ENGINEERED CYCLIC GMP-AMP SYNTHASE (cGAS) VARIANT ENZYMES

20250084421

18/953518

November 20, 2024

Cyclic GMP-AMP synthase (cGAS) enzymes have been engineered to produce polypeptides having increased cGAS activity in the cyclization of modified nucleoside triphosphates, including thiolated or fluorinated nucleoside triphosphates, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing cGAS enzymes, compositions comprising the cGAS enzymes and methods of using the engineered cGAS enzymes are useful for the production of pharmaceutical compounds.

1. An engineered cGAS enzyme comprising a polypeptide sequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 2, 34, 318, 556, 566, and/or 666, wherein the polypeptide sequence of said engineered cGAS enzyme comprises at least one substitution or substitution set and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NOs: 2, 34, 318, 556, 566, and/or 666.

2. The engineered cGAS enzyme of claim 1, wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2, and wherein the polypeptide sequence of said engineered cGAS enzyme comprises at least one substitution or substitution set at one or more positions in said polypeptide sequence selected from 163/201/389, 114/389, 126, 128, 131, 131/161, 135, 161, 163, 163/172/181/201/256/257/389, 163/172/181/256/278/334/389, 163/172/181/257/278, 163/172/256/257/389, 163/172/256/278/389, 163/181, 163/181/201, 163/181/201/256/334/389, 163/181/201/256/389, 163/181/201/257/334, 163/181/201/257/334/389, 163/181/201/334/389, 163/181/201/389, 163/181/256, 163/181/256/257, 163/181/256/257/334/389, 163/181/256/257/389, 163/181/256/334/389, 163/181/256/389, 163/181/257, 163/181/257/334, 163/181/257/334/389, 163/181/278/334, 163/181/334/389, 163/181/389, 163/201/256, 163/201/256/334, 163/201/257/278/334, 163/201/257/278/389, 163/201/257/334, 163/201/257/334/389, 163/201/257/389, 163/201/334, 163/256, 163/256/257, 163/256/257/334, 163/256/257/334/389, 163/256/257/389, 163/256/278, 163/256/334, 163/256/334/389, 163/256/389, 163/257, 163/257/278/334, 163/257/278/389, 163/257/334, 163/257/334/389, 163/257/389, 163/334, 163/334/389, 163/389, 164/177/255, 171, 172, 172/181/201/256/257/278/389, 172/181/256/334/389, 176, 177, 179, 181, 181/201/256/257, 181/201/256/257/389, 181/201/256/278/334/389, 181/201/334, 181/256/257, 181/256/334, 181/257, 181/257/334, 181/257/475, 181/389, 201/334, 255, 256/257/334, 256/334, 256/334/389, 256/389, 257, 257/278/334/389/392, 257/334, 257/334/389, 257/389, 334, 334/389, 338, 341, 364, 378, 388, 389, and 390, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

3. The engineered cGAS enzyme of claim 1, wherein said engineered cGAS enzyme comprises at least one improved property compared to the wild-type Haliaeetus leucocephalus cGAS enzyme.

4. The engineered cGAS enzyme of claim 3, wherein said improved property comprises improved activity on a substrate.

5. The engineered cGAS enzyme of claim 4, wherein said substrate comprises Sp-3′Fluoro-3′-deoxy guanosine-5′-(1-thio)-triphosphate (F-thioGTP or compound (2)) and/or Sp-2′Fluoro-ara-adenosine-5′-1-thio-triphosphate (F-thioATP or compound (3)).

6. The engineered cGAS enzyme of claim 3, wherein said improved property comprises improved production of compound (1).

7. The engineered cGAS enzyme of claim 3, wherein said improved property comprises increased activity, increased substrate tolerance, and/or increased stability.

8. A polynucleotide sequence encoding at least one engineered cGAS enzyme comprising a polypeptide sequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 2, 34, 318, 556, 566, and/or 666, wherein the polypeptide sequence of said engineered cGAS enzyme comprises at least one substitution or substitution set and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NOs: 2, 34, 318, 556, 566, and/or 666.

9. A polynucleotide sequence encoding at least one engineered cGAS enzyme, said polynucleotide sequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 1, 33, 317, 555, 565, and/or 665, wherein the polynucleotide sequence of said engineered cGAS enzyme comprises at least one substitution at one or more positions.

10. The polynucleotide sequence of claim 9 encoding at least one engineered cGAS enzyme wherein said engineered cGAS enzyme comprises a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 2 with the substitutions G163N, Y201F, C389S, R255K and L334I, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

11. The polynucleotide sequence of claim 8, wherein said polynucleotide sequence is operably linked to a control sequence.

12. The polynucleotide sequence of claim 8, wherein said polynucleotide sequence is codon optimized.

13. The polynucleotide sequence of claim 9, wherein said polynucleotide sequence comprises a polynucleotide sequence set forth in the odd numbered sequences of SEQ ID NOs: 3-811.

14. An expression vector comprising at least one polynucleotide sequence of claim 8.

15. A host cell comprising at least one expression vector of claim 14.

16. A host cell comprising at least one polynucleotide sequence of claim 15.

17. A method of producing an engineered cGAS enzyme in a host cell, comprising culturing the host cell of claim 16, under suitable conditions, such that at least one engineered cGAS enzyme is produced.

18. The method of claim 17, further comprising recovering at least one engineered cGAS enzyme from the culture and/or host cell.

19. The method of claim 17, further comprising the step of purifying said at least one engineered cGAS enzyme.

20. The polynucleotide sequence of claim 8, wherein the polypeptide sequence of the engineered cGAS enzyme further comprises at least one substitution at one or more positions selected from 135, 149, 159, 164, 181, 190, 204, 262, 286 and 464 wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 34.

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