Submission declined on 24 July 2024 by Mgp28 (
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Comment: To identify that a topic is sufficiently notable to need a Wikipedia article, we look for "significant coverage in reliable sources that are independent of the subject." Chang C. Liu, leader of the group that developed this method, is listed in as an author in all the references currently on this article. They are therefore not independent of the subject. I would like to see what authors outside this group are saying about the method, both to demonstrate that it is notable, and to make sure that we adopt a
WP:Neutral point of view.
Mgp28 (
talk) 15:08, 24 July 2024 (UTC)
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[2]. 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:
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
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)[2]. 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).[3]
Table 1: mutation rates of selected orthogonal error-prone DNAPs
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[5]. The protein of interest catalyses the synthesis of
L-tryptophan, an
amino acid essential for growth of a
tryptophanauxotrophic 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[6], 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[7]. 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.
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in-depth (not just passing mentions about the subject)
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reliable
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secondary
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independent of the subject
Make sure you add references that meet these criteria before resubmitting. Learn about mistakes to avoid when addressing this issue. If no additional references exist, the subject is not suitable for Wikipedia.