Systems for the expression of orthogonal translation components in eubacterial host cells

1. A method for producing, in a host cell, a polypeptide of interest comprising an unnatural amino acid at a specified position, the method comprising:

(a) providing:

(i) an unnatural amino acid;

(ii) a nucleic acid construct comprising a nucleotide sequence encoding an orthogonal tRNA (O-tRNA) and a nucleotide sequence encoding an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein said O-RS preferentially aminoacylates said O-tRNA with said unnatural amino acid; and,

(iii) a polynucleotide encoding said polypeptide of interest, said polynucleotide comprising at least one selector codon that is recognized by said O-tRNA, and wherein the position of the selector codon correlates to the specified position of the unnatural amino acid in the polypeptide of interest;

(iv) a host cell comprising (i), (ii) and (iii); and

wherein said nucleic acid construct of (ii) and (iii) collectively comprises providing at least one of:

(I) promoter and terminator nucleotide sequences derived from an Escherichia coli proline tRNA gene, wherein said promoter and terminator sequences are both operatively linked to said nucleotide sequence encoding said O-tRNA, and wherein said nucleotide sequence encoding said O-tRNA is heterologous to said promoter and terminator sequences; or

(II) a polycistronic operon comprising a plurality of O-tRNA gene nucleotide sequences, wherein at least one O-tRNA gene is separated from at least one adjacent O-tRNA gene by a heterologous polynucleotide linker derived from a naturally occurring polynucleotide linker from a naturally occurring tRNA operon; and

(III) a promoter nucleotide sequence corresponding to a modified E. coli glnS promoter having the nucleotide sequence of SEQ ID NO: 13, wherein said modified E. coli glnS nucleotide sequence is operatively linked to said nucleotide sequence encoding said O-RS;

(b) growing said host cell; and

(c) incorporating said unnatural amino acid at said specified position in said polypeptide during translation of said polypeptide in said host cell, thereby producing said polypeptide of interest comprising said unnatural amino acid at the specified position.

2. The method of claim 1 , wherein:

said providing a nucleic acid construct comprises providing a nucleotide sequence encoding an O-tRNA derived from one or more Archaea tRNA; or

said providing a nucleic acid construct comprises providing a polycistronic operon comprising a plurality of nucleotide sequences encoding one or more O-tRNA species; or

said providing a nucleic acid construct comprises providing a nucleotide sequence encoding an O-tRNA comprising the nucleotide sequence of SEQ ID NO: 1 (MjtRNA-Tyr(CUA)); or

said providing a nucleic acid construct comprises providing a polycistronic operon comprising a plurality of the nucleotide sequence of SEQ ID NO: 1 (MjtRNA-Tyr(CUA)).

3. The method of claim 1 , wherein:

said providing a nucleic acid construct comprises providing a nucleotide sequence encoding an O-RS derived from a Methanococcus jannaschii aminoacyl-tRNA synthetase; or

said providing a nucleic acid construct comprises providing a nucleotide sequence encoding an O-RS derived from a Methanococcus jannaschii tyrosyl-tRNA synthetase; or

said providing a nucleic acid construct comprises providing a nucleotide sequence encoding an O-RS having aspartic acid to arginine substitution at amino acid position 286 or at a position analogous to position 286, relative to the amino acid sequence of wild-type Methanococcus jannaschii tyrosyl-tRNA synthetase provided in SEQ ID NO: 2 (wild type Mj-tRNATyrRS).

4. The method of claim 1 , wherein:

said providing a nucleic acid construct of (I) comprises providing nucleotide sequences corresponding to an Escherichia coli proline tRNA promoter and terminator sequences, said E. coli proline tRNA selected from E. coli proK, proL and proM tRNA; or

said providing a nucleic acid construct of (I) comprises providing nucleotide sequences corresponding to an Escherichia coli proline tRNA promoter and terminator sequences, said E. coli proline tRNA promoter and terminator sequences comprising the promoter and terminator sequences of E. coli proK provided in SEQ ID NOS: 32 (promoter) and 33 (terminator), respectively.

5. The method of claim 1 , wherein said providing a polycistronic operon of (II) comprises providing a plurality of identical heterologous polynucleotide linkers; or

said providing a polycistronic operon of (II) comprises providing a plurality of heterologous polynucleotide linkers, wherein at least two of the heterologous polynucleotide linkers are different; or

said providing a polycistronic operon of (II) comprises providing a heterologous polynucleotide linker comprising a 5′ terminal thymidine nucleotide, and a 3′ terminal adenosine nucleotide; or

said providing a polycistronic operon of (II) comprises providing a heterologous polynucleotide linker derived from the naturally occurring polynucleotide linker located between the endogenous Escherichia coli tRNA genes selected from: valU and valX; ileT and alaT; serV and argV; valV and valW; glyT and thrT; metT and leuW; glnW and metU; hisR and leuT; glnU and glnW; leuP and leuV; glnV and glnX; alaW and alaX; ileU and alaU; ileV and alaV; metU and glnV; glvW and cysT; argX and hisR; and argY and argZ; or said providing a polycistronic operon of (II) comprises providing a heterologous polynucleotide linker derived from the nucleotide sequence of SEQ ID NO: 14 (valU/valX linker) or 15 (ileT/alaT linker).

6. The method of claim 1 , wherein:

said providing a host cell comprises providing a eubacterial host cell; or

said providing a host cell comprises providing an Escherichia coli host cell.