Phage-assisted continuous evolution

In the initial paper pioneering this technique, T7 RNA polymerases were evolved to recognize different promoters, such as the T3 or SP6 promoters.[4] This was done by making the target promoter the sole promoter for gIII.[5] Hence, mutant polymerases with greater specificity for the desired promoter caused greater pIII production. This resulted in polymerases with ~3-4 orders of magnitude greater activity for the target promoter than the original T3 promoter.[4] While this original PACE system only performed positive selection, a variant was developed that allowed for negative selection as well. This is done by linking undesired activity to the production of non-functional pIII, which decreases the amount of infectious phage made.[6]

A scheme for evolving polymerase promoter specificity using PACE. T7 RNAP containing mutations unfavorable to T3 promoter binding results in no gIII transcription. However, mutations that allow for the binding to a T3 promoter lead to increased gIII transcription.

Proteases have been evolved to cut different peptides using PACE. In these systems, the desired protease cut site is used to link a T7 RNA polymerase and a T7 lysozyme. The T7 lysozyme prevents the T7 polymerase from transcribing gIII. When the peptide linker is cleaved, the T7 polymerase is activated, allowing for the transcription of the pIII gene. This method was used to create a TEV protease with a significantly different peptide substrate.[6][7]

A scheme of using PACE to evolve protease activity. When the T7 RNAP and the T7 lysozyme are linked, transcription of gIII is blocked. When the protease is active for the cleavage site, the T7 polymerase is liberated, allowing for transcription of gIII.

Using PACE, aminoacyl-tRNA synthetases (aaRSs) were evolved for noncanonical amino acids as well. Activity of an aaRS is linked to pIII production by the addition of a TAG stop codon in the middle of gIII. Synthetases that aminoacylate the TAG codon's suppressor tRNA prevents stop codon activity, allowing for production of functional pIII. Using this system, aaRSs were evolved that utilize non-canonical amino acids p-nitro-phenyalanine, iodophenylalanine, and Boc-lysine.[8]

A scheme of using PACE to evolve orthogonal aminoacyl-tRNA synthetases. Without synthetase activity, T7 RNAP cannot be fully translated due to the presence of an Amber stop codon. Upon introduction of a functioning synthetase for the Amber codon tRNA, this Amber stop codon now encodes a non-canonical amino acid, allowing for the transcription of the full T7 RNAP and hence allowing gIII transcription.

Protein-protein interactions have been evolved using PACE as well. Under this scheme, the target protein is fused with a DNA binding protein, which binds to a target sequence placed upstream of the gIII promoter. The protein undergoing evolution is fused with an RNA polymerase. The better the protein-protein interaction, the more transcription of pIII occurs, allowing the evolution of the protein-protein interaction under PACE conditions.[6] This method was used to evolve Bacillus thuringiensis endotoxin variants that can overcome insect toxin resistance.[6][9]

A scheme for using PACE to evolve protein-protein interactions. The target protein, in purple, is fused to a DNA binding protein, in blue. The evolving protein, in red, is bound to a functioning RNAP, in green. By having the DNA binding protein bind upstream of the gIII promoter, more favorable binding between the target and evolving protein leads to higher transcription of gIII.

PACE was used to evolve APOBEC1 for greater soluble expression. APOBEC1 is a cytidine deaminase that has found use in base editors to catalyze the single nucleotide edit C-->T.[10] In E. coli, APOBEC1 usually falls out of solution into the insoluble fraction.[11] To evolve APOBEC1 for better soluble expression, the N-terminus of a T7 polymerase was fused to APOBEC1, with the remaining portion of the polymerase separately expressed. The T7 polymerase can only function when the N-terminus portion can bind to the rest of the polymerase. Since APOBEC1 must be properly folded for the N-terminus portion to be exposed properly, T7 polymerase activity is correlated to APOBEC1 folding. As follows, pIII transcription and production is linked with APOBEC1 soluble expression via the T7 polymerase. Using this approach, the soluble expression of APOBEC1 was increased by 4 fold with no change in function.[7][9]

A scheme for the engineering of higher soluble expression. POI = protein of interest. When the POI is properly folded, the T7n portion is exposed, allowing for the binding to the T7c portion to form a fully functional T7 RNAP. This T7 RNAP can then transcribe gIII. If the POI is misfolded, then the T7 RNAP doesn't form, leading to no gIII transcription.

PACE was also used to create a more catalytically active deoxyadenosine deaminase. Deoxyadenosine deaminase is used in base editors to perform the single nucleotide edit A-->T. This was done by placing adenosine-containing stop codons in the gene for T7 polymerase. If the base editor is able to correct the error, functional T7 polymerase is produced, allowing production of pIII. Using this system, they evolved a deoxyadenosine deaminase with 590 fold activity compared to wild type.[12]

Evolution of higher activity deoxyadenosine deaminases using PACE. Here, the T7 RNAP gene contains two stop codons, both containing adenosine nucleotides. Without deoxyadenosine deaminase activity, the resulting T7 RNAP is truncated and hence nonfunctional. However, with a functional deoxyadenosine deaminase, the stop codons are converted into amino acid encoding codons, which allows for the production of functional T RNAP, which can go on to transcribe gIII.