Part:BBa_K5466020

Policistronic response to detection of AFB1 by split-ubiquin system

This device elicit three response upon AFB1 detection, activated by (BBa_K5466018) and (BBa_K5466019):

1. Expression of Nb28-S102D Aga2P, a VHH with high affinity to AFB1 anchored to the cell wall, capturing the AFB1 present in the environment.
2. A positive feedback loop amplifies the response, ensuring that upon the first detection of aflatoxin, the maximum response is triggered. This allows the yeast cell wall to become saturated with Nband ensuring that the response is sustained over time.This positive feedback occurs because the transcription factor, that activates the expression of this device, is synthesized.
3. A reporter RFP that allows us to visualize the presence of aflatoxin in the environment and confirms that the response is occurring.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1272
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

IGG6/NAL10

To reduce the length and complexity of genetic constructs, systems for polycistronic expression are required, permitting the expression of several genes from a single promoter and avoiding a multiple promoter strategy.

That why we use IGG6, to enable polycistronic gene expression in yeast and generate a complex response.

Display of Anti-AFB1 Nb

Displaying the NB28-S102D nanobody (Nb) on the yeast cell wall via Aga2P allows the engineered yeast to maximize its surface area with Nbs ready to capture AFB1. Due to its high sensitivity and binding capacity, this method is highly efficient for capturing AFB1 from the environment. We opted to anchor the nanobody to the yeast surface because our goal is to use this in a probiotic yeast to capture AFB1 in the intestine.

If the VHHs were secreted, they could dilute in the vast intestinal environment. By exposing them on the cell, the yeast retains the proteins on its surface, ensuring they do not disperse or degrade easily in the intestinal environment. We expect to prevent degradation by proteolytic enzymes in the digestive tract, keeping the proteins more protected thanks to robutness of the cell wall. Additionally, secreting proteins could disrupt the balance of the intestinal microbiome or trigger an unwanted immune response. By displaying it, we minimize their impact on the intestinal environment and reduce the risk of adverse effects.

This same approach could be applied to capture other toxins or molecules.

AntiAFB1- Nb28-S102D

We choose a sdAb, specifically a VHH, also known as a nanobody (Nb), to capture AFB1 because it is particularly effective due to its high solubility, exceptional stability, and high affinity. Nbs are significantly smaller than conventional antibodies. Recent studies indicate that Nbs can form concave-shaped binding sites for small molecules, similar to conventional antibodies.

The half-maximal inhibitory concentration (IC50) is a metric used to assess the potency of a substance in inhibiting a biological or biochemical function. It represents the concentration of an inhibitory agent (such as a drug) required to reduce a specific biological process or target by 50% in vitro. This target could be an enzyme, a cell, or a cell receptor. In this case, the lower the IC50, the less AFB1 is required to bind the 50% of the nanobodies. Nb 28-S102D has an IC50 of 1.18 ng/mL.

Protein displays

Aga2p can utilize either the N- or C-terminus for surface protein display, and it can also use both termini to display two heterologous proteins as part of one fusion protein. We demonstrate that various proteins can be anchored in this manner while retaining their functional activity. In one instance, Lim et al. (2017) achieved dual expression of a fluorescent protein alongside a ligand, receptor, or antibody fragment. This approach reduces both time and cost, streamlining the determination of equilibrium binding constants compared to conventional yeast surface display methods. Additionally, Lim et al. (2017) demonstrate 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.

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-terminal end of the scFv.

LexA-VP16

This device is activated by LexA-VP16 and also, it produce LexA-VP16. This create a positive feedback ensuring that the response is sustained, and aumented, over time. This allows the yeast cell wall to become saturated with Nb against AFB1.

The LexA-VP16 is a ortholog synthetic receptor. An orthogonal system should deliver its intended functions with minimal, or ideally no, cross-talk with the host organism. The concept of orthogonality is critical in this system because if the positive feedback loop affected another gene, it could compromise the yeast's metabolism and endanger its integrity.

Reporter

We use a reporter RFP to visually indicate the presence of aflatoxin in the environment. When AFB1 is detected, the expression of RFP provides a clear signal, allowing us to monitor the response in real time. This serves as both a confirmation that the detection system is actively responding to the toxin and a convenient method to track the process without the need for complex or time-consuming assays. The fluorescence of RFP acts as a reliable indicator, ensuring that the system is functioning as intended in various environments.

References

Harmsen, M. M., & De Haard, H. J. (2007). Properties, production, and applications of camelid single-domain antibody fragments. Applied Microbiology and Biotechnology, 77(1), 13–22. https://doi.org/10.1007/s00253-007-1142-2

He, T., Wang, Y., Li, P., Zhang, Q., Lei, J., Zhang, Z., Ding, X., Zhou, H., & Zhang, W. (2014). Nanobody-Based Enzyme Immunoassay for Aflatoxin in Agro-Products with High Tolerance to Cosolvent Methanol. Analytical Chemistry, 86(17), 8873-8880. https://doi.org/10.1021/ac502390c

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

Rantasalo, A., Czeizler, E., Virtanen, R., Rousu, J., Lähdesmäki, H., Penttilä, M., Jäntti, J., & Mojzita, D. (2016). Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae. PLoS ONE, 11(2), e0148320. https://doi.org/10.1371/journal.pone.0148320

Wang, Z., Mathias, A., Stavrou, S., & Neville, D. M. (2005). A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities. Protein Engineering Design And Selection, 18(7), 337-343. https://doi.org/10.1093/protein/gzi036

Yue, Q., Meng, J., Qiu, Y., Yin, M., Zhang, L., Zhou, W., An, Z., Liu, Z., Yuan, Q., Sun, W., Li, C., Zhao, H., Molnár, I., Xu, Y., & Shi, S. (2023). A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi. Nature Communications, 14(1). https://doi.org/10.1038/s41467-023-40027-0