CONTRIBUTION

Bacterial systems

Among the options for the construction of novel synthetic receptors in bacterial cells, are the engineering of one-component and two-component systems.

Two component systems (TCSs) consist of two elements: a transmembrane sensor histidine kinase (HK) domain, and a cytosolic response regulator (RR) domain. The signaling capacity of these systems relies on the phosphorylation exerted upon stimulus by the histidine kinase on the response regulator, which is then activated and induces expression of effector genes (Lazar & Tabor, 2021). Most of the studies engineering this kind of systems focus on rewiring sensor or RR domains from TCSs of other species, allowing to increase the range of inputs and outputs of the receptor. However, this approach is limited by the availability of characterized TCSs since, to our knowledge, no sdAbs or scFvs have been applied as sensor domains within this framework.

One component systems consist of single proteins containing both sensor and output domains. The most studied system is Escherichia coli’s CadC transmembrane transcriptional activator, on which modification relies the technology of EMeRALD (Engineered Modularized Receptor Activated via Ligand-induced Dimerization).

CadC is a bitopic transmembrane protein with a cytoplasmic split-DNA binding domain and a periplasmic pH-sensor in which dimerization of the sensor domain triggers dimerization of the cytoplasmic DBD and transcriptional activation (Chang et al., 2021).

EMeRALD exploits the modular structure of this one-component system to generate new receptors by swapping sensor domains and introducing desired ligand binding domains (LBDs). This approach has been proven compatible with LBDs from other one-component systems as well as antibody-derived domains including VHH and scFvs (Chang et al., 2017), boosting the range of ligands of application.

In view of this system’s scalability and modularity, we conceived the idea of taking this property a step further and designed a strategy for the rapid assembly of EMeRALD based-receptors. By integrating a new fusion site to the MoClo standards, the different receptors can be assembled by Golden Gate thanks to the fusion of its forming domains: the LBDs on one side and the DBD together with the transmembrane and linker regions on the other. The mentioned fusion site, defined based on the sequence of the later fragment (unaltered in all receptors) consists of the nucleotides TGGC, which are also appended to the LBDs to maintain the reading frame.

This strategy allows for teams to easily construct new receptors for novel ligands once the CadC fragment has been cloned and verified, making it a safe bet for future projects undertaken by the group.

A Visualization of the designed standard fusion site for EMeRALD receptor assembly

Mammalian systems

Mammals possess by far the greatest diversity of synthetic receptors. A large number of synthetic receptor platforms have been developed for use in mammalian systems. Despite not being the key point of our discussion, we do pay attention to GEMS (Generalized Extracellular Molecule Sensor).

The GEMS platform was presented by Scheller et al. (2018) as a versatile strategy for designing receptor scaffolds in mammalian cells that facilitate specific molecular inputs to activate various signaling pathways. The GEMS platform is based on ligand induced dimerization of antibody-derived LBDs, which trigger activation of intracellular signaling domains. The core element of the system consists of the transmembrane and extracellular D2 domain from the Erythropoietin Receptor (EpoR), which was demonstrated to provide robust signaling and high signal-to-noise ratio in all the combinations constructed.

This included scFvs and VHH against epitopes of different nature, including a synthetic dye, nicotine, a peptide tag, and the prostate-specific antigen (PSA) biomarker; which where used to activate different pathways upon dimerization, ranging from JAK/STAT to MAPK, PLCG, and PI3K/Akt. Therefore, the system was established as a highly versatile tool.

For teams further interested in developing synthetic receptors in mammalian systems, we recommend the following readings: Manhas et al. (2022) "The Evolution of Synthetic Receptor Systems" and Teng et al. (2024) "Programmable Synthetic Receptors: The Next Generation of Cell and Gene Therapies."

Yeast systems

Regarding yeast hosts, many efforts have been made to produce synthetic receptors in the model Saccharomyces cerevisiae, including the engineering of G-protein-coupled receptors (Lengger & Jensen, 2019).

A widely applied tool in these eukaryotic organisms is the Yeast Two-Hybrid System (Y2H). This technique was originally designed to detect whether two proteins, referred to as "prey" and "bait", interacted with each other, thanks to their fusion to the DNA-binding (DB) or activation domain (AD) of a split transcription factor. This system has also been re-engineered for assessment of membrane protein interactions (mbY2H) through the split-ubiquitin system (SUS), which is based on the release of an attached transcription factor upon interaction and ubiquitin reconstitution.

Both of this techniques have been repurposed for application as synthetic receptors (Brückner et al., 2009; Su et al., 2022), by substituting the “bait” and “prey” proteins with scFvs or sdAbs , which can promote ligand-induced dimerization of fused domains, restoring their ability to activate gene expression and producing a response to the target.

One of this approaches, named Patrol Yeast (Su et al., 2022), drew inspiration from GEMS on the use of EpoR as a receptor scaffold to create a signaling platform in S. cerevisiae based on the split-ubiqutin system. This resulted in a system composed of LBD-EpoR-NubG and LBD-EpoR-Cub-TF proteins which was demonstrated to admit scFvs and sdAbs (including VL and VH chains) as ligand binding domains.

Similarly, due to this system’s modularity and versatility, we designed an extended-MoClo standard by adding two novel fusion sites in order to assemble this kind of receptors, with compatibility with the Open Yeast Collection (OYC). Both fusion sites are taken from the sequence of the central EpoR scaffold and admit interchangeability of both LBDs and intracellular domains. This not only permits rapid generation of new receptors once EpoR, NubG and Cub-TF have been cloned, but also allows for assessment of the dimerization capacity of the LBDs prior to the implementation of the receptor. This is possible thanks to the attachment of split-fluorescent proteins, which rapidly produce a signal upon reconstitution.

A Visualization of the designed standard fusion site for Patrol Yeast receptor assembly

Conclusion

We reviewed available systems for the construction of synthetic transmembrane receptors and identified two promising systems for application in bacteria and yeasts, namely EMeRALD and GEMS-split-ubiquitin.

We generated new parts for the application of both systems to AFB1 detection, which can be taken by teams as a starting point for their own designs:

• CadC (BBa_K5466012)
• EpoR (BBa_K5466007)
• NubG (BBa_K5466008)
• Cub (BBa_K5466009)
• Split mCerulean N-terminal (BBa_K5466031)
• Split mCerulean C-terminal (BBa_K5466032)

Furthermore, we propose a new standard to assemble this kind of receptors through Golden Gate Assembly, which we expect to facilitate the generation of novel receptors. The adhesion to this standard eliminates the need to resynthesize whole receptors upon exchange of a single domain and easily admits parts pre-owned by teams by amplification with primers harboring the proper overhangs for addition of the corresponding fusion site.

A bit of context

Recombinant proteins are expressed on the cell surface to facilitate the high-throughput measurement of biophysical and biochemical properties. Since the first surface expression system was introduced on bacteriophages in the 1980s, various methods have been developed for displaying proteins on the surfaces of bacteria, yeast, insects, and mammalian cells. In these systems, cells express a protein variant of interest, fused to an anchor protein, which is directed by a signal sequence to the cell wall or membrane, making it accessible to the extracellular space (Lim et al., 2007).

Yeast surface display has been widely used to engineer proteins with enhanced binding affinity, thermal stability, or modified catalytic properties. It offers key advantages over other platforms, such as eukaryotic post-translational modifications, ease of culturing and genetic manipulation, and compatibility with flow cytometry (Lim et al., 2007).

Why Aga2p?

Traditional yeast surface display vectors fuse the protein of interest to either the N- or C-terminus of a cell wall-anchored protein. These proteins are often tagged with epitopes for flow cytometry detection using fluorescent antibodies. A common anchor protein, Aga2p, is covalently linked to Aga1p on the yeast cell surface via disulfide bonds, ensuring proper display (Lim et al., 2007).

A optimiez strategy allows both the N- and C-termini of Aga2p to be used for displaying two heterologous proteins in a single fusion construct while maintaining their functionality. For instance, Lim et al. (2017) demonstrated this method by co-expressing a fluorescent protein along with a ligand, receptor, or antibody fragment. This approach reduces both time and cost, facilitating the determination of equilibrium binding constants compared to conventional yeast surface display methods. Additionally, Lim et al. (2017) demonstrated that dual expression of the bioconjugation enzyme Staphylococcus aureus sortase A and its corresponding peptide substrate, within the same Aga2p construct, allows for the measurement of catalytic activity on a non-natural substrate. This method is simpler and more versatile than previously reported approaches (Wang et al., 2005).

A Protein fusion with Aga2p N-terminus. B Protein fusion with Aga2p C-terminus. C Protein fusion with Aga2p N- and C-termini

Optimizing sdAb and scFv display

Firstly, the N-termini of the heavy and light chain variable domains in all immunoglobulin subtypes are located near the CDRs, which are responsible for antigen recognition. Therefore, fusions to the N-terminus of these binding domains may interfere with antigen binding (Lim et al., 2007).

Previously applied display strategies involved fusion of AGA2P and its signal peptide to the N-terminus of the protein of interest. Wang et al. (2005) showed increased affinity constants for displayed scFvs when AGA2 signal peptide was attached to the N-terminal and the rest of the protein was fused to the C-terminus end of the scFv (Lim et al., 2007).

They observed that when the scFv had low affinity, fusing it to the C-terminus of Aga2p through its N-terminal end further reduced its affinity to the point where it couldn't be measured. However, fusing the scFv to the N-terminus of Aga2p through its C-terminal end allowed measurements in the micromolar range. To directly compare how the fusion orientation affects affinity, they repeated the experiments with a higher-affinity scFv. Fusing the scFv through the C-terminal end resulted in a threefold increase in affinity, and additionally, the maximum binding capacity was increased by 20% (Lim et al., 2007).

Wang et al. (2005) demonstrated the importance of specific amino acids for proper peptide cleavage, and proposed to add AG to the C-terminus of Aga2p signal peptide based on their findings. After cloning the scFv insert directly between the Aga2p leader sequence and the Aga2P protein, the fusion protein was not properly cleaved, resulting in no detectable signal. It was later discovered that, without the scFv insert, the EF dipeptide—derived from the EcoRI recognition site used to fuse the desired displayed protein with Aga2P—was positioned at the terminus of the Aga2 signal peptide. Previous studies indicated that the +1 and +2 amino acids at the N-terminus of the fusion protein are critical for signal peptidase processing. Several sequences (EF, EA, EAEA, and AG) were tested, all can be cut away by correct peptidase processing and successfully induced display. AG, being neutral and minimally bulky, was chosen as the default peptide spacer.

Our parts

Here we present the parts for other teams to get inspired:

• AGA2P Signal Peptide (BBa_K5466002) with an AG tail to optimize peptide cleavage.
• A flexible linker (BBa_K5466003) to connect the protein of interest to Aga2p.
• AGA2P without N-terminal signal peptide (BBa_K5466005) for fusion with the protein of interest.

As an example from our project, we fused an anti-aflatoxin sdAb to Aga2p for expression on the cell wall: Nb28-S102D Aga2P (BBa_K5466017). We hope this facilitates the work of future iGEM teams, whether for capturing ligands from the environment or studying proteins.

Conclusion

Thanks to these discoveries, yeast surface display can be optimized by reducing the complexity of many investigations. The ability to fuse proteins at both the N- and C-termini, specifically fusing scFvs and sdAbs via the C-terminus to avoid reducing their affinity, along with understanding critical amino acids in the signal peptide for proper signal peptidase processing, can greatly enhance the efficiency of yeast surface display systems. This information can be applied to develop more efficient systems for capturing toxins in the environment or other target molecules, as we have done in our project.

References

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Chang, H., Mayonove, P., Zavala, A., De Visch, A., Minard, P., Cohen-Gonsaud, M., & Bonnet, J. (2017). A modular receptor platform to expand the sensing repertoire of bacteria. ACS Synthetic Biology, 7(1), 166–175. https://doi.org/10.1021/acssynbio.7b00266

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He, T., Nie, Y., Yan, T., Zhu, J., He, X., Li, Y., Zhang, Q., Tang, X., Hu, R., Yang, Y., & Liu, M. (2021). Enhancing the detection sensitivity of nanobody against aflatoxin B1 through structure-guided modification. International Journal of Biological Macromolecules, 194, 188–197. https://doi.org/10.1016/j.ijbiomac.2021.11.182

Lazar, J. T., & Tabor, J. J. (2021). Bacterial two-component systems as sensors for synthetic biology applications. Current Opinion in Systems Biology, 28, 100398. https://doi.org/10.1016/j.coisb.2021.100398

Lengger, B., & Jensen, M. K. (2019). Engineering G protein-coupled receptor signalling in yeast for biotechnological and medical purposes. FEMS Yeast Research, 20(1). https://doi.org/10.1093/femsyr/foz087

Lim, S., Glasgow, J. E., Interrante, M. F., Storm, E. M., & Cochran, J. R. (2017). Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions. Biotechnology Journal, 12(5). https://doi.org/10.1002/biot.201600696

Manhas, J., Edelstein, H. I., Leonard, J. N., & Morsut, L. (2022). The evolution of synthetic receptor systems. Nature Chemical Biology, 18(3), 244–255. https://doi.org/10.1038/s41589-021-00926-z

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