Transgenic bacteria with expanded amino acid usage and nucleic acid molecules for use in the same
View Patent ↗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.
1. A recombinant nucleic acid molecule, wherein the molecule encodes a tRNA and is at least 90% identical to SEQ ID NO: 18, and comprising one or more of the following features:
(i) a G or C at a position corresponding to position 7;
(ii) a T at a position corresponding to position 49;
(iii) a A or C at a position corresponding to position 50;
(iv) a T at a position corresponding to position 64;
(v) a G or A at a position corresponding to position 65; and/or
(vi) a G, T or C at a position corresponding to position 66.
2. The recombinant nucleic acid molecule of claim 1 , wherein the molecule encodes a tRNA and is at least 90% identical to SEQ ID NO: 18; and comprises one or more of the following features:
(i) a G at a position corresponding to position 7;
(ii) a T at a position corresponding to position 49;
(iii) a C at a position corresponding to position 50;
(iv) a T at a position corresponding to position 64;
(v) a G at a position corresponding to position 65; and/or
(vi) a C at a position corresponding to position 66.
3. The recombinant nucleic acid molecule of claim 2 , wherein the molecule comprises 2, 3, 4, 5 or 6 of the following features:
(i) a G at a position corresponding to position 7;
(ii) a T at a position corresponding to position 49;
(iii) a C at a position corresponding to position 50;
(iv) a T at a position corresponding to position 64;
(v) a G at a position corresponding to position 65; and/or
(vi) a C at a position corresponding to position 66.
4. The recombinant nucleic acid molecule of claim 2 , wherein the molecule encodes a tRNA and is at least 95% identical to SEQ ID NO: 18.
5. The recombinant nucleic acid molecule of claim 4 , wherein the molecule encodes a tRNA and comprises the sequence of SEQ ID NO: 18.
6. A transgenic bacterial cell, said cell comprising the recombinant nucleic acid molecule in accordance with claim 1 .
7. A method of producing a commercial polypeptide comprising at least a first selenocysteine position comprising:
(i) obtaining a bacterial cell according to claim 6 and an expression cassette encoding the commercial polypeptide; and
(ii) incubating the bacterial cell in conditions that allow expression of the recombinant nucleic acid molecule and the commercial polypeptide.