Design

Chassis Organism

We selected the Yarrowia lipolytica Po1h strain as our chassis organism. Yarrowia lipolytica is a generally regarded as safe (GRAS) non-conventional yeast with several irreplaceable advantages:

Firstly, it possesses exceptionally strong protein secretion capability and remarkable tolerance, enabling it to efficiently secrete target proteins and withstand the harsh conditions of high oxidative stress in the diabetic wound environment(Sanya & Onésime, 2022). Moreover, it can utilize glucose produced by diabetic wounds as a carbon source, allowing it to thrive in this environment. This is the foundation for the chassis organism to perform its therapeutic functions(Arnesen & Borodina, 2022).

Secondly, the Po1h strain is deficient in major extracellular proteases (acidic protease AEP and alkaline protease AXP), which greatly reduces the degradation of heterologous secreted proteins, ensuring the normal expression and maintained bioactivity of the therapeutic proteins.

Based on extensive literature review, we believe Yarrowia lipolytica is the optimal chassis organism for achieving our goal of building a robust and efficient "living therapeutic factory".

Therapy System

We aim to provide a dynamically responsive, programmable, and integrated therapeutic solution for diabetic chronic wounds. Therefore, we designed an intelligent therapy system controlled by multiple signals from the wound microenvironment.

Imparting Long-term Antimicrobial Function to Yeast

We employed a constitutive promoter (hp4d pro) to continuously express the antimicrobial peptide Pexiganan and efficiently display it on the yeast cell surface via a GPI-anchored cell wall protein (GPI-CWPs, YlCWP110) system(Jaafar & Zueco, 2004; Yue et al., 2008).

Img.1 Schematic of the Pexiganan expression plasmid. A constitutive promoter drives the surface display of the antimicrobial peptide Pexiganan. Pexiganan has been reported to exhibit potent antibacterial activity against a variety of bacterial strains isolated from diabetic wounds. For the delivery system, the hp4d promoter was selected to initiate Pexiganan expression during the early stable growth phase of yeast, ensuring tight encapsulation upon application. For details, please refer to our Encapsulation and Delivery System section.

Enabling Yeast to Exert Anti-inflammatory Function upon Glucose Stimulation

To resolve chronic inflammation, we designed an anti-inflammatory circuit triggered by the high-glucose wound microenvironment. Our team analyzed the expression of glycolysis-related genes in Yarrowia lipolytica under high glucose conditions and selected the most responsive PGK promoter to drive the expression of the anti-inflammatory factor IL-4. When the glucose concentration in the wound exudate is high, this circuit is automatically activated, secreting IL-4 to induce macrophage polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, thereby promoting the resolution of wound inflammation(Hassanshahi et al., 2022).

Img.2 Schematic of the IL-4 expression plasmid. The PGK promoter, located upstream of the phosphoglycerate kinase gene—which is essential for glycolysis—demonstrates a strong response to 2% glucose stimulation (for details, get from Results). We propose that upon induction by glucose present in wound exudate, the engineered yeast will produce IL-4 to exert anti-inflammatory effects, enabling dynamic adjustment of cytokine levels based on the volume of exudate.

Using Heat Shock to Promote Angiogenesis

We placed the pro-angiogenic factor VEGF under the control of a heat-sensitive promoter, HSP90. To precisely initiate the proliferation phase, our hardware team designed ROS and pH sensors. When ROS levels decrease below a certain threshold, the sensor will signal the patient. The patient can then visit a hospital where an infrared light irradiation process heats the hydrogel, stimulating massive VEGF expression to promote angiogenesis at the wound site and accelerate healing(Crawford & Ferrara, 2009).

Img.3 Schematic of the VEGF expression plasmid. HSP90 is a heat shock protein in Yarrowia lipolytica. Its promoter can drive heterologous gene expression with nearly a 200% increase at 39°C (for details, see Results). We envision that after being alerted by the sensor integrated within the hydrogel, patients could activate VEGF expression autonomously using an infrared lamp at home or seek clinical assistance for activation.

Experimental Validation Strategy

To ensure the reliability of each functional module, the wet lab team adopted a step-by-step validation strategy:

At the genetic level, PCR and electrophoresis were used to detect successful transformation and the correctness of the target genes. At the phenotypic level, the efficacy of the three expression products was validated separately: Oxford cup assays for antimicrobial activity of transformants displaying the peptide, macrophage polarization assays for IL-4, and cell proliferation assays for VEGF.

Img.4 Overall design of the Yeast Medics therapeutic system. We propose that patients can self-apply the hydrogel containing engineered yeast at home (see Implementation for the instruction manual). With the support of sensors and the robotic arm provided by our hardware team, patients will be empowered to monitor wound healing status autonomously and perform automated disinfection and drug administration.

For details, please proceed to our Results page.

Encapsulation and Delivery System

To ensure that the engineered yeast can function stably, safely, and persistently at the wound site, we developed an encapsulation and delivery system based on a thermosensitive hydrogel.

Thermosensitive Hydrogel Matrix

We selected hydroxybutyl chitosan (HBC) to construct the hydrogel matrix. HBC possesses unique thermosensitive properties: it is liquid at 4°C and transitions to a solid gel at room temperature. The liquid state at low temperatures allows us to inject and fill irregular, deep wounds with the engineered yeast-loaded hydrogel. Its phase transition property enables it to form a solid gel at body temperature, perfectly conforming to the wound bed, providing physical protection and maintaining a moist wound environment(Sun et al., 2020).

Enhanced Adhesion and Photothermal Conversion

To achieve efficient heating via infrared light irradiation for VEGF activation while solving the problem of dressing detachment, we modified HBC with L-DOPA biomimetic modification. The catechol groups of L-DOPA endow the hydrogel with strong tissue adhesion, preventing it from detaching during daily activities. Simultaneously, the polydopamine (PDA) nanoparticles formed by the in-situ oxidative polymerization of L-DOPA significantly enhance the photothermal conversion efficiency of the hydrogel, making the infrared light-triggered VEGF expression control strategy faster and more efficient(Waite & Tanzer, 1981).

Non-covalent and Gentle Encapsulation

We utilize non-covalent interactions, such as multiple hydrogen bonds formed between the antimicrobial peptides displayed on the surface of Yarrowia lipolytica and the hydrogel network, to gently yet firmly encapsulate the engineered yeast cells within the hydrogel network. Experimental verification confirmed that this strategy ensures high encapsulation efficiency while maximizing the maintenance of yeast cell viability, with extremely low leakage rates, guaranteeing safety for clinical application. This encapsulation system not only provides a stable proliferation microenvironment for the engineered yeast but also integrates multiple functions including exudate absorption, physical barrier, active adhesion, and enhanced photothermal control. It is a key guarantee for achieving integrated therapy.

Img.5 The technical path of Yeast Medics

References

[1] Arnesen, J. A., & Borodina, I. (2022). Engineering of Yarrowia lipolytica for terpenoid production. Metabolic Engineering Communications, 15, e00213. https://doi.org/10.1016/j.mec.2022.e00213

[2] Crawford, Y., & Ferrara, N. (2009). VEGF inhibition: Insights from preclinical and clinical studies. Cell and Tissue Research, 335(1), 261–269. https://doi.org/10.1007/s00441-008-0675-8

[3] Hassanshahi, A., Moradzad, M., Ghalamkari, S., Fadaei, M., Cowin, A. J., & Hassanshahi, M. (2022). Macrophage-Mediated Inflammation in Skin Wound Healing. Cells, 11(19), 2953. https://doi.org/10.3390/cells11192953

[4] Jaafar, L., & Zueco, J. (2004). Characterization of a glycosylphosphatidylinositol-bound cell-wall protein (GPI-CWP) in Yarrowia lipolytica. Microbiology, 150(1), 53–60. https://doi.org/10.1099/mic.0.26430-0

[5] Sanya, D. R. A., & Onésime, D. (2022). New roles for Yarrowia lipolytica in molecules synthesis and biocontrol. Applied Microbiology and Biotechnology, 106(22), 7397–7416. https://doi.org/10.1007/s00253-022-12227-z

[6] Sun, M., Wang, T., Pang, J., Chen, X., & Liu, Y. (2020). Hydroxybutyl Chitosan Centered Biocomposites for Potential Curative Applications: A Critical Review. Biomacromolecules, 21(4), 1351–1367. https://doi.org/10.1021/acs.biomac.0c00071

[7] Waite, J. H., & Tanzer, M. L. (1981). Polyphenolic Substance of Mytilus edulis: Novel Adhesive Containing L-Dopa and Hydroxyproline. Science, 212(4498), 1038–1040. https://doi.org/10.1126/science.212.4498.1038

[8] Yue, L., Chi, Z., Wang, L., Liu, J., Madzak, C., Li, J., & Wang, X. (2008). Construction of a new plasmid for surface display on cells of Yarrowia lipolytica. Journal of Microbiological Methods, 72(2), 116–123. https://doi.org/10.1016/j.mimet.2007.11.011