Genetic Compatibility and Extensibility of Orthogonal Replication

Area of Science:

• Molecular Biology
• Yeast Genetics
• Synthetic Biology

Background:

• The Orthogonal Replication (OrthoRep) system enables rapid, continuous plasmid mutagenesis in yeast without affecting the host genome.
• Previous applications of OrthoRep have shown success in protein and enzyme evolution.
• The general applicability of OrthoRep across different yeast strains and its limitations required systematic investigation.

Purpose of the Study:

• To systematically assess the generality and compatibility of the OrthoRep system in various Saccharomyces cerevisiae strains.
• To determine the maximum genetic material capacity of the OrthoRep plasmid.
• To develop efficient methods for genetic manipulation of the OrthoRep system.

Main Methods:

• Compatibility testing of OrthoRep across diverse Saccharomyces cerevisiae strains.
• Plasmid capacity assessment for encoding large genetic constructs (≥22 kb).
• Development and application of a CRISPR/Cas9-based system for OrthoRep genetic modifications.
• Investigation of the biological incompatibility previously observed with respiration-deficient (ρ0) strains.

Main Results:

• OrthoRep demonstrated full compatibility with all tested Saccharomyces cerevisiae strains.
• The orthogonal plasmid successfully encoded genetic material up to at least 22 kb.
• A CRISPR/Cas9 method was established for streamlined genetic manipulation of OrthoRep.
• The incompatibility with ρ0 strains was attributed to a toxin/antitoxin system on the wild-type plasmid, which is replaced in practical applications.

Conclusions:

• OrthoRep is a generally compatible platform for continuous in vivo evolution in Saccharomyces cerevisiae.
• The system's versatility is enhanced by its broad strain compatibility and efficient genetic manipulation capabilities.
• The identified incompatibility factor can be circumvented, further solidifying OrthoRep's utility for directed evolution studies.