Engineered RNA polymerase variants

Abstract

The present disclosure relates to engineered RNA polymerase polypeptides and compositions thereof, and polynucleotides encoding the engineered RNA polymerase polypeptides. The present disclosure also provides methods of producing RNA using the engineered RNA polymerase polypeptides or compositions thereof.

Description

Cross Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 502,006, filed May 12, 2023, which is incorporated herein by reference.

[0002] Reference to a Sequence Listing, Table, or Computer Program The Sequence Listing submitted with this filing, on the computer readable medium, created on May 10, 2024, and having a file size of 419,237 bytes, is hereby incorporated by reference herein and is hereby made a part of this specification. TECHNICAL FIELD

[0003] The present disclosure relates to engineered RNA polymerase variants, compositions thereof, and methods of using the engineered RNA polymerase variants. BACKGROUND

[0004] RNA polymerases transcribe DNA (and in some cases RNA) into RNA transcripts. These enzymes represent the primary mechanism driving transcription. RNA polymerases have been isolated and purified to a degree sufficient for use in the in vitro production of RNA. In vitro transcription allows the synthesis of RNA molecules of any sequence, ranging in size from short oligonucleotides to several thousand bases, guided by a polynucleotide template. Typically, in vitro transcription involves engineering a template that includes a promoter sequence upstream of the sequence of interest, followed by transcription using a corresponding RNA polymerase. RNA transcripts can be further modified by capping, splicing, and / or addition of a poly-A tail, among other ways. Some modifications, such as capping and addition of a poly-A tail, can occur as part of the transcription reaction, for example, by co-transcriptional capping by the RNA polymerase and by inclusion of appropriate poly-dT sequences in the polynucleotide template. In other cases, capping and addition of a poly-A tail can occur post-transcriptionally, for example, by using RNA capping enzymes (e.g., a Fousty or vaccinia virus capping enzyme) and poly(A) polymerase (e.g., E. coli poly(A) polymerase) for poly-A tailing. In vitro generated transcripts can be used in analytical techniques (e.g., hybridization assays), structural studies (e.g., NMR and X-ray crystallography), biochemical and genetic studies (e.g., as antisense reagents), as functional molecules (e.g., ribozymes and aptamers), and as therapeutic agents.

[0005] RNA has become a focus for therapeutic applications, including, among others, mRNA-based vaccines, cancer immunotherapeutics, genomic engineering (e.g., CRISPR), and enzyme replacement therapies. For such applications, it is important that a stable RNA polymerase be available, enabling the production of RNA with high yield and high fidelity, with minimal amounts of unwanted products, including, among others, incomplete transcripts and double-stranded RNA products. SUMMARY

[0006] The present disclosure provides engineered RNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered RNA polymerase polypeptides. The present disclosure also provides methods of using the engineered RNA polymerase polypeptides and compositions thereof for nucleic acid synthesis and other purposes.

[0007] In one aspect, the present disclosure provides an engineered RNA polymerase or a functional fragment thereof, comprising an amino acid sequence that corresponds to a reference sequence of residues 8 to 890 of SEQ ID NO: 2 or 4 or that has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence of SEQ ID NO: 2 or 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence of residues 8 to 890 of SEQ ID NO: 2 or 4 or relative to the reference sequence of SEQ ID NO: 2 or 4.

[0008] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence that corresponds to a reference sequence of residues 8 to 890 of SEQ ID NO: 2 or that has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence of SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence of residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence of SEQ ID NO: 2.

[0009] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence that corresponds to a reference sequence of residues 8 to 890 of SEQ ID NO: 4 or that has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence of SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence of residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence of SEQ ID NO: 2.

[0010] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to residues 8 to 890 of an even numbered SEQ ID NO. of SEQ ID NOS: 4-138, or to the sequence corresponding to residues 8 to 890 of a reference sequence of SEQ ID NO: 2, or to the sequence corresponding to residues 8 to 890 of a reference sequence of SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, or relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2.

[0011] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to residues 8 to 890 of an even numbered SEQ ID NO. of SEQ ID NOS: 4-138, or to the sequence corresponding to residues 8 to 890 of a reference sequence of SEQ ID NO: 2, or to the sequence corresponding to residues 8 to 890 of a reference sequence of SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, or relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2.

[0012] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to residues 8 to 890 of an even numbered SEQ ID NO. of SEQ ID NOS: 4-138, or to the sequence corresponding to residues 8 to 890 of a reference sequence of SEQ ID NO: 2, or to the sequence corresponding to residues 8 to 890 of a reference sequence of SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, or relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2.

[0013] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 38 / 143 / 664 / 793, 38 / 340 / 364 / 640 / 664 / 782 / 793, 38 / 143 / 246 / 340 / 399 / 640 / 782 / 793, 38 / 379 / 437 / 664 / 779, 38 / 379 / 517 / 664, 664 / 782 / 793, 38 / 143 / 340 / 640 / 664, 38 / 607 / 664 / 720, 38 / 246 / 399 / 640 / 782 / 856, 340 / 664 / 751 / 793 / 856, 143 / 340 / 437 / 664 / 779, 340 / 664 / 751 / 856, 38 / 437, 143 / 517 / 664, 38 / 664, 38 / 416 / 664, 143 / 246 / 793, 399 / 640, 640 / 782 / 793, 38 / 310 / 437, 246 / 416 / 664, 246 / 793, 38 / 310 / 379, 38 / 246 / 340 / 437, 38 / 340 / 664, 664 / 720, 38 / 143 / 340 / 640, 664, 517 / 664 / 720, 38 / 340 / 399 / 416 / 664 / 751 / 793, 246 / 437, 38 / 244 / 340 / 379 / 437 / 664 / 720 / 779, 133 / 134 / 135 / 136 / 246 / 340 / 379 / 437 / 517 / 607 / 664 / 720, 38 / 782, 38 / 379, 38 / 244 / 246 / 310 / 379, 38 / 340 / 720 or 38 / 379 / 437 / 517, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0014] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence that is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4.

[0015] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or higher sequence identity with the sequence corresponding to the even-numbered SEQ ID NOs of ...

[0016] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0017] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or higher sequence identity with SEQ ID NO. corresponding to even-numbered residues of SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0018] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 38, 133, 134, 135, 136, 143, 244, 246, 310, 340, 364, 379, 399, 401, 404, 416, 437, 517, 607, 640, 664, 670, 720, 751, 756, 779, 782, 793, 856, or 876, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0019] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 38 / 143 / 340 / 437 / 640 / 670 / 751 / 856, 640 / 670 / 856, 399 / 640, 399 / 640 / 670 / 782 / 856, 143 / 340 / 399 / 640 / 782, 437, 143 / 340 / 437 / 751 / 782 / 856 / 876, 399 / 640 / 670 / 782, 437 / 670 / 779 / 782 / 856, 437 / 782, 38 / 437, 437 / 779 / 782, 143 / 399 / 437 / 751 / 782, 399 / 437 / 640 / 670 / 751 / 779 / 782 / 856, 38 / 143 / 340 / 670 / 856, 38 / 437 / 640 / 779, 437 / 751, 38 / 399 / 437 / 640 / 670, 143 / 340 / 399 / 640 / 779 / 782, 38 / 143 / 399 / 437 / 640 / 670 / 751, 340 / 437 / 782, 640 / 751 / 856, 143 / 640 / 782, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0020] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4, or relative to a reference sequence corresponding to SEQ ID NO: 2 or 4.

[0021] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions of the engineered RNA polymerase as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4 or relative to a reference sequence corresponding to SEQ ID NO: 2 or 4.

[0022] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence of residues 8 to 890 of SEQ ID NO. containing even-numbered SEQ ID NO. of SEQ ID NO: 4-138, wherein optionally the amino acid sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions.

[0023] In some embodiments, the engineered RNA polymerase has RNA polymerase activity and is characterized by at least one improved property compared to a reference RNA polymerase. In some embodiments, the improved property is selected from, for example, i) increased activity, ii) increased stability, and iii) increased thermostability, or any combination of i), ii) and iii), compared to a reference RNA polymerase, wherein the reference RNA polymerase has a sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4, or a sequence corresponding to SEQ ID NO: 2 or 4.

[0024] In some further embodiments, the engineered RNA polymerase is purified. In some embodiments, the engineered RNA polymerase is provided in solution, as a lyophilized product, or immobilized on the surface of a substrate such as a solid substrate, membrane, or particle.

[0025] On the other hand, this disclosure provides recombinant polynucleotides encoding the engineered RNA polymerase disclosed herein.

[0026] In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence of nucleotide residues 21 to 2670 of SEQ ID NO. corresponding to odd-numbered SEQ ID NO: 3-137 or a polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO. corresponding to odd-numbered SEQ ID NO: 3-137, wherein the recombinant polynucleotide encodes an RNA polymerase.

[0027] In some embodiments, the polynucleotide sequence encoding the recombinant polynucleotide of the engineered RNA polymerase is codon-optimized. In some embodiments, the polynucleotide sequence is codon-optimized for expression in bacterial, fungal, insect, or mammalian cells.

[0028] In some embodiments, the recombinant polynucleotide comprises nucleotide residues 21 to 2670 of SEQ ID NO. containing odd numbers of SEQ ID NO: 3-137 or a polynucleotide sequence containing odd numbers of SEQ ID NO: 3-137.

[0029] In another aspect, this disclosure provides expression vectors comprising recombinant polynucleotides encoding the engineered RNA polymerase described herein. In some embodiments, the recombinant polynucleotides of the expression vector are operatively linked to a control sequence. In some embodiments, the control sequence comprises a promoter, particularly a heterologous promoter.

[0030] In another aspect, this disclosure also provides host cells transformed using the expression vectors provided herein. In some embodiments, the host cells are prokaryotic or eukaryotic cells. In some embodiments, the host cells are bacterial cells, such as Escherichia coli or Bacillus subtilis.

[0031] In another aspect, this disclosure provides a method for producing an engineered RNA polymerase polypeptide, the method comprising culturing the host cells described herein under suitable culture conditions to express the encoded engineered RNA polymerase. In some embodiments, the method further includes recovering or isolating the expressed engineered RNA polymerase from the culture and / or host cells. In some embodiments, the method further includes purifying the expressed engineered RNA polymerase.

[0032] In another embodiment, this disclosure provides a composition comprising an engineered RNA polymerase disclosed herein. In some embodiments, the composition further comprises at least a buffer. In some embodiments, the composition further comprises a reducing agent, such as dithiothreitol or mercaptoethanol. In some embodiments, the composition further comprises one or more nucleoside triphosphate (NTP) substrates, particularly rNTP substrates. In some embodiments, the composition further comprises a cap analogue. In some embodiments, the composition further comprises a target DNA template. In some embodiments, the target DNA template comprises a promoter recognized by the engineered RNA polymerase. In some embodiments, the composition further comprises an additive, such as a molecular congesting agent.

[0033] In another aspect, this disclosure provides a method for producing RNA, the method comprising contacting a target DNA template with an engineered RNA polymerase described herein in the presence of one or more nucleoside triphosphates, under conditions suitable for transcribing all or part of a target DNA template. In some embodiments, the method further includes providing a cap analogue. In some embodiments, suitable conditions include temperatures from about 25°C to about 50°C. In some embodiments, suitable conditions include additives, such as molecular congestors.

[0034] On the other hand, this disclosure also provides a kit comprising at least one engineered RNA polymerase disclosed herein. In some embodiments, the kit further includes a buffer, one or more rNTPs, and Mg... +2 One or more of a reducing agent and one or more molecular crowding agents. Detailed Implementation

[0035] This disclosure provides engineered RNA polymerase peptides and compositions thereof, wherein the engineered RNA polymerase exhibits one or more improved properties, particularly including enhanced activity, enhanced stability, and / or enhanced thermostability. This disclosure also provides recombinant polynucleotides encoding the engineered RNA polymerase peptides and methods for producing RNA using the engineered RNA polymerase. In some embodiments, the expressed RNA-encoding peptide may be a non-coding RNA (ncRNA), such as shRNA, miRNA, or guide RNA.

[0036] Abbreviations and definitions For the purposes of this disclosure, unless otherwise expressly defined, the technical and scientific terms used in the description herein will have the meanings commonly understood by one of ordinary skill in the art.

[0037] It should be understood that the present invention is not limited to the specific methods, schemes, and reagents described, as these may vary depending on the context in which they are used by those skilled in the art. Therefore, the terms defined below are described more fully by reference to the application as a whole.

[0038] Furthermore, the headings provided herein are not intended to limit the various aspects or embodiments of the invention, which can be obtained by referring to the application as a whole.

[0039] As used herein, the singular “a / kind (a, an)” and “the / said” include plural referents unless the context clearly indicates otherwise.

[0040] As used herein, the term “include” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).

[0041] It should also be understood that, where the description of the implementation scheme uses the term "comprising" and its cognates, the implementation scheme may also be described using the language "substantially composed of" or "consisting of".

[0042] Furthermore, the numerical range includes the number that defines the range. Therefore, each numerical range disclosed herein is intended to encompass every narrower numerical range falling within such a wider numerical range, as if such a narrower numerical range were explicitly stated herein. Each maximum (or minimum) numerical limit disclosed herein is also intended to include every lower (or higher) numerical limit, as if such lower (or higher) numerical limits were explicitly stated herein.

[0043] "Approximately" refers to the acceptable error for a specific value. In some cases, "approximately" means within 0.05%, 0.5%, 1.0%, or 2.0% of the given value. In other cases, "approximately" means within 1, 2, 3, or 4 standard deviations of the given value.

[0044] "ATCC" refers to the American Type Culture Collection, whose biobank collection includes genes and strains.

[0045] "NCBI" refers to the National Center for Biotechnology Information and the sequence database it provides.

[0046] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably to refer to polymers of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation).

[0047] Amino acids are referred to in this document by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. The abbreviations used for gene-encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glycine (Gly or G), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When using a three-letter abbreviation, unless specifically preceded by "L" or "D" or clearly indicated from the context of its use, amino acids can refer to α-carbon (C) αThe amino acid sequence is represented by its L- or D-configuration. For example, "Ala" indicates alanine without specifying the configuration with respect to the α-carbon, "D-Ala" and "L-Ala" indicate D-alanine and L-alanine, respectively. When using single-letter abbreviations, uppercase letters indicate amino acids with the L-configuration with respect to the α-carbon, and lowercase letters indicate amino acids with the D-configuration with respect to the α-carbon. For example, "A" indicates L-alanine, and "a" indicates D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or mixtures thereof), the sequence is conventionally presented in the direction from amino (N) to carboxyl (C).

[0048] "RNA polymerase" or "RNAP" refers to an enzyme that catalyzes RNA synthesis from the 5' to 3' direction using a polynucleotide as a template. In some embodiments, RNA polymerase uses a DNA template and is referred to as DNA-directed or DNA-dependent RNA polymerase. In some embodiments, RNA polymerase uses an RNA template and is referred to as RNA-directed or RNA-dependent RNA polymerase. In some embodiments, RNA polymerase is capable of synthesizing RNA using both DNA and RNA as templates. In some embodiments, the RNA product is referred to as an "RNA transcript."

[0049] As used herein, "cap" refers to a nucleoside whose 5' carbon is bonded to a triphosphate group, which in turn bonds to the 5' carbon of the 5' nucleotide of the RNA transcript. In some embodiments, the cap nucleoside is guanine. In some embodiments, the nitrogen at the 7-position of guanine in the cap is methylated and denoted as m. 7 G. In some implementations, the cap is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. The terms "capped RNA," "5'-capped RNA," and "capped mRNA" refer to the RNA and mRNA that make up the cap, respectively.

[0050] "Fusion protein," "chimeric protein," and "chimera" refer to hybrid proteins produced by the conjugation of two or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are produced using recombinant technologies (e.g., molecular biology techniques known in the art).

[0051] The terms “polynucleotide,” “nucleic acid,” or “oligonucleotide” are used herein to refer to polymers comprising at least two nucleotides, wherein the nucleotides are deoxyribonucleotides or ribonucleotides or a mixture of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations for gene-encoding nucleosides are conventional and follow the following: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically described, the abbreviation for nucleosides may be ribonucleoside or 2'-deoxyribonucleoside. Nucleosides may be designated as ribonucleoside or 2'-deoxyribonucleoside on a single or collective basis. When a polynucleotide, nucleic acid, or oligonucleotide sequence is presented as a string of single-letter abbreviations, the sequence is presented in the 5' to 3' orientation according to common practice and does not indicate phosphate esters. The term “DNA” refers to deoxyribonucleic acid. The term “RNA” refers to ribonucleic acid. Polynucleotides or nucleic acids may be single-stranded or double-stranded, or may include both single-stranded and double-stranded regions.

[0052] "Double strand" and "ds" refer to a double-stranded nucleic acid (e.g., DNA or RNA) molecule composed of two single-stranded polynucleotides that are complementary in sequence (A paired with T or U, C paired with G), arranged in an antiparallel 5' to 3' orientation, and held together by hydrogen bonds between nucleobases (e.g., adenine [A], guanine [G], cytosine [C], thymine [T], uridine [U]).

[0053] When used in relation to cells, polynucleotides, or peptides, “engineered,” “recombinant,” “non-natural,” and “variant” refer to a material or a material corresponding to the natural or native form of that material that has been modified in a manner not present in or similar to that found in nature, but produced or derived from synthetic materials and / or manipulated using recombinant techniques.

[0054] "Wild-type" and "naturally occurring" refer to forms found in nature. For example, wild-type polypeptide or polynucleotide sequences are sequences that exist in organisms, can be isolated from natural sources, and have not been intentionally modified by humans.

[0055] "Coding sequence" refers to the part of a nucleic acid (e.g., a gene) that encodes the amino acid sequence of a protein or polypeptide.

[0056] "Sequence identity percentage (%)" refers to a comparison between polynucleotides and peptides, and is determined by comparing two optimally aligned sequences within a comparison window. For optimal alignment, the portion of the polynucleotide or peptide sequence in the comparison window may contain additions or deletions (i.e., vacancies) compared to the reference sequence. The percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to generate a matching position number, dividing the matching position number by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Alternatively, the percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears, or by aligning the nucleic acid base or amino acid residue with vacancies to obtain a matching position number, dividing the matching position number by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Those skilled in the art will understand that many established algorithms exist for aligning two sequences. The optimal alignment of sequences for comparison can be performed as is known in the art, for example, by Smith and Waterman’s local homology algorithm (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by Needleman and Wunsch’s homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by Pearson and Lipman’s similarity search method (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection. Examples of algorithms suitable for determining sequence identity percentages and sequence similarity include, but are not limited to, BLAST and BLAST 2.0 algorithms (see, for example, Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucleic Acids Res., 1977, 3389-3402). Software for performing BLAST analysis is publicly available from the website of the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length “W” in the query sequence. These short words match or satisfy a positive threshold score “T” when compared to words of the same length in a database sequence. T is called the neighborhood word score threshold (see Altschul et al., IdemThese initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence, as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters “M” (reward score for a pair of matching residues; always >0) and “N” (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Word hit extension in each direction stops when: the cumulative alignment score decreases by an amount “X” from its maximum realized value; the cumulative score becomes zero or lower due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word length of 11 (W), an expected value of 10 (E), M=5, N=-4, and a comparison of two strands. For amino acid sequences, the BLASTP program defaults to a word length of 3 (W), an expected value of 10 (E), and a BLOSUM62 scoring matrix (see, for example, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and sequence identity percentage can be performed using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) with the provided default parameters.

[0057] A “reference sequence” is a defined sequence used as the basis for sequence comparison. A reference sequence can be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, at least 100 residues long, or the full length of a nucleic acid or polypeptide. Since each of two polynucleotides or polypeptides can (1) contain sequences similar to the two sequences (i.e., a portion of the complete sequence) and (2) also contain sequences different from the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing the sequences of the two polynucleotides or polypeptides on a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, wherein the reference sequence is a sequence that may have one or more variations in the primary sequence. For example, the phrase “a reference sequence corresponding to SEQ ID NO: 2 containing valine at the residue corresponding to X143” (or “a reference sequence corresponding to SEQ ID NO: 2 containing arginine at the residue corresponding to position 143”) refers to a reference sequence in which the corresponding residue (e.g., alanine) at position X38 in SEQ ID NO: 2 has been changed to valine.

[0058] A “comparison window” refers to a conceptual segment of adjacent nucleotide positions or amino acid residues, where the sequence can be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 consecutive nucleotides or amino acids, and wherein, for optimal alignment of the two sequences, the sequence portion within the comparison window may contain 20% or less of additions or deletions (i.e., vacancies) compared to the reference sequence (which does not contain additions or deletions). In some embodiments, the comparison window may be longer than 15-20 consecutive residues, and optionally include windows of 30, 40, 50, 100, or longer.

[0059] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "reference," and "relative to" refer to specifying the number of residues in the reference sequence when the given amino acid or polynucleotide sequence is compared to a reference sequence. In other words, the residue numbering or position of a given polymer is specified relative to the reference sequence, rather than by the actual numerical position of residues within the given amino acid or polynucleotide sequence. For example, residue matching between two sequences can be optimized by introducing vacancies, comparing a given amino acid sequence (e.g., the amino acid sequence of an engineered RNA polymerase) to a reference sequence. In these cases, the numbering of residues in the given amino acid or polynucleotide sequence is relative to the reference sequence with which it is compared, despite the presence of vacancies. In some embodiments, the sequence is tagged (e.g., with histidine tags).

[0060] A "mutation" refers to an alteration in the sequence of a nucleic acid. In some embodiments, a mutation results in a change in the sequence of the encoded polypeptide (i.e., compared to the original sequence without the mutation). In some embodiments, a mutation includes substitution, resulting in a different amino acid. In some alternative embodiments, a mutation includes addition, resulting in an amino acid being added (e.g., inserted) into the original polypeptide sequence. In some further embodiments, a mutation includes deletion, resulting in an amino acid being removed from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0061] "Amino acid difference" and "residue difference" refer to the difference of an amino acid residue at a position in a polypeptide sequence relative to the corresponding amino acid residue at a position in a reference sequence. The position of the amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "residue difference at position X143 compared to SEQ ID NO:2" (or "residue difference at position 143 compared to SEQ ID NO:2") refers to the difference of an amino acid residue at the polypeptide position corresponding to position 143 of SEQ ID NO:2. Therefore, if the reference polypeptide of SEQ ID NO:2 has valine at position 143, then "residue difference at position X143 compared to SEQ ID NO:2" refers to the substitution of any residue other than valine at the position of the polypeptide corresponding to position 143 of SEQ ID NO:2. In some cases herein, a specific amino acid residue difference at a position is represented as "XnY," where "Xn" specifies the corresponding residue and position of the reference polypeptide (as described above), and "Y" is a single-letter identifier for the amino acid found in the engineered polypeptide (i.e., a residue different from that in the reference polypeptide). In some instances (e.g., in the tables of the embodiments), this disclosure also provides specific amino acid differences represented by the conventional symbol “AnB”, where A is a single-letter identifier of a residue in the reference sequence, “n” is the position number of the residue in the reference sequence, and B is a single-letter identifier of a residue substitution in the sequence of the engineered polypeptide. In some embodiments, amino acid differences (e.g., substitutions) are represented by the abbreviation “nB” without an identifier of a residue in the reference sequence. In some embodiments, the phrase “amino acid residue nB” indicates the presence of an amino residue in the engineered polypeptide, which may or may not be a substitution in the context of the reference sequence. In some embodiments, “substitution” includes the deletion of an amino acid and may be represented by the “-” symbol.

[0062] In some cases, the polypeptides of this disclosure may include one or more amino acid residue differences relative to a reference sequence, indicated by a list of designated positions where residue differences exist relative to the reference sequence. In some embodiments, where more than one amino acid may be used at a particular residue position of the polypeptide, the various amino acid residues that may be used are separated by " / " (e.g., X664K / X664R, X664K / R, or 664K / R). This disclosure includes engineered polypeptide sequences comprising one or more amino acid differences, including one or both of conserved and non-conserved amino acid substitutions and amino acid insertions and deletions in the sequence.

[0063] "Amino acid substitution set" and "substitution set" refer to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, the substitution set contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, the substitution set refers to the set of amino acid substitutions present in any of the variant RNA polymerase polypeptides listed in any of the tables in the examples. In these substitution sets, individual substitutions are separated by semicolons (e.g., Q246A; S437P) or forward slashes (" / "; e.g., Q246A / S437P or 246A / 437P).

[0064] "Conservative amino acid substitution" refers to the substitution of a residue with a different residue having a similar side chain, and therefore generally involves the substitution of an amino acid in a polypeptide with an amino acid from the same or similar defined amino acid class. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain may be substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain may be substituted with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain may be substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain may be substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid may be substituted with another hydrophobic or hydrophilic amino acid, respectively.

[0065] "Non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitution can use amino acids between rather than within groups and affect: (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine); (b) charge or hydrophobicity; and / or (c) the body of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include acidic amino acids substituted with basic or aliphatic amino acids; aromatic amino acids substituted with small amino acids; and hydrophilic amino acids substituted with hydrophobic amino acids.

[0066] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletions may include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, representing up to 10% or up to 20% of the total number of amino acids constituting the reference peptide, while preserving biological activity and / or the improved properties of the engineered RNA polymerase. Deletions may target internal and / or terminal portions of the peptide. In various embodiments, deletions may include continuous segments or may be discontinuous. Deletions are indicated by "-" and may be present in a substitution set.

[0067] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. Insertion can occur within the polypeptide itself, or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins known in the art. Insertions can be continuous segments of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0068] The terms “functional fragment” and “bioactive fragment” are used interchangeably herein and refer to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion and / or an internal deletion, but wherein the remaining amino acid sequence is identical to the corresponding position in the sequence being compared (e.g., the full-length engineered RNA polymerase of this disclosure) and substantially retains all the activities of the full-length polypeptide.

[0069] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other naturally occurring contaminants, such as proteins, lipids, and polynucleotides. This term includes polypeptides that have been removed or purified from their natural environment or expression system (e.g., host cells or in vitro synthesis). Recombinant RNA polymerase polypeptides may be present intracellularly, in cell culture media, or prepared in various forms, such as lysates or isolated formulations. Therefore, in some embodiments, the recombinant RNA polymerase polypeptides provided herein are isolated polypeptides.

[0070] "Substantially pure polypeptide" refers to a composition in which the polypeptide is the dominant substance (i.e., it is more abundant than any other individual macromolecule in the composition, based on molar or weight), and is typically substantially pure when the target substance constitutes at least about 50% (in molar or weight %) of the present macromolecules. Typically, a substantially pure RNA polymerase composition will contain about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecules present in the composition, based on molar or weight %. In some embodiments, the target substance is purified to substantially homogeneity (i.e., contaminant substances are not detectable in the composition by conventional detection methods), wherein the composition consists substantially of a single macromolecule. Solvent substances, small molecules (<500 Daltons), and elemental ions are not considered macromolecules. In some embodiments, the isolated recombinant RNA polymerase polypeptide is a substantially pure polypeptide composition.

[0071] "Improved properties" refers to an engineered RNA polymerase peptide that exhibits improved properties in any RNA polymerase property compared to a reference RNA polymerase peptide, such as a wild-type RNA polymerase peptide or another engineered RNA polymerase peptide. Improved properties include, but are not limited to, properties such as: increased protein expression, enhanced activity, improved stability, improved thermal stability, improved pH stability, improved chemical stability, improved solvent stability, and improved solubility.

[0072] "Enzyme activity enhancement" and "enzyme activity improvement" refer to an improvement in the properties of an engineered RNA polymerase peptide, which can be represented by an increase in specific activity compared to a reference RNA polymerase peptide (e.g., wild-type RNA polymerase and / or other engineered RNA polymerases). Exemplary methods for determining enzyme activity are provided in the examples. An improvement in enzyme activity can be from about 1.1 times the enzyme activity of the corresponding wild-type or reference peptide to about 1.5 times, 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times, or more of the enzyme activity of a naturally occurring RNA polymerase from which the RNA polymerase peptide is derived or another engineered RNA polymerase.

[0073] "Codon optimization" refers to changing the codons of polynucleotides encoding proteins to codons that are preferentially used in a particular organism, making the encoded protein more effectively expressed in that organism. Although the genetic code is degenerate, as most amino acids are represented by a few codons (called "synonyms"), codon usage in a particular organism is known to be non-random and biased towards specific codon triples. This codon bias can be even higher for a given gene, genes with a common function or ancestral origin, highly expressed proteins versus low-copy-number proteins, and aggregated protein-coding regions of an organism's genome. In some implementations, polynucleotides encoding RNA polymerase polypeptides are codon-optimized for optimal production from a selected host organism for expression.

[0074] "Control sequence" herein refers to all components that are essential or advantageous for the expression of the polynucleotides and / or polypeptides disclosed herein. Each control sequence may be native or exogenous to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. In some embodiments, the control sequence includes at least a promoter and transcription and translation termination signals. In some embodiments, the control sequence is provided with an adapter to introduce a specific restriction site that facilitates the attachment of the control sequence to the coding region of the nucleic acid sequence encoding the polypeptide.

[0075] "Operably linked" refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) relative to the polynucleotide of interest, such that the control sequence directs or regulates the expression of the polynucleotide, and, where appropriate, directs or regulates the expression of the encoded polypeptide of interest.

[0076] A “promoter” or “promoter sequence” is a nucleic acid sequence, such as a coding sequence, that is recognized by a host cell for the expression of a polynucleotide of interest. A promoter sequence contains a transcriptional control sequence that mediates the expression of the polynucleotide of interest. A promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell. In some embodiments, a “promoter” refers to a nucleic acid sequence that is recognized by an engineered RNA polymerase.

[0077] "Suitable reaction conditions" or "suitable conditions" refers to those conditions (e.g., temperature, pH, buffer, salt, solubilizer, etc.) under which the RNA polymerase of this disclosure can synthesize RNA transcripts. Exemplary "suitable conditions" are provided herein (see Examples).

[0078] “Cultivation” refers to the growth of a population of cells (e.g., host cells) under suitable conditions using any suitable culture medium (e.g., liquid, gel, or solid).

[0079] A "vector" is a recombinant construct used to introduce a polynucleotide of interest into a cell. In some embodiments, the vector is an expression vector operatively linked to a suitable control sequence capable of expressing the polynucleotide or the polypeptide encoded in the polynucleotide in a suitable host. In some embodiments, the "expression vector" has a connection with the polynucleotide (e.g., ， The transgene (transgene) is operatively linked to a promoter sequence to drive expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0080] "Expression" includes any step involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also covers peptide secretion from cells.

[0081] "Production" refers to the production of proteins and / or other compounds by cells. This term is intended to encompass any steps involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses the secretion of peptides from cells.

[0082] "Heterogeneous" or "recombinant" refers to a relationship between two or more nucleic acid or polypeptide sequences (e.g., promoter sequences, signal peptides, terminator sequences, etc.) that are derived from different sources and are essentially unrelated.

[0083] "Host cell" and "host strain" refer to a suitable host for an expression vector containing a polynucleotide (e.g., a polynucleotide sequence encoding at least one RNA polymerase variant) provided herein. In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA technology, as is known in the art.

[0084] Engineered RNA polymerase polypeptides In one aspect, this disclosure provides RNA polymerases engineered to have improved properties, including, in particular, enhanced activity, improved stability, and enhanced thermostability. In some embodiments, the engineered RNA polymerase variants can be used to prepare RNA and other molecular biology techniques.

[0085] In some embodiments, the engineered RNA polymerase or a functional fragment thereof comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4.

[0086] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0087] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0088] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or higher sequence identity with the even-numbered SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0089] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 38, 133, 134, 135, 136, 143, 244, 246, 310, 340, 364, 379, 399, 416, 437, 517, 607, 640, 664, 670, 720, 751, 779, 782, 793, 856, or 876, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0090] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substituted or amino acid residue 38G / R, 133Q, 134H, 135R, 136E, 143A / V, 244Y, 246A, 310A, 340E / L, 364H, 379S, 399M, 416V, 437P, 517R / Y, 607Y, 640P, 664K / R, 670N, 720E / Q / R, 751R, 779R, 782G / V, 793L, 856I, or 876K, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0091] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution of R38G, L133Q, T134H, S135R, A136E, A143V, V244Y, Q246A, K310A / E / L, R364H, E379S, K399M, A416V, S437P, C517R / Y, E607Y, S640P, W664K / R, K670N, K720E / Q / R, Q751R, H779R, E782G / V, Q793L, F856I, or N876K or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0092] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 38, 143, 664, or 793 or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0093] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substituted or amino acid residue 38G, 143V, 664K, or 793L or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0094] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution of R38G, A143V, W664K or Q793L or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0095] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 38 / 143 / 664 / 793, 38 / 340 / 364 / 640 / 664 / 782 / 793, 38 / 143 / 246 / 340 / 399 / 640 / 782 / 793, 38 / 379 / 437 / 664 / 779, 38 / 379 / 517 / 664, 664 / 782 / 7 93, 38 / 143 / 340 / 640 / 664, 38 / 607 / 664 / 720, 38 / 246 / 399 / 640 / 782 / 856, 340 / 664 / 751 / 793 / 856, 143 / 340 / 437 / 664 / 779, 340 / 664 / 751 / 856, 38 / 437, 143 / 517 / 664, 38 / 664, 38 / 416 / 664, 143 / 246 / 793, 399 / 640, 640 / 782 / 793, 38 / 310 / 437, 246 / 416 / 664, 246 / 793, 38 / 310 / 379, 38 / 246 / 340 / 437, 38 / 340 / 664, 664 / 720, 38 / 143 / 340 / 640, 664, 517 / 664 / 720, 38 / 340 / 399 / 416 / 664 / 751 / 793, 246 / 437, 38 / 244 / 340 / 379 / 437 / 664 / 720 / 779, 133 / 134 / 135 / 136 / 246 / 340 / 379 / 437 / 517 / 607 / 664 / 720, 38 / 782, 38 / 379, 38 / 244 / 246 / 310 / 379, 38 / 340 / 720 or 38 / 379 / 437 / 517, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0096] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions or amino acid residues: 38G / 143V / 664K / 793L, 38G / 340E / 364H / 640P / 664R / 782V / 793L, 38G / 143V / 246A / 340E / 399M / 640P / 782G / 793L, 38G / 379S / 437P / 664K / 779R, 38G / 379S / 517R / 664K, 664R / 782V / 793L, 38G / 143V / 34 0L / 640P / 664R, 38G / 607Y / 664K / 720R, 38G / 246A / 399M / 640P / 782G / 856I, 340L / 664R / 751R / 793L / 856I, 143V / 340L / 437P / 664K / 779R, 340E / 664R / 751R / 856I, 38G / 437P, 143V / 517R / 664K, 38G / 664K, 38G / 416V / 664R, 143V / 246A / 793L, 399M / 640P, 640P / 782G / 793L, 38G / 310A / 437P, 246A / 416V / 664R, 246A / 793L, 38G / 310A / 379S, 38G / 246A / 340L / 437P, 38G / 340E / 664R, 664R / 720Q, 38G / 143V / 340L / 640P, 664R, 664K / 720E, 517R / 664R / 720E, 38G / 340E / 399M / 416V / 664K / 751R / 793L, 246A / 437P 38G / 244Y / 340L / 379S / 437P / 664K / 720R / 779R, 133Q / 134H / 135R / 136E / 246A / 340E / 379S / 437P / 517Y / 607Y / 664R / 720Q, 38G / 782V, 38G / 379S, 38G / 379S, 38G / 244Y / 246A / 310A / 379S, 38G / 340E / 720Q, or 38G / 379S / 437P / 517Y, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0097] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions: R38G / A143V / W664K / Q793L, R38G / K340E / R364H / S640P / W664R / E782V / Q793L, R38G / A143V / Q246A / K340E / K399M / S640P / E782G / Q793L, R38G / E379S / S437P / W664K / H779R, R38G / E379S / C517R / W664K, W664R / E782V / Q793L, and R38G / A143V / K340L / S6 40P / W664R, R38G / E607Y / W664K / K720R, R38G / Q246A / K399M / S640P / E7 82G / F856I, K340L / W664R / Q751R / Q793L / F856I, A143V / K340L / S437P / W 664K / H779R, K340E / W664R / Q751R / F856I, R38G / S437P, A143V / C517R / W 664K, R38G / W664K, R38G / A416V / W664R, A143V / Q246A / Q793L, K399M / S6 40P, S640P / E782G / Q793L, R38G / K310A / S437P, Q246A / A416V / W664R, Q 246A / Q793L, R38G / K310A / E379S, R38G / Q246A / K340L / S437P, R38G / K34 0E / W664R, W664R / K720Q, R38G / A143V / K340L / S640P, W664R, W664K / K72 0E, C517R / W664R / K720E, R38G / K340E / K399M / A416V / W664K / Q751R / Q79 3L, Q246A / S437P, R38G / V244Y / K340L / E379S / S437P / W664K / K720R / H77 9R、L133Q / T134H / S135R / A136E / Q246A / K340E / E379S / S437P / C517Y / E6 07Y / W664R / K720Q, R38G / E782V, R38G / E379S, R38G / E379S, R38G / V244Y / Q246A / K310A / E379S, R38G / K340E / K720Q or R38G / E379S / S437P / C517Y,The amino acid positions are relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0098] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least the following sets of substitutions at the following amino acid positions: 340 / 437 / 640 / 664 / 670 / 751 / 793 / 856, 38 / 143 / 640 / 664 / 670 / 793 / 856, 38 / 143 / 399 / 640 / 664 / 793, 38 / 143 / 399 / 640 / 664 / 670 / 782 / 793 / 856, 38 / 340 / 399 / 640 / 664 / 782 / 793, 38 / 143 / 437 / 664 / 793, 38 / 143 / 437 / 664 / 793, 38 / 340 / 437 / 664 / 751 / 782 / 793 / 856 / 876, 38 / 143 / 399 / 640 / 664 / 670 / 782 / 793, 38 / 143 / 437 / 664 / 670 / 779 / 782 / 793 / 856, 38 / 143 / 437 / 664 / 793, 38 / 143 / 437 / 664 / 782 / 793, 38 / 143 / 399 / 640 / 664 / 670 / 782 / 793, 143 / 437 / 664 / 793, 38 / 143 / 437 / 664 / 779 / 782 / 793, 38 / 399 / 437 / 664 / 751 / 782 / 793, 38 / 143 / 399 / 437 / 640 / 664 / 670 / 751 / 779 / 782 / 793 / 856, 38 / 143 / 437 / 664 / 793, 340 / 664 / 670 / 793 / 856, 143 / 437 / 640 / 664 / 779 / 793, 38 / 143 / 437 / 664 / 751 / 793, 1 43 / 399 / 437 / 640 / 664 / 670 / 793, 38 / 143 / 437 / 664 / 751 / 793, 38 / 340 / 399 / 640 / 664 / 779 / 782 / 793, 399 / 437 / 640 / 664 / 670 / 751 / 793, 38 / 143 / 340 / 437 / 664 / 782 / 793, 38 / 143 / 640 / 664 / 751 / 793 / 856 or 38 / 640 / 664 / 782 / 793, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0099] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 340L / 437P / 640P / 664K / 670N / 751R / 793L / 856I, 38G / 143V / 640P / 664K / 670N / 793L / 856I, 38G / 143V / 399M / 640P / 664K / 793L, 38G / 143V / 399M / 640P / 664K / 670N / 782V / 793L / 856I, 38G / 340E / 399M / 640P / 664K / 782G / 793L, 38G / 143V / 437P / 664K / 793L、38G / 143V / 437P / 664K / 793L、38G / 340L / 437P / 664K / 751R / 782V / 79 3L / 856I / 876K, 38G / 143V / 399M / 640P / 664K / 670N / 782V / 793L, 38G / 143V / 437P / 664K / 670N / 779R / 782G / 793L / 856I, 38G / 143V / 437P / 664K / 793L, 38 G / 143V / 437P / 664K / 782V / 793L, 38G / 143V / 399M / 640P / 664K / 670N / 782V / 7 93L, 143V / 437P / 664K / 793L, 38G / 143V / 437P / 664K / 779R / 782V / 793L, 38G / 399M / 437P / 664K / 751R / 782V / 793L、38G / 143V / 399M / 437P / 640P / 664K / 6 70N / 751R / 779R / 782V / 793L / 856I, 38G / 143V / 437P / 664K / 793L, 340E / 664 K / 670N / 793L / 856I, 143V / 437P / 640P / 664K / 779R / 793L, 38G / 143V / 437P / 664K / 751R / 793L, 143V / 399M / 437P / 640P / 664K / 670N / 793L, 38G / 143V / 43 7P / 664K / 751R / 793L, 38G / 340E / 399M / 640P / 664K / 779R / 782G / 793L, 399M / 437P / 640P / 664K / 670N / 751R / 793L, 38G / 143V / 340L / 437P / 664K / 782V / 793L, 38G / 143V / 640P / 664K / 751R / 793L / 856I or 38G / 640P / 664K / 782V / 793LThe amino acid positions are relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0100] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: K340L / S437P / S640P / W664K / K670N / Q751R / Q793L / F856I, R38G / A143V / S640P / W664K / K670N / Q793L / F856I, R38G / A143V / K399M / S640P / W664K / Q793L, R38G / A143V / K399M / S640P / W664K / K670N / E782V / Q793L / F856I, R38G / K340E / K399M / S 640P / W664K / E782G / Q793L, R38G / A143V / S437P / W664K / Q793L, R38G / A14 3V / S437P / W664K / Q793L, R38G / K340L / S437P / W664K / Q751R / E782V / Q793L / F856I / N876K、R38G / A143V / K399M / S640P / W664K / K670N / E782V / Q793L、R 38G / A143V / S437P / W664K / K670N / H779R / E782G / Q793L / F856I, R38G / A143 V / S437P / W664K / Q793L, R38G / A143V / S437P / W664K / E782V / Q793L, R38G / A143V / K399M / S640P / W664K / K670N / E782V / Q793L, A143V / S437P / W664K / Q 793L, R38G / A143V / S437P / W664K / H779R / E782V / Q793L, R38G / K399M / S437 P / W664K / Q751R / E782V / Q793L, R38G / A143V / K399M / S437P / S640P / W664K / K670N / Q751R / H779R / E782V / Q793L / F856I, R38G / A143V / S437P / W664K / Q 793L, K340E / W664K / K670N / Q793L / F856I, A143V / S437P / S640P / W664K / H7 79R / Q793L, R38G / A143V / S437P / W664K / Q751R / Q793L, A143V / K399M / S437 P / S640P / W664K / K670N / Q793L, R38G / A143V / S437P / W664K / Q751R / Q793L,R38G / K340E / K399M / S640P / W664K / H779R / E782G / Q793L, K399M / S437P / S640P / W664K / K670N / Q751R / Q793L, R38G / A143V / K340L / S437P / W664K / E782V / Q793L, R38G / A143V / S640P / W664K / Q751R / Q793L / F856I or R38G / S640P / W664K / E782V / Q793L, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0101] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the amino acid positions described in Tables 4.1 and 5.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0102] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0103] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at amino acid positions as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0104] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions of the variants described in Tables 4.1 and 5.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0105] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference sequence comprising the substitutions or sets of substitutions described in Tables 4.1 and 5.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0106] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence that is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4.

[0107] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or higher sequence identity with the sequence corresponding to the even-numbered SEQ ID NOs of ...

[0108] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0109] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or higher sequence identity with SEQ ID NO. corresponding to even-numbered residues of SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0110] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 38, 133, 134, 135, 136, 143, 244, 246, 310, 340, 364, 379, 399, 416, 437, 517, 607, 640, 664, 670, 720, 751, 779, 782, 793, 856, or 876, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0111] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substituted or amino acid residue 38G / R, 133Q, 134H, 135R, 136E, 143A / V, 244Y, 246A, 310A, 340E / L, 364H, 379S, 399M, 416V, 437P, 517R / Y, 607Y, 640P, 664K / R, 670N, 720E / Q / R, 751R, 779R, 782G / V, 793L, 856I, or 876K, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0112] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 38, 143, 664, or 793 or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0113] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substituted or amino acid residue 38G, 143V, 664K, or 793L or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0114] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 38 / 143 / 340 / 437 / 640 / 670 / 751 / 856, 640 / 670 / 856, 399 / 640, 399 / 640 / 670 / 782 / 856, 143 / 340 / 399 / 640 / 782, 437, 143 / 340 / 437 / 751 / 782 / 856 / 876, 399 / 640 / 670 / 782, 437 / 670 / 779 / 782 / 856, 437 / 782, 38 / 437, 437 / 77 9 / 782, 143 / 399 / 437 / 751 / 782, 399 / 437 / 640 / 670 / 751 / 779 / 782 / 856, 38 / 143 / 340 / 670 / 856, 38 / 437 / 640 / 779, 437 / 751, 38 / 399 / 437 / 640 / 670, 143 / 340 / 399 / 640 / 779 / 782, 38 / 143 / 399 / 437 / 640 / 670 / 751, 340 / 437 / 782, 640 / 751 / 856, 143 / 640 / 782, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0115] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions: 38R / 143A / 340L / 437P / 640P / 670N / 751R / 856I, 640P / 670N / 856I, 399M / 640P, 399M / 640P / 670N / 782V / 856I, 143A / 340E / 399M / 640P / 782G、437P、437P、143A / 340L / 437P / 751R / 782V / 856I / 876K、399M / 640P / 670N / 782V、43 7P / 670N / 779R / 782G / 856I, 437P, 437P / 782V, 399M / 640P / 670N / 782V, 38R / 437P, 437P / 77 9R / 782V, 143A / 399M / 437P / 751R / 782V, 399M / 437P / 640P / 670N / 751R / 779R / 782V / 856I, 437P、38R / 143A / 340E / 670N / 856I、38R / 437P / 640P / 779R、437P / 751R、38R / 399M / 437P / 6 40P / 670N, 437P / 751R, 143A / 340E / 399M / 640P / 779R / 782G, 38R / 143A / 399M / 437P / 640P / 670N / 751R, 340L / 437P / 782V, 640P / 751R / 856I or 143A / 640P / 782V, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0116] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains substitutions at amino acid positions as described in Table 5.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0117] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution as described in Table 5.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to a reference sequence corresponding to SEQ ID NO: 4.

[0118] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the amino acid positions described in Table 5.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0119] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions of the RNA polymerase variants as described in Table 5.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to a reference sequence corresponding to SEQ ID NO: 4.

[0120] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference sequence comprising the substitutions or substitution sets described in Table 5.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0121] In some embodiments, the engineered RNA polymerase comprises the components corresponding to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 1 The reference sequence of residues 8 to 890 of 14, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138 has an amino acid sequence identity of at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher.

[0122] In some embodiments, the engineered RNA polymerase comprises the components corresponding to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 1 The reference sequence 12, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138 has an amino acid sequence identity of at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher.

[0123] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises residues 8 to 890 of SEQ ID NO. with even numbers from SEQ ID NO: 4-138, or comprises SEQ ID NO. with even numbers from SEQ ID NO: 4-138. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid substitutions. In some embodiments, the amino acid substitutions comprise non-conserved or conserved substitutions. In some embodiments, the amino acid substitutions comprise conserved substitutions.

[0124] In some embodiments, the engineered RNA polymerase contains SEQ ID NO: The amino acid sequence of residues 8 to 890 of 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 10 amino acid substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid substitutions. In some embodiments, the amino acid substitutions comprise non-conserved or conserved substitutions. In some embodiments, the amino acid substitutions comprise conserved substitutions.

[0125] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence comprising SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 10 amino acid substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid substitutions. In some embodiments, the amino acid substitutions comprise non-conserved or conserved substitutions. In some embodiments, the amino acid substitutions comprise conserved substitutions.

[0126] In some embodiments, the engineered RNA polymerase of this disclosure has RNA polymerase activity. In some embodiments, the engineered RNA polymerase has RNA polymerase activity and at least one improved or enhanced property compared to a reference RNA polymerase.

[0127] In some embodiments, the engineered RNA polymerase has increased RNA polymerase activity compared to a reference RNA polymerase. In some embodiments, the engineered RNA polymerase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, 30, 35, 40 times or more of activity compared to a reference RNA polymerase. Exemplary methods for enhancing activity and analyses for measuring such activity are provided in the examples.

[0128] In some implementations, the engineered RNA polymerase exhibits improved stability compared to a reference RNA polymerase.

[0129] In some embodiments, the engineered RNA polymerase exhibits improved thermal stability compared to a reference RNA polymerase. In some embodiments, the engineered RNA polymerase exhibits improved thermal stability at temperatures of 35°C or higher, 40°C or higher, 45°C or higher, or 50°C or higher compared to a reference RNA polymerase. In some embodiments, the engineered RNA polymerase exhibits improved thermal stability over a temperature range of 35°C to 50°C compared to a reference RNA polymerase.

[0130] In some embodiments, the reference RNA polymerase has a sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4, or a sequence corresponding to SEQ ID NO: 2 or 4. In some embodiments, the reference RNA polymerase has a sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO: 2.

[0131] In some embodiments, the engineered RNA polymerase has one or more improved properties selected from the group consisting of: i) increased polymerase activity, ii) increased stability, and iii) increased thermostability, or any combination of i), ii), and iii), compared to a reference RNA polymerase. In some embodiments, the reference RNA polymerase has a sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4, or a sequence corresponding to SEQ ID NO: 2 or 4. In some embodiments, the reference RNA polymerase has a sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO: 2.

[0132] In some embodiments, the engineered RNA polymerase is expressed as a fusion protein. In some embodiments, the engineered RNA polymerase described herein may be fused to a variety of polypeptide sequences (e.g., polypeptide tags, by way of example but not limited to, that can be used for detection and / or purification). In some embodiments, the fusion protein of the engineered RNA polymerase comprises a glycine-histidine or histidine-tag (His-tag). In some embodiments, the engineered RNA polymerase comprises polylysine, such as 2-12 lysine units, for example, to conjugate with a support medium. In some embodiments, the fusion protein of the engineered RNA polymerase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the fusion protein of the engineered RNA polymerase comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, the fusion is fused to the amino (N-) terminus of the engineered RNA polymerase polypeptide. In some embodiments, the fusion is fused to the carboxyl (C-) terminus of the engineered RNA polymerase polypeptide.

[0133] In some embodiments, the engineered RNA polymerase peptide described herein is an isolated composition. In some embodiments, as further discussed herein, the engineered RNA polymerase peptide is purified.

[0134] In some embodiments, this disclosure also provides functional or bioactive fragments of the engineered RNA polymerase polypeptide described herein. Therefore, for each and every embodiment of the engineered RNA polymerase herein, a functional or bioactive fragment of the engineered RNA polymerase is provided herein. In some embodiments, the functional or bioactive fragment of the engineered RNA polymerase comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the activity of the RNA polymerase polypeptide from which it is derived (i.e., the parental RNA polymerase). In some embodiments, the functional or bioactive fragment comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parental sequence of the RNA polymerase. In some implementations, the functional fragments will be truncated to fewer than 5, 10, 15, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino acids.

[0135] In some embodiments, the functional or bioactive fragment of the engineered RNA polymerase polypeptide described herein includes at least a mutation or set of mutations in the amino acid sequence of the parental engineered RNA polymerase described herein. Therefore, in some embodiments, the functional or bioactive fragment of the engineered RNA polymerase exhibits enhancing or improved properties associated with the mutation or set of mutations in the parental RNA polymerase.

[0136] Polynucleotides encoding engineered polypeptides, expression vectors, and host cells In another aspect, this disclosure provides recombinant polynucleotides encoding the engineered RNA polymerase described herein. In some embodiments, the recombinant polynucleotide is operatively linked to one or more heterologous regulatory sequences controlling gene expression to produce a recombinant polynucleotide construct capable of expressing the engineered RNA polymerase. In some embodiments, an expression construct containing at least one heterologous polynucleotide encoding an engineered RNA polymerase polypeptide is introduced into a suitable host cell to express the corresponding RNA polymerase polypeptide.

[0137] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of codons corresponding to a variety of amino acids provide a description of all polynucleotides capable of encoding the subject polypeptide. The degeneracy of the genetic code (where the same amino acid is encoded by substitutional or synonymous codons) allows for the preparation of extremely large quantities of nucleic acids, all of which encode the engineered RNA polymerase of this disclosure. Therefore, this disclosure provides methods and compositions for producing each and every possible variant of the polynucleotide encoding the engineered RNA polymerase polypeptide described herein, which can be prepared by selecting combinations based on possible codon selection, and all such polynucleotide variants should be considered to be specifically disclosed for any polypeptide described herein, including the examples (e.g., Tables 4.1 and 5.1) and the amino acid sequences presented in the sequence listing.

[0138] In some embodiments, the codons are preferably optimized for use by the selected host cell in protein production. In some embodiments, preferred codons in bacteria are used for expression in bacteria. In some embodiments, preferred codons in fungi are used for expression in fungal cells. In some embodiments, preferred codons in insect cells are used for expression in insect cells. In some embodiments, preferred codons in mammalian cells are used for expression in mammalian cells. In some embodiments, the codon-optimized polynucleotide encoding the engineered RNA polymerase polypeptide described herein contains preferred codons at approximately 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full-length coding region.

[0139] Therefore, in some embodiments, the recombinant polynucleotide of this disclosure comprises a polynucleotide sequence encoding the engineered RNA polymerase polypeptide described herein. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon-optimized. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon-optimized for expression in bacterial or fungal cells.

[0140] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 2 or 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4.

[0141] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0142] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the even-numbered sequences of SEQ ID NO: 4-138, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0143] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substituted amino acid sequence at the following amino acid positions: 38, 133, 134, 135, 136, 143, 244, 246, 310, 340, 364, 379, 399, 416, 437, 517, 607, 640, 664, 670, 720, 751, 779, 782, 793, 856, or 876, or combinations thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0144] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substituted amino acid sequence at the following amino acid positions: 38, 143, 664, or 793 or combinations thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0145] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence containing at least one set of substitutions at the following amino acid positions: 38 / 143 / 664 / 793, 38 / 340 / 364 / 640 / 664 / 782 / 793, 38 / 143 / 246 / 340 / 399 / 640 / 782 / 793, 38 / 379 / 437 / 664 / 779 38 / 379 / 517 / 664, 664 / 782 / 793, 38 / 143 / 340 / 640 / 664, 38 / 607 / 664 / 720, 38 / 246 / 399 / 640 / 782 / 856, 340 / 664 / 751 / 793 / 856, 143 / 340 / 437 / 664 / 779, 340 / 664 / 751 / 856, 38 / 437, 143 / 517 / 664, 38 / 664, 38 / 416 / 664, 143 / 246 / 793, 399 / 640, 640 / 782 / 793, 38 / 310 / 437, 246 / 416 / 664, 246 / 793, 38 / 310 / 379, 38 / 246 / 340 / 437, 38 / 340 / 664, 664 / 720, 38 / 143 / 340 / 640, 664, 517 / 664 / 720, 38 / 340 / 399 / 416 / 664 / 751 / 793, 246 / 437, 38 / 244 / 340 / 379 / 437 / 664 / 720 / 779, 133 / 134 / 135 / 136 / 246 / 340 / 379 / 437 / 517 / 607 / 664 / 720, 38 / 782, 38 / 379, 38 / 244 / 246 / 310 / 379, 38 / 340 / 720 or 38 / 379 / 437 / 517, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0146] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence containing at least one set of substitutions at the following amino acid positions: 340 / 437 / 640 / 664 / 670 / 751 / 793 / 856, 38 / 143 / 640 / 664 / 670 / 793 / 856, 38 / 143 / 399 / 640 / 664 / 793, 38 / 143 / 399 / 640 / 664 / 670 / 782 / 793 / 856, 38 / 340 / 399 / 640 / 664 / 782 / 793, 38 / 143 / 437 / 664 / 793, 38 / 143 / 437 / 664 / 793, 38 / 340 / 437 / 664 / 751 / 782 / 793 / 856 / 876, 38 / 143 / 399 / 640 / 664 / 670 / 782 / 793, 38 / 143 / 437 / 664 / 670 / 779 / 782 / 793 / 856, 38 / 143 / 437 / 664 / 793, 38 / 143 / 437 / 664 / 782 / 793, 38 / 143 / 399 / 640 / 6 64 / 670 / 782 / 793, 143 / 437 / 664 / 793, 38 / 143 / 437 / 664 / 779 / 782 / 793, 38 / 399 / 437 / 664 / 751 / 782 / 793, 38 / 143 / 399 / 437 / 640 / 664 / 670 / 751 / 779 / 782 / 793 / 856, 38 / 143 / 437 / 664 / 793, 340 / 664 / 670 / 793 / 856, 143 / 437 / 640 / 664 / 779 / 793, 38 / 143 / 437 / 664 / 751 / 793, 143 / 399 / 437 / 640 / 664 / 670 / 793, 38 / 143 / 437 / 664 / 751 / 793, 38 / 340 / 399 / 640 / 664 / 779 / 782 / 793, 399 / 437 / 640 / 664 / 670 / 751 / 793, 38 / 143 / 340 / 437 / 664 / 782 / 793, 38 / 143 / 640 / 664 / 751 / 793 / 856 or 38 / 640 / 664 / 782 / 793, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0147] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an RNA polymerase comprising at least one substituted amino acid sequence at an amino acid position as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0148] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an RNA polymerase comprising at least one substituted amino acid sequence as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0149] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at an amino acid position as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0150] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an RNA polymerase comprising at least one substituted or substituent set of amino acid sequences of RNA polymerase variants as described in Tables 4.1 and 5.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0151] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference sequence comprising the substitutions or sets of substitutions as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO:2 or relative to the reference sequence corresponding to SEQ ID NO:2.

[0152] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4.

[0153] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the sequence corresponding to the even-numbered SEQ ID NOs of ...

[0154] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0155] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the even-numbered SEQ ID NOs of ...

[0156] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substituted amino acid at the following amino acid positions: 38, 133, 134, 135, 136, 143, 244, 246, 310, 340, 364, 379, 399, 416, 437, 517, 607, 640, 664, 670, 720, 751, 779, 782, 793, 856, or 876, or combinations thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to a reference sequence corresponding to SEQ ID NO: 4.

[0157] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence containing at least one substituted amino acid at the following amino acid positions: 38 / 143 / 340 / 437 / 640 / 670 / 751 / 856, 640 / 670 / 856, 399 / 640, 399 / 640 / 670 / 782 / 856, 143 / 340 / 399 / 640 / 782, 437, 143 / 340 / 437 / 751 / 782 / 856 / 876, 399 / 640 / 670 / 782, 437 / 670 / 779 / 782 / 856, 437 / 7 82, 38 / 437, 437 / 779 / 782, 143 / 399 / 437 / 751 / 782, 399 / 437 / 640 / 670 / 751 / 779 / 782 / 856, 38 / 143 / 340 / 670 / 856, 38 / 437 / 640 / 779, 437 / 751, 38 / 399 / 437 / 640 / 670, 143 / 340 / 399 / 640 / 779 / 782, 38 / 143 / 399 / 437 / 640 / 670 / 751, 340 / 437 / 782, 640 / 751 / 856, 143 / 640 / 782, wherein the amino acid position is relative to the position corresponding to SEQ. Reference sequence of residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0158] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising at least one substituted amino acid sequence at an amino acid position as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4, or relative to a reference sequence corresponding to SEQ ID NO: 2 or 4.

[0159] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising at least one substituted amino acid sequence as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4, or relative to a reference sequence corresponding to SEQ ID NO: 2 or 4.

[0160] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase containing at least one substitution or set of substitutions at an amino acid position as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4, or relative to a reference sequence corresponding to SEQ ID NO: 2 or 4.

[0161] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising at least one substitution or set of substitutions of engineered RNA polymerase variants as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4 or relative to a reference sequence corresponding to SEQ ID NO: 2 or 4.

[0162] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference sequence comprising the substitutions or sets of substitutions as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

[0163] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising residues 8 to 890 of SEQ ID NO. containing even numbers from SEQ ID NO: 4-138 or an amino acid sequence containing even numbers from SEQ ID NO: 4-138.

[0164] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising SEQ ID NO: The amino acid sequence of residues 8 to 890 of 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138.

[0165] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising SEQ ID NO: Amino acid sequences of 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 1334, 136, or 138.

[0166] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference polynucleotide sequence corresponding to odd-numbered SEQ ID NOs of SEQ ID NOs: 3-137, wherein the recombinant polynucleotide encodes an RNA polymerase.

[0167] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference polynucleotide sequence corresponding to nucleotide residues 21 to 2670: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, or 137, where the recombinant polynucleotide encodes RNA polymerase.

[0168] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference polynucleotide sequence corresponding to the following: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, or 137, where the recombinant polynucleotide encodes RNA polymerase.

[0169] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence containing nucleotide residues 21 to 2670: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135 or 137.

[0170] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising the following: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, or 137.

[0171] In some embodiments, the recombinant polynucleotide encoding any of the RNA polymerases described herein is manipulated in a variety of ways to promote the expression of the RNA polymerase polypeptide. Therefore, in some embodiments, this disclosure provides an expression vector comprising a recombinant polynucleotide encoding the engineered RNA polymerase described herein. In some embodiments, the expression vector comprises one or more control sequences operatively linked to the recombinant polynucleotide to regulate the expression of the RNA polymerase encoding the polynucleotide and / or the expression of the corresponding polypeptide. In some embodiments, the control sequences particularly include a leader sequence, a polyadenylated sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. In some embodiments, the choice of control sequences depends on the host type to which the expression vector is to be introduced.

[0172] In some embodiments, suitable promoters are selected based on the host cell. For bacterial host cells, suitable promoters for directing the transcription of the nucleic acid constructs of this disclosure include, but are not limited to, promoters derived from the *Escherichia coli* lac operon, the *Streptomyces agarase* gene (dagA), the *Bacillus subtilis* fructan sucrase gene (sacB), the *Bacillus licheniformis* α-amylase gene (amyL), the *Bacillus thermophilus* maltose amylase gene (amyM), the *Bacillus amyloliquefaciens* α-amylase gene (amyQ), the *Bacillus licheniformis* penicillinase gene (penP), the *Bacillus subtilis* xylA and xylB genes, and prokaryotic β-lactamase genes (see, for example, Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA, 1978, 75:3727-3731), and the tac promoter (see, for example, DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80:21-25). Exemplary promoters for filamentous fungal host cells include, but are not limited to, promoters derived from the genes of Aspergillus oryzae TAKA amylase, Rhizopus oryzae aspartic protease, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus avocado glucosylase (glaA), Rhizopus oryzae lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamipridase, and Fusarium oxysporum trypsin-like protease (see, for example, WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters of the Aspergillus niger neutral α-amylase and Aspergillus oryzae triose phosphate isomerase genes), and their mutant, truncated, and hybrid promoters. Exemplary yeast cell promoters may be derived from the genes of *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Other useful promoters for yeast host cells are known in the art (see, for example, Romanos et al., Yeast, 1992, 8:423-488). Exemplary promoters for insect cells include, but are not limited to, polyhedrosis proteins, p10, ELT, OpIE2, and hr5 / ie1 promoters. Exemplary promoters for mammalian cells include, but are not limited to, promoters derived from cytomegalovirus (CMV), chicken β-actin promoters fused with CMV enhancers, simian virus 40 (SV40), and promoters derived from... Homo sapiens Phosphoglycerate kinase, β-actin, elongation factor-1a, or glyceraldehyde-3-phosphate dehydrogenase or derived from Gallus The promoter of β-actin.

[0173] In some embodiments, the control sequence is a suitable transcription terminator sequence (i.e., a sequence recognized by the host cell to terminate transcription). In some embodiments, the terminator sequence is operatively linked to the 3' end of a nucleic acid sequence encoding an RNA polymerase polypeptide. Any suitable terminator that is functional in the selected host cell can be used in this invention. For bacterial expression, the transcription terminator can be a Rho-dependent terminator that depends on the Rho transcription factor, or a Rho-independent or intrinsic terminator that does not require the transcription factor. Exemplary bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412(5):793-813. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucosylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes of Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, for example, Romanos et al., ibid.). Exemplary terminators for insect and mammalian cells include, but are not limited to, terminators derived from cytomegalovirus (CMV), simian virus 40 (SV40), human growth hormone hGH, bovine growth hormone BGH, and human or rabbit β-globulin.

[0174] In some embodiments, the control sequence is a suitable leader sequence, i.e., an untranslated region of mRNA important for translation in the host cell. In some embodiments, the leader sequence is operatively linked to the 5' end of a nucleic acid sequence encoding an RNA polymerase polypeptide. Any suitable leader sequence that is functional in the selected host cell can be used in this disclosure. Exemplary leader sequences for filamentous fungal host cells are obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leader sequences for yeast host cells are obtained from the genes of Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leader sequences for mammalian host cells include, but are not limited to, those present in orthopoxvirus mRNA. element.

[0175] In some embodiments, the control sequence is a polyadenylated sequence (i.e., a sequence operatively linked to the 3' end of a nucleic acid sequence and recognized by the host cell during transcription as a signal to add polyadenylated residues to the transcribed mRNA). Any suitable polyadenylated sequence functional in a selected host cell can be used in this invention. Exemplary polyadenylated sequences for filamentous fungal host cells include, but are not limited to, genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucosylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase. Useful polyadenylated sequences for yeast host cells are known (see, for example, Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylated and 3' UTR sequences for mammalian host cells include, but are not limited to, the 3'-UTR of α- and β-globin mRNAs, which contain several sequence elements that increase mRNA stability and translation.

[0176] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region encoding an amino acid sequence linked to the amino terminus of a polypeptide and directing the encoded polypeptide into the cellular secretion pathway). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in the translation reading frame to a segment encoding the coding region of the secretory polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is exogenous to the coding sequence. Any suitable signal peptide coding region directing the expressed polypeptide into the secretion pathway of a selected host cell can be used to express the engineered polypeptide. Effective signal peptide coding regions of bacterial host cells are signal peptide coding regions, including but not limited to those obtained from the genes of Bacillus NClB 11837 maltose amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilis protease, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Other signal peptides are known in the art (see, for example, Simonen and Palva, Microbiol. Rev., 1993, 57:109-137). In some embodiments, the effective signal peptide coding regions of filamentous fungal host cells include, but are not limited to, signal peptide coding regions derived from the genes of *Aspergillus oryzae* TAKA amylase, *Aspergillus niger* neutral amylase, *Aspergillus niger* glucosylamylase, *Rhizopus oryzae* aspartic protease, *Polytrichum spp.* cellulase, and *Polytrichum spp.* lipase. Useful signal peptides of yeast host cells include, but are not limited to, those derived from the genes of *Saccharomyces cerevisiae* α-factor and *Saccharomyces cerevisiae* invertase. Useful signal peptides of mammalian host cells include, but are not limited to, signal peptides derived from the genes of immunoglobulin γ (IgG) or other human secreted proteins (e.g., human β-galactosidase polypeptide).

[0177] In some embodiments, the control sequence is a propeptide coding region encoding an amino acid sequence located at the N-terminus of the polypeptide. The resulting polypeptide is referred to as a "proenzyme," "propeptide," or "zymogen." The propeptide can be converted from a propeptide into a mature, active polypeptide by catalytic or autocatalytic cleavage of the propeptide. The propeptide coding region can be obtained from any suitable source, including but not limited to the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizopus oryzae aspartic protease, and Myceliophthora thermophila lactase (see, for example, WO95 / 33836). When both the signal peptide and the propeptide region are present at the N-terminus of the polypeptide, the propeptide region is located near the N-terminus of the polypeptide, and the signal peptide region is located near the N-terminus of the propeptide region.

[0178] In some implementations, the control sequences include one or more regulatory sequences that facilitate the regulation of polynucleotide and / or corresponding encoded polypeptide expression in response to the growth status of the host cell. Examples of regulatory systems are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac, and trp operon systems. In yeast host cells, suitable regulatory systems include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA α-amylase promoter, the Aspergillus niger glucosylase promoter, and the Aspergillus oryzae glucosylase promoter. Exemplary inducible promoters regulated by exogenous factors include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex) inducible promoter, the mouse mammary tumor virus (MMTV) promoter, the ecdysone insect promoter, the tetracycline inducible promoter system, the RU486 inducible promoter system, and the rapamycin inducible promoter system.

[0179] Recombinant expression vectors can be any suitable vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA procedures and elicit expression by engineered RNA polymerases. Vector selection is typically based on compatibility between the vector and the host cell into which it is to be introduced. Vectors can be linear or closed circular plasmids.

[0180] In some embodiments, the expression vector is a self-replicating vector (i.e., a vector existing as an extrachromosomal entity whose replication does not depend on chromosome replication, such as plasmids, extrachromosomal elements, mini-chromosomes, or artificial chromosomes). The vector may contain any means to ensure self-replication, such as a replication origin. In some alternative embodiments, the vector is one that, upon introduction into a host cell, is integrated into the genome and replicates along with the chromosome into which it is already integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids, and / or transposons, containing the total DNA to be introduced into the host cell's genome, are used.

[0181] In some embodiments, the recombinant polynucleotide may be provided on a non-replicating expression vector or plasmid. In some embodiments, the non-replicating expression vector or plasmid may be based on a replication-defective viral vector (see, for example, Travieso et al., npj Vaccines, 2022, Vol. 7, Item 75).

[0182] In some implementations, the expression vector contains one or more selectable markers that allow for easy selection of transformed cells. A “selective marker” is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, prototrophic to auxotrophic traits, etc. Examples of bacterial selective markers include, but are not limited to, those derived from… Bacillus subtilis or Ground Bacillus licheniformis of Dal Genes, or markers that confer resistance to antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for filamentous fungal host cells include, but are not limited to, amdS (acetamidinase; e.g., from Aspergillus nidulans or Aspergillus oryzae), argB (ornithine carbamoyltransferase), bar (phosphinicotinic acetyltransferase; e.g., from Streptomyces hygromycin), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotate nucleoside-5'-phosphate decarboxylase; e.g., from Aspergillus nidulans or Aspergillus oryzae), sC (adenosyl sulfate transferase), and trpC (o-aminobenzoic acid synthase) and their equivalents. Optional markers for mammalian cells include, but are not limited to, chloramphenicol acetyltransferase (CAT), nosemynin N-acetyltransferase, blastcin-S deaminase, blastcin-S acetyltransferase, Shble (Zeocin® resistance), aminoglycoside 3'-phosphotransferase (neomycin resistance), hph (hygromycin resistance), thymidine kinase, and puromycin N-acetyltransferase.

[0183] In another aspect, this disclosure provides a host cell comprising a polynucleotide encoding at least one of the engineered RNA polymerase polypeptides of this disclosure, said polynucleotide being operatively linked to one or more control sequences for expression of the engineered RNA polymerase polypeptide in the host cell. In some embodiments, the host cell comprises an expression vector comprising a polynucleotide encoding the engineered RNA polymerase polypeptide described herein, said polynucleotide being operatively linked to one or more control sequences. Host cells suitable for expressing polypeptides encoded by expression vectors of this disclosure are known in the art and include, but are not limited to, bacterial cells such as *Escherichia coli*, *Bacillus subtilis*, *Vibrio fluvialis*, *Streptomyces*, and *Salmonella typhimurium* cells; fungal cells such as yeast cells (e.g., *Saccharomyces cerevisiae* or *Pichia pastoris* (ATCC Registry No. 201178)); insect cells such as *Drosophila S2* and *Noctuidae Sf9* cells; animal cells such as *CHO*, *COS*, *BHK*, 293, and *Bos melanoma* cells; and plant cells. Exemplary host cells also include various *Escherichia coli* strains (e.g., W3110 (ΔfhuA) and BL21).

[0184] In another aspect, this disclosure provides a method for generating an engineered RNA polymerase polypeptide, wherein the method comprises culturing a host cell capable of expressing a polynucleotide encoding the engineered RNA polymerase polypeptide under suitable conditions, thereby expressing or generating an engineered RNA polymerase. In some embodiments, the method further comprises isolating the expressed RNA polymerase from the culture and / or cells. In some embodiments, the method further comprises purifying the expressed RNA polymerase polypeptide.

[0185] Suitable culture media and growth conditions for host cells are known in the art. Any suitable method intended for introducing polynucleotides for expressing RNA polymerase peptides into cells may be used in this invention. Suitable techniques include, but are not limited to, electroporation, bioprojectile particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0186] In some embodiments, recombinant peptides (e.g., engineered RNA polymerase peptides) may be generated using any suitable method known in the art. For example, a variety of different mutagenesis techniques may be utilized by those skilled in the art. Furthermore, mutagenesis kits are also available from many commercial molecular biology vendors. Methods may be used for specific substitutions at defined amino acid sites (site-directed mutagenesis), specific or random mutations in local regions of a gene (region-specific mutagenesis), or random mutagenesis across the entire gene (e.g., saturation mutagenesis). Many methods for generating peptide variants are known to those skilled in the art, including, but not limited to, site-directed mutagenesis of single-stranded or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods for DNA and protein engineering are provided in the following patents: U.S. Patent No. 6,117,679; U.S. Patent No. 6,420,175; U.S. Patent No. 6,376,246; U.S. Patent No. 6,586,182; U.S. Patent No. 7,747,391; U.S. Patent No. 7,747,393; U.S. Patent No. 7,783,428; and U.S. Patent No. 8,383,346. After generating variants, they can be screened for any desired properties (e.g., high or enhanced activity, or low or reduced activity, enhanced thermal activity, enhanced stability, increased substrate range, enhanced fidelity, enhanced salt tolerance and / or pH stability, etc.).

[0187] In some embodiments, engineered RNA polymerase peptides having the properties disclosed herein can be obtained by subjecting a polynucleotide encoding a naturally occurring or engineered RNA polymerase peptide to suitable mutagenesis and / or directed evolution methods known in the art, such as those described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, for example, Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91:10747-10751; WO 95 / 22625; WO97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Patent 6,537,746). Other directed evolution procedures that can be used include staggered extension process (StEP), in vitro recombination (see, for example, Zhao et al., Nat. Biotechnol., 1998, 16:258–261), mutagenesis PCR (see, for example, Caldwell et al., PCR Methods Appl., 1994, 3:S136–S140), and cassette mutagenesis (see, for example, Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525–3529).

[0188] Mutagenesis and directed evolution methods can be applied to polynucleotides encoding RNA polymerases to generate variant libraries that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution methods are available in this disclosure and are known in the art (see, for example, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,88). 3, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406, 910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603, 6,518,065, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,57 6,467, 6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297, 7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477、7,462,469、7,534,564、7,620,500、7,620,502、7,629,170、7,702,464、7,747,391、7,747,393、7,751,986、7,776,598、7,783,428、7,795,030、7,853,410、7,868,138、7,783,428、7,873,477、7,873,499、7,904,249、7,957,912、7,981,614、8 ,014,961, 8,029,988, 8,048,674, 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, 9,665,694, 9,684,771 and all relevant PCT and non-US corresponding patents; Ling et al., Anal. Biochem., 1997, 254(2):157-78; Dale et al., Meth. Mol. Biol., 1996, 57:369-74; Smith, Ann. Rev. Genet., 1985, 19:423-462; Botstein et al., Science, 1985, 229:1193-1201; Carter, Biochem. J., 1986, 237:1-7; Kramer et al., Cell, 1984, 38:879-887; Wells et al., Gene, 1985, 34:315-323; Minshull et al., Curr. Op. Chem. Biol., 1999, 3:284-290; Christians et al., Nat. Biotechnol., 1999, 17:259-264; Crameri et al., Nature, 1998, 391:288-291; Crameri et al., Nat. Biotechnol., 1997, 15:436-438; Zhang et al., Proc. Nat. Acad. Sci. USA, 1997, 94:4504-4509; Crameri et al., Nat. Biotechnol., 1996, 14:315-319; Stemmer, Nature, 1994, 366:389-391; Stemmer,Proc. Nat. Acad. Sci. USA, 1994, 91:10747-10751; EP 3 049 973; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767; WO 2009 / 152336; and WO 2015 / 048573, all of which are incorporated herein by reference.

[0189] In some embodiments, clones obtained after mutagenesis are screened by subjecting the peptide preparation to defined treatment or assay conditions (e.g., buffer, temperature, pH conditions, DNA template, etc.) and measuring peptide activity after treatment or other suitable assay conditions. Clones containing polynucleotides encoding the peptide of interest are then isolated, the polynucleotides are sequenced to identify nucleotide sequence changes (if any), and used for expression of the peptide in host cells. Measurement of peptide activity from the expression library can be performed using any suitable method known in the art and as described in the examples.

[0190] For engineered peptides with known sequences, the polynucleotides encoding the peptides can be prepared using standard solid-phase methods according to known synthetic approaches. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then conjugated (e.g., by enzymatic or chemical ligation, or polymerase-mediated methods) to form any desired continuous sequence (see, for example, Hughes et al., Cold Spring Harb Perspect Biol. Jan 2017; 9(1):a023812). For example, the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classic phosphoramidite method (see, for example, Beaucage et al., Tet. Lett., 1981, 22:1859-69; and Mattes et al., EMBO J., 1984, 3:801-05), as it is commonly practiced in automated synthetic methods. According to the phosphoramidite method, the oligonucleotides are synthesized (e.g., in an automated DNA synthesizer, purified, annealed, ligated, and cloned into a suitable vector).

[0191] In some embodiments, a method for preparing an engineered RNA polymerase polypeptide may include: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence of any variant as described herein, and (b) expressing the RNA polymerase polypeptide encoded by the polynucleotide. In some embodiments of this method, the amino acid sequence encoded by the polynucleotide may optionally have one or more (e.g., up to 3, 4, 5, or up to 10) amino acid residues deleted, inserted, and / or substituted. In some embodiments, the amino acid sequence may optionally have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residues deleted, inserted, and / or substituted. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residues deleted, inserted, and / or substituted. In some embodiments, the substitution is conserved or non-conserved.

[0192] In some embodiments, any one or more known techniques for protein purification are used to recover and / or purify any engineered RNA polymerase peptide expressed in host cells from cells and / or culture media, including in particular lysozyme treatment, sonication, filtration, salting out, selective precipitation, ultracentrifugation, and chromatography.

[0193] Chromatographic techniques used for the separation and purification of RNA polymerase peptides include, in particular, reversed-phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, gel electrophoresis, and affinity chromatography. The conditions used for purifying a specific peptide depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, and molecular shape, and will be apparent to those skilled in the art. In some embodiments, affinity techniques can be used to separate improved RNA polymerase peptides. For affinity chromatography purification, any antibody that specifically binds to the RNA polymerase peptide of interest can be used. To generate antibodies, various host animals (including, but not limited to, rabbits, mice, rats, etc.) can be immunized by injecting the RNA polymerase peptide or a fragment thereof. In some embodiments, the RNA polymerase peptide or fragment is linked to a suitable vector, such as BSA, via a side-chain functional group or a linker connected to the side-chain functional group. If the engineered RNA polymerase includes a fusion peptide (e.g., a His tag) that can be affinity-purified, standard affinity methods for the specific fusion protein can be used.

[0194] Compositions of engineered RNA polymerases In another aspect, this disclosure provides compositions of the RNA polymerases disclosed herein. In some embodiments, the engineered RNA polymerase peptides in the composition are isolated or purified. In some embodiments, the RNA polymerase is combined with other components and compounds to provide compositions and formulations comprising engineered RNA polymerase peptides suitable for various applications and uses.

[0195] In some embodiments, the composition comprises at least one engineered RNA polymerase described herein. For example, the composition comprises at least one engineered RNA polymerase provided in Tables 4.1 and 5.1. In some embodiments, the composition comprising the engineered RNA polymerase is an aqueous solution. In some embodiments, the composition comprising the engineered RNA polymerase is a lyophilized product.

[0196] In some embodiments, the composition further comprises one or more of the following: (i) a buffer solution, (ii) one or more rNTPs, and (iii) Mg +2 (iv) Target DNA substrate / template. In some embodiments, the target DNA substrate is a double-stranded DNA template and includes a promoter recognized by an engineered RNA polymerase. As further described herein, in some embodiments, the promoter sequence is a naturally occurring T7 RNA polymerase promoter, such as a class II or class III promoter. In some embodiments, the RNA polymerase promoter is a synthetic or hybrid T7 RNA polymerase promoter.

[0197] In some embodiments, the composition further comprises a reducing agent, such as dithiothreitol or mercaptoethanol. In some embodiments, the composition further comprises additives, such as molecular crowding agents or promoters of RNA polymerase activity. In some embodiments, molecular crowding agents particularly include bovine serum albumin (BSA), polyethylene glycol, dextran, and Ficoll. In some embodiments, additives that promote RNA polymerase activity are surfactants, particularly including nonionic detergents (e.g., Triton X-100, Tween 20, or NP-40), and polyamines, such as spermidine or spermine.

[0198] In some embodiments, the composition further comprises one or more oligonucleotide primers, which may be sequence-specific primers and / or “random” or “universal” primers that can serve as primers for engineered RNA polymerase extension. In some embodiments, the primers are oligonucleotide primers (i.e., RNA primers).

[0199] In some embodiments, the composition further comprises a "cap" analogue, for example, for co-transcriptional production of capped RNA transcripts. In some embodiments, the cap analogue is a dinucleotide or trinucleotide cap analogue. In some embodiments, the cap analogue is a dinucleotide cap analogue, particularly including α,γ-bis(N... 7 2-methylguanosine triphosphate (m 7 G(5')ppp(5')m 7 G) or anti-reverse cap analogue 3'-O-Me-m 7 G(5')ppp(5')G. In some embodiments, the dinucleotide cap analog is α,γ-bis(N7-methylguanosine) triphosphate. In some embodiments, the cap analog is a trinucleotide cap analog, particularly including m 7 G(5')ppp(5')AmpG (GAG) and m 7 ,3'-O-propyne G(5')ppp(5')AmpG、m 7 GpppApA, m 7 GpppApC, m 7 GpppApG, m 7 GpppApU.m 7 GpppCpA、m 7 GpppCpC, m 7 GpppCpG、m 7 GpppCpU, m 7 GpppGpA, m 7 GpppGpC, m 7 GpppGpG, m 7 GpppGpU, m 7CpppUpA、m 7 GpppUpC, m 7 GpppUpG and m 7 GpppUpU. In some embodiments, the cap analog is a tetranucleotide cap, particularly including (m 7 GpppNmpGmpG) and cap2-1 (m 7 GpppNpGmpG). Dinucleotide and trinucleotide cap analogs are described in particular in Shanmugasundaram et al., ChemRec., 2022, 22(8): e202200005 and PCT patent publication WO20172239, which are incorporated herein by reference. Tetranucleotide caps are described in particular in Drazkowska et al., Nucl Acids Res., 2022, 50(16):9051–9071. In some embodiments, the cap analog is a commercially available cap analog sold under Trilink® or CleanCap®.

[0200] In some implementations, the trinucleotide cap is m 7 G 3’OMe pppApA、m 7 G 3’OMe pppApC, m 7 G 3’OMe pppApG, m 7 G 3’OMe pppApU, m 7 G 3’Ome pppCpA、m 7 G 3’OMe pppCPC, m 7 G 3’OMe pppCpG, m 7 G 3’OMe pppCpU、m 7 G 3’ OMe pppGpA, m 7 G 3’OMe pppGpC, m 7 G 3’OMe pppGpG, m 7 C 3’OMe pppGpU, m 7 G 3’OMe pppUpA、m 7 C 3’OMe pppUpC、m 7 G 3’OMe pppUpG or m 7 G 3’OMepppUpU. In some implementations, the trinucleotide cap is m 7 G 3’OMe pppA 2’OMe pA, m 7 G 3’OMe pppA 2’OMe pC, m 7 G 3’OMe pppA 2’OMe pG, m 7 C 3’OMe pppA 2’OMe pU, m 7 G 3’OMe pppC 2’OMe pA, m 7 Ci 3’ OMe ppppC 2’OMe pC, m 7 C 3’OMe pppC 2’OMe pCi.m 7 G 3’OMe pppC 2’OMe pU, m 7 G 3’OMe pppG 2’OMe pA. m 7 G 3’ OMe ppppG 2’OMe pC, m 7 G 3’OMe pppG 2’OMe pG, m 7 G 3’OMe pppC 2’OMe pU, m 7 G 3’OMe pppU 2’OMe pA, m 7 G 3’OMe pppU 2’ OMe pC, m 7 G 3’OMe pppU 2’OMe pG or m 7 G 3’OMe pppU 2’OMe pU. In some implementations, the trinucleotide cap is m 7 GpppA 2’OMe pA, m 7 GpppA 2’OMe pC, m 7 GpppA 2’OMe pG, m 7 GpppA 2’OMe pU, m7 GpppC 2’OMe pA, m 7 GpppC 2’ OMe pC, m 7 GpppC 2’OMe pG, m 7 CpppC 2’OMe pU, m 7 GpppG 2’OMe pA. m 7 GpppG 2’OMe pC, m 7 CpppG 2’OMe pG, m 7 GpppG 2’OMe pU, m 7 GpppU 2’OMe pA, m 7 GpppU 2’OMe pC, m 7 GpppU 2’OMe pG or m 7 GpppU 2’OMe pU.

[0201] In some embodiments, the composition further comprises a pyrophosphatase and / or an RNase inhibitor. In some embodiments, the pyrophosphatase is an inorganic pyrophosphatase. In some embodiments, the RNase inhibitor is a mammalian RNase inhibitor (e.g., a placental RNase inhibitor or a porcine RNase inhibitor) or a synthetic RNase inhibitor, such as RiboGrip®.

[0202] In some embodiments, the engineered RNA polymerase described herein is provided immobilized on a substrate or supporting medium such as a solid substrate, porous substrate, membrane, or particles. The polypeptide may be encapsulated within the matrix or membrane. In some embodiments, the matrix comprises polymeric materials such as calcium alginate, agar, k-carrageenan, polyacrylamide, and collagen. In some embodiments, solid matrices particularly include activated carbon, porous ceramics, and diatomaceous earth. In some embodiments, the matrix is ​​a particle, membrane, or fiber. Types of membranes particularly include nylon, cellulose, polysulfone, or polyacrylate.

[0203] In some embodiments, RNA polymerase is immobilized on the surface of a support material. In some embodiments, peptides are adsorbed onto the support material. In some embodiments, peptides are immobilized on the support material via covalent attachment. Support materials particularly include inorganic materials such as alumina, silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, polyacrylates, agarose or derivatives thereof (e.g., cross-linked agarose), polyacrylamide, polystyrene, and ion exchange resins such as Amberlite, Sephadex, and Dowex.

[0204] Use of engineered RNA polymerase polypeptides and kits On the other hand, this disclosure provides the use of engineered RNA polymerases for preparing RNA of interest. In some embodiments, the engineered RNA polymerase is used in an in vitro transcription reaction. In some embodiments, the engineered RNA polymerase is used to generate (e.g., but not limited to) mRNA, self-replicating RNA, circular RNA, shRNA, miRNA, and CRISPR guide RNA. In some embodiments, the resulting RNA is mRNA encoding a polypeptide of interest (e.g., a polypeptide vaccine or therapeutic polypeptide).

[0205] In some embodiments, the method of generating RNA includes contacting a target DNA template with an engineered RNA polymerase as described herein under suitable reaction conditions in the presence of one or more nucleoside triphosphates (NTPs) to generate all or part of the RNA transcript of the target DNA template. In some embodiments, the method further includes providing a cap analogue, such as a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap, as described herein.

[0206] In some embodiments, the NTP is an rNTP. In some embodiments, the NTP substrate is a modified nucleotide. In some embodiments, the modified nucleotide is a naturally modified nucleotide, such as the selected modified nucleotide being naturally occurring 5'-methylcytidine and / or pseudouridine. In some embodiments, the modified nucleotide is a 2'-ribose-modified nucleotide, such as 2-O-methyl, 2'-O-ethyl, and 2'-halogenated (e.g., 2'-bromo, 2'-fluorine). In some embodiments, the modified nucleotide is a base-modified nucleotide (e.g., see...). et al., Org. Biomol. Chem., 2018, 16, 5800-5807.

[0207] In some embodiments, the concentration of one or more NTPs (e.g., rNTPs) is 0.5-15 mM, 1-12 mM, 2-10 mM, or 4-8 mM. In some embodiments, the concentration of NTPs is about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 15 mM, or 20 mM.

[0208] In some embodiments, suitable conditions include temperatures from about 10°C to about 60°C. In some embodiments, suitable conditions include temperatures from about 20°C to about 55°C. In some embodiments, suitable conditions include temperatures from about 25°C to about 50°C. In some embodiments, suitable conditions include temperatures from about 30°C to about 40°C. In some embodiments, suitable conditions include temperatures from about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C.

[0209] In some implementations, suitable conditions include buffer solution and / or Mg2+. +2 In some embodiments, suitable conditions include a reducing agent, such as dithiothreitol or mercaptoethanol. In some embodiments, suitable reaction conditions also include additives, such as bovine serum albumin (BSA), glycerol, polyethylene glycol (PEG), dextran, Ficoll, spermidine, or spermine, as described above.

[0210] In some embodiments, the target DNA template is a double-stranded DNA template comprising a promoter sequence recognized by the engineered RNA polymerase described herein. In some embodiments, the promoter sequence is a naturally occurring T7 RNA polymerase promoter, such as a class II or class III promoter (see, for example, Ikeda et al., J Biol. Chem., 1992, 267(4):2640-2649). In some embodiments, the promoter is a heterozygous or synthetic T7 promoter recognized by the engineered RNA polymerase of this disclosure (see, for example, Lieber et al., Eur. J. Biochem. 1993, 217:387-394).

[0211] In some embodiments, if no cap is provided for co-transcriptional capping during the transcription reaction, the method further includes a step of capping the RNA transcript. In some embodiments, RNA capping is performed using a capping enzyme (e.g., a Forstovirus or vaccinia virus capping enzyme).

[0212] In some embodiments, the method further includes a step of poly(A) tailing of the RNA transcript. In some embodiments, the poly(A) tailing step is performed when a polynucleotide template is unavailable for generating a poly(A) tail in RNA transcription. In some embodiments, the poly(A) tailing step uses a poly(A) polymerase that catalyzes the incorporation of an adenine residue into the 3' end of the RNA. In some embodiments, the poly(A) polymerase is a bacterial poly(A) polymerase (e.g., *Escherichia coli*), a yeast poly(A) polymerase (e.g., *Saccharomyces cerevisiae*), or a mammalian poly(A) polymerase.

[0213] In some embodiments, the method also includes the presence of a pyrophosphatase conjugated to an engineered RNA polymerase. Generally, pyrophosphatases are inorganic pyrophosphatases.

[0214] In some embodiments, the method further includes the presence of an RNase inhibitor to inhibit any RNase that may be present in the reaction mixture.

[0215] In some embodiments, the method is used to generate an RNA transcript encoding a polypeptide of interest. In some embodiments, the polypeptide of interest is an enzyme, vaccine antigen, cytokine, growth factor, monoclonal antibody, structural polypeptide or protein, or ligand or receptor polypeptide or protein.

[0216] In some embodiments, the DNA template and the corresponding RNA transcripts produced encode a vaccine antigen or a therapeutic peptide. In some embodiments, the DNA template and the produced RNA transcripts encode a vaccine antigen to stimulate an immune response to that antigen, such as treating a disease condition or stimulating immunity against an infectious pathogen.

[0217] In some implementations, the DNA template and corresponding RNA transcripts encode a vaccine antigen of a microbial or viral polypeptide. Exemplary microbial polypeptides include, in particular, streptococcal proteins (e.g., group A or group B antigens) and Yersinia pestis proteins (e.g., F1 protein). Exemplary viral peptides include, in particular, peptides from: human immunodeficiency virus (HIV), hepatitis viruses (e.g., HAV, HBV, HCV, etc.), herpes simplex virus (e.g., HSV-1, HSV-2, etc.), herpes zoster virus, human papillomavirus, respiratory syncytial virus (RSV), coronaviruses (e.g., SARS-CoV-2), measles virus, poxviruses (e.g., smallpox, monkeypox, etc.), rhabdoviruses (e.g., rabies), influenza viruses (e.g., H1N1, H10N8, H7N9, etc.), human metapneumovirus (HMPV) and parainfluenza virus type 3 (PIV3), human cytomegalovirus, Zika virus, Epstein-Barr virus (EBV), etc.

[0218] In some embodiments, the DNA template and corresponding RNA transcripts encode cancer or tumor antigens. Exemplary cancer antigens include, in particular, mRNA vaccines encoding tumor-associated antigens (TAAs), such as overexpressed antigens EGFR, HER2, and cancer testis antigen (CTA); differentiation antigens such as PSA and gp100; carcinoembryonic antigens, such as 5T4 and CEA; and tumor viral antigens, such as HPV E6 and E7 oncogenes. In some embodiments, the cancer or tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA) or a neoantigen derived from a somatic mutation. Exemplary TAAs, TSAs, and neoantigens include, in particular, NY-ESO-1, MAGE-C3, tyrosinase, TPTE, RBL038, RBL039, RBL-040, RBL-041, RBL-045, PSA, PSCA, PSMA, STEAP1, PAP, and MUC1.

[0219] In some embodiments, the DNA template and the corresponding RNA transcript encode a therapeutic peptide. Exemplary therapeutic peptides include, in particular, enzymes (e.g., enzymes associated with disease conditions), cytokines, structural peptides or proteins, and ligand or receptor peptides or proteins.

[0220] Exemplary cytokines include, in particular, IL-1β, IL-6, TNFα, IL-12, IL-15, GM-CSF, IFNα, and GM-CSF. Exemplary ligand proteins or peptides include, in particular, OX40 ligand (OX40L), 4-1BB ligand (4-1BBL), glucocorticoid-induced tumor necrosis factor receptor (GITR) ligand (GITRL), CD40 ligand (CD40L), inducible T cell co-stimulatory ligand (ICOSL), CD70, and caTLR4.

[0221] Exemplary therapeutic peptides include, in particular, glucose-6-phosphatase (glycogen storage defect type 1A); phosphoenolpyruvate carboxylkinase (Pepck deficiency); galactose-1-phosphate uridine transferase (galactosemia); phenylalanine hydroxylase (phenylketonuria); branched-chain α-ketoacid dehydrogenase (maple syrup urine disease); fumarate acetoacetate hydrolase (tyrosinemia type 1); methylmalonyl-CoA mutase (methylmalonic acidemia); medium-chain acyl-CoA dehydrogenase (medium-chain acetyl-CoA deficiency); and ornithine transaminase. Formicylase (ornithine transcarbamate deficiency); argininosuccinate synthase (citrullinemia); low-density lipoprotein receptor protein (familial hypercholesterolemia); UDP-glucuronyltransferase (Kriegler-Najjar disease); adenosine deaminase (severe combined immunodeficiency); hypoxanthine-guanine phosphoribosyltransferase (gout and Lesh-Nian syndrome); biotinase (biotinase deficiency); β-glucocerebrosidase (Gaucher disease); β-glucuronidase (Slay syndrome); peroxisome membrane protein 70 kDa (Zelvig syndrome); bile pigmentogen deaminase (acute intermittent porphyria); α-1 antitrypsin (α-1 antitrypsin deficiency; emphysema); factor VIII (hemophilia A); and erythropoietin (thalassemia).

[0222] In some embodiments, the method is used to generate non-coding RNA (ncRNA), such as shRNA, siRNA, miRNA, iRNA, etc. In some embodiments, for example, but not limited to, the non-coding RNA molecule is miRNA, which regulates gene expression by targeting the cleavage / degradation of RNA transcripts or the translational repression of target messenger RNA (mRNA). In some embodiments, the non-coding RNA is shRNA or siRNA. shRNA or siRNA can target any target RNA (see, for example, Zhang et al., Biochemical Pharmacology, 2021, 189:114432; and Schaefer et al., 2018, PLOS ONE13(1): e0191570).

[0223] In some embodiments, the DNA template and the expressed RNA are one or more components of the gene editing system or recombination system. In some embodiments, the DNA template and the corresponding RNA encode CRISPR-associated protein 9 (Cas9) or express guide RNA (gRNA). In some embodiments, the DNA template and the corresponding RNA encode zinc finger nucleases (ZFNs) or transcription factor-like effector nucleases (TALENs). In some embodiments, the transgene contains a polynucleotide encoding a recombinase (e.g., Cre recombinase or flp recombinase).

[0224] In some embodiments, engineered RNA polymerases are used to produce RNA for use as probes. In some embodiments, labeled nucleoside triphosphate substrates or nucleotide analogs may be used in the RNA polymerase reaction to incorporate the labeled nucleotides or nucleotide analogs into the synthesized RNA.

[0225] In another aspect, this disclosure provides a kit comprising an engineered RNA polymerase or a combination thereof as described herein. In some embodiments, the kit further comprises at least one buffer. In some embodiments, the buffer comprises a reducing agent, such as dithiothreitol or mercaptoethanol. In some embodiments, the kit further comprises a DNA template.

[0226] In some embodiments, the kit further includes additives such as one or more of glycerol, polyethylene glycol (e.g., PEG 6000 and PEG 8000), dextran, bovine serum albumin (BSA), Ficoll, spermidine or spermine, and magnesium acetate or magnesium chloride. In some embodiments, the kit also contains one or more NTPs, particularly rNTPs. In some embodiments, the engineered RNA polymerase in the kit is provided in lyophilized or solution form.

[0227] Example The following embodiments, including experiments and results, are provided for illustrative purposes only and should not be construed as limiting the invention.

[0228] Example 1 E. coli expression host containing recombinant RNA polymerase (RNAP) gene The initial RNA polymerase used to generate variants of the present invention is SEQ ID NO: 2 cloned into the expression vector pCK110900 (see Figure 3 of U.S. Patent Publication 2006 / 0195947), which is operatively linked to the lac promoter under the control of the lac1 repressor. The expression vector also contains the P15a origin of replication and a chloramphenicol resistance gene. The resulting plasmid was transformed into *E. coli* W3110 using standard methods known in the art. Transformants are isolated by subjecting cells to chloramphenicol selection, as is known in the art (see, for example, U.S. Patent Nos. 8,383,346 and WO2010 / 144103).

[0229] Example 2 Preparation of wet cell pellet containing HTP RNA polymerase (RNAP) *E. coli* cells containing a recombinant RNAP-encoding gene from a monoclonal colony were seeded into 180 µl of Luria broth (LB) containing 1% glucose and 30 µg / mL chloramphenicol (CAM) in the wells of a 96-well shallow-well microtiter plate. The plate was sealed with an O2-permeable seal and the cultures were incubated overnight at 30°C, 200 rpm, and 85% humidity. Then, 10 µl of each cell culture was transferred to the wells of a 96-well deep-well plate containing 390 mL of Terrific Broth (TB) and 30 µg / mL CAM. The deep-well plate was sealed with an O2-permeable seal and incubated at 30°C, 250 rpm, and 85% humidity until the OD600 reached 0.6–0.8. The cell cultures were then induced with isopropyl galactothioglycoside (IPTG) to a final concentration of 1 mM and incubated overnight under the same conditions as initially used. Then centrifuge at 4,000 rpm for 10 minutes to pellet the cells. Discard the supernatant and freeze the pellet at -80°C before lysis.

[0230] Example 3 Preparation of HTP-purified RNA polymerase (RNAP) First, 400 µL of lysis buffer (containing 50 mM sodium phosphate pH 7.5, 2 mM magnesium sulfate, 0.1% (v / v) Tween-20, 0.2 g / L lysozyme, 0.5 g / L p-mercurybenzenesulfonic acid (PMBS), and 0.5 mM dithiothreitol (DTT)) was added to the cell paste in each well, as described in Example 2. The cells were resuspended and lysed by shaking on a benchtop shaker at room temperature for 2 hours. Then, 40 µL of 5 M NaCl and 5 µL of 0.8 M imidazole were added to the lysed cells, and the cells were centrifuged at 4,000 rpm for 10 min at 4°C. Load the supernatant onto a HisPur™ Ni-NTA rotating plate (Thermo Fisher) and wash twice with 600 µL of wash buffer containing 50 mM sodium phosphate pH 7.5, 300 mM sodium chloride, 0.1% (v / v) Tween-20, 10 mM imidazole, and 0.5 mM DTT. Elute twice with 90 µL of elution buffer containing 50 mM sodium phosphate pH 7.5, 300 mM sodium chloride, 0.1% (v / v) Tween-20, 250 mM imidazole, and 0.5 mM DTT. Then, following the manufacturer's instructions, the HisPur™ plate eluent was replaced with a 2X storage buffer containing 100 Tris-HCl (pH 8.0), 200 mM sodium chloride, 0.2% (v / v) Triton X-100, 2 mM EDTA (pH 8.0), and 10 mM DTT using a Zeba™ rotary desalting plate (40 kDa MWCO) (Thermo Fisher). 70 µL of the Zeba™ plate eluent was mixed with an equal volume of pure glycerol and stored at -20°C before activity assays were performed.

[0231] Example 4 Improved thermostability of RNA polymerase compared to SEQ ID NO: 2 After screening for wild-type RNA polymerase activity in an in vitro transcription (IVT) assay, the RNA polymerase of SEQ ID NO: 2 was selected as the parental enzyme. Libraries of engineered genes were generated using established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were generated in HTP as described in Example 2 and purified as described in Example 3. Each variant was diluted 4-fold with RNase-free water, pre-incubated at 42°C for 30 min, and screened in 1 µL of a reaction containing 0.05 µg / µL 6 kb DNA template, 30 mM Tris-HCl pH 8.0, 26.9 mM magnesium chloride, 3 mM DTT, 6 mM ATP, 6 mM GTP, 6 mM CTP, 6 mM UTP, 1 U / µL RNase inhibitor (New England Biolabs, catalog number M0314L), and 0.002 U / µL IPPase (New England Biolabs, catalog number M2403L). The reaction mixture was incubated in a Bio-Rad Hard-Shell® 96-well PCR plate (Bio-Rad) at 37°C for 2 h and quenched with 125 nL 0.5 M EDTA. The quenched mixture was diluted 100-fold with RNase-free water, and RNA concentration was measured using the Quant-iT™ RNA Assay Kit (Limited Version) (Thermo Fisher, Catalog No. Q10213). Activity relative to SEQ ID NO: 2 (active FIOP) was calculated as the ratio of RNA produced by the variant to RNA produced by SEQ ID NO: 2, which was much less than 1 and therefore rounded up to 1. The results are shown in Table 4.1.

[0232] Example 5 Improved thermostability of RNA polymerase compared to SEQ ID NO: 4 After screening for wild-type RNA polymerase activity in an in vitro transcription (IVT) assay, the RNA polymerase of SEQ ID NO: 4 was selected as the parental enzyme. Libraries of engineered genes were generated using established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were generated in HTP as described in Example 2 and purified as described in Example 3. Each variant was diluted 4-fold with RNase-free water, pre-incubated at 47°C for 30 min, and screened in 1 µL of a reaction containing 0.05 µg / µL 6 kb DNA template, 30 mM Tris-HCl pH 8.0, 26.9 mM magnesium chloride, 3 mM DTT, 6 mM ATP, 6 mM GTP, 6 mM CTP, 6 mM UTP, 1 U / µL RNase inhibitor (New England Biolabs, catalog number M0314L), and 0.002 U / µL IPPase (New England Biolabs, catalog number M2403L). The reaction mixture was incubated in a Bio-Rad Hard-Shell® 96-well PCR plate (Bio-Rad) at 37°C for 2 h and quenched with 125 nL 0.5 M EDTA.

[0233] The quenched mixture was diluted 100-fold with RNase-free water, and RNA concentration was measured using the Quant-iT™ RNA Assay Kit (Limited Version) (Thermo Fisher, Catalog No. Q10213). Activity relative to SEQ ID NO: 4 (active FIOP) was calculated as the ratio of RNA produced by the variant to RNA produced by SEQ ID NO: 4, which was much less than 1 and therefore rounded up to 1. The results are shown in Table 5.1.

[0234] Although the invention has been described with reference to specific embodiments, various changes and equivalents may be made to suit particular circumstances, materials, composition of substances, processes, one or more process steps, thereby achieving the benefits of the invention without departing from the scope of the claims.

[0235] For all purposes, every publication and patent document cited in this disclosure is incorporated herein by reference as if each such publication or document were specifically and individually indicated to be incorporated herein by reference. The citation of publications and patent documents is not intended to indicate that any such document is relevant prior art, nor does it constitute an admission of its content or date.

Claims

1. An engineered RNA polymerase or a functional fragment thereof, said engineered RNA polymerase or functional fragment comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of said SEQ ID NO: 2 or 4, wherein said amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of said SEQ ID NO: 2 or 4.

2. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or with respect to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions.

3. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions.

4. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the even-numbered SEQ ID NO: 4-138, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

5. The engineered RNA polymerase according to any one of claims 1-4, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution at the following amino acid positions: 38, 133, 134, 135, 136, 143, 244, 246, 310, 340, 364, 379, 399, 416, 437, 517, 607, 640, 664, 670, 720, 751, 779, 782, 793, 856, or 876 or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

6. The engineered RNA polymerase according to any one of claims 1-5, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or amino acid residue 38G, 133Q, 134H, 135R, 136E, 143V, 244Y, 246A, 310A, 340E / L, 364H, 379S, 399M, 416V, 437P, 517R / Y, 607Y, 640P, 664K / R, 670N, 720E / Q / R, 751R, 779R, 782G / V, 793L, 856I, or 876K or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

7. The engineered RNA polymerase according to any one of claims 1-4, wherein the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 38, 143, 664 or 793 or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

8. The engineered RNA polymerase according to any one of claims 1-4 and 7, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substituted or amino acid residue 38G, 143V, 664K or 793L or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

9. The engineered RNA polymerase according to any one of claims 1-4 and 7, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution of R38G, A143V, W664K or Q793L or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

10. The engineered RNA polymerase according to any one of claims 1-4, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions at the following amino acid positions: 38 / 143 / 664 / 793, 38 / 340 / 364 / 640 / 664 / 782 / 793, 38 / 143 / 246 / 340 / 399 / 640 / 782 / 793, 38 / 379 / 437 / 664 / 779, 38 / 379 / 517 / 664, 664 / 782 / 793, 38 / 143 / 340 / 640 / 664, 38 / 607 / 664 / 720, 38 / 246 / 399 / 640 / 782 / 856, 340 / 664 / 751 / 793 / 856, 143 / 340 / 437 / 664 / 779, 340 / 664 / 751 / 856, 38 / 437, 143 / 517 / 664, 38 / 664, 38 / 416 / 6 64, 143 / 246 / 793, 399 / 640, 640 / 782 / 793, 38 / 310 / 437, 246 / 416 / 664, 246 / 793, 38 / 310 / 379, 38 / 246 / 340 / 437, 38 / 340 / 664, 664 / 720, 38 / 143 / 340 / 640, 664, 517 / 664 / 720, 38 / 340 / 399 / 416 / 664 / 751 / 793 246 / 437, 38 / 244 / 340 / 379 / 437 / 664 / 720 / 779, 133 / 134 / 135 / 136 / 246 / 340 / 379 / 437 / 517 / 607 / 664 / 720, 38 / 782, 38 / 379, 38 / 244 / 246 / 310 / 379, 38 / 340 / 720 or 38 / 379 / 437 / 517, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO:2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

11. The engineered RNA polymerase according to any one of claims 1-4 and 10, wherein the amino acid sequence of said engineered RNA polymerase comprises at least one set of substitutions: 38G / 143V / 664K / 793L, 38G / 340E / 364H / 640P / 664R / 782V / 793L, 38G / 143V / 246A / 340E / 399M / 640P / 782G / 793L, 38G / 379S / 437P / 664K / 779R, 38G / 379S / 517R / 664K, 664R / 782V / 7 93L、38G / 143V / 340L / 640P / 664R、38G / 607Y / 664K / 720R、38G / 246A / 399M / 640P / 782G / 856I、340L / 664R / 751R / 793L / 856I、143 V / 340L / 437P / 664K / 779R, 340E / 664R / 751R / 856I, 38G / 437P, 143V / 517R / 664K, 38G / 664K, 38G / 416V / 664R, 143V / 246A / 793L, 3 99M / 640P, 640P / 782G / 793L, 38G / 310A / 437P, 246A / 416V / 664R, 246A / 793L, 38G / 310A / 379S, 38G / 246A / 340L / 437P, 38G / 340E / 664R, 664R / 720Q, 38G / 143V / 340L / 640P, 664R, 664K / 720E, 517R / 664R / 720E, 38G / 340E / 399M / 416V / 664K / 751R / 793L, 246A / 437P, 38G / 244Y / 340L / 379S / 437P / 664K / 720R / 779R, 133Q / 134H / 135R / 136E / 246A / 340E / 379S / 437P / 517Y / 607Y / 664R / 720Q, 38G / 782V, 38G / 379S, 38G / 379S, 38G / 244Y / 246A / 310A / 379S, 38G / 340E / 720Q, or 38G / 379S / 437P / 517Y, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

12. The engineered RNA polymerase according to any one of claims 1-4 and 10, wherein the amino acid sequence of said engineered RNA polymerase comprises at least one set of substitutions: R38G / A143V / W664K / Q793L, R38G / K340E / R364H / S640P / W664R / E782V / Q793L, R38G / A143V / Q246A / K340E / K399M / S640P / E782G / Q793L, R38G / E379S / S437P / W664K / H779R, R38G / E379S / C517R / W664K, W664R / E782V / Q793L , R38G / A143V / K340L / S640P / W664R, R38G / E607Y / W664K / K720R, R38G / Q2 46A / K399M / S640P / E782G / F856I, K340L / W664R / Q751R / Q793L / F856I, A14 3V / K340L / S437P / W664K / H779R, K340E / W664R / Q751R / F856I, R38G / S437 P, A143V / C517R / W664K, R38G / W664K, R38G / A416V / W664R, A143V / Q246A / Q 793L, K399M / S640P, S640P / E782G / Q793L, R38G / K310A / S437P, Q246A / A4 16V / W664R, Q246A / Q793L, R38G / K310A / E379S, R38G / Q246A / K340L / S437P , R38G / K340E / W664R, W664R / K720Q, R38G / A143V / K340L / S640P, W664R, W 664K / K720E, C517R / W664R / K720E, R38G / K340E / K399M / A416V / W664K / Q75 1R / Q793L, Q246A / S437P, R38G / V244Y / K340L / E379S / S437P / W664K / K720R / H779R, L133Q / T134H / S135R / A136E / Q246A / K340E / E379S / S437P / C517Y / E607Y / W664R / K720Q, R38G / E782V, R38G / E379S, R38G / E379S, R38G / V244Y / Q246A / K310A / E379S, R38G / K340E / K720Q or R38G / E379S / S437P / C517Y,The amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

13. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO:

4.

14. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or higher sequence identity with the sequence corresponding to the even-numbered SEQ ID NOs of ...

15. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or with respect to the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions.

16. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the even-numbered SEQ ID NOs of SEQ ID NO: 84-138, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO:

4.

17. The engineered RNA polymerase of claim 15 or 16, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution at the following amino acid positions: 38, 133, 134, 135, 136, 143, 244, 246, 310, 340, 364, 379, 399, 416, 437, 517, 607, 640, 664, 670, 720, 751, 779, 782, 793, 856, or 876, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO:

4.

18. The engineered RNA polymerase of claim 15 or 16, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or amino acid residue 38G / R, 133Q, 134H, 135R, 136E, 143A / V, 244Y, 246A, 310A, 340E / L, 364H, 379S, 399M, 416V, 437P, 517R / Y, 607Y, 640P, 664K / R, 670N, 720E / Q / R, 751R, 779R, 782G / V, 793L, 856I, or 876K or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO:

4.

19. The engineered RNA polymerase of claim 15, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions at the following amino acid positions: 38 / 143 / 340 / 437 / 640 / 670 / 751 / 856, 640 / 670 / 856, 399 / 640, 399 / 640 / 670 / 782 / 856, 143 / 340 / 399 / 640 / 782, 437, 143 / 340 / 437 / 751 / 782 / 856 / 876, 399 / 640 / 670 / 782, 437 / 670 / 779 / 782 / 856, 437 / 782, 38 / 4 37, 437 / 779 / 782, 143 / 399 / 437 / 751 / 782, 399 / 437 / 640 / 670 / 751 / 779 / 782 / 856, 38 / 143 / 340 / 670 / 856, 38 / 437 / 640 / 779, 437 / 751, 38 / 399 / 437 / 640 / 670, 143 / 340 / 399 / 640 / 779 / 782, 38 / 143 / 399 / 437 / 640 / 670 / 751, 340 / 437 / 782, 640 / 751 / 856 or 143 / 640 / 782, wherein the amino acid position is relative to the position corresponding to SEQ. The reference sequence of residues 8 to 890 of ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO:

4.

20. The engineered RNA polymerase of claim 15 or 18, wherein the amino acid sequence of said engineered RNA polymerase comprises at least one set of substitutions: 38R / 143A / 340L / 437P / 640P / 670N / 751R / 856I, 640P / 670N / 856I, 399M / 640P, 399M / 640P / 670N / 782V / 856I, 143A / 340E / 399M / 640P / 782G, 437P, 437P, 143A / 340L / 437P / 751R / 782V / 856I / 876K, 399M / 640P / 6 70N / 782V, 437P / 670N / 779R / 782G / 856I, 437P, 437P / 782V, 399M / 640P / 670N / 782V, 38R / 437 P、437P / 779R / 782V、143A / 399M / 437P / 751R / 782V、399M / 437P / 640P / 670N / 751R / 779R / 782V / 856I、437P、38R / 143A / 340E / 670N / 856I、38R / 437P / 640P / 779R、437P / 751R、38R / 399M / 437 P / 640P / 670N, 437P / 751R, 143A / 340E / 399M / 640P / 779R / 782G, 38R / 143A / 399M / 437P / 640P / 670N / 751R, 340L / 437P / 782V, 640P / 751R / 856I or 143A / 640P / 782V, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO:

4.

21. The engineered RNA polymerase of claim 15 or 18, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions: G38R / V143A / K340L / S437P / S640P / K670N / Q751R / F856I, S640P / K670N / F856I, K399M / S640P, K399M / S640P / K670N / E782V / F856I, V143A / K340E / K3 99M / S640P / E782G, S437P, S437P, V143A / K340L / S437P / Q751R / E782V / F856I / N876K, K399M / S640P / K670N / E 782V, S437P / K670N / H779R / E782G / F856I, S437P, S437P / E782V, K399M / S640P / K670N / E782V, G38R / S437P, S4 37P / H779R / E782V, V143A / K399M / S437P / Q751R / E782V, K399M / S437P / S640P / K670N / Q751R / H779R / E782V / F 856I, S437P, G38R / V143A / K340E / K670N / F856I, G38R / S437P / S640P / H779R, S437P / Q751R, G38R / K399M / S43 7P / S640P / K670N, S437P / Q751R, V143A / K340E / K399M / S640P / H779R / E782G, G38R / V143A / K399M / S437P / S640P / K670N / Q751R, K340L / S437P / E782V, S640P / Q751R / F856I or V143A / S640P / E782V, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO:

4.

22. The engineered RNA polymerase of claim 1, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4 or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

23. The engineered RNA polymerase of claim 1, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions of the engineered RNA polymerase as described in Tables 4.1 and 5.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4 or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

24. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference sequence comprising the substitutions or sets of substitutions described in Tables 4.1 and 5.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

25. The engineered RNA polymerase of claim 1, wherein the amino acid sequence of the engineered RNA polymerase comprises residues 8 to 890 of SEQ ID NO. with even numbers from SEQ ID NO: 4-138, or comprises SEQ ID NO. with even numbers from SEQ ID NO: 4-138, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions.

26. The engineered RNA polymerase of claim 1, wherein the amino acid sequence of the engineered RNA polymerase comprises SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, The amino acid sequence comprises residues 8 to 890 of 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138, and optionally the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions.

27. The engineered RNA polymerase of claim 1, wherein the amino acid sequence of the engineered RNA polymerase comprises SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138, wherein the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions.

28. The engineered RNA polymerase according to any one of claims 1-27, wherein the engineered RNA polymerase has RNA polymerase activity.

29. The engineered RNA polymerase according to any one of claims 1-28, having at least one improved property compared to a reference RNA polymerase.

30. The engineered RNA polymerase of claim 29, wherein the improved property is selected from i) increased activity, ii) increased stability and iii) increased thermostability or any combination of i), ii) and iii) compared to the reference RNA polymerase, wherein the reference RNA polymerase has the sequence of residues 8 to 890 corresponding to SEQ ID NO: 2 or 4 or the sequence corresponding to SEQ ID NO: 2 or 4.

31. The engineered RNA polymerase according to any one of claims 1-30, wherein the engineered RNA polymerase is purified.

32. A recombinant polynucleotide encoding an engineered RNA polymerase according to any one of claims 1-30.

33. The recombinant polynucleotide of claim 32, comprising a reference polynucleotide sequence of nucleotide residues 21 to 2670 of SEQ ID NO. corresponding to odd-numbered SEQ ID NO: 3-137 or a polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO. corresponding to odd-numbered SEQ ID NO: 3-137, wherein the recombinant polynucleotide encodes an RNA polymerase.

34. The recombinant polynucleotide of claim 32 or 33, wherein the polynucleotide sequence is codon-optimized for the expression of the encoded engineered RNA polymerase.

35. The recombinant polynucleotide of claim 32, comprising a polynucleotide sequence comprising nucleotide residues 21 to 2670: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135 or 137.

36. The recombinant polynucleotide of claim 32, comprising a polynucleotide sequence comprising: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, or 137.

37. An expression vector comprising any one of claims 32-36.

38. The expression vector of claim 37, wherein the recombinant polynucleotide is operatively linked to a control sequence.

39. The expression vector of claim 38, wherein the control sequence comprises at least a promoter.

40. A host cell comprising the expression vector according to any one of claims 37-39.

41. The host cell of claim 40, comprising a prokaryotic cell or a eukaryotic cell.

42. The host cell of claim 41, wherein the host cell is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.

43. A method for producing an engineered RNA polymerase polypeptide in a host cell, comprising culturing the host cell according to any one of claims 40-42 under suitable culture conditions to express the encoded engineered RNA polymerase.

44. The method of claim 43, further comprising recovering the expressed engineered RNA polymerase from the culture medium and / or the host cell.

45. The method of claim 43 or 44, further comprising purifying the expressed engineered RNA polymerase.

46. ​​A composition comprising the RNA polymerase according to any one of claims 1-31.

47. The composition of claim 46, further comprising at least one buffer and one or more rNTPs.

48. The composition of claim 46 or 47, further comprising a target DNA template.

49. The composition of any one of claims 46-48, wherein the RNA polymerase is a lyophilized product.

50. A method for producing RNA in vitro, comprising contacting a target DNA template with an engineered RNA polymerase according to any one of claims 1-31 in the presence of one or more nucleoside triphosphates under conditions suitable for transcription of a DNA template.

51. The method of claim 50, wherein the target DNA template comprises a promoter recognized by the engineered RNA polymerase.

52. The method of claim 50 or 51, further comprising providing a cap-like object.

53. The method of any one of claims 50-52, wherein the suitable conditions include a temperature of about 25°C to 50°C.

54. The method according to any one of claims 50-53, further comprising pyrophosphatase and / or RNase inhibitors.

55. A kit comprising the engineered RNA polymerase according to any one of claims 1-31.

56. The kit of claim 55, further comprising one or more of a buffer solution, one or more rNTPs, and cap analogs.

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