Draft:OrthoRep

OrthoRep is a hypermutation system used for the directed evolution of proteins. It was developed by the group of Chang C. Liu at the University of California, Irvine. It is one of the methods used for in vivo continuous evolution, which aims to expand the depth and scale in the evolutionary search for highly efficient proteins compared to conventional directed evolution. The method is based on a natural plasmid system in Kluveromyces lactis, which was ported to Saccharomyces cerevisiae, creating an extrachromosomal orthogonal error-prone replication system in yeast.

Method principle

OrthoRep in vivo hypermutation system. A highly error-prone orthogonal DNA polymerase (DNAP, green) specifically hyper-mutates a gene of interest located on a p1 plasmid (blue) in the cytosol of Saccharomyces cerevisiae through a protein-primed mechanism (red). The host genome is unaffected. The introduced mutations are shown as pink stars.

OrthoRep is based on a highly error-prone orthogonal DNA polymerase (DNAP), which specifically hyper-mutates a gene of interest (GOI) located on a so-called p1 plasmid in the cytosol of S. cerevisiae. Although the GOI experiences a elevated mutation rate, the host genome of the cell is unaffected because of the highly specific protein-primed replication mechanism of p1 recognition by the error-prone polymerase.

As mutations are getting introduced onto the gene of interest, improved variants of the corresponding protein of interest can be identified by two possible approaches:

1. Selection
   • the function of the GOI is linked to cell growth or survival (e.g., drug resistance, formation of an essential nutrient, ...)
   • by passaging of the cells in media with increased selection stringency (e.g., increased drug concentration, decreased nutrient concentration), only the cells bearing improved protein variant will survive
2. High-throughput screening
   • the function of the GOI is linked to an optical signal (e.g., fluorescence)
   • coupling to an in vitro step, e.g., fluorescence-activated cell sorting (FACS), is necessary to identify promising variants of the GOI

Advantages over classical directed evolution

Comparison of classical directed evolution and in vivo continuous evolution using OrthoRep. The main drawback of conventional evolution is that it relies on a manually staged library generation step using in vitro mutagenesis (e.g. error-prone PCR), which limits the sequence diversity that can be generated. In vivo continuous evolution eliminates this limitation by using in vivo hypermutation systems such as OrthoRep, where mutatitions are generated autonomously inside the cells.

OrthoRep is one of the examples of an in vivo hypermutation system (other examples include for instance MutaT7 or EvolvR). Compared to conventional directed evolution, which relies on a manually staged in vitro library generation (e.g., with error-prone PCR, which can generate very limited sequence diversity), such hypermutation systems enable genetic diversity to be generated autonomously inside the cells. When coupled to selection, all of the steps involved in directed evolution campaigns - (i) gene amplification and hypermutation, (ii) protein expression and (iii) selection - happen autonomously as the cells replicate, without any interference from the researcher.

As a consequence, OrthoRep enables multiple evolutionary pathways to be analysed simultaneously and in higher depth compared to conventional directed evolution, increasing the explored sequence space, and potentially leading to better performing protein variants.

Error-prone polymerases

A number of orthogonal error-prone DNAPs have been engineered for the OrthoRep system. Some of the most commonly used ones are listed in Table 1. They vary in their error rates and their preferences for different mutation types (transitions and transversions).

Table 1: mutation rates of selected orthogonal error-prone DNAPs

DNAP	error rate (substitutions per base)	fold increase over genomic mutation rate
TP-DNAP1-4-2	10-5	100,000
BadBoy2	10-4	1,000,000
BB-Tv	10-5	100,000

Examples of use

Tryptophan synthase evolution

In an 2020 article in Nature Communications, Rix et al. reported the evolution of a highly efficient tryptophan synthase 𝛽-subunit (TrpB) from Thermotoga maritima. The protein of interest catalyses the synthesis of L-tryptophan, an amino acid essential for growth of a tryptophan auxotrophic yeast strain. By coupling OrthoRep hypermutation with selection, the authors engineered a TrpB with increased tryptophan-forming activity and broadened secondary promiscuous activities toward related substrates.

Antibody evolution

'Autonomous hypermutation yeast surface display' (AHEAD), a technology based on the OrthoRep system reported in 2021, has been used to generate potent nanobodies against the SARS-Cov-2 S-glycoprotein and other targets.

Optimization of cis,cis-muconic acid biosynthetic pathway

In a 2021 article in Microbial Biotechnology journal, the biosynthetic pathway for the production of cis,cis-muconic acid (CCM, precursor used for bioplastic and coatings) was optimised using OrthoRep. The evolution of the rate-limiting enzyme in the pathway, PCA decarboxylase, was followed in high throughput using a transcription-based biosensor, yielding a fluorescent readout, and enabling coupling to FACS.