Description:

The Chemical and Biological Engineering Department is pleased to present student seminars by Kevin Fitzgerald and Caleb Lay as part of our Spring 2025 seminar series.

Kevin Fitzgerald will present a seminar titled "Engineering Acinetobacter baylyi ADP1 for Enhanced Cyanophycin Production in Wastewater.”

In alignment with global engineering targets to improve supply chain sustainability, contemporary waste streams are increasingly recognized as promising nutrient sources for biological upscaling. Agricultural and municipal wastewaters, in particular, offer attractive opportunities due to cost-negative substrates and existing treatment infrastructure. Among the value-added products of interest, cyanophycin—a non-ribosomal polypeptide composed primarily of arginine and aspartic acid—has emerged as a compelling target for metabolic engineering due to its ease of purification and potential downstream applications as a nutritional supplement or platform chemical. Critically, cyanophycin is natively synthesized by several wastewater-associated microbes, making it a natural candidate for bioproduction in waste-fed systems.

In this seminar, I will present our ongoing efforts to engineer a strain of the naturally competent bacterium Acinetobacter baylyi ADP1 to produce cyanophycin under conditions suitable for industrial application. As an early contributor to this project in the Tyo lab, I have adapted methods for the separation, visualization, and quantification of cyanophycin. Building on this workflow, I have focused on deregulating the biosynthesis of arginine—the limiting precursor in cyanophycin production—to enable product formation without the need for external amino acid supplementation. I will also share preliminary results from our efforts to modulate the rate of cyanophycin synthesis relative to cell growth, with the goal of optimizing key bioprocess metrics including productivity, titer, and substrate utilization efficiency.

Caleb Lay will present a seminar titled " Enhancing Cell-Free Expression and Oxidative Folding of Complex Disulfide-Bonded Protein Therapeutics.”

Cell-free protein synthesis (CFPS) has emerged as a powerful platform for biomanufacturing, offering rapid, scalable, and distributed production of protein therapeutics. However, CFPS remains inefficient for proteins requiring complex disulfide bonds, limiting its ability to produce difficult-to-express biologics such as monoclonal antibodies and thrombolytics. These proteins frequently misfold and aggregate in vitro, severely reducing their bioactivity. Overcoming this challenge requires a deeper understanding of oxidative folding and the development of strategies to enhance disulfide bond formation.

This work presents the largest systematic study to date on improving disulfide-bonded protein folding in cell-free expression systems, evaluating over 300 oxidative folding effectors selected through an exhaustive literature search and computational analysis. These effectors span a broad range of eukaryotic, bacterial, and archaeal proteins, including well-characterized folding factors from E. coli, yeast, and humans, as well as novel candidates identified through sequence similarity networks. By systematically assessing both orthologous effectors (species variants of known folding proteins) and non-orthologous effectors (diverse classes of oxidoreductases, isomerases, and chaperones), we identified key proteins that significantly enhance the yield of properly folded, active proteins such as reteplase and trastuzumab. These FDA-approved therapeutics rely on correct disulfide bond formation for function and efficacy, making these improvements particularly impactful. Notably, a previously unstudied ortholog of DsbC from Photobacterium damselae—which outperformed native E. coli DsbC, the current gold standard oxidative folding effector used in industry and academia—enhanced active reteplase and full-length trastuzumab production by over 50%. Additionally, we are systematically testing combinations of top-performing effectors and developing combinatorial mimics of periplasmic and endoplasmic reticulum folding networks to recapitulate synergistic interactions that exist in vivo. The development of a high-throughput fluorescence-based reteplase activity assay has accelerated this screening effort, enabling rapid quantification of oxidative folding efficiency and guiding optimization strategies.

By systematically mapping the impact of oxidative folding effectors in CFPS, this work advances the field of synthetic biology and provides a framework for rationally engineering cell-free platforms for therapeutic protein production. These findings not only enhance the capabilities of cell-free systems but also lay the groundwork for decentralized, on-demand biomanufacturing of complex biologics.

Bagels and coffee will be provided at 9:30am, and the seminar will start at 9:40am. Please plan to arrive on time to grab a bagel and mingle!

*Please note that there will be no Zoom option for seminars this year.