A method for increasing the production of dsRNA

Abstract

The application belongs to the technical field of bioengineering and particularly relates to a method for improving the yield of dsRNA. The method is suitable for an E. coli expression system carrying a dsRNA expression vector. By optimizing multiple components in the fermentation medium, such as carbon and nitrogen sources, phosphate, metal ion inorganic salt, vitamins and effectors, and combining the regulation of fermentation parameters (including dissolved oxygen, feeding strategy and induction timing), the yield of dsRNA is significantly improved to 728 mg / L. The dsRNA fermentation process has excellent scalability and industrial application potential, and provides a reliable solution for the large-scale and low-cost production of dsRNA.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and particularly relates to a method for improving the yield of dsRNA. BACKGROUND

[0002] Double-stranded RNA (dsRNA) as an important biological macromolecule, has shown great application potential in the fields of agricultural biological pesticides and gene function research. For example, RNA interference (RNAi) technology based on dsRNA has become a core means for developing a new generation of targeted insecticides and new therapeutic drugs. However, to realize the large-scale promotion of these applications, it is necessary to solve the key bottleneck problem of high production cost of dsRNA. At present, the use of recombinant Escherichia coli fermentation to produce dsRNA is one of the most cost-effective solutions.

[0003] Although the E. coli expression system has the advantages of simple operation, rapid growth, high yield, etc., its inherent metabolic network and physiological characteristics are not always matched with the efficient synthesis of exogenous dsRNA. The traditional LB and other basic media are designed to promote rapid growth of bacterial cells, and the nutritional components are not optimized for the accumulation of specific products such as dsRNA. Therefore, the types and proportions of carbon and nitrogen sources, phosphate concentration, metal ions, vitamins, and effectors (such as nucleosides, amino acids) in the medium are insufficient or unbalanced, which will limit the production potential of the strain, resulting in low yield and high cost, and it is difficult to meet the needs of industrial production. In the large-scale fermentation process, the synergistic optimization of medium composition and fermentation process is more critical, but the systematic study of dsRNA medium is relatively scarce. Whether the unification of high-density culture and efficient expression can be achieved at the fermenter level has become a bottleneck hindering the expansion of the dsRNA production line. For example, the highest yield of dsRNA produced by bacterial fermentation reported in 2024 is 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), which is obtained at the scale of a fermenter, but still far from the economic requirements of industrial production.

[0004] Currently, although some studies have attempted to improve the stability of dsRNA by genetically engineering host strains (such as knocking out nuclease genes), there is still a lack of systematic research on directly strengthening the supply of synthesis substrates and energy levels from the perspective of metabolic engineering through medium component optimization. An optimized medium that can provide the "building materials" and "catalytic tools" required for dsRNA synthesis is crucial for releasing the production capacity of engineered bacteria. More importantly, precisely regulated fermentation processes (such as dynamic feeding based on metabolic feedback, dissolved oxygen ladder control, growth-coupled induction, and other strategies) are also key approaches to further break through the yield bottleneck and achieve efficient large-scale production.

[0005] Therefore, the present application aims to provide a method for improving the yield of dsRNA, an optimized fermentation medium strategy for dsRNA production, and a large-scale fermentation process. By systematically studying and optimizing the types and concentrations of carbon and nitrogen sources, phosphates, metal ions, vitamins, and specific effectors in the medium, and combining process control under the scale of a fermenter, a balance is established between the growth of recombinant E. coli and the synthesis of products, significantly improving the yield and productivity of dsRNA, and laying a solid foundation for large-scale, low-cost industrial production of dsRNA. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a method for improving the yield of dsRNA to address the low yield and low production efficiency of dsRNA in the prior art. Specifically, by systematically optimizing and proportioning the carbon and nitrogen sources, inorganic salts, vitamins, and effector components in the fermentation medium, the synergistic effect of the components is utilized to create an extracellular metabolic environment conducive to the efficient synthesis of dsRNA, promote the growth of the host bacteria, and strengthen the metabolic flow of the host bacteria towards the synthesis of product dsRNA. At the same time, carbon source is dynamically supplemented during fermentation to maintain a low sugar concentration, and an inducer is added during the logarithmic growth phase of the bacteria to induce the efficient expression of dsRNA, ultimately achieving high and stable production of dsRNA.

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

[0008] A method for improving the yield of dsRNA, wherein seed liquid of a recombinant strain carrying a dsRNA expression vector is inoculated into an optimized fermentation medium for fermentation culture. When the dissolved oxygen value first rises to more than 80% and the residual sugar concentration is less than 4 g / L, carbon source is supplemented to maintain the residual sugar concentration within the range of 0-5 g / L. An inducer is added to induce the expression of dsRNA when the bacteria ferment to the logarithmic growth phase.

[0009] The dsRNA has been disclosed in Chinese Patent CN118240729A.

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

[0011] In some embodiments of the present application, the recombinant strain carrying the dsRNA expression vector is obtained by overexpressing a BcSAS1 gene fusion fragment, or a DCL1+2 gene fusion fragment, or an ERG gene fusion fragment, using Escherichia coli BL21(DE3)-△C-△rnc as a chassis strain.

[0012] The Escherichia coli BL21(DE3)-△C-△rnc is the host B-△r-△C disclosed in Chinese patent CN118291510A.

[0013] The recombinant strain using Escherichia coli BL21(DE3)-△C-△rnc as a chassis strain and overexpressing the BcSAS1 gene fusion fragment is the recombinant strain B-△r-△C-BcSAS1-CAG disclosed in Chinese patent CN118291510A. 40 -BcSAS1.

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

[0015] In some embodiments of the present application, the DCL1+2 gene has a nucleotide sequence as shown in SEQ ID NO. 1; the CAG 40 tag has a nucleotide sequence as shown in SEQ ID NO. 2; the DCL1+2 reverse complementary gene has a nucleotide sequence as shown in SEQ ID NO. 3; the ERG gene has a nucleotide sequence as shown in SEQ ID NO. 5; and the ERG reverse complementary gene has a nucleotide sequence as shown in SEQ ID NO. 6.

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

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

[0018] Specifically, the metal ion inorganic salts include inorganic salts providing divalent metal ions, wherein the divalent metal ions include Mg2+ Ca 2+ Zn 2+ Fe 2+ Mn 2+ Co 2+ Cu 2+ , or a combination of two or more thereof.

[0019] Specifically, the metal ion inorganic salt comprises any one or a combination of two or more 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.

[0020] In some embodiments of the present application, the metal ion inorganic salt consists 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 comprise any one or a combination of two or more 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.

[0022] In some embodiments of the present application, the vitamins consist 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 two or more groups of amino acids and nucleotides / nucleotide precursors.

[0024] Specifically, the amino acids comprise any one or a combination of two or more 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; and the nucleotides / nucleotide precursors comprise 1.5-5 g / L hypoxanthine and / or 0.05-1 g / L uracil.

[0025] In some embodiments of the present application, the amino acids consist 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, 0.03-1 g / L isoleucine; the nucleotides / nucleotide precursors consist of 1.5-5 g / L hypoxanthine, 0.05-1 g / L uracil.

[0026] In some embodiments of the present application, the initial fermentation parameters of 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, and the final concentration thereof is 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 application, the initial fermentation parameters of 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, and the final concentration thereof is 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 application, when the dissolved oxygen value first rises to above 80% and the residual sugar concentration approaches 0 g / L, the carbon source is supplemented to maintain the residual sugar concentration in the range of 0-2 g / L.

[0029] In some embodiments of the present application, the inducer is added to induce the expression of dsRNA when the bacterial cells are fermented to the logarithmic growth phase, and the timing is 8-14 hours after inoculation or OD 600 fermented to 20.

[0030] Beneficial effects:

[0031] (1) The application provides a method for improving the yield of dsRNA, which is suitable for an E. coli expression system carrying a dsRNA expression vector. By optimizing the carbon and nitrogen sources, phosphate, metal ion inorganic salt, vitamins and effectors and other components in the LB medium, the synergistic effect of each component is fully utilized. The optimized dsRNA fermentation medium significantly improves the dsRNA yield by about 29%, and the highest yield reaches about 151.122 mg / L.

[0032] (2) The application further realizes the process amplification and optimization from laboratory shake flask to 6 L fermenter by precisely controlling the fermentation parameters (including dissolved oxygen, feeding strategy and induction timing). Combined with the dsRNA fermentation medium, the yield reaches 728 mg / L in the LB medium, which is significantly better than the reported yield of 182 mg / L in the existing bacterial expression system. The results fully prove that the medium formula and fermentation process synergistically improve the yield of dsRNA, and have excellent amplification and industrial application potential, providing a reliable solution for large-scale and low-cost production of dsRNA. BRIEF DESCRIPTION OF DRAWINGS

[0033] The above and / or other aspects of the application will become more apparent by describing in detail the preferred embodiments thereof with reference to the attached drawings.

[0034] Figure 1 It is a gel electrophoresis map of recombinant E. coli.

[0035] Figure 2 It is the optimization result of adding different concentrations of carbon / nitrogen sources in LB medium.

[0036] Figure 3 It is the optimization result of adding different phosphates in LB medium.

[0037] Figure 4 It is the optimization result of adding different concentrations of metal ion-containing inorganic salts in LB medium.

[0038] Figure 5 It is the relationship diagram of dsRNA yield and OD value with different concentrations of vitamins added in LB medium. 600

[0039] Figure 6 It is the optimization result of adding different concentrations of effectors (nucleotides) in LB medium.

[0040] Figure 7 It is the experimental result of adding composition in LB medium.

[0041] Figure 8 ​Production of dsRNA in 6 L fermenter in LB medium. DETAILED DESCRIPTION

[0042] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following detailed description, taken in conjunction with the accompanying drawings in which:

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

[0044] Example 1: Screening of recombinant E. coli expressing foreign RNA

[0045] I. Recombinant E. coli pET28a-ERG-CAG 40 -ERG-BL21(DE3)-△C-△rnc, pET28a-DCL1+2-CAG 40 II. Construction of DCL1+2-BL21(DE3)-△C-△rnc

[0046] 1. Fusion gene fragment DCL1+2-CAG40-DCL1+2, ERG-CAG 40 Construction of ERG and corresponding recombinant plasmid

[0047] The DCL1+2 gene fragment, CAG 40 tag, and DCL1+2 reverse complementary gene fragment are connected in the order of DCL1+2, CAG 40 , and DCL1+2 reverse complement, to obtain a fusion gene fragment DCL1+2-CAG 40 -DCL1+2 with a nucleotide sequence as shown in SEQ ID NO. 4. Wherein, the DCL1+2 gene has a nucleotide sequence as shown in SEQ ID NO. 1; the CAG 40 tag has a nucleotide sequence as shown in SEQ ID NO. 2; and the DCL1+2 reverse complementary gene has a nucleotide sequence as shown in SEQ ID NO. 3.

[0048] The ERG gene fragment, CAG 40 tag, and ERG reverse complementary gene fragment are connected in the order of ERG, CAG 40 , and ERG reverse complement, to obtain a fusion gene fragment ERG-CAG 40 -ERG with a nucleotide sequence as shown in SEQ ID NO. 7. Wherein, the ERG gene has a nucleotide sequence as shown in SEQ ID NO. 5; the CAG 40a tag with a nucleotide sequence as shown in SEQ ID NO. 2; and the ERG reverse complementary gene with a nucleotide sequence as shown in SEQ ID NO. 6.

[0049] The fusion gene fragment ERG-CAG 40 ERG, DCL1+2-CAG 40 The DCL1+2 and the plasmid pET28a were respectively digested by XbaI and EcoR I, and then connected, and verified to obtain the recombinant plasmids pET28a-ERG-CAG 40 ERG, pET28a-DCL1+2-CAG 40 DCL1+2.

[0050] The fusion gene fragment and the recombinant plasmid are synthesized by Suzhou Jinyuzhi Technology Biotechnology Co., Ltd.

[0051] 2. Construction of recombinant E. coli

[0052] The synthesized recombinant plasmid is introduced into the BL21(DE3)-ΔC-Δrnc recombinant strain by heat shock transformation, and verified to be correct, that is, the recombinant E. coli pET28a-ERG-CAG based on the small cell system + containing the CAG 40 tag is obtained. 40 ERG-BL21(DE3)-ΔC-Δrnc, pET28a-DCL1+2-CAG 40 DCL1+2-BL21(DE3)-ΔC-Δrnc. Among them, the BL21(DE3)-ΔC-Δrnc recombinant strain is 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 E. coli pET28a-DCL1+2-DCL1+2-BL21(DE3)-Δrnc, pET28a-ERG-ERG-BL21(DE3)-Δrnc

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

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

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

[0057] The fusion gene fragments DCL1+2-DCL1+2 and ERG-ERG are respectively digested with Xba I and EcoR I, and then connected with the plasmid pET28a, and verified to obtain recombinant plasmids pET28a-DCL1+2-DCL1+2 and pET28a-ERG-ERG, respectively.

[0058] The above fusion gene fragments and recombinant plasmids are synthesized by Suzhou Jinyuzhi Technology Biotechnology Co., Ltd.

[0059] 2. Construction of recombinant strains

[0060] The synthesized recombinant plasmids pET28a-DCL1+2-DCL1+2 and pET28a-ERG-ERG are respectively introduced into the BL21(DE3)-△rnc recombinant strain by heat shock transformation, and verified to be correct, i.e., to obtain recombinant Escherichia coli pET28a-DCL1+2-DCL1+2-BL21(DE3)-△rnc and pET28a-ERG-ERG-BL21(DE3)-△rnc. Among them, the BL21(DE3)-△rnc recombinant strain is the strain B-△r disclosed in Example 17 of Chinese Patent CN118291510A.

[0061] III. Fermentation experiment verification of recombinant Escherichia coli expressing exogenous RNA to produce dsRNA

[0062] The recombinant Escherichia coli pET28a-BcSAS1-BcSAS1-BL21(DE3)-△rnc (referred to as BcSAS1 control bacteria), pET28a-DCL1+2-DCL1+2-BL21(DE3)-△rnc (referred to as DCL1+2 control bacteria), pET28a-ERG-ERG-BL21(DE3)-△rnc (referred to as ERG control bacteria), pET28a-BcSAS1-CAG 40-BcSAS1-BL21(DE3)-△C-△rnc (referred to as BcSAS1 experimental bacteria) pET28a-DCL1+2-CAG 40 -DCL1+2-BL21(DE3)-△C-△rnc (referred to as DCL1+2 experimental bacteria), pET28a-ERG-CAG 40 -ERG-BL21(DE3)-△C-△rnc (referred to as ERG experimental bacteria) were inoculated into 5 mL LB medium, and cultured at 37°C, 200 rpm for 10-12 hours. The activated bacterial liquid was inoculated into 50 mL LB liquid medium at a inoculation amount of 1% v / v, and fermented at 37°C, 200 rpm. When the fermentation reached OD 600 0.8, 0.4 mM IPTG was added, and the fermentation was continued for 4 h. After fermentation, the dsRNA of each fermentation liquid was extracted and the dsRNA production of the three recombinant E. coli strains expressing foreign RNA was compared. Each strain was repeated for 3 batches. Among them, the recombinant E. coli pET28a-BcSAS1-BcSAS1-BL21(DE3)-△rnc, pET28a-BcSAS1-CAG 40 -BcSAS1-BL21(DE3)-△C-△rnc has been disclosed in Chinese patent CN118291510A, i.e. the recombinant strains B-△r-BcSAS1-BcSAS1, B-△r-△C-BcSAS1-CAG 40 -BcSAS1.

[0063] From the gel map of Figure 1 It can be seen that BcSAS1 control bacteria, DCL1+2 experimental bacteria, BcSAS1 experimental bacteria, and ERG experimental bacteria all appeared a target single band at the corresponding position; although the bands of DCL1+2 control bacteria and ERG control bacteria were very bright, no target band appeared, which may be due to the instability of the dsRNA produced by DCL1+2 control bacteria and ERG control bacteria, which presented fragmentation. Among the remaining 4 strains, the yield of BcSAS1 control bacteria was the lowest, and the yield difference between BcSAS1 experimental bacteria was large. The yield of ERG experimental bacteria was the highest, which was 79.24 mg / L, followed by BcSAS1 experimental bacteria, and then DCL1+2 experimental bacteria. The recombinant E. coli pET28a-ERG-CAG 40 -ERG-BL21(DE3)-△C-△rnc was used for subsequent fermentation medium verification experiments.

[0064] Example 2: Verification of different concentrations of carbon / nitrogen sources for improving dsRNA yield in recombinant E. coli

[0065] With LB medium as the basic medium, set 5~15 g / L of glucose as the carbon source gradient or 3~9 g / L of ammonium sulfate as the nitrogen source gradient, and evaluate the influence of different concentrations of carbon and nitrogen sources on the dsRNA yield.

[0066] Pick recombinant E. coli pET28a-ERG-CAG 40 Inoculate the single colony of ERG-BL21(DE3)-△C-△rnc into 5 mL of LB medium, and cultivate at 37°C with 200 rpm shaking for 10~12 hours. After activation, inoculate the bacterial liquid into 50 mL of LB liquid medium + different concentrations of carbon / nitrogen sources at a 1% v / v inoculation amount, and continue to ferment at 37°C with 200 rpm shaking. When the fermentation reaches OD 600 0.8, supplement IPTG at a final concentration of 0.4 mM, and continue to ferment for 4 hours. After the fermentation is completed, collect each fermentation liquid to extract dsRNA and compare the dsRNA yield.

[0067] The results, as shown in Figure 2 , show that the addition of a low concentration of carbon source (5 g / L) has a certain positive effect on the dsRNA yield, and the dsRNA yield is increased by 1.8 mg / L, but the increase is not significant. Under a medium concentration of glucose (10 g / L), the dsRNA yield is almost flat compared with the LB control, while a high concentration of glucose (15 g / L) has a reverse effect on fermentation. In addition, the addition of different concentrations of ammonium sulfate has a reverse effect, and the yield is reduced.

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

[0069] With LB medium as the basic medium, investigate the influence of different phosphates (different concentrations of potassium salt and different concentrations of sodium salt, Table 1) on the dsRNA yield in the LB medium. Recombinant E. coli pET28a-ERG-CAG 40 ERG-BL21(DE3)-△C-△rnc cultivation and shake flask fermentation of dsRNA refer to Example 2, and determine the dsRNA yield after fermentation.

[0070] Table 1: Different phosphate concentrations

[0071]

[0072] The results, as shown in Figure 3As shown in the results, the type (potassium salt / sodium salt) and concentration of phosphate significantly affected the dsRNA yield based on LB medium, high concentration of phosphate inhibited the synthesis of dsRNA, and low concentration of phosphate had no significant effect on the synthesis of dsRNA. Only 80.065 mg / L of dsRNA was produced with the addition of 5 g / L of disodium hydrogen phosphate, which was basically the same as the control group of 81.857 mg / L.

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

[0074] Different concentrations of metal ion-containing inorganic salts (Table 2) were added to the LB medium-based medium for shake flask fermentation to investigate their effects on dsRNA yield. Recombinant E. coli pET28a-ERG-CAG 40 ERG-BL21(DE3)-△C-△rnc was cultured and dsRNA was fermented in a shake flask according to Example 2, and the dsRNA yield after fermentation was determined.

[0075] Table 2 Different concentrations of metal ion-containing inorganic salts

[0076]

[0077] As Figure 4 As shown in the results, the addition of metal ions promoted the production of dsRNA. Among them, 10 mg / L calcium chloride promoted the increase of dsRNA yield by 44%, which was the most significant. Further gel electrophoresis found that basically the dsRNA obtained by adding different concentrations of metal ion-containing inorganic salts appeared a band at the target position (751 bp), but the band appeared double bands when adding magnesium sulfate, which affected the dsRNA product, so even though the dsRNA yield was high under 9 g / L magnesium sulfate, the concentration of magnesium sulfate was considered to be selected at 3 g / L. The preferred concentrations of each metal ion inorganic salt 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: Effect of different vitamins on dsRNA accumulation

[0079] Different concentrations of vitamins (Table 3) were added to the LB medium-based medium to investigate their effects on the production of dsRNA in shake flask fermentation. Recombinant E. coli pET28a-ERG-CAG 40 ERG-BL21(DE3)-△C-△rnc was cultured and dsRNA was fermented in a shake flask according to Example 2, and the dsRNA yield and OD600 .

[0080] Table 3 Different vitamin addition concentration configuration

[0081]

[0082] From the yield, except VB5, the rest of the vitamins have a promoting effect on the production of dsRNA, 0.1 mg / L VB2 has the best effect, which can increase about 7 mg / L. From the OD 600 value, VB1, VB5 have a promoting effect on the cell concentration of recombinant E. coli, and vitamin B5 has the best effect. Further investigation of the relationship between dsRNA yield and OD 600 value ( Figure 5 ), it is found that there is no obvious correlation between dsRNA yield and OD. The results of this example show that the optimization of adding vitamins (especially VB2) in LB medium can effectively drive cell metabolism and promote the efficient synthesis of dsRNA, and VB5 can be used as a key factor to promote cell growth. Considering comprehensively, 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, 0.1 mg / L VB12.

[0083] Example 6: Effect of different concentrations of effectors on dsRNA accumulation

[0084] Based on LB medium, the yield of dsRNA produced by shake flask fermentation under different effector addition concentrations (Table 4) was investigated. The recombinant E. coli pET28a-ERG-CAG 40 -ERG-BL21(DE3)-△C-△rnc was cultured and the shake flask fermentation of dsRNA was carried out according to Example 2, and the dsRNA yield and OD 600 .

[0085] Table 4 Different effector addition concentration configuration

[0086]

[0087] It is found that although high concentration of sodium citrate has a negative effect on dsRNA production, the dsRNA yield increases after reducing the concentration of sodium citrate, but in general, organic acids have no effect on improving the yield of dsRNA. L-tryptophan and aspartic acid have no effect on improving the yield of dsRNA, but 0.5 g / L aspartic acid is helpful for growth, 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)-△C-△rnc was performed according to Example 2, and 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 40-ERG-BL21(DE3)-△C-△rnc single colony was inoculated into 5 mL LB medium and cultured at 37°C, 200 rpm for 10-12 hours. The culture after shaking was inoculated into 100 mL fermentation medium LB medium at a 2% v / v inoculation amount and shaken at 37°C, 200 rpm for 10-12 hours to obtain a seed solution. The seed solution was inoculated into a 6 L bioreactor (Infors HT, Switzerland) containing fermentation medium (LB medium + composition) at a 2% v / v inoculation amount, and the initial fermentation volume was 3 L. The initial fermentation parameters were set as follows: stirring speed 500 rpm, aeration amount 2 vvm, tank pressure 0.05 MPa, and the dissolved oxygen was maintained at 30-40% throughout the process. When the fermentation reached the middle-late logarithmic growth phase (about 12 hours), the inducer IPTG was added at 37°C to a final concentration of 0.4 mM, and the induction parameters were as follows: 37°C, stirring speed 500 rpm, aeration amount 2 vvm, tank pressure 0.05 MPa, and the dissolved oxygen was maintained at 20-30% throughout the process, and the fermentation pH was maintained at 7.0. During the period, when the dissolved oxygen value first increased rapidly to more than 80% and the residual glucose concentration was close to zero (usually around 8 hours), the glucose (600 g / L) was supplemented to maintain a low sugar level (residual glucose close to zero). During the entire fermentation process, the residual glucose concentration was continuously monitored and controlled to maintain a level close to zero. The results of large-scale fermentation in a 6 L bioreactor showed that the dsRNA yield reached 728 mg / L in LB medium + composition, which was significantly higher than the highest yield of 182 mg / L in a previously reported bacterial system. Figure 8

[0095] The present application provides a method for improving the yield of dsRNA. There are many ways to achieve this technical solution, and the above description is only the preferred embodiment of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should be considered within the scope of protection of the present application. The components not explicitly described in the embodiments can be implemented using existing technology.​

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 rose to above 80% and the residual sugar concentration was below 4 g / L, carbon source was added to maintain a low sugar level, with 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, using *Escherichia coli* BL21(DE3)-Δmin... C -△ rnc It was obtained by overexpressing the ERG gene fusion fragment from a chassis strain. The nucleotide sequence of the ERG gene fusion fragment is shown in SEQ ID NO.7; The optimized fermentation medium is obtained by adding metal ions, inorganic salts, vitamins, and effectors to LB medium as the base medium. The inorganic salt of metal ions is composed of 10 mg / L anhydrous calcium chloride, 0.15 mg / L cobalt chloride hexahydrate, 1.5 mg / L copper chloride pentahydrate, 21 mg / L ferrous sulfate heptahydrate, 3 g / L magnesium sulfate heptahydrate, 1 mg / L manganese sulfate monohydrate, and 5 mg / L zinc sulfate heptahydrate. The vitamins are composed of 0.5 mg / L VB1, 0.1 mg / L VB2, 0.1 mg / L VB3, 0.1 mg / L nicotinamide, 0.5 mg / L VB6, 0.3 mg / L VB7, and 0.1 mg / L VB12. The effector is composed of 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.

2. 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.

3. 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.

4. 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.

5. The method according to claim 1, characterized in that, 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. Wherein, the CAG 40 The tag, the nucleotide sequence of which is shown in SEQ ID NO.2; 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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