained after verification.

[0058] The aforementioned fusion gene fragment and recombinant plasmid were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0059] 2. Construction of recombinant strains

[0060] The synthesized recombinant plasmids pET28a-DCL1+2-DCL1+2 and pET28a-ERG-ERG were introduced into the BL21(DE3)-Δrnc recombinant strain via heat shock transformation. The results were verified to be correct, thus obtaining recombinant Escherichia coli pET28a-DCL1+2-DCL1+2-BL21(DE3)-Δrnc and pET28a-ERG-ERG-BL21(DE3)-Δrnc. The BL21(DE3)-Δrnc recombinant strain is the strain B-Δr disclosed in Example 17 of Chinese Patent CN118291510A.

[0061] III. Experimental Verification of dsRNA Production by Fermentation of Recombinant E. coli Expressing Exogenous RNA

[0062] Recombinant E. coli expressing exogenous RNA, including pET28a-BcSAS1-BcSAS1-BL21(DE3)-Δrnc (BcSAS1 control), pET28a-DCL1+2-DCL1+2-BL21(DE3)-Δrnc (DCL1+2 control), pET28a-ERG-ERG-BL21(DE3)-Δrnc (ERG control), and pET28a-BcSAS1-CAG, were selected. 40-BcSAS1-BL21(DE3)-ΔC-Δrnc (abbreviated as BcSAS1 experimental bacteria) pET28a-DCL1+2-CAG 40 -DCL1+2-BL21(DE3)-ΔC-Δrnc (abbreviated as DCL1+2 experimental bacteria), pET28a-ERG-CAG 40 Six single colonies of ERG-BL21(DE3)-ΔC-Δrnc (ERG experimental bacteria) were inoculated into 5 mL of LB medium and cultured at 37°C and 200 rpm with shaking for 10–12 hours. The activated bacterial culture was then inoculated into 50 mL of LB liquid medium at a 1% v / v inoculation rate and fermented at 37°C and 200 rpm. When fermentation reached OD... 600 IPTG was added to a final concentration of 0.4 mM at a concentration of 0.8, and fermentation continued for another 4 h. After fermentation, dsRNA was extracted from each fermentation broth, and the yield of dsRNA produced by fermentation of the three recombinant E. coli strains expressing exogenous RNA was compared. Each strain was replicated in 3 batches. Among them, the recombinant E. coli pET28a-BcSAS1-BcSAS1-BL21(DE3)-Δrnc and pET28a-BcSAS1-CAG were compared. 40 -BcSAS1-BL21(DE3)-ΔC-Δrnc has been disclosed in Chinese Patent CN118291510A, namely the recombinant strains B-Δr-BcSAS1-BcSAS1 and B-Δr-ΔC-BcSAS1-CAG disclosed in Chinese Patent CN118291510A, respectively. 40 -BcSAS1.

[0063] from Figure 1 The gel images show that the target bands appeared at their respective positions in the BcSAS1 control, DCL1+2 experimental, and ERG experimental strains. While the bands in the DCL1+2 and ERG control strains were bright, the target bands did not appear. This may be due to the instability and fragmentation of the dsRNA produced by the DCL1+2 and ERG control strains. Considering the dsRNA yield, among the remaining four strains, the BcSAS1 control strain had the lowest yield, with a significant difference in yield between parallel studies of the BcSAS1 experimental strain. The ERG experimental strain had the highest yield at 79.24 mg / L, followed by the BcSAS1 experimental strain, and then the DCL1+2 experimental strain. Subsequent fermentation culture validation experiments used high-yielding and stable recombinant E. coli pET28a-ERG-CAG. 40 -ERG-BL21(DE3)-ΔC-Δrnc proceeds.

[0064] Example 2: Validation of the effect of different concentrations of carbon / nitrogen sources on increasing dsRNA production in recombinant Escherichia coli

[0065] Using LB medium as the basal medium, a carbon source gradient of 5–15 g / L glucose or a nitrogen source gradient of 3–9 g / L ammonium sulfate was set up to evaluate the effects of different concentrations of carbon and nitrogen sources on dsRNA production.

[0066] Select recombinant Escherichia coli pET28a-ERG-CAG 40 A single colony of -ERG-BL21(DE3)-ΔC-Δrnc was inoculated into 5 mL of LB medium and cultured at 37°C with shaking at 200 rpm for 10–12 hours. The activated bacterial culture was then inoculated into 50 mL of LB liquid medium with different concentrations of carbon / nitrogen sources at a 1% v / v inoculation rate and fermented at 37°C with shaking at 200 rpm. When fermentation reached OD... 600 IPTG was added to a final concentration of 0.4 mM when the concentration was 0.8, and fermentation continued for another 4 h. After fermentation was completed, dsRNA was extracted from each fermentation broth and the dsRNA yield was compared.

[0067] The results are as follows Figure 2 As shown, adding a low concentration of carbon source (5 g / L) had a positive effect on dsRNA production, increasing it by 1.8 mg / L, but the increase was not significant. At a medium concentration of glucose (10 g / L), dsRNA production was almost the same as the LB control, while a high concentration of glucose (15 g / L) had the opposite effect on fermentation. Furthermore, adding different concentrations of ammonium sulfate all produced adverse effects, reducing the yield in all cases.

[0068] Example 3: Effects of different phosphates on dsRNA accumulation

[0069] Using LB medium as the basal medium, the effects of different phosphates (different concentrations of potassium salt and sodium salt, Table 1) on dsRNA production in LB medium were investigated. Recombinant *E. coli* pET28a-ERG-CAG 40 The dsRNA culture and shake-flask fermentation process of -ERG-BL21(DE3)-ΔC-Δrnc was performed according to Example 2, and the dsRNA yield after fermentation was measured.

[0070] Table 1. Configurations with different phosphate concentrations

[0071]

[0072] The results are as follows Figure 3As shown, based on LB medium, the type (potassium / sodium) and concentration of phosphate significantly affected dsRNA production. High concentrations of phosphate inhibited dsRNA synthesis, while low concentrations had no significant effect. Only the addition of 5 g / L disodium hydrogen phosphate resulted in 80.065 mg / L dsRNA, which was essentially the same as the control group's 81.857 mg / L.

[0073] Example 4: Effects of different concentrations of metal ion-containing inorganic salts on dsRNA accumulation

[0074] LB medium was used as the basal medium, and different concentrations of inorganic salts containing metal ions were added (Table 2) for shake-flask fermentation to investigate their effect on dsRNA production. Recombinant *E. coli* pET28a-ERG-CAG 40 The dsRNA culture and shake-flask fermentation process of -ERG-BL21(DE3)-ΔC-Δrnc was performed according to Example 2, and the dsRNA yield after fermentation was measured.

[0075] Table 2. Preparation of inorganic salts containing metal ions at different concentrations

[0076]

[0077] like Figure 4 The results showed that the addition of metal ions promoted dsRNA production. Among them, 10 mg / L calcium chloride increased dsRNA production by 44%, exhibiting the most significant effect. Further gel electrophoresis revealed that dsRNAs obtained with different concentrations of inorganic salts containing metal ions generally showed bands at the target position (751 bp). However, the addition of magnesium sulfate resulted in double bands, affecting the dsRNA product. Therefore, even though 9 g / L magnesium sulfate resulted in high dsRNA production, a magnesium sulfate concentration of 3 g / L was considered optimal. The final preferred concentrations of the inorganic salts containing the metal ions were: 10 mg / L calcium chloride, 0.15 mg / L cobalt chloride, 1.5 mg / L copper chloride, 21 mg / L ferrous sulfate, 3 g / L magnesium sulfate, 1 mg / L manganese sulfate, and 5 mg / L zinc sulfate.

[0078] Example 5: Effects of different vitamins on dsRNA accumulation

[0079] Using LB medium as the basal medium, the effects of different vitamin concentrations (Table 3) on the yield of dsRNA produced by shake-flask fermentation were investigated. Recombinant *E. coli* pET28a-ERG-CAG 40 The dsRNA culture and shake-flask fermentation process using ERG-BL21(DE3)-ΔC-Δrnc was performed according to Example 2. The dsRNA yield and OD were measured after fermentation.600 .

[0080] Table 3. Preparation of different vitamin concentrations

[0081]

[0082] In terms of yield, except for VB5, all other vitamins promoted dsRNA production, with 0.1 mg / L VB2 showing the best effect, increasing it by approximately 7 mg / L. From OD... 600 The results showed that vitamins B1 and B5 promoted the cell concentration of recombinant E. coli, with vitamin B5 showing the best effect. Further investigation was conducted on dsRNA production and OD... 600 The relationship between values ​​( Figure 5 The study found no significant correlation between dsRNA production and OD. This example demonstrates that optimized vitamin supplementation (especially VB2) in LB medium effectively drives cell metabolism and promotes efficient dsRNA synthesis, while VB5 acts as a key factor in promoting cell growth. Considering all factors, the preferred vitamins and concentrations are: 0.5 mg / L VB1, 0.1 mg / L VB2, 0.1 mg / L VB3, 0.1 mg / L VB3 derivative (nicotinamide), 0.5 mg / L VB6, 0.3 mg / L VB7, and 0.1 mg / L VB12.

[0083] Example 6: Effects of different concentrations of effector on dsRNA accumulation

[0084] Using LB medium as the basal medium, the yield of dsRNA produced by shake-flask fermentation under different concentrations of effector additives (Table 4) was investigated. Recombinant *E. coli* pET28a-ERG-CAG 40 The dsRNA culture and shake-flask fermentation process using ERG-BL21(DE3)-ΔC-Δrnc was performed according to Example 2. The dsRNA yield and OD were measured after fermentation. 600 .

[0085] Table 4. Configuration of different effector concentrations

[0086]

[0087] The results showed that although high concentrations of sodium citrate had a negative impact on dsRNA production, dsRNA production rebounded after reducing the sodium citrate concentration. However, overall, organic acids had no effect on increasing dsRNA production. L-tryptophan and aspartic acid had no effect on increasing dsRNA production, but 0.5 g / L aspartic acid was beneficial for growth and OD. 600The values ​​increased; 0.03 g / L methionine and 0.03 g / L isoleucine had a significant effect on increasing dsRNA production, and dsRNA production increased with increasing threonine concentration. Additionally, from... Figure 6 As you can see, 0.05–1 g / L uracil promoted dsRNA production, with 1 g / L uracil showing the best effect, increasing it by 20% to 109.948 mg / L. Meanwhile, dsRNA production decreased with increasing hypoxanthine concentration, but OD levels were measured... 600 Adding 5 g / L hypoxanthine to LB medium can promote an increase in the concentration of recombinant *E. coli* cells by approximately 50%. However, due to the insolubility of high-concentration hypoxanthine, the OD of hypoxanthine (5 g / L) is relatively high. Therefore, adding effectors can effectively regulate the metabolic process of the producing bacteria, thereby increasing both dsRNA production and cell concentration. Considering all factors, the preferred formulations are: 0.05 g / L L-tryptophan, 0.5 g / L aspartic acid, 1 g / L threonine, 0.03 g / L methionine, 0.03 g / L isoleucine, 1.5 g / L hypoxanthine, and 1 g / L uracil.

[0088] Example 7: Effects of adding different compositions on dsRNA accumulation

[0089] Using LB medium as the basal medium, shake-flask fermentation experiments were conducted to investigate the effect of adding the composition (Table 5) to the medium on dsRNA production. Recombinant *E. coli* pET28a-ERG-CAG 40 The dsRNA culture and shake-flask fermentation process using ERG-BL21(DE3)-ΔC-Δrnc was performed according to Example 2. The dsRNA yield and OD were measured after fermentation. 600 .

[0090] Table 5 Composition Formulation

[0091]

[0092] like Figure 7 As shown, adding all the above components to LB medium increased the yield of dsRNA by approximately 29%, reaching approximately 151.122 mg / L. Furthermore, this example also found that adding metal ions to LB medium increased the concentration of recombinant E. coli cells producing dsRNA by approximately 20%. Subsequent large-scale fermentation used LB medium + the composition as the fermentation medium.

[0093] Example 7 Large-scale fermentation production of dsRNA

[0094] Select recombinant Escherichia coli pET28a-ERG-CAG 40A single colony of -ERG-BL21(DE3)-ΔC-Δrnc was inoculated into 5 mL of LB medium and cultured at 37°C with shaking at 200 rpm for 10–12 hours. The culture was then transferred to 100 mL of LB fermentation medium at a 2% v / v inoculation rate and cultured again at 37°C with shaking at 200 rpm for 10–12 hours to obtain the seed culture. The seed culture was then inoculated into a 6 L bioreactor (Infors HT, Switzerland) containing fermentation medium (LB medium + composite) at a 2% v / v inoculation rate, with an initial fermentation volume of 3 L. The initial fermentation parameters were set as follows: stirring speed 500 rpm, aeration rate 2 vvm, tank pressure 0.05 MPa, and dissolved oxygen maintained at 30–40% throughout the process. When fermentation reached the mid-to-late logarithmic growth stage (approximately 12 hours), IPTG was added at 37°C to a final concentration of 0.4 mM. The induction parameters were as follows: 37°C, stirring speed 500 rpm, aeration rate 2 vvm, tank pressure 0.05 MPa. Dissolved oxygen was maintained at 20-30% throughout the process, and the fermentation pH was maintained at 7.0. During this period, when the dissolved oxygen level first rapidly increased to above 80% and the residual glucose concentration approached zero (usually around 8 hours), glucose was added (600 g / L) to maintain a low glucose level (residual glucose close to zero). Throughout the fermentation process, the residual glucose concentration was continuously monitored and controlled to maintain it at a near-zero level. Large-scale fermentation results were observed in a 6 L bioreactor, such as... Figure 8 As shown, the dsRNA yield reached 728 mg / L in LB medium + composition, significantly exceeding the highest yield of 182 mg / L previously reported in bacterial systems.

[0095] This invention provides a concept and method for increasing dsRNA yield. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for increasing dsRNA yield, characterized in that, Seed culture of recombinant strains carrying dsRNA expression vector was inoculated into optimized fermentation medium for fermentation culture. When the dissolved oxygen value first increased to above 80% and the residual sugar concentration was below 4 g / L, carbon source was added to maintain the residual sugar concentration in the range of 0-5 g / L. When the cell fermentation reached the logarithmic growth phase, an inducer was added to induce dsRNA expression. The recombinant strain carrying the dsRNA expression vector was constructed using Escherichia coli as the expression system. Specifically, it was obtained by overexpressing the BcSAS1 gene fusion fragment, the DCL1+2 gene fusion fragment, or the ERG gene fusion fragment using Escherichia coli BL21(DE3)-ΔC-Δrnc as the chassis strain.

2. The method according to claim 1, characterized in that, The optimized fermentation medium is obtained by adding metal ions, inorganic salts, vitamins, and effectors to LB medium as the base medium.

3. The method according to claim 2, characterized in that, The inorganic salt containing the metal ion includes an inorganic salt that provides divalent metal ions, wherein the divalent metal ions include Mg. 2+ Ca 2+ Zn 2+ Fe 2+ Mn 2+ Co 2+ Cu 2+ Any one or more of the following combinations.

4. The method according to claim 3, characterized in that, The metal ion inorganic salt includes any one or a combination of several of the following: 10-30 mg / L calcium chloride, 0.15-1.5 mg / L cobalt chloride, 0.15-1.5 mg / L copper chloride, 7-21 mg / L ferrous sulfate, 3-9 g / L magnesium sulfate, 0.5-1.5 mg / L manganese sulfate, and 5-15 mg / L zinc sulfate.

5. The method according to claim 2, characterized in that, The vitamins include any one or a combination of several of the following: 0.1-5 mg / L VB1, 0.1-1 mg / L VB2, 0.1-1 mg / L VB3, 0.1-1 mg / L nicotinamide, 0.1-1 mg / L VB6, 0.03-3 mg / L VB7, and 0.1-1 mg / L VB12.

6. The method according to claim 2, characterized in that, The effector is selected from any one or a combination of several of amino acids and nucleotides / nucleotide precursors; The amino acids include any one or a combination of several of the following: 0.05-1 g / L L-tryptophan, 0.05-1 g / L aspartic acid, 0.03-1 g / L threonine, 0.03-1 g / L methionine, and 0.03-1 g / L isoleucine; the nucleotides / nucleotide precursors include 1.5-5 g / L hypoxanthine and / or 0.05-1 g / L uracil.

7. The method according to claim 1, characterized in that, The initial fermentation parameters for the fermentation culture are set as follows: temperature 35~40°C, stirring speed 100~800 rpm, aeration rate 0.5~4 vvm, tank pressure 0.02~0.1 MPa, dissolved oxygen maintained at 20~60% throughout the process, and fermentation pH maintained at 6.0~8.

0.

8. The method according to claim 1, characterized in that, The carbon source is 100~1000 g / L glucose; the inducer is IPTG with a final concentration of 0.01~10 mM.

9. The method according to claim 1, characterized in that, The induction parameters are set as follows: temperature 20~37°C, stirring speed 100~800 rpm, aeration rate 0.5~4 vvm, tank pressure 0.02~0.1 MPa, dissolved oxygen maintained at 20~60% throughout the process, and fermentation pH maintained at 6.0~8.

0.

10. The method according to claim 1, characterized in that, The DCL1+2 gene fusion fragment is obtained using CAG. 40 The tag is obtained by linking the DCL1+2 gene and the DCL1+2 reverse complement gene. The ERG gene fusion fragment is obtained using CAG. 40 The tag is obtained by linking the ERG gene and the ERG reverse complement gene. The DCL1+2 gene has the nucleotide sequence shown in SEQ ID NO.1; the CAG 40 The tag, the nucleotide sequence of which is shown in SEQ ID NO.2; the DCL1+2 reverse complement gene, the nucleotide sequence of which is shown in SEQ ID NO.3; the ERG gene, the nucleotide sequence of which is shown in SEQ ID NO.5; the ERG reverse complement gene, the nucleotide sequence of which is shown in SEQ ID NO.6.

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