Transgenic bacteria with expanded amino acid usage and nucleic acid molecules for use in the same

Patent metadata

Field	Value
Publication number	US-12344879-B2
Application number	US-202318324843-A
Country	US
Kind code	B2
Filing date	May 26, 2023
Priority date	Dec 15, 2015
Publication date	Jul 1, 2025
Grant date	Jul 1, 2025

Abstract

Recombinant bacterial cells are provided that comprise a stable non-canonical amino acid translation pathway. In some aspects, the bacteria comprise nucleic acids encoding a non-canonical amino acid translation pathway (e.g., a tRNA for incorporation of a non-canonical amino acid, such selenocysteine); a marker polypeptide that includes the non-canonical amino acid. Recombinant tRNA and selection marker coding sequences are likewise provided.

First claim

What is claimed is:

1. A recombinant nucleic acid molecule, wherein the molecule encodes a tRNA and is at least 92% identical to SEQ ID NO: 19 or SEQ ID NO: 20; and wherein the tRNA encoded by the recombinant nucleic acid molecule comprises a U at a position corresponding to position 49.

2. The recombinant nucleic acid molecule of claim 1 , wherein the molecule encodes a tRNA and is at least 95% identical to SEQ ID NO: 19 or SEQ ID NO: 20; and wherein the tRNA encoded by the recombinant nucleic acid molecule comprises a U at a position corresponding to position 49.

3. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (i) a G at a position corresponding to position 7.

4. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (i) a C at a position corresponding to position 7.

5. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (iii) a A at a position corresponding to position 50.

6. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (iii) a C at a position corresponding to position 50.

7. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (iv) a U at a position corresponding to position 64.

8. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (v) a A at a position corresponding to position 65.

9. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (v) a G at a position corresponding to position 65.

10. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (vi) a C at a position corresponding to position 66.

11. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (vi) a G at a position corresponding to position 66.

12. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises (vi) a U at a position corresponding to position 66.

13. The recombinant nucleic acid molecule of claim 1 , wherein the molecule comprises two or more of the following features:
(i) a G or C at a position corresponding to position 7;
(ii) a U at a position corresponding to position 49;
(iii) a A or C at a position corresponding to position 50;
(iv) a U at a position corresponding to position 64;
(v) a G or A at a position corresponding to position 65;
(vi) a G, U or C at a position corresponding to position 66.

14. The recombinant nucleic acid molecule of claim 1 , wherein the molecule comprises three or more of the following features:
(i) a G or C at a position corresponding to position 7;
(ii) a U at a position corresponding to position 49;
(iii) a A or C at a position corresponding to position 50;
(iv) a U at a position corresponding to position 64;
(v) a G or A at a position corresponding to position 65;
(vi) a G, U or C at a position corresponding to position 66.

15. The recombinant nucleic acid molecule of claim 1 , wherein the tRNA encoded by the recombinant nucleic acid molecule comprises: (i) a G at a position corresponding to position 7; (ii) a U at a position corresponding to position 49; (iii) a C at a position corresponding to position 50; (iv) a U at a position corresponding to position 64; (v) a G at a position corresponding to position 65; and (vi) a C at a position corresponding to position 66.

16. The recombinant nucleic acid molecule of claim 1 , wherein the recombinant nucleic acid molecule comprises a sequence with at least 95% sequence identity to SEQ ID NO: 19.

17. The recombinant nucleic acid molecule of claim 1 , wherein the recombinant nucleic acid molecule comprises a sequence with at 18 least 95% sequence identity to SEQ ID NO: 20.

18. A transgenic bacterial cell, said cell comprising the recombinant nucleic acid molecule according to claim 1 .

19. A method of producing a recombinant polypeptide comprising a selenocysteine comprising:
(i) providing a bacterial cell comprising (a) the recombinant nucleic acid molecule according to claim 1 and (b) an expression cassette encoding the recombinant polypeptide; and
(ii) incubating the bacterial cell under conditions that allow expression of the polypeptide comprising the selenocysteine.

Assignees

• Univ Texas

Inventors

• Ellington Andrew D
• THYER Ross

Classifications

• C12N15/67

  General methods for enhancing the expression · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C12Y305/02006

  Beta-lactamase (3.5.2.6) · CPC title

• C12N9/86

  acting on amide bonds in cyclic amides, e.g. penicillinase {(3.5.2)} · CPC title