T7 RNA polymerase mutant with improved specific enzyme activity and 3 '-consistency and application thereof

Abstract

The invention discloses a T7RNA polymerase mutant with improved specific enzyme activity and 3 '-consistency and application of the T7RNA polymerase mutant, and belongs to the field of gene engineering and enzyme engineering. Starting from a wild T7RNAP protein structure, a mutant with improved specific enzyme activity and 3 '-consistency is obtained. Compared with the prior art, the T7RNAP mutant provided by the invention has higher specific enzyme activity and thermal stability, generates lower dsRNA by-products, can tolerate higher reaction temperature, and has wide application prospects in the aspects of in-vitro transcription, circular RNA, mRNA vaccines, isothermal amplification, acellular in-vitro transcription and translation systems, gene editing and drug production research.

Description

TECHNICAL FIELD

[0001] The present application relates to T7 RNA polymerase mutants with improved specific activity and 3'-uniformity and applications thereof, and belongs to the field of biotechnology. BACKGROUND

[0002] T7 RNA polymerase (T7 RNAP) is an enzyme that specifically catalyzes the formation of RNA in the 5'→3' direction, with a total of 883 amino acids and a molecular weight of about 99 kDa. It has high promoter specificity and can only transcribe DNA downstream of the T7 promoter. This enzyme can be used not only for in vitro RNA synthesis, such as gRNA, siRNA, and isotope-labeled or non-isotope-labeled RNA probe synthesis, synthesis of Capped mRNA with Cap analog for intracellular or in vivo expression, and mRNA vaccine and drug research; but also for isothermal amplification, such as sequence-specific nucleic acid in vitro amplification (NASBA) and transcription-mediated amplification (TMA).

[0003] Although wild-type T7 RNAP has the advantages of high transcription efficiency and strong elongation ability, there are still some non-negligible shortcomings in practical application research, such as the optimal reaction temperature is around 37℃, which is difficult to be compatible with other high-temperature-resistant enzymes to establish an efficient "one-pot" multifunctional enzyme catalytic system; and many by-products may be produced during RNA synthesis, including short abortive fragments produced during transcription initiation, 3' end extension products, dsRNA, etc., which are very unfavorable for mRNA vaccine and drug research and production. However, with the rapid development of mRNA drug and molecular diagnostic technology, the wild-type T7 RNA polymerase currently on the market cannot fully meet the growing research needs. Therefore, it is urgent to optimize and improve T7 RNAP so that it can not only tolerate a wider range of reaction temperatures and maintain efficient transcription, but also reduce the problem of RNA product heterogeneity.

[0004] Among existing technologies, CN 111212905 A provides a T7 RNAP mutant that reduces dsRNA contaminants and linked transcripts generated during in vitro transcription reactions; and CN 112831484 A provides a mutant suitable for synthesizing RNA containing internal termination signals and RNA with hairpin structures at the ends, which can synthesize Cas9 sgRNA with higher purity compared to T7 RNAP-WT. However, the thermal stability and heat resistance of these mutants lack supporting data, making it difficult to meet the research needs of high-temperature reactions. Although other technologies (such as CN 108779446 A and US 9193959 B2) provide T7 RNAP mutants with improved thermal stability, there is still no complete data chain to support their application performance. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a T7 RNAP mutant with enhanced enzyme activity, exhibiting higher reactivity and tolerance to higher reaction temperatures. The 3' end uniformity of mRNA synthesized under both room temperature (37°C) and high-temperature conditions is significantly improved, resulting in lower concentrations of dsRNA. Furthermore, it can be applied to one-step synthesis of circular RNA. This is achieved through the following techniques.

[0006] The following mutants were obtained through directed evolution. The mutant library was constructed into the pQE-80L vector through homologous recombination and transformed into the BL21(DE3) host. The mutants with enhanced specific enzyme activity were obtained using the FADS high-throughput screening platform.

[0007] Recombinant strains containing wild-type and mutant T7 RNAP were obtained, and T7 RNAP mutants were prepared by inducing expression and protein purification. Then, using 1000 nt RNA as a template, γ-IPPA and PNP were coupled via pyrophosphate, and the absolute specific enzyme activity of different mutants compared to T7 RNAP-WT was indirectly determined by the change in the final 2-amino-6-mercapto-7-methylpurine at 360 nm. The application performance of the T7 RNAP mutants was assessed by analyzing the products synthesized through in vitro transcription (IVT), including IVT yield, product purity, dsRNA content in the product, 3' end concordance index, and the ability to synthesize circular RNA.

[0008] The first objective of this invention is to provide a performance-enhancing RNA polymerase mutant, wherein the RNA polymerase mutant is based on the amino acid sequence corresponding to the parental RNA polymerase, and has an amino acid mutation at position 397, or an amino acid mutation at an equivalent position in the parental RNA polymerase; and has an amino acid mutation at any one of positions 43, 45, or 47, or an amino acid mutation at an equivalent position in the parental RNA polymerase.

[0009] The parent RNA polymerase has an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.1 and has RNA polymerase activity.

[0010] In one embodiment, the performance is selected from enzyme activity, 3' end uniformity, and dsRNA byproducts.

[0011] In one embodiment, the parental RNA polymerase is a wild-type T7 RNA polymerase with the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.14.

[0012] In one embodiment, the parental RNA polymerase having at least 80% identity with the amino acid shown in SEQ ID NO.1 is derived from different *E. coli* phages or other phages, including but not limited to: *Escherichia phage* T7, *Enterobacteria phage* vB EcoP IMME390, *Enterococcus phage* EFA-2, *Escherichia phage* 13a, *Escherichia phage* 64795ec1, *Escherichia phage* ClCC80001, *Escherichia phage* Ebrios, *Escherichia phage* EG1, *Escherichia phage* HZ2R8, *Escherichia phage* HZP2, *Escherichia phage* JacobBurckhardt, *Escherichia phage* JeanTinguely, *Escherichia phage* LL2, *Escherichia phage* N30, *Escherichia phage* NC-A, and *Klebsiella phage*. Patroon, Salmonella phage 3A_8767, Salmonella phage Vi06, Serratia phage 2050H2, Serratia phage Pila, Serratia phage SlM9-3Y, Escherichiaphage JB01, Yersinia phage phiA1122, Yersinia phage vB_YpYeO9, Yersinia phageYpP-R, Aeromonas phage PZL-Ah1, Citrobacter phage SH2, Escherichia phage E-2, Yersinia phage phiYe-F10, Citrobacter phage phiCFP-1, Citrobacter phage SH1, Salmonella phage phiSG-JL2, Yersinia phiYeO3-12, Klebsiella The amino acid sequence of any RNA polymerase in phage 31 or Enterobacteria phage T3.

[0013] In one embodiment, the RNA polymerase mutant also has one or more amino acid mutations at positions 430, 633, 786, and 744.

[0014] In one embodiment, corresponding to the amino acid sequence shown in SEQ ID NO.1,

[0015] The 43rd amino acid is mutated to Y, E, or A; preferably, it is mutated to Y or E;

[0016] The 45th amino acid is mutated to A;

[0017] The amino acid at position 47 is mutated to Y, E, or R; preferably, it is mutated to Y.

[0018] The amino acid at position 397 is mutated to W, A, or P; preferably, it is mutated to W.

[0019] In one embodiment, the amino acid sequence shown in SEQ ID NO.1 should be used.

[0020] The 43rd amino acid is mutated to Y, E, or A; preferably, it is mutated to Y or E;

[0021] The 430th amino acid is mutated to a nonpolar hydrophobic amino acid; optionally, it is mutated to P, L, V, M, or F; preferably, it is mutated to P.

[0022] The amino acid at position 633 is mutated to a nonpolar hydrophobic amino acid; optionally, it is mutated to P, L, V, M or F, A, I, W; preferably, it is mutated to P;

[0023] The amino acid at position 786 is mutated to a nonpolar hydrophobic amino acid or Y; optionally, it is mutated to P, I, A, V, Y, L, M, W or F; preferably, it is mutated to M;

[0024] The 744th amino acid is mutated to R, P, or L; preferably, it is mutated to R.

[0025] Preferably, the RNA polymerase mutant contains at least the following mutations equivalent to or corresponding to the parental polymerase mutant: S43Y, S397W, S430P, S633P, Q744R, and Q786M, named as follows.

[0026] S43Y / S397W / S430P / S633P / Q744R / Q786M (hereinafter referred to as T7-M30), the amino acid sequence is shown in SEQ ID NO.2;

[0027] Preferably, the RNA polymerase mutant contains at least the following mutations equivalent to or corresponding to the parental polymerase mutant: S43E, S397W, S430P, S633P, Q744R, and Q786M, named as follows.

[0028] S43E / S397W / S430P / S633P / Q744R / Q786M (hereinafter referred to as T7-M31), the amino acid sequence is shown in SEQ ID NO.3;

[0029] Preferably, the RNA polymerase mutant contains at least the following mutations equivalent to or corresponding to the parental polymerase mutant: E45A, S397W, S430P, S633P, Q744R, and Q786M, named as follows.

[0030] E45A / S397W / S430P / S633P / Q744R / Q786M (hereinafter referred to as T7-M32), the amino acid sequence is shown in SEQ ID NO.4;

[0031] Preferably, the RNA polymerase mutant contains at least the following mutations equivalent to or corresponding to the parental polymerase mutant: G47Y, S397W, S430P, S633P, Q744R, and Q786M, named as follows.

[0032] G47Y / S397W / S430P / S633P / Q744R / Q786M (hereinafter referred to as T7-M33), the amino acid sequence is shown in SEQ ID NO.5;

[0033] Preferably, the RNA polymerase mutant contains at least the following mutations equivalent to or corresponding to the parental polymerase mutant: G47E, S397W, S430P, S633P, Q744R, and Q786M, named as follows.

[0034] G47E / S397W / S430P / S633P / Q744R / Q786M (hereinafter referred to as T7-M34), the amino acid sequence is shown in SEQ ID NO.6;

[0035] Preferably, the RNA polymerase mutant contains at least the following mutations equivalent to or corresponding to the parental polymerase mutant: G47R, S397W, S430P, S633P, Q744R, and Q786M, named as follows.

[0036] G47R / S397W / S430P / S633P / Q744R / Q786M (hereinafter referred to as T7-M35), the amino acid sequence is shown in SEQ ID NO.7.

[0037] A second objective of this invention is to provide a polynucleotide encoding any of the aforementioned RNA polymerase mutants.

[0038] In one embodiment, the nucleotide sequence of T7-M30 is shown in SEQ ID NO.15; the nucleotide sequence of T7-M31 is shown in SEQ ID NO.16; the nucleotide sequence of T7-M32 is shown in SEQ ID NO.17; the nucleotide sequence of T7-M33 is shown in SEQ ID NO.18; the nucleotide sequence of T7-M34 is shown in SEQ ID NO.19; and the nucleotide sequence of T7-M35 is shown in SEQ ID NO.20.

[0039] The present invention also provides a vector carrying the above-mentioned polynucleotides or a cell expressing any of the above-mentioned RNA polymerase mutants.

[0040] Optionally, the carrier is pQE-80L, or it can be pET-28a(+) or pPIC9K, etc.

[0041] The present invention also provides a method for synthesizing polynucleotides using any of the above-mentioned RNA polymerase mutants; wherein the synthesis method includes in vitro transcription synthesis, isothermal amplification, or transcription-mediated amplification.

[0042] In one embodiment, the synthesis method includes catalytic synthesis of polynucleotides using an enzyme system containing the RNA polymerase mutant or whole-cell catalytic synthesis of polynucleotides using the aforementioned cells.

[0043] Optionally, the synthesis method includes in vitro transcription synthesis, isothermal amplification, or transcription-mediated amplification.

[0044] The present invention also provides the application of any of the above-mentioned RNA polymerase mutants or the above-mentioned polynucleotide synthesis methods in the preparation of mRNA vaccines, nucleic acid drugs, gene editing or protein expression systems.

[0045] A third objective of this invention is to provide a polynucleotide synthesis kit comprising any of the aforementioned RNA polymerase mutants.

[0046] A fourth object of the present invention is to provide a method for improving at least one performance of an RNA polymerase, said performance being selected from enzyme activity, 3' end uniformity, and dsRNA byproducts, wherein the RNA polymerase mutant is based on the amino acid sequence corresponding to the parental RNA polymerase, having an amino acid mutation at position 397, or an amino acid mutation at an equivalent position in the parental RNA polymerase; and having an amino acid mutation at any one of positions 43, 45, or 47, or an amino acid mutation at an equivalent position in the parental RNA polymerase;

[0047] The parent RNA polymerase has an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.1 and has RNA polymerase activity.

[0048] The beneficial effects of this invention are:

[0049] (1) The T7 RNAP mutant provided by the present invention has a good temperature adaptability range, and can react not only under the conventional 37℃ condition, but also under the high temperature 50℃ condition.

[0050] (2) The T7 RNAP mutant provided by the present invention has high specific enzyme activity at 37℃ and 50℃. With wild-type specific enzyme activity as a reference, the specific enzyme activities of the combined mutants T7-M30, T7-M31, T7-M32, T7-M33, T7-M34 and T7-M35 are superior to those of the prior art at 37℃ and 50℃. At 37℃, the specific enzyme activity reaches more than 35764 U / mg, with a maximum of 137327 U / mg; at 50℃, the specific enzyme activity reaches more than 44451 U / mg, with a maximum of 98174 U / mg.

[0051] (3) The T7 RNAP mutant provided by this invention has excellent application performance at 37℃ and 50℃. At 50℃, the yield of 1μg template for 1h reaches more than 169.1μg, with a maximum of 240.4μg. The 3' end uniformity at 50℃ reaches more than 79.38%, with a maximum of 86.80%. The RNA yield synthesized by this invention is significantly better than that of the prior art, the purity of the product is no worse than that of the prior art, and the synthesized mRNA has fewer dsRNA byproducts, which will produce lower immunogenicity. The 3' uniformity of the product is greatly improved compared with the prior art. These indicators are very beneficial for the research and production of mRNA vaccines and drugs.

[0052] (4) The T7 RNAP mutant provided by the present invention can synthesize circular RNA in one step at high temperature, which is faster and simpler than the two-step method for synthesizing circular RNA and produces a lower content of dsRNA. Attached Figure Description

[0053] Figure 1 SDS-PAGE images of different T7 RNAP mutants after purification by nickel column.

[0054] Figure 2 The results show the dsRNA content of the T7 RNAP mutant.

[0055] Figure 3The image shows the consistency test results for the 3' variant of the T7 RNAP mutant.

[0056] Figure 4 A one-step method was used to synthesize circular RNA for the T7 RNAP mutant. Detailed Implementation

[0057] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] Some definitions or terms involved in this invention:

[0059] Performance enhancement: The term "performance enhancement" refers to characteristics associated with variants that are improved relative to their parents. Such performance enhancement includes, but is not limited to, one or more of the following: increased enzyme activity of RNA polymerase mutants, improved thermostability, higher in vitro transcription yield in in vitro transcription (IVT) reactions, improved 3' end uniformity, fewer dsRNA byproducts, or lower immunogenicity of RNA transcripts produced by RNA polymerase mutants.

[0060] Corresponding to: As used herein, the term "corresponding to" refers to the manner in which a specific amino acid in a sequence is identified (where a specific amino acid sequence is referenced). For example, for the purposes of this invention, when referring to a specific amino acid position, a person skilled in the art can compare another amino acid sequence with the referenced amino acid sequence to determine which specific amino acid might be of interest in the other amino acid sequence. Alternative alignment methods can be used, and such methods are well known to those skilled in the art.

[0061] Mutant: As used herein, when referring to variations of the invention, the terms "mutant," "peptide variant," "peptide," or "RNA polymerase mutant" mean a polypeptide having RNA polymerase activity and containing alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the "parental" RNA polymerase. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. In the description of variations of the invention, the nomenclature described below has been adapted for ease of reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are used. Substitution: For amino acid substitution, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, the substitution of glutamic acid at position 49 with arginine is represented as "E49R." Multiple mutations are separated by a symbol (" / "), for example, "E49R / M358F" means that glutamic acid (E) and methionine (M) at positions 49 and 358 are replaced by arginine (R) and phenylalanine (F), respectively.

[0062] Parental RNA polymerase: As used herein, the term "parental" RNA polymerase refers to an RNA polymerase that has been modified to produce the RNA polymerase mutant of the present invention. The term also refers to a polypeptide to which the mutant of the present invention is compared. The parent can be a naturally occurring (wild-type) polypeptide, or it can be, or even a variant thereof, prepared by any suitable means. For example, the parental protein can be a variant of a naturally occurring polypeptide whose amino acid sequence has been modified or altered. Thus, the parental RNA polymerase can have one or more (or one or more) amino acid substitutions, deletions, and / or insertions. Therefore, the parental RNA polymerase can be a variant of the parental RNA polymerase. The parent can also be an allelic variant, which is a polypeptide encoded by any of two or more alternative forms of a gene occupying the same chromosomal locus.

[0063] Wild-type enzyme: When referring to an amino acid or nucleic acid sequence, the term "wild-type" means that the amino acid or nucleic acid sequence is a naturally occurring or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences). When the parent enzyme is not a variant enzyme, the terms "wild-type enzyme" and "parent enzyme" are used interchangeably.

[0064] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0065] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453) is used to determine sequence identity between two amino acid sequences. This algorithm is implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16:276-277) (preferably version 5.0.0 or later). The parameters used can be a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of "longest identity" marked by Needle (obtained using the -nobrief option) is used as the identity percentage and calculated as follows:

[0066] (identical residues x 100) / (alignment length - total number of vacancies in the alignment)

[0067] Alternatively, the parameters used can be a vacancy open penalty of 10, a vacancy extension penalty of 0.5, and EDNAFULL (the EMBOSS version of NCBI NUC4.4) to replace the matrix. The output of "Longest Identity" marked with Needle (obtained using the -nobrief option) is used as the identity percentage and calculated as follows:

[0068] (identical deoxyribonucleotides × 100) / (alignment length – total number of vacancies in the alignment)

[0069] Expression: As used in this article, “expression” refers to any step involving variant generation, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0070] Expression vector: As used herein, the term “expression vector” refers to a linear or circular DNA molecule that contains a polynucleotide encoding a variant and is operatively linked to a control sequence that provides for its expression.

[0071] Host cell: The term "host cell" means any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "host cell" encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication, along with recombinant host cells, isolated host cells (e.g., isolated recombinant host cells), and heterologous host cells.

[0072] Recombination: When used to refer to cells, nucleic acids, proteins, or vectors, the term "recombination" means that the cell has been modified from its natural state. Thus, for example, recombinant cells express genes not found in the natural (non-recombinant) form of the cell, or express natural genes at different levels or under different conditions compared to those found in nature. The difference between recombinant nucleic acids and their natural sequences lies in the operative linking of one or more nucleotides and / or a foreign sequence (e.g., a foreign promoter in an expression vector). The difference between recombinant proteins and their natural sequences may lie in the fusion of one or more amino acids and / or a foreign sequence. A vector containing nucleic acids encoding a polypeptide is a recombinant vector. The term "recombination" is synonymous with "genetically modified" and "transgenic."

[0073] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0074] Example 1: Construction of a recombinant expression vector carrying the nucleotide sequence encoding the T7 RNA polymerase mutant

[0075] Mutants were obtained through directed evolution. A mutant library was constructed into the pQE-80L vector using homologous recombination and transformed into the BL21(DE3) host. Mutants with enhanced specific enzyme activity were obtained using the FADS high-throughput screening platform. The specific steps are as follows:

[0076] 1. Construction of mutant libraries

[0077] The wild-type T7 RNA polymerase gene nucleotide sequence (corresponding amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.14) was optimized according to the E. coli codon to obtain the nucleotide sequence. The optimized nucleotide sequence was then inserted between the BamHI and HindIII restriction endonuclease sites of the vector pQE-80L (Qiagen, catalog number HG-VYQ0254), resulting in the recombinant vector pQE-80L-T7 RNAP containing the wild-type T7 RNAP gene. The T7 RNAP gene was amplified using the recombinant vector pQE-80L-T7 RNAP as a template. The degree of mutation was mainly determined by altering the Mn content in the reaction system. 2+This is achieved through concentration. Mn was set at concentrations of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, and 0.6 mM respectively. 2+ Gene amplification is performed using a concentration gradient, with the optimal mutation rate being 1 to 3 mutation points per gene.

[0078] The amplification system was as follows: pQE-80L-T7 RNAP plasmid (0.2–1 ng / μL), dATP (0.25 mM), dGTP (0.25 mM), dCTP (1.05 mM), dTTP (1.05 mM), DreamTaq Buffer (Takara), DreamTaq (Takara, 0.1 U / μL), MnCl2 (0.1 mM–0.6 mM), T7 RNAP-F (0.2 μM), T7 RNAP-R (0.2 μM), and the total PCR reaction volume was 50 μL.

[0079] The DNA sequences of the amplification primers T7 RNAP-F and T7 RNAP-R are shown in Table 1, and the amplification PCR program is shown in Table 2.

[0080] Table 1. Primer sequences for mutant library construction and amplification

[0081] Primer name Sequence (5' - 3') Sequence number T7 RNAP-F ACGAGCTCATGAACACGATTAACATCGCTAAGAAC SEQ ID NO. 21 T7 RNAP-R CGAAGCTTTTACGCGAACGCGAAGTCC SEQ ID NO. 22

[0082] Table 2 PCR amplification program

[0083]

[0084]

[0085] The size of the target fragment was analyzed by agarose gel electrophoresis, and the fragment was recovered using a kit. It was then digested with BamHI and HindIII, and the digested products were purified and recovered. The fragments were then ligated overnight with linearized pQE-80L vector (digested with BamHI and HindIII) using T4 DNA ligase.

[0086] The recovered ligation products were electroporated into electroporated competent E. coli 10G cells. The library size was calculated based on the grown clones, and the library size reached 10^6 cells / year. 6 The above. In 0.2mM Mn 2+ Under the condition of introducing a frequency of 1.7 base mutations / gene, at 0.3mM Mn 2+ The introduction frequency under these conditions is 3.5 base mutations per gene. Therefore, this example uses 0.2mM Mn 2+ and 0.3mMMn 2+ The mutant plasmids obtained under the conditions were mixed to obtain a mutant library in which an average of 2.6 mutant bases were introduced into each gene.

[0087] 2. Screening of mutant libraries

[0088] The obtained hybrid plasmid mutant library was electrotransfected into fresh E. coli BL21(DE3) competent cells to obtain a sample containing 10... 7 A library of approximately 100 clone mutants was prepared. Following the protein expression protocol, *E. coli* was induced and expressed. Single cells and the reaction system were encapsulated using the FADS platform (Fluorescence-Activated Droplet Sorting, a microfluidic microfluidic cell sorting platform based on ultra-high throughput fluorescence activated droplet sorting). The reaction system, as disclosed in patent CN 115896213A, contains single clones, 40-100 nM iSpinach DNA template, 100 μM DFHBI, 0.5-6 mM NTPs, 30 mM MgCl2, 5 mM NaCl, 20 mM DTT, 0.002 U / μL inorganic pyrophosphatase, and 0.2 U / μL murine ribonuclease inhibitor. The single-cell encapsulation rate was controlled at approximately 10% to improve screening accuracy. During in vitro incubation, the droplets are heat-treated at 45°C for 7 minutes, which reduces their activity to about one-third of its original value, achieving the effect of high-temperature treatment. They are then incubated at 37°C for 3 hours. Before sorting the mutant library using FADS, the optimal laser spot position and sorting speed are determined by sorting FITC and ink. The incubated microdroplets are then re-injected into the detection and sorting chip.

[0089] Based on the signal distribution generated by the mutant library, the PMT was set to 0.43V, the small and large sorting thresholds were set to 0.2V and 0.6V respectively, and the sorting voltage was 1.6V. Droplets exhibiting the highest activity (approximately 0.42%) were collected in 1.5mL centrifuge tubes. Positive genes were recovered by PCR. The target fragment was then digested with SacI and HindIII, reconstructed into the pQE-80L linearized vector, and transformed again into BL21(DE3) competent cells. Single clones grown on the plates were picked for secondary screening. The identified mutation sites were at positions 43, 45, 47, and 397.

[0090] In this invention, S430P, S633P, Q786M, and Q744R are combined with newly discovered mutations in order to obtain a T7 RNAP mutant that exhibits superior enzyme activity and application performance compared to existing technologies.

[0091] 3. Construct a reorganization vehicle

[0092] Starting with the amino acid sequence of wild-type T7 RNAP (WT) SEQ ID NO.1, directed evolution ultimately yielded five mutants with enhanced overall activity: the combinatorial mutant S43Y / S397W / S430P / S633P / Q744R / Q786M (abbreviated as T7-M30, amino acid sequence as shown in SEQ ID NO.2), the combinatorial mutant S43E / S397W / S430P / S633P / Q744R / Q786M (abbreviated as T7-M31, amino acid sequence as shown in SEQ ID NO.3), and the combinatorial mutant E45A / S397W / S430P / S633P / Q744R / Q786M (abbreviated as T7-M32, amino acid sequence as shown in SEQ ID NO.3). The following mutants are listed: (as shown in SEQ ID NO.4), G47Y / S397W / S430P / S633P / Q744R / Q786M (abbreviated as T7-M33, amino acid sequence as shown in SEQ ID NO.5), G47E / S397W / S430P / S633P / Q744R / Q786M (abbreviated as T7-M34, amino acid sequence as shown in SEQ ID NO.6), and G47R / S397W / S430P / S633P / Q744R / Q786M (abbreviated as T7-M35, amino acid sequence as shown in SEQ ID NO.7).

[0093] Among them, S430P (US 7507567B2), S633P (US 7507567B2), and Q786M (CN102177236B) are mutations reported in the prior art that can improve the thermostability of T7 RNAP, Q744R (US2015 / 0024435 A1) is a mutation reported in the prior art that can improve the activity of T7 RNAP, and S43Y (CN112831484B) is a mutation reported in the prior art that can reduce the RdRp activity of T7 RNAP.

[0094] Based on the mutation points reported in existing technologies that improve thermal stability, single-point mutants S43Y (amino acid sequence as shown in SEQ ID NO. 8), S430P (amino acid sequence as shown in SEQ ID NO. 9), S633P (amino acid sequence as shown in SEQ ID NO. 10), Q786M (amino acid sequence as shown in SEQ ID NO. 11), Q744R (amino acid sequence as shown in SEQ ID NO. 12), and combined mutants S430P / S633P / Q744R / Q786M (amino acid sequence as shown in SEQ ID NO. 13) were designed.

[0095] The nucleotide sequence corresponding to the above mutant is inserted between the two restriction endonuclease sites, BamHI and HindIII, of the vector pQE-80L to form a recombinant plasmid vector of mutant RNAP.

[0096] Example 2: Construction of recombinant microbial cells expressing T7 RNA polymerase mutant

[0097] The recombinant plasmid vector carrying the T7 RNA polymerase mutant obtained in Example 1 was transformed into BL21(DE3) Escherichia coli competent cells to obtain T7 RNAP wild-type and mutant recombinant strains (with ampicillin resistance), which were named as follows:

[0098] WT (used to express wild-type T7 RNAP, the amino acid sequence of the mutant is shown in SEQ ID NO.1);

[0099] E. coil-T7-M30 (used to express the combinatorial mutant T7-M30, the amino acid sequence of which is shown in SEQ ID NO.2);

[0100] E. coil-T7-M31 (used to express the combinatorial mutant T7-M31, the amino acid sequence of which is shown in SEQ ID NO.3);

[0101] E. coil-T7-M32 (used to express the combinatorial mutant T7-M32, the amino acid sequence of which is shown in SEQ ID NO.4);

[0102] E. coil-T7-M33 (used to express the combinatorial mutant T7-M33, the amino acid sequence of which is shown in SEQ ID NO. 5);

[0103] E. coil-T7-M34 (used to express the combinatorial mutant T7-M34, the amino acid sequence of which is shown in SEQ ID NO. 6);

[0104] E. coil-T7-M35 (used to express the combinatorial mutant T7-M35, the amino acid sequence of which is shown in SEQ ID NO. 7);

[0105] E.coil-T7-S43Y (used to express the single mutant T7-S43Y, the amino acid sequence of which is shown in SEQ ID NO. 8);

[0106] E.coil-T7-S430P (used to express the single mutant T7-S430P, the amino acid sequence of which is shown in SEQ ID NO.9);

[0107] E. coil-T7-S633P (used to express the single mutant T7-S633P, the amino acid sequence of which is shown in SEQ ID NO. 10);

[0108] E. coil-T7-Q786M (used to express the single mutant T7-Q786M, the amino acid sequence of which is shown in SEQ ID NO. 11);

[0109] E. coil-T7-Q744R (used to express the single mutant T7-Q744R, the amino acid sequence of which is shown in SEQ ID NO. 12);

[0110] E. coil-T7-S430P / S633P / Q744R / Q786M (used to express the combinatorial mutant T7-S430P / S633P / Q744R / Q786M, the amino acid sequence of which is shown in SEQ ID NO.13).

[0111] Example 3: Preparation of T7 RNAP mutant

[0112] The recombinant strains obtained in Example 2 were cultured in LB medium until the absorbance (OD) was measured. 600 When the concentration reaches approximately 0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM, and continue culturing at 37°C for 4 hours to induce T7 RNAP mutant expression.

[0113] After culturing, the cells were centrifuged at 4°C and 5000 rpm for 20 minutes to collect the cell sludge. The sludge was resuspended in a lysis buffer containing 300 mM NaCl, 20 mM Tris HCl (pH 7.5), 0.5 mg / mL lysozyme, and 0.5 mM DTT, and the cells were homogenized using an autoclave. The homogenized cell suspension was then centrifuged at 4°C and 12000 rpm for 1 hour using a high-speed refrigerated centrifuge to separate the supernatant containing the target protein. The supernatant was filtered to remove impurities and then temporarily stored on ice.

[0114] Protein purification was performed using a nickel column. Before purification, the nickel column was equilibrated with elution buffer (20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 0.5 mM DTT). The filtered supernatant was added to the nickel column, and after the protein solution had completely passed through the column, impurities were washed with Triton buffer, followed by elution with 300 mM imidazole solution. The eluted protein solution was concentrated using ultrafiltration tubes, with the concentration process involving replacement with buffer (50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 1 mM EDTA). The collected protein samples were analyzed by SDS-PAGE electrophoresis and Coomassie brilliant blue staining to assess protein purity and concentration. The concentrated protein solution was diluted and stored using an enzyme storage buffer. This buffer consisted of 50 mM Tris-HCl, 100 mM NaCl, 20 mM 2-Mercaptoethanol, 1 mM EDTA, 50% Glycerol, and 0.1% Triton X-100, with the pH adjusted to 7.9 to ensure protein stability and activity. T7 RNAP and its mutants were then prepared. The gel electrophoresis results are shown below. Figure 1 As shown.

[0115] Example 4: Detection of enzyme activity in T7 RNAP mutants

[0116] The T7 RNAP mutant prepared in Example 3 was used to detect its enzyme activity. The steps are as follows:

[0117] Take 270 μL of the reaction premix (system shown in Table 3) into a 1.5 mL EP tube, add 30 μL of diluted enzyme solution (concentration 0.5 mg / mL), mix well, and place in a cuvette. Record the absorbance value at 360 nm in real time for 7 min. Capture the reaction change between 200 s and 400 s, and calculate the specific enzyme activity using the following formula. The measurement method is the same for 37℃ and 50℃, only the reaction temperature differs. All reagents in this reaction system must be prepared with Nuclease-free H2O, and all consumables used must be RNase-free to prevent the generated RNA from being degraded by RNase. The formula for calculating the specific enzyme activity is as follows:

[0118] Enzyme activity specific to enzyme solution (U / mg) = (8181.81 × ΔA × df) / C

[0119] Where ΔA represents the change in absorbance; df represents the dilution factor; and C represents the concentration of the enzyme before dilution.

[0120] Table 3. Reaction system for determining specific enzyme activity.

[0121] Reagent Volume (270 μL) [ N F H2O ] 117 μL 10 x reaction buffer 30 μL NTPs (25 mM) 12 μL PNP (2 KU / mL) 15 μL MESG (1 mM) 60 μL Y-IPPA (0.1 U / μL) 3 μL RNase inhibitor (40 U / μL) 3 μL Assay template (500 ng / μL) 30 μL

[0122] The specific enzyme activities of different mutant combinations are shown in Table 4. The results show that, compared with the wild type, the specific enzyme activities of all mutants were increased to varying degrees at 37℃ and 50℃. Among them, the specific enzyme activity of wild-type WT was 11884 U / mg at 37℃ and approximately 756 U / mg at 50℃. The combined mutant T7-M30 showed a 3.00-fold and 68.15-fold increase in activity compared to WT at 37℃ and 50℃, respectively; the combined mutant T7-M31 showed an 11.56-fold and 112.76-fold increase compared to WT at 37℃ and 50℃, respectively; the combined mutant T7-M32 showed a 4.50-fold and 58.79-fold increase compared to WT at 37℃ and 50℃, respectively; the combined mutant T7-M33 showed a 9.61-fold and 129.85-fold increase compared to WT at 37℃ and 50℃, respectively; the combined mutant T7-M34 showed a 4.92-fold and 75.02-fold increase compared to WT at 37℃ and 50℃, respectively; and the combined mutant T7-M35 showed a 5.01-fold and 77.17-fold increase compared to WT at 37℃ and 50℃, respectively. That is, the T7 RNAP mutant provided by the present invention has higher specific enzyme activity than the prior art under both room temperature (37°C) and high temperature (50°C) conditions.

[0123] Table 4. Mutant Enzyme Activity

[0124]

[0125]

[0126] Example 5: Comparison of IVT production in T7 RNAP mutant and wild type at different temperatures

[0127] The in vitro transcription (IVT) reaction system is shown in Table 5. The reaction system can be scaled up proportionally. After the system is prepared, it is reacted at 37℃ and 50℃ for 1 h respectively. Then, for each reaction, 10U DNase I is added according to 1μg template and reacted at 37℃ for 30 min to digest the DNA template. Then, the IVT product is recovered by LiCl precipitation method, the RNA concentration is measured, and the mRNA yield (μg) corresponding to 1μg template DNA is calculated as IVT yield.

[0128] Table 5 In vitro transcription reaction system

[0129] Reagent Component DNA template 1 μg T7 RNAP mutant (0.5 mg / mL) 2 μL ATP / CTP / GTP / UTP (100 mM each) 2 μL each RNase inhibitor (40 U / μL) 1 μL Inorganic pyrophosphatase (0.1 U / μL) 0.4 μL 10 x in vitro transcription Buffer 2 μL Nuclease-free (NF) H2O Make up to 20 μL

[0130] The IVT yield results are shown in Table 6. Compared with the wild type, T7-M30 yielded approximately 4.67 times the wild type at 37°C, while the IVT yield of T7-M30 at 50°C was almost the same as that at 37°C. T7-M31 had a higher yield, approximately 5.23 times the wild type at 37°C, and a yield as high as 240.4g at 50°C.

[0131] The above results demonstrate that the T7 RNAP mutant provided by this invention exhibits better IVT application performance under high-temperature conditions compared to existing technologies, and can generate higher yields of products under the same conditions. This indicates that the purity of the IVT product from the T7 RNAP mutant provided by this invention is significantly improved compared to existing technologies.

[0132] Table 6 IVT Production

[0133] T7 RNAP mutant 37℃ 50℃ WT 40.6 μg -- T7-M30 189.5 μg 169.1 μg T7-M31 212.5 μg 240.4 μg

[0134] Note: "--" indicates not detected.

[0135] Example 6: Comparison of dsRNA content in IVT products of T7 RNAP mutant and wild type at different temperatures

[0136] The dsRNA content in IVT products was detected by ELISA.

[0137] Take the IVT RNA sample recovered after LiCl precipitation from Example 5, and follow the instructions of the HanHai New Enzyme dsRNA Content Detection Kit (Catalog No.: HBP003801). Serially dilute the known concentration of dsRNA standard as a positive control. Add 100 μL of each mutant transcription sample and the serially diluted standard to the microplate at two replicates per well. The operation must be rapid and completed within 15 minutes. After sealing the plate, incubate at 500 rpm for 1 hour at room temperature. Discard the liquid in the wells, add 250 μL of 1×Wash Buffer, let stand for 30 seconds, then blot dry the microplate. Repeat this step five times. Add 100 μL of the pre-prepared biotin-conjugated detection antibody to the microplate and incubate at 500 rpm for 1 hour at room temperature. Wash five times again with 1×Wash Buffer. Then add 100 μL of the pre-prepared Streptavidin-HRP to the microplate and incubate at 500 rpm for 30 minutes at room temperature. Wash five more times with 1×Wash Buffer. Add 100 μL of the room-temperature TMB substrate solution to the microplate and incubate at room temperature in the dark for 30 min. Add 50 μL of stop solution and gently shake the microplate until the color development is uniform. Use a microplate reader to read the absorbance values ​​at 450 nm and 650 nm wavelengths (450 nm as the detection wavelength and 650 nm as the reference wavelength).

[0138] dsRNA content detection results are as follows Figure 2As shown, when the mRNA loading amount was consistent, the dsRNA content of the combined mutants T7-M30 and T7-M31 was significantly lower than that of the wild type at both 37℃ and 50℃. At 37℃, it decreased to about one-tenth of the wild type, while at 50℃ it decreased to one-thousandth of the wild type. The results indicate that the dsRNA content in the IVT product of the T7 RNAP mutant provided by this invention is significantly improved compared to the wild type in the prior art.

[0139] Example 7: 3' concordance detection of IVT products between T7 RNAP mutant and wild type at different temperatures

[0140] The 3' consistency of IVT products was determined by RNase T1 digestion assay.

[0141] RNase T1 specifically degrades single-stranded RNA at the G residue by cleaving the phosphodiester bond between the 3'-guanosine and 5'-OH residues of adjacent nucleotides through the formation of the corresponding intermediate 2',3'-cyclic phosphate (-mP) salt. The reaction products are 3'-GMP and oligonucleotides containing terminal 3'-GMP. The molecular weight of the 3'-terminal products was then analyzed by LC-MS and compared with the theoretical molecular weight. If the normal mRNA product has a G base ending in -OH at the 3' end, then the heterogeneous product has a G base ending in -mP at the 3' end, and the heterogeneous product has a molecular weight 80% greater than the normal product. The 3'-terminal homogeneity (%) was calculated by analyzing the proportion of products with the theoretical molecular weight, using the following formula: 3'-terminal homogeneity = (homogeneous product / (homogeneous product + heterogeneous product)) × 100%.

[0142] Experimental results are as follows Figure 3 As shown, the molecular weight of the 3' end homogeneous product is 9663.1, and the molecular weight of the 3' end heterogeneous product is 9743.1. Data analysis is shown in Table 7. The 3' end homogeneous product reacted at 50℃ is significantly superior to the WT product reacted at 37℃. This indicates that the T7 RNAP mutant provided by this invention not only improves 3' end homogeneity at normal temperatures, but also further enhances the uniformity of the IVT product through high-temperature reaction, representing a significant improvement over the wild type in existing technologies.

[0143] Table 7. 3' End Consistency of IVT Products

[0144] Mutant Reaction temperature Homogeneity percentage Heterogeneity percentage Uniformity (%) WT 37℃ 5.83 90.65 6.02 T7-M30 37℃ 33.47 41.63 44.60 T7-M30 50℃ 79.38 20.62 79.38 T7-M31 37℃ 60.21 38.84 60.79 T7-M31 50℃ 79.87 12.16 86.80

[0145] Example 8: One-step transcription synthesis of circular RNA from T7 RNAP mutants and wild-type at different temperatures

[0146] Using self-circulated RNA as a template, the reactions were carried out at 37℃ (WT, T7-M31), 47℃ (T7-M31), and 50℃ (T7-M31) for 90 min, respectively. Since wild-type WT was inactive at 47℃, no experimental groups were set up at temperatures above 47℃. The specific one-step synthesis system of circular RNA is shown in Table 8.

[0147] The reaction was then immediately terminated on ice. 10 U of DNase I was added to the one-step transcription system and incubated at 37°C for 1 hour. The product was purified using a column-based RNA purification kit. Nine volumes of Buffer A were added and inverted to mix. Half a volume of anhydrous ethanol was then added, and the entire volume was transferred to the column. The column was centrifuged at 12,000 rpm for 1 minute, and the liquid was discarded. Elution was performed with 700 μL of Buffer RPE, centrifuged at 12,000 rpm for 1 minute, and the liquid was discarded. Another 300 μL of Buffer RPE was added for washing, centrifuged at 12,000 rpm for 1 minute, and the liquid was discarded. The column was centrifuged at 12,000 rpm for 2 minutes, 30 μL of RNase-free water was added, and the mixture was allowed to stand for 2 minutes. Then, it was centrifuged at 12,000 rpm for 2 minutes, and RNA quantification was performed using Nanodrop. Linear RNA and circular RNA in the RNA product were analyzed by nucleic acid electrophoresis.

[0148] Table 8 One-step circular RNA synthesis system

[0149]

[0150]

[0151] Experimental results are as follows Figure 4 As shown, mRNA represents linear RNA and circRNA represents circular RNA. The results indicate that the proportion of circular RNA transcribed at high temperatures (47℃ and 50℃) is significantly higher than that at 37℃, demonstrating that the mutant T7-M31 can indeed be directly used for circular RNA synthesis at high temperatures. However, because the reaction buffer was not optimized and was not the optimal reaction condition for self-circulating RNA, the circularization rate was affected. With further optimization, the circularization rate should be able to reach over 80%.

[0152] The sequences used in this application:

[0153] The amino acid sequence of WT is SEQ ID NO.1

[0154] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0155] Amino acid sequence SEQ ID NO.2 of the combined mutant T7 - M30:

[0156] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHEYYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKP EDIDMNPEALTAWKRAAAAVYRKDKARKSRRIWLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVPMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRPVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIRTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSMDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0157] Amino acid sequence SEQ ID NO.3 of the combined mutant T7 - M31:

[0158] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHEEYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRIWLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVPMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRPVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIRTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSMDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0159] Amino acid sequence SEQ ID NO.4 of the combined mutant T7-M32:

[0160] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYAMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKK ALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRIWLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVPMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRPVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIRTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSMDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0161] Amino acid sequence of the combined mutant T7-M33: SEQ ID NO.5

[0162] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMYEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRIWLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVPMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRPVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIRTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSMDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0163] Amino acid sequence SEQ ID NO.6 of the combined mutant T7-M34:

[0164] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMEEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDS ETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRIWLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVPMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRPVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIRTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSMDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0165] The amino acid sequence of the combined mutant T7-M35 is as shown in SEQ ID NO.7:

[0166] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMREARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIK PEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEML IESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKI NKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRIWLEFMLEQANKFANKAIWFPYNMDWRGRVYAVPMFNPQGNDMTK GLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGCSSGIQHFSAMLR DEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRPVMTLAYGSKEFGFRQQVLEDTIQPAIDSG KGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIRTRLNLMFLGQFRLQPTINTNKDSEIDAH KQESGIAPNFVHSMDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNRRDILESDFAFA

[0167] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A performance-enhanced mutant RNA polymerase, characterized in that, The RNA polymerase mutant has an amino acid mutation at position 397 based on the amino acid sequence of the parent RNA polymerase, or an amino acid mutation has occurred at the amino acid residue at the equivalent position in the parent RNA polymerase; and has an amino acid mutation at any one of positions 43, 45 or 47, or an amino acid mutation has occurred at the amino acid residue at any one of the equivalent positions in the parent RNA polymerase; The amino acid sequence of the parent RNA polymerase has at least 80% identity to the amino acid sequence shown in SEQ ID NO. 1 and has RNA polymerase activity.

2. The RNA polymerase mutant of claim 1, wherein, The RNA polymerase mutant also has one or more amino acid mutations at positions 430, 633, 786, 744.

3. The RNA polymerase mutant according to any one of claims 1 to 2, wherein corresponding to the amino acid sequence shown in SEQ ID NO. 1, The amino acid mutation at position 43 is Y, E or A; preferably, it is Y or E; The amino acid mutation at position 45 is A; The amino acid mutation at position 47 is Y, E or R; preferably, it is Y; The amino acid mutation at position 397 is W, A or P; preferably, it is W.

4. The RNA polymerase mutant of claim 2, wherein corresponding to the amino acid sequence shown in SEQ ID NO. 1, The amino acid mutation at position 430 is a non-polar hydrophobic amino acid; optionally, it is P, L, V, M, F; preferably, it is P; The amino acid mutation at position 633 is a non-polar hydrophobic amino acid; optionally, it is P, L, V, M or F, A, I, W; preferably, it is P; The amino acid mutation at position 786 is a non-polar hydrophobic amino acid or Y; optionally, it is P, I, A, V, Y, L, M, W or F; preferably, it is M; The amino acid mutation at position 744 is R, P or L; preferably, it is R; Preferably, the RNA polymerase mutant at least simultaneously has the mutations equivalent to or corresponding to the following mutations of the parent polymerase mutant: S43Y, S397W, S430P, S633P, Q744R, Q786M, named S43Y / S397W / S430P / S633P / Q744R / Q786M; preferably, the RNA polymerase mutant at least simultaneously has the mutations equivalent to or corresponding to the following mutations of the parent polymerase mutant: S43E, S397W, S430P, S633P, Q744R, Q786M, named S43E / S397W / S430P / S633P / Q744R / Q786M; Preferably, the RNA polymerase mutant at least simultaneously has the mutations equivalent to or corresponding to the following mutations of the parent polymerase mutant: E45A, S397W, S430P, S633P, Q744R, Q786M, named E45A / S397W / S430P / S633P / Q744R / Q786M; Preferably, the RNA polymerase mutant at least simultaneously exists equivalent to or corresponding to the following mutations of the parent polymerase mutant: G47Y, S397W, S430P, S633P, Q744R, Q786M, named as G47Y / S397W / S430P / S633P / Q744R / Q786M. Preferably, the RNA polymerase mutant at least simultaneously exists equivalent to or corresponding to the following mutations of the parent polymerase mutant: G47E, S397W, S430P, S633P, Q744R, Q786M, named as G47E / S397W / S430P / S633P / Q744R / Q786M. Preferably, the RNA polymerase mutant at least simultaneously exists equivalent to or corresponding to the following mutations of the parent polymerase mutant: G47R, S397W, S430P, S633P, Q744R, Q786M, named as G47R / S397W / S430P / S633P / Q744R / Q786M.

5. A polynucleotide encoding the RNA polymerase mutant according to any one of claims 1-4.

6. A vector carrying the polynucleotide according to claim 5 or a cell expressing the RNA polymerase mutant according to any one of claims 1-4.

7. A method of synthesizing a polynucleotide, comprising, The RNA polymerase mutant according to any one of claims 1-4 is used; wherein the synthesis method comprises in vitro transcription synthesis, isothermal amplification or transcription-mediated amplification.

8. Use of the RNA polymerase mutant according to any one of claims 1-4 or the polynucleotide according to claim 7 in the preparation of mRNA vaccines, nucleic acid drugs, gene editing or protein expression systems.

9. A polynucleotide synthesis kit, characterized in that, The RNA polymerase mutant according to any one of claims 1-4 is used.

10. A method of improving at least one property of an RNA polymerase, the property selected from the group consisting of specific enzyme activity, 3' end uniformity, dsRNA byproducts, characterized in that, The RNA polymerase mutant has an amino acid mutation at the 397th position based on the amino acid sequence of the parent RNA polymerase, or an amino acid mutation at the equivalent position of the amino acid residue in the parent RNA polymerase; and has an amino acid mutation at any one of the 43rd, 45th or 47th position, or an amino acid mutation at any one of the equivalent position of the amino acid residue in the parent RNA polymerase. The amino acid sequence of the parent RNA polymerase has at least 80% identity with the amino acid shown in SEQ ID NO. 1 and has RNA polymerase activity. The amino acid sequence of the parent RNA polymerase has at least 80% identity with the amino acid shown in SEQ ID NO. 1 and has RNA polymerase activity.

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