Grubbe, W. S. | bioRxiv Science

bioRxiv2025

Geometric Tuning of Cytokine Receptor Association Modulates Synthetic Agonist Signaling

Cytokines signal by bringing receptor subunits together, but the role of receptor geometry in shaping signaling remains unclear because natural ligands enforce fixed assemblies. Here, we present a de novo protein design platform that rigidly scaffolds receptor-binding domains into defined spatial arrangements. Applying this across IL-7, type I and III interferons, IL-10, gp130, {beta} common, and synthetic receptor pairs, we show that by varying geometry, we can bias pSTAT pathway usage and tune functional outcomes. Geometric control allowed us to decouple pSTAT1 from pSTAT5 in IL-7, separate antigen presentation (MHC-I) from checkpoint induction (PD-L1) in type I interferons, and suppress pro-inflammatory IFN{gamma} secretion while retaining anti-inflammatory activity in IL-10. We further created minimal IL-6 and IL-3 agonists and strengthened synthetic receptor pairings inaccessible with present cytokines. These results establish receptor geometry as a central determinant of cytokine activity and provide a platform for programmable immune modulation.

synthetic biology↗

bioRxiv2020

Cell-free styrene biosynthesis at high titers

Styrene is an important petroleum-derived molecule that is polymerized to make versatile plastics, including disposable silverware and foamed packaging materials. Finding more sustainable methods, such as biosynthesis, for producing styrene is essential due to the increasing severity of climate change as well as the limited supply of fossil fuels. Recent metabolic engineering efforts have enabled the biological production of styrene in Escherichia coli, but styrene toxicity and volatility limit biosynthesis in cells. To address these limitations, we have developed a cell-free styrene biosynthesis platform. The cell-free system provides an open reaction environment without cell viability constraints, which allows exquisite control over reaction conditions and greater carbon flux toward product formation rather than cell growth. The two biosynthetic enzymes required for styrene production were generated via cell-free protein synthesis and mixed in defined ratios with supplemented L-phenylalanine and buffer. By altering the time, temperature, pH, and enzyme concentrations in the reaction, this approach increased the cell-free titer of styrene from 5.36 {+/-} 0.63 mM to 40.33 {+/-} 1.03 mM, an order of magnitude greater than cellular synthesis methods. Cell-free systems offer a complimentary approach to cellular synthesis of small molecules, which can provide particular benefits for producing toxic molecules. HighlightsA cell-free system for styrene biosynthesis was established. This in vitro system achieved styrene titers an order of magnitude greater than the highest reported concentration in vivo.

synthetic biology↗