CN112980811B

CN112980811B - RNA polymerase mutant and application thereof, recombinant vector and preparation method and application thereof, recombinant engineering bacteria and application thereof

Info

Publication number: CN112980811B
Application number: CN202110247557.5A
Authority: CN
Inventors: 马富强; 白雪; 杨晟; 郭天杰; 王志耘; 杨广宇
Original assignee: Suzhou Hanyuan New Enzyme Biotechnology Co ltd
Current assignee: Suzhou Hanyuan New Enzyme Biotechnology Co ltd
Priority date: 2021-03-06
Filing date: 2021-03-06
Publication date: 2022-02-25
Anticipated expiration: 2041-03-06
Also published as: CN112980811A

Abstract

The invention relates to an RNA polymerase mutant and application thereof, a recombinant vector and preparation method and application thereof, and recombinant engineering bacteria and application thereof. The RNA polymerase mutant comprises: (a) polypeptide obtained by deleting, substituting or adding one or more amino acids in the amino acid sequence shown as SEQ ID No. 2; or (b) a polypeptide having at least 90% homology with the polypeptide consisting of the amino acid sequence shown as SEQ ID No. 2. The RNA polymerase mutant has good thermal stability.

Description

RNA polymerase mutant and application thereof, recombinant vector and preparation method and application thereof, recombinant engineering bacteria and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an RNA polymerase mutant and application thereof, a recombinant vector and preparation method and application thereof, and recombinant engineering bacteria and application thereof.

Background

In molecular biology, RNA polymerase is an enzyme that synthesizes RNA from a DNA template. Using helicase, RNAP opens double-stranded DNA locally, making one of the exposed nucleotide strands available as a template for RNA synthesis, a process called transcription. Transcription factors and their associated transcription intermediary complexes must be attached to a DNA binding site called the promoter region where RNAP can initiate DNA unfolding. RNAP not only initiates RNA transcription, but also directs the positioning of nucleotides, facilitates attachment and extension, has intrinsic proofreading and replacement capabilities, and terminates recognition capabilities. In eukaryotes, RNAP can construct strands up to 240 ten thousand nucleotides in length. However, the existing rnases have poor thermal stability and are difficult to meet practical requirements.

Disclosure of Invention

Based on this, it is necessary to provide an RNA polymerase mutant having a good thermostability.

In addition, an application of the RNA polymerase mutant, a recombinant vector, a preparation method and an application thereof, a recombinant engineering bacterium and an application thereof are also needed to be provided.

An RNA polymerase mutant, the RNA polymerase mutant comprising:

(a) polypeptide obtained by deleting, substituting or adding one or more amino acids in the amino acid sequence shown as SEQ ID No. 2; or

(b) And a polypeptide having at least 90% homology with the polypeptide consisting of the amino acid sequence shown as SEQ ID No. 2.

The research finds that the polypeptide comprises one or more amino acids which are obtained by deleting, replacing or adding one or more amino acids in the amino acid sequence shown as SEQ ID No. 2; or, the RNA polymerase mutant of the polypeptide with at least 90% homology with the polypeptide consisting of the amino acid sequence shown as SEQ ID No.2 has better thermal stability. Experiments prove that the half-life of the RNA polymerase mutant is longer than that of wild type RNA polymerase, and the half-life of the combined mutant with four site mutations is about 5 times of that of the wild type RNA polymerase at 65 ℃, so that the mutant has better thermal stability.

In one embodiment, the RNA polymerase mutant comprises: a polypeptide obtained by mutating one of amino acid 423, amino acid 447, amino acid 506, amino acid 531, amino acid 568 and amino acid 833 of an amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: a polypeptide obtained by performing at least one of L447F mutation, V568P mutation, M833F mutation, Q506A mutation, C531S mutation and W423F mutation on an amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: a polypeptide obtained by mutating an amino acid sequence shown as SEQ ID No.2 by L447F.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by V586P mutation of amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by M833F mutation of amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by Q506A mutation of amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation and V586P mutation of the amino acid sequence shown in SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation and M833F mutation of the amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation and Q506A mutation of the amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation and C531S mutation of the amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation, V586P mutation and M833F mutation of the amino acid sequence shown in SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation, V586P mutation and Q506A mutation of the amino acid sequence shown in SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation, V586P mutation and C531S mutation of the amino acid sequence shown in SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation, V586P mutation, C531S mutation and M833F mutation of the amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the coding sequence of the RNA polymerase mutant comprises:

(a) a polynucleotide having at least 90% homology with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 1; or the like, or, alternatively,

(b) and a polynucleotide obtained by deleting, replacing or adding one or more bases in the nucleotide sequence shown as SEQ ID No. 1.

In one embodiment, the RNA polymerase mutant is a soluble enzyme or an immobilized enzyme.

A recombinant vector containing the coding sequence of the RNA polymerase mutant.

The preparation method of the recombinant vector comprises the following steps: and carrying out PCR amplification on the first vector by adopting a mutation amplification primer pair to obtain a recombinant vector, wherein the mutation amplification primer pair contains a nucleotide sequence corresponding to a mutation site of the RNA polymerase mutant, and the first vector contains a coding sequence corresponding to an amino acid sequence shown as SEQ ID No. 2.

A recombinant engineering bacterium contains the recombinant vector.

The RNA polymerase mutant, the recombinant vector or the recombinant engineering bacterium are applied to RNA synthesis.

Drawings

FIG. 1 is a schematic diagram of the homology modeling structure of wild-type T3RNA polymerase.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.

One embodiment of the present invention provides an RNA polymerase mutant comprising:

The research finds that the polypeptide comprises one or more amino acids which are obtained by deleting, replacing or adding one or more amino acids in the amino acid sequence shown as SEQ ID No. 2; or, the RNA polymerase mutant of the polypeptide with at least 90% homology with the polypeptide consisting of the amino acid sequence shown as SEQ ID No.2 has better thermal stability. Experiments prove that the half-life of the combined mutant of the four site mutations in the RNA polymerase mutant at 65 ℃ is about 5 times of that of wild type RNA polymerase, and the RNA polymerase mutant has better thermal stability.

Specifically, the sequence shown as SEQ ID No.2 is: MNIIENIEKNDFSEIELAAIPFNTLADHYGSALAKEQLALEHESYELGERRFLK MLERQAKAGEIADNAAAKPLLATLLPKLTTRIVEWLEEYASKKGRKPSAYAP LQLLKPEASAFITLKVILASLTSTNMTTIQAAAGMLGKAIEDEARFGRIRDLEA KHFKKHVEEQLNKRHGQVYKKAFMQVVEADMIGRGLLGGEAWSSWDKET TMHVGIRLIEMLIESTGLVELQRHNAGNAGSDHEALQLAQEYVDVLAKRAG ALAGISPMFQPCVVPPKPWVAITGGGYWANGRRPLALVRTHSKKGLMRYED VYMPEVYKAVNLAQNTAWKINKKVLAVVNEIVNWKNCPVADIPSLERQELPP KPDDIDTNEAALKEWKKAAAGIYRLDKARVSRRISLEFMLEQANKFASKKAI WFPYNMDWRGRVYAVPMFNPQGNDMTKGLLTLAKGKPIGEEGFYWLKIHG ANCAGVDKVPFPERIAFIEKHVDDILACAKDPINNTWWAEQDSPFCFLAFCFE YAGVTHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQD IYGIVAQKVNEILKQDAINGTPNEMITVTDKDTGEISEKLKLGTSTLAQQWLA YGVTRSVTKRSVMTLAYGSKEFGFRQQVLDDTIQPAIDSGKGLMFTQPNQAA GYMAKLIWDAVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTKEILRHRCA VHWTTPDGFPVWQEYRKPLQKRLDMIFLGQFRLQPTINTLKDSGIDAHKQES GIAPNFVHSQDGSHLRMTVVYAHEKYGIESFALIHDSFGTIPADAGKLFKAVR ETMVITYENNDVLADFYSQFADQLHETQLDKMPPLPKKGNLNLQDILKSDFA FA are provided.

Wherein, the RNA polymerase mutant comprises: a polypeptide obtained by mutating one of amino acid 423, amino acid 447, amino acid 506, amino acid 531, amino acid 568 and amino acid 833 of an amino acid sequence shown as SEQ ID No. 2.

Further, RNA polymerase mutants include: a polypeptide obtained by performing at least one of L447F mutation, V568P mutation, M833F mutation, Q506A mutation, C531S mutation and W423F mutation on an amino acid sequence shown as SEQ ID No. 2.

Wherein, the L447F mutation is that the leucine at position 447 of the amino acid sequence shown as SEQ ID No.2 is replaced by phenylalanine; the V568P mutation is a substitution of proline for valine at position 568 of the amino acid sequence shown in SEQ ID No. 2; M833F mutation is that the methionine at position 833 of the amino acid sequence shown as SEQ ID No.2 is replaced by phenylalanine; the Q506A mutation is that the 506 th glutamic-propionamide of the amino acid sequence shown as SEQ ID No.2 is replaced by alanine; the C531S mutation is that the 531 st cysteine of the amino acid sequence shown as SEQ ID No.2 is replaced by serine; the W423F mutation is that the tryptophan at the 423 th site of the amino acid sequence shown as SEQ ID No.2 is replaced by phenylalanine.

In one embodiment, the RNA polymerase mutant comprises: a polypeptide obtained by mutating an amino acid sequence shown as SEQ ID No.2 by L447F. Alternatively, the RNA polymerase mutant is a polypeptide obtained by performing L447F mutation on an amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by V586P mutation of amino acid sequence shown as SEQ ID No. 2. Alternatively, the RNA polymerase mutant is a polypeptide obtained by performing V586P mutation on an amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by M833F mutation of amino acid sequence shown as SEQ ID No. 2. Alternatively, the RNA polymerase mutant is a polypeptide obtained by mutation of M833F in the amino acid sequence shown in SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by Q506A mutation of amino acid sequence shown as SEQ ID No. 2. Alternatively, the RNA polymerase mutant is a polypeptide obtained by performing Q506A mutation on an amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation and V586P mutation of the amino acid sequence shown in SEQ ID No. 2. Alternatively, the RNA polymerase mutant is a polypeptide obtained by performing L447F mutation and V586P mutation on an amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation and M833F mutation of the amino acid sequence shown as SEQ ID No. 2. Alternatively, the RNA polymerase mutant is a polypeptide obtained by simultaneously carrying out L447F mutation and M833F mutation on an amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation and Q506A mutation of the amino acid sequence shown as SEQ ID No. 2. Alternatively, the RNA polymerase mutant is a polypeptide obtained by simultaneously carrying out an L447F mutation and a Q506A mutation on an amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation and C531S mutation of the amino acid sequence shown as SEQ ID No. 2. Alternatively, the RNA polymerase mutant is a polypeptide obtained by simultaneously carrying out an L447F mutation and a C531S mutation on an amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation, V586P mutation and M833F mutation of the amino acid sequence shown in SEQ ID No. 2. Alternatively, the RNA polymerase mutant is a polypeptide obtained by simultaneously carrying out an L447F mutation, a V586P mutation and an M833F mutation on an amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation, V586P mutation and Q506A mutation of the amino acid sequence shown in SEQ ID No. 2. Alternatively, the RNA polymerase mutant is a polypeptide obtained by simultaneously performing L447F mutation, V586P mutation and Q506A mutation on the amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation, V586P mutation and C531S mutation of the amino acid sequence shown in SEQ ID No. 2. Alternatively, the RNA polymerase mutant is a polypeptide obtained by simultaneously performing L447F mutation, V586P mutation and C531S mutation on the amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the RNA polymerase mutant comprises: polypeptide obtained by L447F mutation, V586P mutation, C531S mutation and M833F mutation of the amino acid sequence shown as SEQ ID No. 2. Alternatively, the RNA polymerase mutant is a polypeptide obtained by simultaneously carrying out an L447F mutation, a V586P mutation, a C531S mutation and an M833F mutation on an amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the coding sequence of the RNA polymerase mutant comprises:

Specifically, the sequence shown as SEQ ID No.1 is: AAAGCGTTTATGCAGGTGGTGGAAGCGGATATGATTGGCCGCGGCCTGCTG GGCGGCGAAGCGTGGAGCAGCTGGGATAAAGAAACCACCATGCATGTGG GCATTCGCCTGATTGAAATGCTGATTGAAAGCACCGGCCTGGTGGAACTGC AGCGCCATAACGCGGGCAACGCGGGCAGCGATCATGAAGCGCTGCAGCTG GCGCAGGAATATGTGGATGTGCTGGCGAAACGCGCGGGCGCGCTGGCGGG CATTAGCCCGATGTTTCAGCCGTGCGTGGTGCCGCCGAAACCGTGGGTGG CGATTACCGGCGGCGGCTATTGGGCGAACGGCCGCCGCCCGCTGGCGCTG GTGCGCACCCATAGCAAAAAAGGCCTGATGCGCTATGAAGATGTGTATATG CCGGAAGTGTATAAAGCGGTGAACCTGGCGCAGAACACCGCGTGGAAAAT TAACAAAAAAGTGCTGGCGGTGGTGAACGAAATTGTGAACTGGAAAAAC TGCCCGGTGGCGGATATTCCGAGCCTGGAACGCCAGGAACTGCCGCCGAA ACCGGATGATATTGATACCAACGAAGCGGCGCTGAAAGAATGGAAAAAAG CGGCGGCGGGCATTTATCGCCTGGATAAAGCGCGCGTGAGCCGCCGCATTA GCCTGGAATTTATGCTGGAACAGGCGAACAAATTTGCGAGCAAAAAAGCG ATTTGGTTTCCGTATAACATGGATTGGCGCGGCCGCGTGTATGCGGTGCCGA TGTTTAACCCGCAGGGCAACGATATGACCAAAGGCCTGCTGACCCTGGCG AAAGGCAAACCGATTGGCGAAGAAGGCTTTTATTGGCTGAAAATTCATGG CGCGAACTGCGCGGGCGTGGATAAAGTGCCGTTTCCGGAACGCATTGCGT TTATTGAAAAACATGTGGATGATATTCTGGCGTGCGCGAAAGATCCGATTA ACAACACCTGGTGGGCGGAACAGGATAGCCCGTTTTGCTTTCTGGCGTTTT GCTTTGAATATGCGGGCGTGACCCATCATGGCCTGAGCTATAACTGCAGCC TGCCGCTGGCGTTTGATGGCAGCTGCAGCGGCATTCAGCATTTTAGCGCGA TGCTGCGCGATGAAGTGGGCGGCCGCGCGGTGAACCTGCTGCCGAGCGA AACCGTGCAGGATATTTATGGCATTGTGGCGCAGAAAGTGAACGAAATTCT GAAACAGGATGCGATTAACGGCACCCCGAACGAAATGATTACCGTGACCG ATAAAGATACCGGCGAAATTAGCGAAAAACTGAAACTGGGCACCAGCACC CTGGCGCAGCAGTGGCTGGCGTATGGCGTGACCCGCAGCGTGACCAAACG CAGCGTGATGACCCTGGCGTATGGCAGCAAAGAATTTGGCTTTCGCCAGC AGGTGCTGGATGATACCATTCAGCCGGCGATTGATAGCGGCAAAGGCCTGA TGTTTACCCAGCCGAACCAGGCGGCGGGCTATATGGCGAAACTGATTTGGG ATGCGGTGAGCGTGACCGTGGTGGCGGCGGTGGAAGCGATGAACTGGCTG AAAAGCGCGGCGAAACTGCTGGCGGCGGAAGTGAAAGATAAAAAAACCA AAGAAATTCTGCGCCATCGCTGCGCGGTGCATTGGACCACCCCGGATGGCT TTCCGGTGTGGCAGGAATATCGCAAACCGCTGCAGAAACGCCTGGATATGA TTTTTCTGGGCCAGTTTCGCCTGCAGCCGACCATTAACACCCTGAAAGATA GCGGCATTGATGCGCATAAACAGGAAAGCGGCATTGCGCCGAACTTTGTG CATAGCCAGGATGGCAGCCATCTGCGCATGACCGTGGTGTATGCGCATGAA AAATATGGCATTGAAAGCTTTGCGCTGATTCATGATAGCTTTGGCACCATTC CGGCGGATGCGGGCAAACTGTTTAAAGCGGTGCGCGAAACCATGGTGATT ACCTATGAAAACAACGATGTGCTGGCGGATTTTTATAGCCAGTTTGCGGAT CAGCTGCATGAAACCCAGCTGGATAAAATGCCGCCGCTGCCGAAAAAAGG CAACCTGAACCTGCAGGATATTCTGAAAAGCGATTTTGCGTTTGCG are provided.

Since the same amino acid can be determined by several different codons, the same amino acid can correspond to different nucleotide sequences. Thus, the amino acid sequence of the RNA polymerase mutant in the present application includes the nucleotide sequence encoded by the codon-synonymous mutation obtained by 1 or several nucleotide substitutions of the nucleotide sequence shown in SEQ ID NO. 1. The RNA polymerase mutant of the present application can be obtained by a person skilled in the art by a method of cDNA cloning and site-directed mutagenesis or other suitable methods based on the amino acid sequence of the RNA polymerase mutant disclosed in the present application, and thus, the nucleotide sequence encoding the above RNA polymerase mutant is not limited to the nucleotide sequence shown in SEQ ID NO. 1. If the encoded protein has no significant functional difference from the RNA polymerase mutant, the protein is also included in the scope of the present invention.

In addition, due to polymorphism and variation of protein coding sequences, naturally occurring proteins may have genetic mutations, in which bases are deleted, substituted or added, or amino acids are deleted, inserted, substituted or otherwise varied in the coding sequences, resulting in deletion, substitution or addition of one or more amino acids in the amino acid sequence of the protein. Thus, there are some proteins that are substantially equivalent to the non-mutated proteins in terms of their physiological and biological activities. These polypeptides or proteins which differ structurally from the corresponding protein, but which do not differ significantly in function from the protein, are referred to as functionally equivalent variants.

Functionally equivalent variants are also suitable for polypeptides made by introducing such variations into the amino acid sequence of a protein by altering one or more codons by artificial means such as deletions, insertions, and mutations. Although this allows more variant variants to be obtained, the resulting variants are functionally equivalent variants provided that their physiological activity is substantially equivalent to that of the original non-variant protein.

Generally, functionally equivalent variants are homologous to the coding sequence, and thus polypeptides or proteins resulting from at least one alteration, such as a deletion, insertion or substitution of one or more bases in the coding sequence of the protein or a deletion, insertion or substitution of one or more amino acids in the amino acid sequence of the protein, generally have functionally equivalent activity to the protein, and thus polypeptides encoded by the above nucleotide sequences or polypeptides consisting of the above amino acid sequences are also included within the scope of the present invention if the encoded protein does not significantly differ in function from the RNA polymerase mutant.

The RNA polymerase mutant has higher thermal stability and excellent catalytic activity, and can be applied to RNA synthesis. Alternatively, the RNA polymerase mutant described above can be used in the synthesis of RNA in vitro.

Furthermore, the RNA polymerase mutant has a single-point mutant or a combined mutant, and the half life of the combined mutant with four site mutations is about 5 times of that of wild type RNA polymerase at 65 ℃, so that the mutant has better thermal stability.

One embodiment of the present invention provides a recombinant vector comprising the coding sequence of the RNA polymerase mutant.

Wherein, the recombinant vector is a cloning vector or an expression vector.

Specifically, the recombinant vector is pQE80L vector containing the coding sequence of the RNA polymerase mutant. The recombinant vector is not limited to the pQE80L vector containing the coding sequence of the RNA polymerase mutant, and the RNA polymerase gene may be integrated into another host cell group for expression.

An embodiment of the present invention provides a method for preparing the recombinant vector, including the steps of: and carrying out PCR amplification on the first vector by adopting a mutation amplification primer pair to obtain a recombinant vector, wherein the mutation amplification primer pair contains a nucleotide sequence corresponding to a mutation site of the RNA polymerase mutant, and the first vector contains a coding sequence corresponding to an amino acid sequence shown as SEQ ID No. 2.

Wherein the mutation amplification primer pair comprises:

L447F-F: CTGCTGACCTTCGCGAAAGGCA (shown as SEQ ID No. 3);

L447F-R: TGCCTTTCGCGAAGGTCAGCAG (shown as SEQ ID No. 4);

V568P-F: GAGCGAAACCCCTCAGGATATTTAT (shown as SEQ ID No. 5);

V568P-R: ATAAATATCCTGAGGGGTTTCGCTC (shown as SEQ ID No. 6);

M833F-F: TGCGCGAAACCTTCGTGATTACCTA (shown as SEQ ID No. 7);

M833F-R: TAGGTAATCACGAAGGTTTCGCGCA (shown as SEQ ID No. 8);

Q506A-F: GTGGGCGGAAGCTGATAGCC (shown as SEQ ID No. 9);

Q506A-R: GGCTATCAGCTTCCGCCCAC (shown as SEQ ID No. 10);

C531S-F: GCTATAACAGCAGCCTGCCGCT (shown as SEQ ID No. 11);

C531S-R: AGCGGCAGGCTGCTGTTATAGC (shown as SEQ ID No. 12);

W423F-F: GTTTCCGTATAACATGGATTCGCGC (shown as SEQ ID No. 13);

W423F-R: GCGCGAATCCATGTTATACGGAAAC (shown as SEQ ID No. 14);

N872D-F: AAAAGGCAACCTGGACCTGC (shown as SEQ ID No. 15);

N872D-R: GCAGGTCCAGGTTGCCTTTT (shown as SEQ ID No. 16);

I818H-F: TAGCTTTGGCACCCATCCG (shown as SEQ ID No. 17);

I818H-R: CGGATGGGTGCCAAAGCTA (shown as SEQ ID No. 18);

W416Y-F: AGCGATTACGTTTCCGTATAACATG (shown as SEQ ID No. 19);

W416Y-R: CATGTTATACGGAAACGTAATCGCT (shown as SEQ ID No. 20);

M750T-F: GCCTGGATACGATTTTTCTGGG (shown as SEQ ID No. 21);

M750T-R: CCCAGAAAAATCGTATCCAGGC (shown as SEQ ID No. 22).

In the construction of single-site mutants of RNA polymerase mutants, PCR amplification was performed using one of the above-described mutation amplification primer pairs. In the construction of the multipoint mutant of the RNA polymerase mutant, after single-point mutation is carried out on one mutation site, mutation is carried out on a second mutation site, and the mutation is obtained by sequential superposition. Specifically, the following procedure was followed: after amplification of the amplification primer pair of one mutation site, amplification products containing the prior mutation are amplified by the amplification primer pair of the other mutation site.

In one embodiment, the step of PCR amplifying the first vector with the mutant amplification primer pair further comprises the step of constructing the first vector.

Wherein the step of constructing the first vector comprises: sequences such as upstream primers were used: 5' -CGCGGATCCGCGAAAGCGTTTATGCAG-3' (underlined bases are restriction enzyme BamHI recognition sites as shown in SEQ ID No. 23), downstream primer: 3' -GCGATTTTGCGTTTGCGAAAAGTACTAn amplification primer shown as TTT-5' (shown as SEQ ID No.24, with underlined base being a recognition site of a restriction enzyme ScaI)For the amplified target gene, the target gene is a coding sequence corresponding to the amino acid sequence shown as SEQ ID No. 2; connecting the target gene to an empty vector, transforming, and extracting positive plasmids to obtain a first vector.

In one embodiment, before the step of performing PCR amplification on the first vector using the mutant amplification primer pair, the following steps are further included: the mutant RNA polymerase was screened for mutation sites.

Specifically, the screening of the mutation site of the RNA polymerase mutant comprises the following steps: the method comprises the steps of searching an NCBI database (http:// www.ncbi.nlm.nih.gov /) for a T3RNA polymerase amino acid sequence (shown as SEQ ID No. 2), removing repeated identical sequences, selecting a protein sequence with identity (identity) more than 50% of a target protein sequence, then performing multi-sequence alignment through Clusalx1.83 software, uploading a fasta file to a Consensus Maker v2.0.0 server, modifying set parameters according to needs, generating a Consensus sequence which can be edited later by the online software, and screening out mutation sites related to stability: L447F, V568P, M833F, Q506A, C531S, W423F.

The bacteriophage T3RNA polymerase (NP-523301.1) is a DNA-dependent RNA polymerase with strict specificity as its respective double-stranded promoter. The enzyme catalyzes the synthesis of 5 'to 3' end RNA on single-stranded DNA or double-stranded DNA downstream of the promoter. T3RNA polymerase (NP-523301.1) accepts modified nucleotides as substrates for RNA synthesis. However, since natural enzymes all function in a relatively mild environment in vivo, and industrial application processes require enzymes to function in a relatively harsh environment (such as high temperature, extreme pH value, organic solvent, non-natural substrate, product inhibition, etc.), natural enzymes often suffer from poor stability in application. For this purpose, enzymes with good stability must be selected to meet the requirements of industrial production. The key to solve the problem is to improve the thermal stability of T3RNA polymerase by means of protein engineering.

The protein engineering is based on the relationship between the structural rule and the biological function of protein molecules, and carries out gene modification or gene synthesis by means of chemistry, physics and molecular biology to modify the existing protein or manufacture a new protein to meet the requirements of human on production and life. Rational design is the most common method in protein engineering, and utilizes computer-aided molecular model combined with site-directed mutagenesis to realize protein function optimization, such as improvement of catalytic activity, thermal stability, acid and alkali resistance, etc. To effectively optimize the thermal stability of proteins, Markus Wys et al proposed the Consenssus Concept in 2001. Different from the conventional rational protein design method based on the precise structure-function relationship of protein, the Consensus Concept is based on the amino acid sequence information of homologous protein, and the information capable of improving the thermal stability of enzyme is analyzed from the evolutionary point of view. The invention takes the Consensus theory as a guiding idea, integrates and analyzes the RNA polymerase family sequence, and combines the assistance of bioinformatics and crystallography methods to obtain the RNA polymerase mutant with high stability.

The construction method of the recombinant vector is different from the rational design based on the precise structure-function relationship of protein, and the invention takes the Consensus Concept as a guiding idea, analyzes the information capable of improving the thermal stability of the enzyme from the evolutionary angle, performs the integration analysis on the RNA polymerase sequence, and combines the assistance of bioinformatics and crystallography methods to obtain the recombinant vector capable of expressing the RNA polymerase mutant with high stability.

The recombinant vector can be used for producing RNA polymerase mutants to be applied to RNA synthesis.

An embodiment of the present invention provides a recombinant engineered bacterium containing the recombinant vector of the above embodiment.

The recombinant engineering bacteria can produce RNA polymerase mutants and can be applied to RNA synthesis; the constructed recombinant engineering bacteria with high expression efficiency have the advantages of short culture period, simple culture condition, high target protein yield and simple purification.

Further, the recombinant engineered bacterium is Escherichia coli containing the recombinant vector of the above embodiment. Alternatively, the recombinant engineered bacterium is a BL21(DE3) engineered bacterium containing the recombinant vector of the above embodiment. The recombinant engineered bacterium is not limited to Escherichia coli containing the recombinant vector of the above embodiment, and expression of a target protein may be performed using a microbial host such as gram-positive bacteria, gram-negative bacteria, yeast, or fungi.

The following are specific examples.

Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, are usually carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer of the kits. The reagents used in the examples are all commercially available.

In the following examples, primers were prepared by synthesis by Shanghai bioengineering, Inc., as specifically described; KOD high fidelity enzyme was purchased from toyobo; restriction enzymes were purchased from NEB; t4 DNA ligase, DNA gel recovery kit and plasmid mini kit were purchased from Takara.

Example 1

Cloning of wild T3RNA polymerase Gene

The wild type T3RNA polymerase gene is codon optimized by using Escherichia coli as a host, synthesized by Jinwei corporation, Suzhou, and the T3RNA polymerase gene sequence is accessed into pQE80L plasmid, named recombinant plasmid pQE80L-T3 RNAP.

Example 2

Expression, purification and Activity determination of T3RNAP

50 μ L of Amp antibiotics (final concentration of Amp antibiotics is 50 μ g/mL) and 20 μ L T3RNA polymerase engineering bacteria were added to 50mL of LB medium, and shaken at 37 ℃ and 200rpm for 8 h. 1mL of Amp antibiotic (the final concentration of Amp antibiotic is 50 mu g/mL) and 10mL of shaken bacterial liquid are added into 1L of LB culture medium, the bacteria are shaken at 37 ℃ and 200rpm, and when the absorbance A of the bacterial liquid at the wavelength of 600nm is measured by an ultraviolet spectrophotometer to be between 0.6 and 0.8, the next step of induction can be carried out. 1mL of IPTG (IPTG final concentration of 50. mu.g/mL) was added to 1L of LB medium, and the mixture was shaken at 37 ℃ and 200rpm for 6.5 hours. Subpackaging the bacterial liquid into centrifuge cups, centrifuging at 5000rpm and 8 ℃ for 10min, discarding the supernatant, adding a small amount of Buffer A (the component is 50mM Tris-HCl, the pH is 8.0,300mM NaCl), suspending and precipitating by using a pipette or a vortex oscillator, transferring the bacterial liquid into centrifuge tubes (the centrifuge tubes are weighed before transferring, the mass is m1), centrifuging at 8000 rpm-10000 rpm and 8 ℃ for 10min, discarding the supernatant, weighing, the mass of the bacterial is m2, and the mass of the wet bacterial is m2-m 1. Adding 10mL of Buffer A into each 1g of bacteria, suspending and precipitating by using a pipettor or a vortex oscillator, transferring the bacteria liquid into a 100mL beaker (the beaker is embedded in ice), crushing the bacteria by using an ultrasonic cell crusher until the bacteria liquid is clear, centrifuging at 12000rpm and 4 ℃ for 15-30 min, and transferring the supernatant into a centrifuge tube.

Extraction of T3RNA polymerase: adding imidazole (final concentration: 3mM), Ni-NTA (1.5 mL per 1L of bacteria) into a centrifuge tube, mixing with the supernatant of the previous step, incubating at 4 ℃ for 10min to 15min, transferring to a chromatography column, wherein the T3RNA polymerase with histidine tag has bound to Ni-NTA, passing the liquid through the chromatography column, discarding the liquid, washing the column with Buffer A (5 times the volume of Ni-NTA), discarding the liquid, washing the column with Buffer B (50mM Tris-HCl, pH 8.0,300mM NaCl, 10% glycerol, 100mM imidazole) (5 times the volume of Ni-NTA), discarding the liquid, washing the column with 600. mu.L to 700. mu.L Buffer C (composition: 50mM Tris-HCl, pH 8.0,100Mm NaCl, 10% (w/w) glycerol, 300mM imidazole) to replace Buffer B, collecting the liquid with RNA (T3) at a concentration higher than that of RNA polymerase, the column was then washed with 1mL of Buffer C each time and the flow-through was collected into 1.5mL ep tubes, with the highest concentration of T3RNA polymerase in the second and third tubes. The collected flow-through was transferred to an ultrafiltration tube with a pore size of 30KD, centrifuged at 4500rpm for 10min, Buffer was replaced with FPLC Buffer 3 times, and finally T3RNA polymerase was diluted to 3-5 mg/mL with Buffer D (composition: 50mM Tris-HCl, pH 8.0,100mM NaCl, 100mM EDTA, 10% (w/w) glycerol).

The T3RNA polymerase assay method comprises the following steps: DNA template with the concentration of 200nM is used as reaction substrate, in 100ul buffer system, 40mM Tris-HCl buffer (pH7.8),20mM NaCl,6mM MgCl₂2mM Spermidine HCl (spermine tetrahydrate), 10mM DTT, enzyme activity was measured in real time. Measuring enzyme activity at 30-60 deg.C every 5 deg.C, increasing temperature density at the position close to optimum temperature, and analyzing optimum reaction temperature of T3 RNAP. Respectively keeping the purified enzyme solution at 10, 20, 30, 40, 50 and 60 deg.CAfter 15min of heat preservation, enzyme activity is determined, and the thermal stability of the enzyme is analyzed.

Example 3

The T3RNAP homologous protein multi-sequence alignment and Consensus analysis are specifically performed as follows:

1. the amino acid sequence of T3RNAP (shown in SEQ ID No. 2) was entered into the NCBI protein database, and all protein sequences having greater than 50% identity (identity) to the target protein sequence were found using the BLASTP tool. Deleting the repeated identical sequence, sorting the rest sequence into fasta format, and inputting into Clustalx1.83 software for multi-sequence alignment. The results of the alignment are output in the format of aln, dnd, and fasta. The dnd file is used for constructing an evolutionary tree file, and the aln and fasta files are sequence files in different forms. Uploading the fasta. file to a Weblogo 3(http:// Weblogo. threeplusone. com /) server, and after setting parameters are modified according to needs, displaying the amino acid abundance of each site of a protein sequence in a multi-sequence alignment result by the online software in a form of a histogram. Uploading the fasta. file to a Consensus Maker v2.0.0 (http:// www.hiv.lanl.gov/content/sequence/CONSENSUS/Consensus. html) server, and after setting parameters are modified as required, generating a Consensus sequence which can be edited later by the online software.

2. The amino acid sequence of the target protein T3RNAP was compared against the consensus sequence of the family and the amino acid abundance map at each position.

Example 4

T3RNAP Structure and selection of mutational hotspots

1. Homology modeling was performed using Swiss-model with 1H38 having 82.2% homology as a template, T3RNA polymerase was performed, and the homology modeling structure of T3RNAP was downloaded. The homology modeling structure of wild-type T3RNA polymerase is shown in FIG. 1.

2. Using PyMOL to observe a homologous modeling structure, and according to structural information, rechecking the mutation site to be selected and the mutation form to screen out a mutant which is most likely to improve the thermal stability of T3RNAP, wherein the screening conditions are as follows:

(1) the standard for judging a certain locus as a candidate locus is as follows: the amino acid abundance of most proteins in the family at the position is high overall; ② the amino acid at the site is conserved; and the amino acid with higher occurrence frequency at the site has larger physical and chemical property difference with the amino acid at the site of T3RNAP, such as charge difference, polarity intensity, steric hindrance and the like.

(2) Removal of active sites in the vicinity, i.e. from catalytic residues

Amino acid residues within the range, excluding amino acid residues in the embedded or semi-embedded state.

After the above two-step screening, 10 mutation sites were remained, and most of them were located on the surface of the protein molecule. Among them, 10 single point mutants: L447F, V568P, M833F, Q506A, C531S, W423F, N872D, I818H, W416Y, M750T.

(3) According to the T3RNAP homology modeling structure, the 10 mutation forms are analyzed in detail one by one, and the mutant which can improve the T3RNAP thermal stability is screened out.

The main judgment criteria are: firstly, the mutation eliminates the original acting force form which is not beneficial to thermal stability, such as electrostatic repulsion, charge aggregation and the like; secondly, the mutation does not damage the existing acting force form which is beneficial to thermal stability and the stable protein structure; and thirdly, new acting force forms which are beneficial to thermal stability, such as hydrogen bonds, salt bridges, hydrophobic interaction and the like, are introduced into the mutation.

Example 5

Construction, expression, purification and property characterization of mutants

1. Construction of T3 site-directed mutants of RNAP

The recombinant plasmid pQE80L-T7RNAP was used as a template, a pair of complementary oligonucleotides having mutation sites was used as primers, and whole plasmid PCR amplification was performed with KOD high fidelity enzyme (Takara) to obtain a recombinant plasmid having a specific mutation site.

The KOD high-fidelity polymerase of Toyobo is used for PCR amplification, and the amplification conditions are as follows: 95 ℃ for 2min, then 55 ℃ for 20sec, 72 ℃ for 100sec for 30 cycles, and finally 72 ℃ for 10 min. The PCR product was recovered from the gel, and the gel-recovered product was digested with DpnI enzyme (Fermentas Corp.) at 37 ℃ for 2h to degrade the original template. The digestion products were transformed into BL21(DE3), spread on LB agar plates containing 50. mu.g/mL ampicillin resistance, cultured overnight at 37 ℃, screened for positive clones, and verified by sequencing. Recombinant bacteria of the T3RNAP enzyme mutant are obtained.

Wherein, the primer pair of complementary oligonucleotides with mutation sites corresponding to each mutation site in the embodiment 5 is specifically:

L447F-F: CTGCTGACCTTCGCGAAAGGCA (shown as SEQ ID No. 3);

L447F-R: TGCCTTTCGCGAAGGTCAGCAG (shown as SEQ ID No. 4);

V568P-F: GAGCGAAACCCCTCAGGATATTTAT (shown as SEQ ID No. 5);

V568P-R: ATAAATATCCTGAGGGGTTTCGCTC (shown as SEQ ID No. 6);

M833F-F: TGCGCGAAACCTTCGTGATTACCTA (shown as SEQ ID No. 7);

M833F-R: TAGGTAATCACGAAGGTTTCGCGCA (shown as SEQ ID No. 8);

Q506A-F: GTGGGCGGAAGCTGATAGCC (shown as SEQ ID No. 9);

Q506A-R: GGCTATCAGCTTCCGCCCAC (shown as SEQ ID No. 10);

C531S-F: GCTATAACAGCAGCCTGCCGCT (shown as SEQ ID No. 11);

C531S-R: AGCGGCAGGCTGCTGTTATAGC (shown as SEQ ID No. 12);

W423F-F: GTTTCCGTATAACATGGATTCGCGC (shown as SEQ ID No. 13);

W423F-R: GCGCGAATCCATGTTATACGGAAAC (shown as SEQ ID No. 14);

N872D-F: AAAAGGCAACCTGGACCTGC (shown as SEQ ID No. 15);

N872D-R: GCAGGTCCAGGTTGCCTTTT (shown as SEQ ID No. 16);

I818H-F: TAGCTTTGGCACCCATCCG (shown as SEQ ID No. 17);

I818H-R: CGGATGGGTGCCAAAGCTA (shown as SEQ ID No. 18);

W416Y-F: AGCGATTACGTTTCCGTATAACATG (shown as SEQ ID No. 19);

W416Y-R: CATGTTATACGGAAACGTAATCGCT (shown as SEQ ID No. 20);

M750T-F: GCCTGGATACGATTTTTCTGGG (shown as SEQ ID No. 21);

M750T-R: CCCAGAAAAATCGTATCCAGGC (shown as SEQ ID No. 22).

2. Characterization of the Properties of the mutants

Pure enzyme solutions of site-directed mutant T3RNA polymerase were obtained and the 10 single-point mutants were characterized as described in example 2. The results show that the thermal stability of 6 mutants in the 10 single-point mutants is obviously improved, and the mutants are respectively L447F, V568P, M833F, Q506A, C531S and W423F.

Mutants with improved stability were additively combined: and (2) cumulatively combining the single-point mutants with improved stability by using a construction method similar to the single-point mutants, selecting a plurality of mutation sites for combination in the amino acid sequence shown in SEQ ID No.2, for example, selecting 2-4 mutation sites from the 6 mutation sites for combination to respectively obtain different RNA polymerase mutants, and expressing, purifying and characterizing the RNA polymerase mutants one by one. Wherein, the RNA polymerase mutant is:

(1) 2 mutation sites are selected for combination, 4 RNA polymerase mutants can be constructed, and the combined mutation sites are respectively: L447F/V586P, L447F/M833F, L447F/Q506A and L447F/C531S, wherein the L447F/V586P indicates that two mutations of L447F and V586P occur at the same time in the amino acid shown in SEQ ID No.2, and the rest mutation types also have similar meanings and are not repeated.

(2) 3 mutation sites are selected for combination, so that 3RNA polymerase mutants can be constructed, and the combined mutation sites are respectively: L447F/V586P/M833F, L447F/V586P/Q506A, L447F/V586P/C531S. Wherein, L447F/V586P/M833F indicates that three mutations of L447F, V586P and M833F occur at the same time in the amino acid shown in SEQ ID No.2, and the rest mutation types also have similar meanings and are not described again.

(3) 4 mutation sites are selected for combination, 1 RNA polymerase mutant can be constructed, and the combined mutation sites are respectively: L447F/V586P/C531S/M833F. Wherein, L447F/V586P/C531S/M833F shows that the amino acid shown as SEQ ID No.2 simultaneously generates four mutations of L447F, V586P, C531S and M833F.

Test example 1

Characterization of enzymatic Properties of RNA polymerase mutants

Performing thermal stability test on wild type RNA polymerase (the amino acid sequence of which is shown as SEQ ID No. 2) and 12 RNA polymerase mutants provided in example 5, and specifically performing the following steps according to a conventional RNA polymerase activity determination method:

incubating the enzyme solution at a certain temperature, sampling at different treatment times, determining the residual activity percentage of the wild-type RNA polymerase or RNA polymerase mutant, plotting the ln value of the residual activity percentage to the time t (min), wherein the slope of a straight line is the inactivation constant kinect, and the half-life period of the wild-type RNA polymerase or RNA polymerase mutant at the temperature is obtained from t1/2 ═ ln 2/kinect. The results are detailed in Table 1.

TABLE 1 characterization of enzymatic Properties of wild-type RNA polymerase, RNA polymerase mutants

T3 RNAP	Half life (min) at 65 DEG C
		Wild type	10
L447F	15
		V568P	18
M833F	17
		Q506A	20
L447F/V586P	26
		L447F/M833F	29
L447F/Q506A	33
		L447F/C531S	20
L447F/V586P/M833F	35
		L447F/V586P/Q506A	30
L447F/V586P/C531S	36
		L447F/V586P/C531S/M833F	49

As can be seen from Table 1, the 12 RNA polymerase mutants include single-point mutants and combination mutants, and compared with the wild type RNA polymerase, the single-point mutants and the combination mutants have longer half-lives at 65 ℃; in particular, the L447F/V586P/C531S/M833F combined mutant has a half-life about 5 times that of the wild type, and shows the additive effect of the thermal stability of a single-point mutant. In conclusion, the RNA polymerase mutant has higher thermal stability and higher catalytic activity, can be used for synthesizing RNA, and has larger application potential.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> New enzyme Biotech Limited of Han Source, Suzhou

<120> RNA polymerase mutant and application thereof, recombinant vector and preparation method and application thereof, recombinant engineering bacteria and application thereof

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aaagcgttta tgcaggtggt ggaagcggat atgattggcc gcggcctgct gggcggcgaa 60

gcgtggagca gctgggataa agaaaccacc atgcatgtgg gcattcgcct gattgaaatg 120

ctgattgaaa gcaccggcct ggtggaactg cagcgccata acgcgggcaa cgcgggcagc 180

gatcatgaag cgctgcagct ggcgcaggaa tatgtggatg tgctggcgaa acgcgcgggc 240

gcgctggcgg gcattagccc gatgtttcag ccgtgcgtgg tgccgccgaa accgtgggtg 300

gcgattaccg gcggcggcta ttgggcgaac ggccgccgcc cgctggcgct ggtgcgcacc 360

catagcaaaa aaggcctgat gcgctatgaa gatgtgtata tgccggaagt gtataaagcg 420

gtgaacctgg cgcagaacac cgcgtggaaa attaacaaaa aagtgctggc ggtggtgaac 480

gaaattgtga actggaaaaa ctgcccggtg gcggatattc cgagcctgga acgccaggaa 540

ctgccgccga aaccggatga tattgatacc aacgaagcgg cgctgaaaga atggaaaaaa 600

gcggcggcgg gcatttatcg cctggataaa gcgcgcgtga gccgccgcat tagcctggaa 660

tttatgctgg aacaggcgaa caaatttgcg agcaaaaaag cgatttggtt tccgtataac 720

atggattggc gcggccgcgt gtatgcggtg ccgatgttta acccgcaggg caacgatatg 780

accaaaggcc tgctgaccct ggcgaaaggc aaaccgattg gcgaagaagg cttttattgg 840

ctgaaaattc atggcgcgaa ctgcgcgggc gtggataaag tgccgtttcc ggaacgcatt 900

gcgtttattg aaaaacatgt ggatgatatt ctggcgtgcg cgaaagatcc gattaacaac 960

acctggtggg cggaacagga tagcccgttt tgctttctgg cgttttgctt tgaatatgcg 1020

ggcgtgaccc atcatggcct gagctataac tgcagcctgc cgctggcgtt tgatggcagc 1080

tgcagcggca ttcagcattt tagcgcgatg ctgcgcgatg aagtgggcgg ccgcgcggtg 1140

aacctgctgc cgagcgaaac cgtgcaggat atttatggca ttgtggcgca gaaagtgaac 1200

gaaattctga aacaggatgc gattaacggc accccgaacg aaatgattac cgtgaccgat 1260

aaagataccg gcgaaattag cgaaaaactg aaactgggca ccagcaccct ggcgcagcag 1320

tggctggcgt atggcgtgac ccgcagcgtg accaaacgca gcgtgatgac cctggcgtat 1380

ggcagcaaag aatttggctt tcgccagcag gtgctggatg ataccattca gccggcgatt 1440

gatagcggca aaggcctgat gtttacccag ccgaaccagg cggcgggcta tatggcgaaa 1500

ctgatttggg atgcggtgag cgtgaccgtg gtggcggcgg tggaagcgat gaactggctg 1560

aaaagcgcgg cgaaactgct ggcggcggaa gtgaaagata aaaaaaccaa agaaattctg 1620

cgccatcgct gcgcggtgca ttggaccacc ccggatggct ttccggtgtg gcaggaatat 1680

cgcaaaccgc tgcagaaacg cctggatatg atttttctgg gccagtttcg cctgcagccg 1740

accattaaca ccctgaaaga tagcggcatt gatgcgcata aacaggaaag cggcattgcg 1800

ccgaactttg tgcatagcca ggatggcagc catctgcgca tgaccgtggt gtatgcgcat 1860

gaaaaatatg gcattgaaag ctttgcgctg attcatgata gctttggcac cattccggcg 1920

gatgcgggca aactgtttaa agcggtgcgc gaaaccatgg tgattaccta tgaaaacaac 1980

gatgtgctgg cggattttta tagccagttt gcggatcagc tgcatgaaac ccagctggat 2040

aaaatgccgc cgctgccgaa aaaaggcaac ctgaacctgc aggatattct gaaaagcgat 2100

tttgcgtttg cg 2112

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Leu Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Ser Ala

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Leu Ala Lys Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Leu Gly

35 40 45

Glu Arg Arg Phe Leu Lys Met Leu Glu Arg Gln Ala Lys Ala Gly Glu

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Ile Ala Asp Asn Ala Ala Ala Lys Pro Leu Leu Ala Thr Leu Leu Pro

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Lys Leu Thr Thr Arg Ile Val Glu Trp Leu Glu Glu Tyr Ala Ser Lys

85 90 95

Lys Gly Arg Lys Pro Ser Ala Tyr Ala Pro Leu Gln Leu Leu Lys Pro

100 105 110

Glu Ala Ser Ala Phe Ile Thr Leu Lys Val Ile Leu Ala Ser Leu Thr

115 120 125

Ser Thr Asn Met Thr Thr Ile Gln Ala Ala Ala Gly Met Leu Gly Lys

130 135 140

Ala Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala

145 150 155 160

Lys His Phe Lys Lys His Val Glu Glu Gln Leu Asn Lys Arg His Gly

165 170 175

Gln Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Ile

180 185 190

Gly Arg Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp Asp Lys Glu

195 200 205

Thr Thr Met His Val Gly Ile Arg Leu Ile Glu Met Leu Ile Glu Ser

210 215 220

Thr Gly Leu Val Glu Leu Gln Arg His Asn Ala Gly Asn Ala Gly Ser

225 230 235 240

Asp His Glu Ala Leu Gln Leu Ala Gln Glu Tyr Val Asp Val Leu Ala

245 250 255

Lys Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys

260 265 270

Val Val Pro Pro Lys Pro Trp Val Ala Ile Thr Gly Gly Gly Tyr Trp

275 280 285

Ala Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys

290 295 300

Gly Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala

305 310 315 320

Val Asn Leu Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu

325 330 335

Ala Val Val Asn Glu Ile Val Asn Trp Lys Asn Cys Pro Val Ala Asp

340 345 350

Ile Pro Ser Leu Glu Arg Gln Glu Leu Pro Pro Lys Pro Asp Asp Ile

355 360 365

Asp Thr Asn Glu Ala Ala Leu Lys Glu Trp Lys Lys Ala Ala Ala Gly

370 375 380

Ile Tyr Arg Leu Asp Lys Ala Arg Val Ser Arg Arg Ile Ser Leu Glu

385 390 395 400

Phe Met Leu Glu Gln Ala Asn Lys Phe Ala Ser Lys Lys Ala Ile Trp

405 410 415

Phe Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Pro Met

420 425 430

Phe Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala

435 440 445

Lys Gly Lys Pro Ile Gly Glu Glu Gly Phe Tyr Trp Leu Lys Ile His

450 455 460

Gly Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile

465 470 475 480

Ala Phe Ile Glu Lys His Val Asp Asp Ile Leu Ala Cys Ala Lys Asp

485 490 495

Pro Ile Asn Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe

500 505 510

Leu Ala Phe Cys Phe Glu Tyr Ala Gly Val Thr His His Gly Leu Ser

515 520 525

Tyr Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile

530 535 540

Gln His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val

545 550 555 560

Asn Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala

565 570 575

Gln Lys Val Asn Glu Ile Leu Lys Gln Asp Ala Ile Asn Gly Thr Pro

580 585 590

Asn Glu Met Ile Thr Val Thr Asp Lys Asp Thr Gly Glu Ile Ser Glu

595 600 605

Lys Leu Lys Leu Gly Thr Ser Thr Leu Ala Gln Gln Trp Leu Ala Tyr

610 615 620

Gly Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr

625 630 635 640

Gly Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Asp Asp Thr Ile

645 650 655

Gln Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn

660 665 670

Gln Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Asp Ala Val Ser Val

675 680 685

Thr Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala

690 695 700

Lys Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Lys Glu Ile Leu

705 710 715 720

Arg His Arg Cys Ala Val His Trp Thr Thr Pro Asp Gly Phe Pro Val

725 730 735

Trp Gln Glu Tyr Arg Lys Pro Leu Gln Lys Arg Leu Asp Met Ile Phe

740 745 750

Leu Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Leu Lys Asp Ser

755 760 765

Gly Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val

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His Ser Gln Asp Gly Ser His Leu Arg Met Thr Val Val Tyr Ala His

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Glu Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly

805 810 815

Thr Ile Pro Ala Asp Ala Gly Lys Leu Phe Lys Ala Val Arg Glu Thr

820 825 830

Met Val Ile Thr Tyr Glu Asn Asn Asp Val Leu Ala Asp Phe Tyr Ser

835 840 845

Gln Phe Ala Asp Gln Leu His Glu Thr Gln Leu Asp Lys Met Pro Pro

850 855 860

Leu Pro Lys Lys Gly Asn Leu Asn Leu Gln Asp Ile Leu Lys Ser Asp

865 870 875 880

Phe Ala Phe Ala

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ctgctgacct tcgcgaaagg ca 22

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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tgcctttcgc gaaggtcagc ag 22

<210> 5

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gagcgaaacc cctcaggata tttat 25

<210> 6

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<213> Artificial Sequence (Artificial Sequence)

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ataaatatcc tgaggggttt cgctc 25

<210> 7

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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tgcgcgaaac cttcgtgatt accta 25

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<213> Artificial Sequence (Artificial Sequence)

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taggtaatca cgaaggtttc gcgca 25

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gtgggcggaa gctgatagcc 20

<210> 10

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<213> Artificial Sequence (Artificial Sequence)

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ggctatcagc ttccgcccac 20

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gctataacag cagcctgccg ct 22

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

agcggcaggc tgctgttata gc 22

<210> 13

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gtttccgtat aacatggatt cgcgc 25

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gcgcgaatcc atgttatacg gaaac 25

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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aaaaggcaac ctggacctgc 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gcaggtccag gttgcctttt 20

<210> 17

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tagctttggc acccatccg 19

<210> 18

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cggatgggtg ccaaagcta 19

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

agcgattacg tttccgtata acatg 25

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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catgttatac ggaaacgtaa tcgct 25

<210> 21

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gcctggatac gatttttctg gg 22

<210> 22

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

cccagaaaaa tcgtatccag gc 22

<210> 23

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

cgcggatccg cgaaagcgtt tatgcag 27

<210> 24

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gcgattttgc gtttgcgaaa agtactttt 29

Claims

1. An RNA polymerase mutant, wherein the RNA polymerase mutant is: a polypeptide obtained by L447F mutation of an amino acid sequence shown as SEQ ID No. 2;

alternatively, the RNA polymerase mutant is: polypeptide obtained by V586P mutation of amino acid sequence shown as SEQ ID No. 2;

alternatively, the RNA polymerase mutant is: polypeptide obtained by M833F mutation of amino acid sequence shown as SEQ ID No. 2;

alternatively, the RNA polymerase mutant is: a polypeptide obtained by Q506A mutation of an amino acid sequence shown as SEQ ID No. 2;

alternatively, the RNA polymerase mutant is: polypeptide obtained by L447F mutation and V586P mutation of the amino acid sequence shown as SEQ ID No. 2;

alternatively, the RNA polymerase mutant is: polypeptide obtained by L447F mutation and M833F mutation of an amino acid sequence shown as SEQ ID No. 2;

alternatively, the RNA polymerase mutant is: a polypeptide obtained by carrying out L447F mutation and Q506A mutation on an amino acid sequence shown as SEQ ID No. 2;

alternatively, the RNA polymerase mutant is: a polypeptide obtained by carrying out L447F mutation and C531S mutation on an amino acid sequence shown as SEQ ID No. 2;

alternatively, the RNA polymerase mutant is: a polypeptide obtained by carrying out L447F mutation, V586P mutation and M833F mutation on an amino acid sequence shown as SEQ ID No. 2;

alternatively, the RNA polymerase mutant is: a polypeptide obtained by carrying out L447F mutation, V586P mutation and Q506A mutation on an amino acid sequence shown as SEQ ID No. 2;

alternatively, the RNA polymerase mutant is: a polypeptide obtained by carrying out L447F mutation, V586P mutation and C531S mutation on an amino acid sequence shown as SEQ ID No. 2;

alternatively, the RNA polymerase mutant is: polypeptide obtained by L447F mutation, V586P mutation, C531S mutation and M833F mutation of the amino acid sequence shown as SEQ ID No. 2.

2. The RNA polymerase mutant of claim 1, wherein the coding sequence of the RNA polymerase mutant is: the polynucleotide is obtained by replacing a polynucleotide sequence consisting of the nucleotide sequence shown as SEQ ID No.1, and the mutation site of the RNA polymerase mutant corresponds to the corresponding nucleotide sequence.

3. The RNA polymerase mutant according to any one of claims 1 to 2, wherein the RNA polymerase mutant is a soluble enzyme or an immobilized enzyme.

4. A recombinant vector comprising the coding sequence of the RNA polymerase mutant of any one of claims 1 to 2.

5. The method for producing the recombinant vector according to claim 4, comprising the steps of: and carrying out PCR amplification on the first vector by adopting a mutation amplification primer pair to obtain a recombinant vector, wherein the mutation amplification primer pair contains a nucleotide sequence corresponding to a mutation site of the RNA polymerase mutant, and the first vector contains a coding sequence corresponding to an amino acid sequence shown as SEQ ID No. 2.

6. A recombinant engineered bacterium comprising the recombinant vector according to claim 4.

7. Use of the RNA polymerase mutant of any one of claims 1 to 2, the recombinant vector of claim 4 or the recombinant engineered bacterium of claim 6 for RNA synthesis.