Project Abstract
The aim of our project is to develop a powerful directed evolution tool by combining the PANCE technology (Phage-Assisted Non-Continuous Evolution) which allow directed evolution with a self-selecting screening of the best variants 1,2 and the Evolution.T7 tool developed by the iGEM Evry Paris-Saclay 2021 team 3 which allows targeted mutagenesis on a specific gene of interest. The PANCE technology is very useful to avoid a long and fastidious step of screening but the evolution part relies on random mutagenesis on the whole system and not only on our gene of interest. It can result in loss of interesting variants if the system becomes non-functional due to mutations in essential genes. In contrast, Evolution.T7 only mutates on a specific gene sequence but does not have an auto-screening method such as PANCE. By combining these two methods in PANCE-EVO.T7, we aim to fasten and improve the discovery of new interesting variants without losing some because of mutations that disrupt the system. In parallel, we aim to compare the efficiency of the PANCE-EVO.T7 system to find the best variants of a protein and the ability of an AI based model to find such variants. We could resume it as “ Who is the best engineer between nature and AI ? “ Our project will first focus on the evolution of a transcription factor, XylS, recently engineered to detect plastic degradation metabolites 4. This mutated XylS could then be used to characterize the activity of plastic degradation enzymes, solving a lack of efficient detection system for their activity. It could also be used to detect small quantities of plastic pollution in water, where our actual techniques cannot detect plastic particles inferior to 100 nm 5. Finally, our new directed evolution system and model (we will see who is the best) could be applied to many other biological topics and fields that involve proteins.
The PANCE-EVO.T7 technology
PACE is a continuous directed-evolution system based on M13 phages 1, and PANCE is the non-continuous version 2,6 (Figure 1). We will choose the non continuous version as it is easier to set up in the lab and is high throughput. Infectivity of M13 phages is dependent on filamentous phage protein III encoded by gene III (pIII / gIII), that binds specifically to F-pilus for entry into the bacteria cytoplasm. In PANCE, the phage genome is depleted from gIII which is replaced by the gene of the protein to evolve (protein of interest, POI) (Helper phage, HP Figure 1). gIII is expressed from another plasmid (accessory plasmid, AP on Figure 1). In order to evolve the POI toward the desired function, the expression level of gIII must be linked to an increase in POI fitness (for example, activation of an activator protein or a polymerase). Therefore, the only phages to harbor gIII on their capsid (and therefore only infective phages) will be the ones carrying a genome (Helper phage, HP Figure 1) with an active POI. Deleterious mutations of the POI will be eliminated because phages carrying inactive POI will lack gIII and be unable to infect bacteria. Over several cycles of infection, the medium is enriched in phage expressing a POI with beneficial mutations thanks to the natural selection process between the different phages. In classical PANCE, to increase mutation rates, bacteria are transfected with a mutagenesis plasmid (MP in Figure 1). The most potent one is MP6 which expresses dnaQ926, emrR, PmCDA1 blocking DNA repair system, as well as dam and seqA involved in DNA methylation system.
Figure 1.PANCE system with the 3 main parts, HP for helper phage, AP for accessory plasmid, MP for mutagenesis plasmid and POI for protein of interest. The purple stars represent random mutations that occurred during the replication. Phages are periodically isolated and introduced in a medium with new host cells (adapted from Brödel et al., 2018 7).
Figure 2. The mutation mechanism of the Evolution.T7 system with a cytosine deaminase (CD) as the base deaminase (adapted from Moore et al., 2018 8).
This mutation system finds its limit through the fact that mutations can occur everywhere in the plasmids and bacteria genome. Some of these mutations could affect and alter the replicative functions of the plasmid or the antibiotic gene resistance (selection gene) and then limit the selection of new potential interesting variants. To contain and focus these mutations only into the targeted gene CDS, and therefore considerably increase the recovery of new variants with the PANCE system, we plan to combine it to Evolution.T7 3. This tool is based on the orthogonal T7 RNA polymerase (T7RNAP) linked to a base deaminase (BD) either a cytosine or an adenosine deaminase (respectively CD and AD), which allows for the rapid generation of genetic diversity in target genes in vivo in E. coli. When BD-T7RNAP fusion protein is expressed, the sequence flanked by the T7 promoter and the T7 terminator(s) gets mutated as the CD or AD randomly deaminates the nucleotides on the non template strand of the T7RNAP. Upon DNA replication, these deaminated bases lead to C→T or A→G transition mutations, depending on whether CD or AD was used (Figure 2).
To be able to introduce also T→C and G→A, Evolution.T7 uses also a mutated T7RNAPCGG-R12-KIRV specific to an altered T7CGG promoter sequence which was placed in the reverse orientation downstream of the target region in order to compensate for the above mentioned bias of deaminations occurring mainly on the non template strand (Figure 3).
Figure 3. The general design of the Evolution.T7 system. The gene to be evolved is placed under the control of the T7 promoter and followed by the T7CGG promoter in the reverse orientation, flanked upstream and downstream by four T7 terminators.
Evolution of the XylS transcription factor as a proof of concept Plastic polymer, such as phtalate esters (PAEs) and polyethylene-terephtalate (PET), degradation in water can take many forms: into toxic microplastics due to physical constraints or into non-toxics monomers of phtalic acid (PA) and terephatlic acid (TPA) thanks to bacterial enzymes PAE hydrolases 9 or the PETases 10. The detection could be achieved thanks to recent new variants of the transcriptional activator XylS of Pseudomonas putida 4 initially activated by benzoic derivatives (Figure 4). These variants are sensitive to PA and TPA but with only low affinity and specificity. Enhancing XylS specificity toward plastic degradation products would be promising for the design of new generation biosensors that can be further used for the evolution of PAE hydrolases and the PETases enzymes for efficient plastic degradation.
Figure 4. Fluorimetric detection of PA and TPA using XylS variant proteins via whole-cell biosensors (adapted from Li et al., 2022 4).
AI-based modeling The bioinformatic parts consist of the development of a computational tool that could predict new variant XylS proteins for desired synthetic ligands (here PA and TPA), based on the PocketGen model as a baseline 11. For now, a first prototype, called XylSevo, has been developed during the D4GEN hackathon organized by Genopole in Paris (March 22-24, 2024) and received the hackathon jury’s Coup de Coeur Prize. By refining this model we aim to add new constraints - such as the nature of the mutagenic domain used by the PANCE.T7 tool. The predicted variant sequences will be then compared to the results obtained in the wet lab through directed evolution.
Conclusion
This project proposal describes a cutting-edge approach for the evolution of and selection of novel proteins, with XylS variants having enhanced binding affinities for PA and TPA as our first example. By succeeding in creating this system, our team aims to provide a new XylS with improved sensitivity for the detection of plastic monomers in water that can be further used for the evolution of enzymes for enhanced plastic degradation. Furthermore, the methodology developed in this project could be extended to other applications in biosensors with other proteins that interact directly or indirectly with nucleic acids such as other transcription factors or DNA polymerases.
References
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