Abstract
We recently developed an orthogonal DNA replication (OrthoRep) system capable of driving the rapid continuous evolution of genes in vivo. However, OrthoRep uses a special transcription system, the components of which (e.g., promoters) have previously limited the strength with which OrthoRep-encoded genes can be expressed. Here, we report a collection of synthetic and evolved OrthoRep expression parts that allow OrthoRep-encoded genes to span expression levels matching those of endogenous Saccharomyces cerevisiae genes. Specifically, we found that various promoter mutations as well as a genetically encoded poly(A) tail enable us to tune the expression level of OrthoRep-encoded genes over a large range and up to levels 43-fold higher than were previously attained, reaching at least ∼40% of the strength of the genomic TDH3 promoter. We further show that expression level gains using our new parts are stable over passaging and consistent across multiple genes and OrthoRep systems of different mutation rates. This new set of expression parts further expands OrthoRep's applicability to the continuous in vivo evolution of proteins and pathways.