Tunable Expression Systems for Orthogonal DNA Replication

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 whose components (e.g. promoters) have previously limited the strength with which OrthoRep-encoded genes can be expressed. Here, we report a collection of synthetic or 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.

Potential For Applying Continuous Directed Evolution To Plant Enzymes

“…In previous work, target gene expression in yeast was enhanced by placing a poly-A tail/selfcleaving ribozyme (RZ) sequence downstream of the target gene [39]. We therefore built a multipurpose p1 integration vector from 172-YTK-P4 (containing the poly-A/RZ self-cleaving ribozyme cassette) and GR306 (used as backbone, with the fluorescent reporter mKate2 in its cloning region), as shown in Figure 3.…”

“…We adapted OrthoRep for high-throughput use by constructing a multi-purpose p1 integration vector based on the GR306 plasmid ( Figure 3A), to which we added a convenient directional cloning site for target genes (with Nsil and SphI sites) and a downstream poly-A/RZ module to enhance expression [39]. Because the poly-A/RZ module contains 75 consecutive adenosine residues it is inherently unstable and shortens with repeated propagation.…”

Winner-Takes-All Resource Competition Redirects Cascading Cell Fate Transitions

“…Several approaches have been proposed to counteract the effects of resource competition, either by finetuning the parameters in the gene circuit 5,7 or manipulating the size of the orthogonal resource pools [31][32][33][34] . Additionally, a burden-driven negative feedback loop was implemented to control gene expression by monitoring the cellular burden [35][36][37] .…”

Scalable, continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

“…To ensure that evolved TmTrpB variants, and not potential host genomic mutations, were primarily responsible for each population's adaptation, we cloned individual TmTrpBs into a standard low copy yeast nuclear plasmid under a promoter that approximates expression from p1, 23,24 transformed the variants into a fresh Dtrp5 strain, and tested for their ability to support indole-dependent growth in the absence of Trp TmTrpB variants are first evolved in replicate for Trp production in yeast. OrthoRep enables replicate evolution through error-prone replication of an orthogonal plasmid by an orthogonal polymerase, maintaining low error rates in genome replication.…”