Manipulated RNA polymerase variant

JP2026516495APending Publication Date: 2026-05-25CODEXIS INC
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Patent Information

Authority / Receiving Office
JP · JP
Patent Type
Applications
Current Assignee / Owner
CODEXIS INC
Filing Date
2024-05-10
Publication Date
2026-05-25

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Abstract

This disclosure relates to engineered RNA polymerase polypeptides and compositions thereof, as well as polynucleotides encoding engineered RNA polymerase polypeptides. The disclosure also provides methods for using engineered RNA polymerase polypeptides or compositions thereof to produce RNA. In another embodiment, the disclosure provides recombinant polynucleotides encoding the engineered RNA polymerases disclosed herein. In a further embodiment, the disclosure provides expression vectors comprising recombinant polynucleotides encoding the engineered RNA polymerases described herein.
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Claims

1. An engineered RNA polymerase or a functional fragment thereof 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 more sequence identity with a reference sequence corresponding to residues 8-890 of SEQ ID NO: 2 or 4, or with a reference sequence corresponding to SEQ ID NO: 2 or 4, wherein the amino acid sequence comprises one or more substitutions with respect to the reference sequence corresponding to residues 8-890 of SEQ ID NO: 2 or 4, or with respect to the reference sequence corresponding to SEQ ID NO: 2 or 4.

2. The manipulated RNA polymerase according to 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 more sequence identity with the reference sequence corresponding to residues 8-890 of SEQ ID NO: 2, or with the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions with respect to the reference sequence corresponding to residues 8-890 of SEQ ID NO: 2, or with respect to the reference sequence corresponding to SEQ ID NO:

2.

3. The manipulated RNA polymerase according to 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 more sequence identity with the reference sequence corresponding to residues 8-890 of SEQ ID NO: 4, or the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions with respect to the reference sequence corresponding to residues 8-890 of SEQ ID NO: 2, or the reference sequence corresponding to SEQ ID NO:

2.

4. The manipulated RNA polymerase according to 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 more sequence identity with a sequence corresponding to an even-numbered sequence number among sequence numbers 4 to 138, wherein the amino acid sequence comprises one or more substitutions with respect to the reference sequence corresponding to residues 8 to 890 of sequence number 2, or with respect to the reference sequence corresponding to sequence number 2.

5. The manipulated RNA polymerase according to any one of claims 1 to 4, wherein the amino acid sequence of the manipulated RNA polymerase includes at least one substitution at 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 a combination thereof, and the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO:

2.

6. The manipulated RNA polymerase according to any one of claims 1 to 5, wherein the amino acid sequence of the manipulated RNA polymerase comprises at least one substitution or one 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, and 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 manipulated RNA polymerase according to any one of claims 1 to 4, wherein the amino acid sequence of the manipulated RNA polymerase includes at least one substitution at amino acid positions 38, 143, 664, or 793, or a combination thereof, and the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO:

2.

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

2.

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

2.

10. The amino acid sequence of the manipulated RNA polymerase has at least one substitution set at 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 / 66 4, 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 / 5 The manipulated RNA polymerase according to any one of claims 1 to 4, comprising 17 / 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 amino acid sequence of the manipulated RNA polymerase is at least one substitution set: 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 / 340L / 640P / 664R, 38G / 607Y / 664 K / 720R, 38G / 246A / 399M / 640P / 782G / 856I, 340L / 664R / 751R / 793L / 856I, 143V / 340L / 437P / 664K / 779R, 340E / 664R / 751R / 856I, 38G / 437 P, 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, 38 G / 340E / 399M / 416V / 664K / 751R / 793L, 246A / 437P, 38G / 244Y / 340L / 379S / 437P / 664K / 720R / 779R, 133Q / 134H / 135R / 136E / 246A / 340E / 37 The manipulated RNA polymerase according to any one of claims 1 to 4 and 10, comprising 9S / 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 positions are relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO:

2.

12. The amino acid sequence of the manipulated RNA polymerase is at least one substitution set: 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 / Q246A / K399M / S640P / E782G / F856I, K34 0L / W664R / Q751R / Q793L / F856I, A143V / K340L / S437P / W664K / H779R, K340E / W664R / Q751R / F856I, R38G / S437P, A143V / C517R / W664K, R38G / W664K, R38 G / A416V / W664R, A143V / Q246A / Q793L, K399M / S640P, S640P / E782G / Q793L, R 38G / K310A / S437P, Q246A / A416V / W664R, Q246A / Q793L, R38G / K310A / E379S , R38G / Q246A / K340L / S437P, R38G / K340E / W664R, W664R / K720Q, R38G / A143 V / K340L / S640P, W664R, W664K / K720E, C517R / W664R / K720E, R38G / K340E / K 399M / A416V / W664K / Q751R / 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, wherein the amino acid positions correspond to the reference sequence corresponding to residues 8-890 of SEQ ID NO: 2,Alternatively, the manipulated RNA polymerase according to any one of claims 1 to 4 and 10, relating to the reference sequence corresponding to sequence number 2.

13. The manipulated RNA polymerase according to 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 more sequence identity with the reference sequence corresponding to residues 8-890 of SEQ ID NO: 4, or with the reference sequence corresponding to SEQ ID NO:

4.

14. The manipulated RNA polymerase according to 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 more sequence identity with the sequence corresponding to the even-numbered sequence numbers among sequence numbers 4 to 138.

15. The manipulated RNA polymerase according to 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 more sequence identity with the reference sequence corresponding to residues 8-890 of SEQ ID NO: 4, or the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions with respect to the reference sequence corresponding to residues 8-890 of SEQ ID NO: 4, or the reference sequence corresponding to SEQ ID NO:

4.

16. The manipulated RNA polymerase according to 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 more sequence identity with a reference sequence corresponding to residues 8 to 890 of even-numbered sequence numbers among sequence numbers 84 to 138, wherein the amino acid sequence comprises one or more substitutions with respect to the reference sequence corresponding to residues 8 to 890 of sequence number 4, or with respect to the reference sequence corresponding to sequence number 4.

17. The manipulated RNA polymerase according to claim 15 or 16, wherein the amino acid sequence of the manipulated RNA polymerase includes at least one substitution at 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 a combination thereof, and the amino acid position is relative to the reference sequence corresponding to residues 8-890 of SEQ ID NO: 4, or to the reference sequence corresponding to SEQ ID NO:

4.

18. The manipulated RNA polymerase according to claim 15 or 16, wherein the amino acid sequence of the manipulated RNA polymerase comprises at least one substitution or one amino acid residue of 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, and the amino acid position is relative to the reference sequence corresponding to residues 8-890 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO:

4.

19. The amino acid sequence of the manipulated RNA polymerase has at least one substitution set at 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 The manipulated RNA polymerase according to claim 15, comprising / 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 positions are relative to the reference sequence corresponding to residues 8-890 of SEQ ID NO: 4, or to the reference sequence corresponding to SEQ ID NO:

4.

20. The amino acid sequence of the manipulated RNA polymerase is at least one substitution set: 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, 437P / 670N / 779R / 782G / 856I, 437P, 437P / 782V, 399M / 640P / 670N / 782V, 38R / 437P, 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 / 7 51R, 38R / 399M / 437P / 640P / 670N, 437P / 751R, 143A / 340E / 399M / 640P / 779R / 782G, 38R / 143A / 399M / 437 The manipulated RNA polymerase according to claim 15 or 18, comprising P / 640P / 670N / 751R, 340L / 437P / 782V, 640P / 751R / 856I or 143A / 640P / 782V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 8-890 of SEQ ID NO: 4, or to the reference sequence corresponding to SEQ ID NO:

4.

21. The amino acid sequence of the manipulated RNA polymerase is at least one substitution set: G38R / V143A / K340L / S437P / S640P / K670N / Q751R / F856I, S640P / K670N / F856I, K399M / S640P, K399M / S640P / K670N / E782V / F856I, V143A / K340E / K399M / S640P / E782G, S437P, S437P, V143A / K 340L / S437P / Q751R / E782V / F856I / N876K, K399M / S640P / K670N / E782V, S437P / K670N / H779R / E782G / F856I, S437P, S43 7P / E782V, K399M / S640P / K670N / E782V, G38R / S437P, S437P / H779R / E782V, V143A / K399M / S437P / Q751R / E782V, K399M / S437P / S640P / K670N / Q751R / H779R / E782V / F856I, S437P, G38R / V143A / K340E / K670N / F856I, G38R / S437P / S640P / H779 R, S437P / Q751R, G38R / K399M / S437P / S640P / K670N, S437P / Q751R, V143A / K340E / K399M / S640P / H779R / E782G, G38R / V1 The manipulated RNA polymerase according to claim 15 or 18, comprising 43A / K399M / S437P / S640P / K670N / Q751R, K340L / S437P / E782V, S640P / Q751R / F856I or V143A / S640P / E782V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 8-890 of SEQ ID NO: 4, or to the reference sequence corresponding to SEQ ID NO:

4.

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

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

24. The manipulated RNA polymerase according to 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 more sequence identity with a reference sequence comprising one substitution or set of substitutions as described in Tables 4.1 and 5.1, wherein the amino acid positions are relative to the reference sequence corresponding to residues 8-890 of SEQ ID NO: 2 or 4, or relative to the reference sequence corresponding to SEQ ID NO: 2 or 4.

25. The manipulated RNA polymerase according to claim 1, wherein the amino acid sequence of the manipulated RNA polymerase comprises residues 8 to 890 of even sequence numbers from sequence numbers 4 to 138, or comprises even sequence numbers from sequence numbers 4 to 138, and optionally the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

26. The amino acid sequence of the manipulated RNA polymerase is sequence numbers 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, The manipulated RNA polymerase according to claim 1, comprising residues 8 to 890 of the order 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.

27. The amino acid sequence of the manipulated RNA polymerase is sequence numbers 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 manipulated RNA polymerase according to claim 1, comprising 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138, wherein optionally, the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

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

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

30. The modified RNA polymerase according to claim 29, wherein the improved properties are selected from i) improved activity, ii) improved stability, and iii) improved thermal stability, or any combination of i), ii), and iii), compared to the reference RNA polymerase, and the reference RNA polymerase has the sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or 4, or the sequence corresponding to SEQ ID NO: 2 or 4.

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

32. Recombinant polynucleotide encoding the manipulated RNA polymerase according to any one of claims 1 to 30.

33. The recombinant polynucleotide according to claim 32, comprising 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 more sequence identity with a reference polynucleotide sequence corresponding to an odd-numbered sequence number among sequence numbers 3 to 137, wherein the recombinant polynucleotide encodes RNA polymerase.

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

35. The recombinant polynucleotide according to claim 32, comprising a polynucleotide sequence containing nucleotide residues 21 to 2670 of sequence numbers 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 according to claim 32, comprising a polynucleotide sequence including sequence numbers 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 a recombinant polynucleotide according to any one of claims 32 to 36.

38. The expression vector according to claim 37, wherein the recombinant polynucleotide is operably ligated to a control sequence.

39. The expression vector according to claim 38, wherein the control sequence includes at least one promoter.

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

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

42. The host cell according to 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 to 42 under suitable culture conditions to express the encoded engineered RNA polymerase.

44. The method according to claim 43, further comprising recovering the expressed manipulated RNA polymerase from the culture medium and / or host cells.

45. The method according to claim 43 or 44, further comprising purifying the expressed manipulated RNA polymerase.

46. A composition comprising the RNA polymerase described in any one of claims 1 to 31.

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

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

49. The composition according to any one of claims 46 to 48, wherein the RNA polymerase is a freeze-dried product.

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

51. The method according to claim 50, wherein the target DNA template includes a promoter recognized by the manipulated RNA polymerase.

52. The method according to claim 50 or 51, further comprising providing a cap analogue.

53. The method according to any one of claims 50 to 52, wherein the preferred conditions include a temperature of approximately 25°C to 50°C.

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

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

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