RNA polymerase intracellular mutagenesis system and construction method thereof

Abstract

The invention discloses an RNA polymerase intracellular mutagenesis system and a construction method thereof. The mutagenesis system comprises a mutagenesis plasmid, a target gene sequence plasmid (GOI plasmid) and a reporter plasmid, the mutagenic plasmid carries an inducible promoter, a DNA polymerase gene sequence, an error-prone DNA polymerase gene sequence and an auxiliary protein gene sequence thereof; the GOI plasmid carries a constitutive strong promoter and a T7 RNA (Ribonucleic Acid) polymerase; the report system carries an inducible promoter and a resistance gene sequence. The plasmid system can perform continuous mutagenesis on a target sequence, the mutagenesis condition is visualized through report plasmids, the whole mutagenesis screening process has the characteristics of high throughput, low cost, simplicity in operation and the like, flora capable of growing in a high-concentration resistant environment can be obtained through simple passage screening, and the plasmid system has a wide application prospect. The construction difficulty of a traditional mutant library is greatly reduced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an RNA polymerase intracellular mutagenesis system and a construction method thereof. Background Art

[0002] Enzymes are protein catalysts produced by organisms that are highly efficient, specific, and controllable. Natural enzymes can catalyze chemical reactions under mild conditions (such as room temperature, normal pressure, and neutral pH), but their industrial applications face many limitations: 1. Insufficient stability: Most natural enzymes are easily inactivated at high temperatures, extreme pH, or in organic solvents; 2. Narrow substrate spectrum: Natural enzymes only have catalytic activity on specific substrates; 3. Low catalytic efficiency: Some industrial scenarios require faster reaction rates; 4. Poor compatibility: It is difficult to adapt to multi-enzyme cascade reactions or non-aqueous reaction systems. Therefore, the modification of enzymes has become a hot topic in the field of biotechnology.

[0003] RNA polymerase (RNAP) is a key enzyme responsible for RNA synthesis in cells and plays an essential role in gene transcription. In both prokaryotes and eukaryotes, RNAP is responsible for transcribing DNA into RNA. With the advancement of research, researchers have characterized numerous types of RNAPs, their applications, and their protein structures. This research has led to the development of RNA in vitro transcription systems, significantly advancing the application of RNA in disease prevention, diagnosis, and treatment. However, RNAPs still face numerous challenges, including promoter dependency: RNAPs must rely on specific promoter sequences, limiting their application in non-engineered systems (e.g., in eukaryotic cells, requiring the construction of additional promoters); byproducts: the production of dsRNA, which can trigger cellular immune responses and requires purification and removal; and template sensitivity: complex templates, fragmentation, or contamination can easily lead to nonspecific transcription. These issues have hindered the application of in vitro RNA transcription, making it difficult to scale up and scale up. Therefore, the development of new RNAPs and in vitro transcription systems is urgently needed.

[0004] Protein engineering, or enzyme engineering, is one of the best strategies for improving enzyme performance. Traditional protein modification relies primarily on random mutagenesis, which requires large mutant libraries and is time-consuming and labor-intensive to screen. With breakthroughs in structural biology and computational biology, protein engineering, or enzyme engineering, has evolved into a precise design model that integrates computer simulations and structural biological information, forming two major technical approaches: rational design and semi-rational design.

[0005] Rational protein design requires a multi-dimensional understanding of the target protein, including: ① amino acid sequence analysis; ② precise determination of its three-dimensional conformation; and ③ systematic elucidation of its functional mechanisms. Functional remodeling can be achieved through precise manipulation of specific amino acid sites. However, a key challenge facing this approach is the lag in structural resolution—only approximately 1% of known protein structures are currently included in the PDB database. This leaves researchers with a limited number of reference protein structures, making precise site design impossible. Multiple mutations must be designed simultaneously at a single site, which then requires extensive wet lab testing for validation. In recent years, the rapid development of AI technology has provided a variety of methods for protein structure prediction and analysis, providing an excellent platform for protein sequence design and novel enzyme development. Using a protein engineering strategy, Magnesium Futai Biotechnologies, through virtual screening, identified over 20 hotspot amino acids in T7 RNA polymerase. Combined with a dual-probe system (monitoring both RNA production and dsRNA content), they identified the mutant M17 (A70Q / F162S / K180E). Its dsRNA content is only 1.8% of that of the wild type, significantly reducing immunogenicity. In addition, Moderna developed the T7 double mutant (G47A+884G) through rational design, reducing dsRNA production without affecting yield and simplifying the mRNA purification process.

[0006] Semi-rational design, as a fusion of directed evolution and rational design, significantly improves transformation efficiency by focusing on key functional domains: Different from the random scanning of the entire sequence by traditional directed evolution (building a 106-108 mutation library), this technology locks the active center based on structural information. For the targeted areas within the range, degenerate codon strategies such as NNK / NND are used to construct streamlined mutation libraries (usually on the order of 103-104). Combined with modern screening technologies such as microfluidic sorting or fluorescence-activated cell sorting (FACS), the efficiency of functional optimization per unit time is increased by 2-3 orders of magnitude. The latest progress shows that the use of intelligent codon compression technologies such as 22c-Trick can further reduce the library capacity by 80% while maintaining amino acid coverage. For example, Chen Tingjian's team modified the Stoffel fragment of Taq DNA polymerase to obtain mutants SFM5-7 and SFM5-46, which can efficiently synthesize fully modified 2'-OMe-DNA and realize the mutual transcription of XNA and RNA, expanding the application dimension of the central dogma.

[0007] Furthermore, with the booming development of synthetic biology, significant progress has been made in the in vivo modification of enzymes. Examples include: phage-assisted continuous evolution: leveraging the genetic mutations naturally occurring during phage replication within a bacterial host, combined with artificial selection pressure, to screen for phage variants that meet the desired function; and error-prone reverse transcription-directed evolution: a technique that combines error-prone reverse transcription with in vivo directed evolution, leveraging the host's own replication and selection mechanisms to obtain optimized enzyme variants. In vivo modification offers the potential for efficient mutation and continuous iteration, a screening environment close to actual function, the ability to handle complex functions and multi-target optimization, and automated processes that reduce the complexity of experimental setup. It is currently one of the most promising strategies for efficient enzyme modification. Summary of the Invention

[0008] In response to the shortcomings of the existing technology and actual needs, the present invention provides an important working principle based on the continuous directed evolution system of Escherichia coli. It uses molecular biology techniques to construct an RNA polymerase intracellular mutagenesis system, uses error-prone DNA polymerase to continuously induce mutagenesis of T7 RNA polymerase, and screens the mutant library of the enzyme through antibiotic resistance, reducing the difficulty of traditional mutant library construction.

[0009] In a first aspect, the present invention provides an RNA polymerase intracellular mutagenesis system, comprising a mutagenic plasmid, a GOI plasmid, and a reporter plasmid; the mutagenic plasmid carries an inducible promoter, a DNA polymerase gene sequence, an error-prone DNA polymerase gene sequence, and an auxiliary protein gene sequence thereof; the GOI plasmid carries a constitutive strong promoter and T7 RNA polymerase; and the reporter system carries an inducible promoter and a resistance gene sequence.

[0010] The gene sequence of the mutagenic plasmid of the present invention is a DNA polymerase gene sequence induced by arabinose, an error-prone DNA polymerase gene sequence induced by rhamnose, and an auxiliary protein gene sequence. The integration of the above elements can induce mutagenesis of the T7 RNA polymerase sequence.

[0011] The GOI plasmid of the present invention is a linear plasmid carrying a T7 RNA polymerase gene sequence. The 23119 strong promoter on the plasmid can express the T7 RNA polymerase, and the kana resistance gene maintains the stable propagation of the linear plasmid.

[0012] The reporter plasmid described in the present invention is a pET series plasmid carrying a chloramphenicol resistance gene, which can visualize the mutagenesis efficiency of the mutant library after screening with high-concentration antibiotics.

[0013] The mutagenic plasmid sequence of the present invention is the sequence shown in SEQ ID NO.1.

[0014] The GOI plasmid sequence of the present invention is the sequence shown in SEQ ID NO.2;

[0015] The reporter plasmid sequence of the present invention is the sequence shown in SEQ ID NO.3.

[0016] In a second aspect, the present invention provides a method for constructing an RNA polymerase intracellular mutagenesis system, comprising the following steps:

[0017] Step 1: Amplify the T7 RNA polymerase gene sequence using PCR;

[0018] Step 2: The gene sequence amplified in step 1 is spliced ​​into a linear plasmid fragment containing a constitutive promoter and a resistance gene using PCR, and sequencing analysis is performed to obtain a verified correct linear plasmid;

[0019] Step 3: Amplify the chloramphenicol resistance gene using PCR;

[0020] Step 4: Insert the gene sequence amplified in step 3 into pET21a containing an inducible promoter + The recombinant plasmid was verified to be correct.

[0021] In a third aspect, the present invention provides a method for continuous evolutionary passage of bacteria.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention utilizes a novel continuous directed evolution system within bacteria, based on the coupled transcription-translation mechanism of Escherichia coli. In this mutagenesis system, the T7 RNA polymerase expressed by the GOI plasmid specifically recognizes the T7 promoter on the reporter plasmid, driving transcription of the chloramphenicol acetyltransferase (CAT) gene. CAT protein expression confers chloramphenicol resistance to the host bacteria, enabling successful mutations to grow in chloramphenicol-containing media (resistance levels are positively correlated with RNA polymerase activity). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the map of the mutagenic plasmid;

[0025] Figure 2 The figure is the restriction enzyme digestion gel image of the mutagenized plasmid;

[0026] Figure 3 is the GOI plasmid map;

[0027] Figure 4 This is the gel image of PCR amplification of GOI linear plasmid;

[0028] Figure 5 To report the plasmid map;

[0029] Figure 6Gel image of colony PCR identification for the construction of reporter plasmid;

[0030] Figure 7 This is a gel image of enzyme digestion identification of the reporter plasmid construction;

[0031] Figure 8 Schematic diagram of growth verification of the intracellular mutagenesis system;

[0032] Figure 9 Schematic diagram of continuous passage and dilution coating of bacterial solution;

[0033] Figure 10 This is a schematic diagram of the chloramphenicol resistance and tolerance growth of the primary bacterial solution;

[0034] Figure 11 This is the iterative growth diagram of the bacterial solution continuously passaged;

[0035] Figure 12 This is a schematic diagram of the 24-hour growth curve of the terminal bacterial solution in different chloramphenicol concentrations;

[0036] Figure 13 Plate the terminal bacterial suspension with chloramphenicol gradient dilutions. DETAILED DESCRIPTION

[0037] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0038] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0039] The present invention utilizes an in vivo protein engineering strategy to perform targeted modification of RNA polymerase to address the problems of low transcription efficiency and high impurities of existing RNA polymerases in ultra-long transcription lengths (up to 12k or even 20-30k) and complex sequences (high GC, hairpin structures, HP / polyG sequences).

[0040] The present invention constructs an RNA polymerase intracellular mutagenesis system in bacteria, uses error-prone DNA polymerase to continuously mutate T7 RNA polymerase, and screens out a mutant library of the enzyme through antibiotic resistance and high-throughput fluorescence screening, thereby reducing the difficulty of traditional mutant library construction. In this way, a large number of RNA polymerase mutants are obtained, and subsequent high-throughput resistance and fluorescence system screening is performed to obtain efficient RNA polymerases with higher transcription intensity, adaptability to complex sequences, and no promoter sequence dependence.

[0041] An RNA polymerase intracellular mutagenesis system comprises a mutagenic plasmid, a GOI plasmid and a reporter plasmid; the mutagenic plasmid carries an inducible promoter, a DNA polymerase gene sequence, an error-prone DNA polymerase gene sequence and an auxiliary protein gene sequence thereof; the GOI plasmid carries a constitutive strong promoter and T7 RNA polymerase; and the reporter system carries an inducible promoter and a resistance gene sequence.

[0042] The gene sequence of the mutagenic plasmid is a DNA polymerase gene sequence induced by arabinose, an error-prone DNA polymerase gene sequence induced by rhamnose, and an auxiliary protein gene sequence. The integration of the above elements can induce mutagenesis of the T7 RNA polymerase sequence.

[0043] The GOI plasmid is a linear plasmid carrying a T7 RNA polymerase gene sequence. The 23119 strong promoter on the plasmid can express the T7 RNA polymerase, and the kana resistance gene maintains the stable propagation of the linear plasmid.

[0044] The reporter plasmid is a pET series plasmid carrying a chloramphenicol resistance gene, and can visualize the mutagenesis efficiency of the mutant library after screening with high-concentration antibiotics.

[0045] The mutagenic plasmid sequence is the sequence shown in SEQ ID NO.1.

[0046] The GOI plasmid sequence is the sequence shown in SEQ ID NO.2;

[0047] The reporter plasmid sequence is the sequence shown in SEQ ID NO.3.

[0048] A method for constructing an RNA polymerase intracellular mutagenesis system comprises the following steps:

[0049] Step 1: Amplify the T7 RNA polymerase gene sequence using PCR;

[0050] Step 2: The gene sequence amplified in step 1 is spliced ​​into a linear plasmid fragment containing a constitutive promoter and a resistance gene using PCR, and sequencing analysis is performed to obtain a verified correct linear plasmid;

[0051] Step 3: Amplify the chloramphenicol resistance gene using PCR;

[0052] Step 4: Insert the gene sequence amplified in step 3 into pET21a containing an inducible promoter + The recombinant plasmid was verified to be correct.

[0053] Example 1

[0054] Construction of an intracellular mutagenesis system for RNA polymerase in Escherichia coli.

[0055] The molecular biology experiments herein, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were performed primarily with reference to Molecular Cloning: A Laboratory Manual (3rd edition), edited by J. Sambrook and D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. For example, the competent cell transformation method and competent cell preparation method were performed with reference to Chapter 1, page 96 of Molecular Cloning: A Laboratory Manual (3rd edition). Specific experimental conditions can be determined through simple experiments if necessary.

[0056] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. These conditions can be adjusted through simple experiments if necessary.

[0057] Experimental materials: Buffers and enzymes involved in the enzyme digestion system were purchased from Thermo Fisher Scientific; buffers, dNTPs, and PrimeSTAR involved in the PCR system were purchased from Toyobo Co., Ltd.; plasmid miniprep kits and gel recovery kits were purchased from Suzhou Uniland Biotechnology Co., Ltd.; gene synthesis was commissioned by Shanghai Sangon Biotechnology Co., Ltd.

[0058] 1. Construction of mutagenic plasmid:

[0059] Shanghai Sangon Biotechnology Co., Ltd. was commissioned to complete the full gene sequence synthesis. The synthesized product was transformed into DH5α competent cells via heat shock and, after recovery at 37°C, plated onto LB plates containing the appropriate antibiotics. A single clone was selected for expansion and plasmid purification using a plasmid extraction kit. The following verification procedures were performed:

[0060] Enzyme digestion verification: Use restriction endonucleases SpeI / HindIII / XbaI for digestion, and confirm the fragment size by 2% agarose gel electrophoresis;

[0061] Sequencing: Full-length Sanger sequencing was performed and sequence accuracy was ensured by BLAST comparison (e.g. Figure 1 、 2 shown).

[0062] 2. GOI plasmid construction:

[0063] Preparation of target fragments: PCR amplification was performed using two specific oligonucleotides as templates;

[0064] Product verification and purification: Take 5 μL of PCR product and perform electrophoresis on 1% agarose gel. Purify the target band using gel recovery kit and send it for sequencing to verify the correctness of the sequence (such as Figure 3 、 4 shown).

[0065] 3. Construction of reporter plasmid:

[0066] Preparation of chloramphenicol resistance gene (CAT) fragment: Using a plasmid containing the CAT gene as a template, primers designed to introduce NdeI / XhoI restriction sites were designed for PCR amplification. The amplified product was confirmed by gel electrophoresis and then purified (same method as in 2).

[0067] Linearization of the pET21a+ vector: Double digest the pET21a+ vector with NdeI / XhoI (reaction system: 1 μg plasmid, 1 μL of each 10 U / μL enzyme, incubate at 37°C for 2 h), and recover the linearized vector by gel purification;

[0068] Ligation and transformation: Perform T4 DNA ligase reaction (16°C overnight) at a molar ratio of 3:1 (insert fragment:vector). Transform the ligation product into DH5α competent cells and plate on LB plates containing ampicillin (100 μg / mL).

[0069] Positive clone screening: randomly pick 10 single colonies for plasmid extraction, followed by enzyme digestion verification: use NdeI / PstI double enzyme digestion plasmid, electrophoresis to confirm the fragment; sequencing verification: use T7 promoter / terminator primers for bidirectional sequencing (such as Figure 5 、 6 , as shown in 7).

[0070] Table 1 Plasmid construction primer sequence list

[0071]

[0072] Example 2

[0073] Passage screening of RNA polymerase intracellular mutagenesis system in bacteria.

[0074] Experimental materials: All components of the culture medium, including additional antibiotics and sugars, were purchased from Shanghai Sangon (Bioengineering) Co., Ltd.

[0075] Medium components: 5g / L yeast extract, 10g / L tryptone, 10g / L sodium chloride, 50ug / ml kanamycin sulfate, 100ug / ml streptomycin sulfate, 100ug / ml ampicillin sodium, 10g / L glucose. (For solid medium, add 20g / L agar powder.)

[0076] 1. Construction of RNA polymerase intracellular mutagenesis system:

[0077] The mutagenesis plasmid, target gene (GOI) plasmid and reporter plasmid were co-transformed into 100 μL of DH5α competent cells at a ratio of 200 ng, 400 ng and 200 ng, respectively. After cell recovery, the cells were evenly spread on the surface of a solid medium containing 1% arabinose and incubated in an inverted manner at 37°C overnight (e.g. Figure 8 shown).

[0078] 2. Passaging of the intracellular mutagenesis system in bacteria:

[0079] Pick a single transformed colony and inoculate it into LB medium containing 1% arabinose (add 1mM IPTG when cultured at 37℃ with shaking until OD600=0.4-0.6) and induce the expression of error-prone DNA polymerase at 16℃. Take the induced bacterial solution and inoculate it into the following four groups of culture media (such as Figure 9 、 10 shown):

[0080] a. Contains 1% arabinose + gradient chloramphenicol (0, 25, 50, 75 μg / mL)

[0081] b. Containing 1% rhamnose + gradient chloramphenicol (0, 25, 50, 75 μg / mL)

[0082] c. Containing 1% arabinose + 1 mM IPTG + gradient chloramphenicol (0, 25, 50, 75, 100, 150, 200, 250, 300, 350 μg / mL)

[0083] d. Containing 1% rhamnose + 1 mM IPTG + gradient chloramphenicol (0, 25, 50, 75, 100, 150, 200, 250, 300, 350 μg / mL)

[0084] The bacterial solution of group d (culture system number d-2) was transferred to a screening medium (1% rhamnose + 1mM IPTG) containing 25μg / mL chloramphenicol at a 2% inoculum volume and passaged 10 times to drive the accumulation of RNA polymerase gene mutations. Then, a chloramphenicol concentration step-up strategy (50→300μg / mL) was used to obtain a high-activity mutant library (such as Figure 11 shown).

[0085] 3. Verification and screening of intracellular mutagenesis system in bacteria:

[0086] The final passage bacterial solution was taken and inoculated with 2% inoculum into the verification medium (1% rhamnose + 1 mM IPTG) containing 200, 250, and 300 μg / mL chloramphenicol, respectively, and the growth curve was determined (such as Figure 12 shown).

[0087] The final bacterial suspension was diluted in a gradient manner and then spread on a solid screening plate (1% arabinose + 1 mM IPTG) containing 200, 250, and 300 μg / mL chloramphenicol. Highly resistant single colonies were picked for PCR amplification of the target gene and Sanger sequencing to screen for potential functional mutation sites (such as Figure 13 shown).

[0088] The present invention discloses an RNA polymerase intracellular mutagenesis system and a method for constructing the same. The mutagenesis system comprises a mutagenic plasmid, a target gene sequence plasmid (GOI plasmid), and a reporter plasmid; the mutagenic plasmid carries an inducible promoter, a DNA polymerase gene sequence, an error-prone DNA polymerase gene sequence, and its auxiliary protein gene sequence; the GOI plasmid carries a constitutively strong promoter and T7 RNA polymerase; and the reporter system carries an inducible promoter and a resistance gene sequence. The plasmid system of the present invention is capable of continuously mutagenizing the target sequence and visualizing the mutagenesis status through the reporter plasmid. The entire mutagenesis screening process is characterized by high throughput, low cost, and simple operation. A bacterial colony capable of growing in a high-concentration resistance environment can be obtained through simple subculture screening, greatly reducing the difficulty of constructing a traditional mutant library.

[0089] The invention discloses an RNA polymerase intracellular mutagenesis system, which can efficiently and simply obtain a mutant library of a target enzyme.

Claims

1. An RNA polymerase intracellular mutagenesis system, characterized in that: The mutagenesis system includes a mutagenic plasmid, a GOI plasmid and a reporter plasmid; The mutagenic plasmid carries an inducible promoter, a DNA polymerase gene sequence, an error-prone DNA polymerase gene sequence and an auxiliary protein gene sequence thereof; The GOI plasmid carries a constitutive strong promoter and T7 RNA polymerase; The reporter system carries an inducible promoter and a resistance gene sequence.

2. The RNA polymerase intracellular mutagenesis system according to claim 1, wherein The gene sequence of the mutagenic plasmid carries a DNA polymerase gene sequence initiated by induction of arabinose, an error-prone DNA polymerase gene sequence initiated by induction of rhamnose, and an auxiliary protein gene sequence.

3. The RNA polymerase intracellular mutagenesis system according to claim 1, wherein The GOI plasmid is a linear plasmid carrying a T7 RNA polymerase gene sequence.

4. The RNA polymerase intracellular mutagenesis system according to claim 1, wherein The reporter plasmid carries a pEt series plasmid containing a chloramphenicol resistance gene.

5. The RNA polymerase intracellular mutagenesis system according to any one of claims 1 to 2, characterized in that The mutagenic plasmid sequence is the sequence shown in SEQ ID NO.

1.

6. The RNA polymerase intracellular mutagenesis system according to any one of claims 1-2, characterized in that The GOI plasmid sequence is the sequence shown in SEQ ID NO.

2.

7. The RNA polymerase intracellular mutagenesis system according to any one of claims 1-2, characterized in that The reporter plasmid sequence is the sequence shown in SEQ ID NO.

3.

8. A method for constructing the RNA polymerase intracellular mutagenesis system according to any one of claims 1 to 4, characterized in that: The following steps are involved: Step 1: Amplify the T7 RNA polymerase gene sequence using PCR; Step 2: The gene sequence amplified in step 1 is spliced ​​into a linear plasmid fragment containing a constitutive promoter and a resistance gene using PCR, and sequencing analysis is performed to obtain a verified correct linear plasmid; Step 3: Amplify the chloramphenicol resistance gene using PCR; Step 4: Insert the gene sequence amplified in step 3 into pET21a containing an inducible promoter + The recombinant plasmid was verified to be correct.

Images