Saccharomyces cerevisiae

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Sensing module

Living systems, from bacteria to multicellular organisms, use specialized receptors to detect environmental stimuli and trigger adaptive responses for survival. However, evolutionary constraints limit the range of inputs organisms can process.

Synthetic receptors offer a solution to expand their sensing capabilities. While many have been engineered by repurposing sensor domains from other species, antibody-derived ligand binding domains (LBDs) provide a more versatile tool for creating highly specific detection platforms.

Based on this premise, we engineered a transmembrane AFB1 synthetic receptor inspired in the GEMS platform (Scheller, et al., 2018), where activation is caused by ligand-induced dimerization of extracellular LBDs, which in turn brings together dimerization-dependent signal transduction domains in the intracellular side.

Our receptor is composed of two separate subunits which are succesfully targeted to the plasma membrane thanks to the attachment of the signal peptide from SUC2, a secreted invertase. There, it can come in contact with extracellular molecules including aflatoxin B1. Both receptors subunits exhibit a similar and highly modular structure with the following domains:

Extracellular LBDs

Scaffold region

Intracellular signaling domains

Output module

The output module permits a maintained and effective cellular response against aflatoxin B1. The key regulatory element is a promoter formed by four LexA operators and the core promoter from Saccharomyces cerevisiae TDH3 gene (~200 bp), which is activated by the sensing module.

Upon receptor dimerization, the transcription factor released binds to the mentioned promoter and activates the transcription of three components involved in the cellular response:

High levels of synthesis of these three proteins is achieved by using a single regulated promoter thanks to the addition of the IGG6 DNA sequence.

IGG6 is a 9-bp motif that resulted from the optimization of a fungal intergenic sequence, IGG1, known to generate bicistronic expression in Glarea lozoyensis. The resulting IGG6 has been proven to permit polycistronic expression in other fungal species and is thought to act by mediating translation re-initiation by the ribosome, producing separate proteins from each of the coding sequences of a transcript (Yue et al., 2023). This element allows us to support the production of our three proteins while avoiding exccesive complexity of the design.

Integrated circuit

Altogether, the genetic circuit implemented is able to give an effective response to aflatoxin B1 exposure, following a simple logic:

• AFB1 receptors are constitutively expressed. However when no AFB1 is present, there is low affinity between the receptors. Therefore ubiquitin is not reconstituted and the synthetic transcription factor is not produced. No further genetic expression in the output module occurs.
• Once AFB1 appears, two receptor subunits can simultaneously bind molecules of the toxin, leading to receptor heterodimerization which is transmitted to the intracellular side.
• Reconstitution of the split-ubiquitin permits recognition of the protein by cellular deubiquitinases, which hydrolyze a peptide bond in the C-terminal end of Cub, releasing the synthetic transcription factor attached.
• The sTF binds to its regulatory promoter in the output module, promoting the synthesis of the IGG6-mediated polycistronic transcript.
• A surface VHH and a yeast enhanced-RFP are produced from the transcript, permitting toxin adsorption and detection, respectively.
• Finally, the synthetic transcription factor is also produced and provides positive feedback, locking the system in an activated state in which the yeast turns into an AFB1-trapping “sponge”.

Animated overview of Saccharomyces cerevisiae's circuit behaviour

Testing designs

Apart from the genetic constructs designed to function in the final version of our organism, we generated three modules aimed to test the functionality of each part of our circuit independently.

As a first approach to demonstrate the AFB1-dependent dimerization capacity of scFv1 and scFv2 in the context of a transmembrane receptor based on the GEMS platform, we applied the technique of bimolecular fluorescence complementation (BiFC).

We designed 2 receptor subunits harboring the scFvs and EpoR scaffold, but with a split mCerulean as intracellular domain. Assessment of the fluorescence intensity of this receptor in response to aflatoxin B1 will provide information on the capacity of reconstitution of split proteins upon AFB1-induced dimerization with the receptor architecture proposed.

To evaluate the detoxification potential of our yeast, the same surface VHH intented for expression upon receptor activation was introduced in a constitutively expressed unit harboring the strong TDH3 promoter.

Finally, to check the functionality of IGG6, the key component of our output module, we constructed a polycistronic gene containing the coding sequences for three fluorescent proteins: yeast-enhanced monomeric RFP (yEmRFP), yeast-enhanced GFP (yeGFP) and yeast enhanced CFP (yECFP), all of them expressed under the same TEF1 promoter.

Escherichia coli

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As mentioned, despite not being the final organism utilized for assays in the laboratory, we still designed a sensing-response platform in the model organism Escherichia coli, whose viability is supported by a wide literature.

Sensing module

In this case, we constructed a synthetic receptor built upon the EMeRALD system, which is based on the engineering of E. coli’s CadC protein. CadC is a transmembrane transcriptional activator from the ToxR family which harbors cytosolic split-DNA binding domains (split-DBDs) which can be activated by receptor dimerization.

CadC counts with a periplasmic pH-sensitive domain and is also responsive to lysine concentration due to interaction of its transmembrane region with the lysine permease (LysP). Substitution of these regions with desired alternative domains renders a receptor unresponsive to its natural regulators and with specificity to a novel ligand, in our case: AFB1 (Chang et al., 2018).

Extracellular LBDs

Transmembrane region

Intracellular signaling domains

Output module

After AFB1 exposure and signaling through the sensing module, expression of genes downstream of the CadBA promoter is triggered, deploying the following two molecular responses:

Integrated circuit

Overall, both modules acting together generate a circuit which allows aflatoxin B1 sensing and a rapid response to the toxin with the following expected behavior:

• Due to the leakiness of bacterial inducible promoters, the receptor subunits are expressed at a low rate downstream of pCadBA, providing an enough basal amount of receptors to monitor AFB1 in the environment.
• Once the toxin appears, receptor heterodimerization is produced thanks to the scFvs in the extracellular side of the membrane.
• Reconstitution of CadC’s DNA binding domain allows binding and transcriptional activation under the CadBA promoter.
• The red fluorophore is produced at high concentrations, making a visually identifiable signal.
• The expression of more receptor proteins is induced, maximizing anti-AFB1 paratopes on the surface and increasing the amount of activated receptors, which in turn promote the synthesis of more effector and reporter molecules, providing the same “sponge” effect described above.

Animated overview of Escherichia coli's circuit behaviour

Scalability

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As it has been exposed, both our genetic designs exhibit a highly modular nature. This valuable property allows for re-engineering of the constructions through exchange of ligand binding domains or effector molecules.

This poses a valuable strategy to tackle toxins or molecules different from AFB1, as well as to deploy novel or more complex responses upon stimulus. We propose to facilitate this adaptability through the implementation of an extended MoClo for the construction of GEMS and EMeRALD-based receptors, as described in our Contribution page.

Conclusion

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Therefore, we propose two alternative designs to incorporate a new AFB1-sensitive pathway that allows detection and adsorption of the toxin, both in Saccharomyces cerevisiae and Escherichia coli. From both of the functions achieved, arise several applications to combat the hazard posed by this mycotoxin.

With Saccharomyces cerevisiae being the most applicable to treat aflatoxin B1 contamination, as describen in our Engineering, both of the designs offer a versatile architecture which can be repurposed for coping with other toxins or molecules of interest.