# An integrated cell and medium engineering approach for production of a nanobody fusion in Saccharomyces cerevisiae

## TL;DR

Researchers improved yeast's ability to produce a nanobody fusion protein by combining genetic modifications and optimized growth conditions.

## Contribution

A novel integrated approach combining cell engineering and medium optimization to enhance production of high-molecular-weight proteins in yeast.

## Key findings

- Deleting PEP1, VPS30, MON2, and ALG3 genes reduced protein degradation and improved production.
- A small-scale fed-batch system with amino acid-rich medium and chemical chaperones increased secreted protein titers.
- The engineered yeast strain and cultivation system enabled production of a nanobody-Fc fusion at 2.5 µg/mL.

## Abstract

Saccharomyces cerevisiae is an established production host for therapeutic proteins; many of those are small proteins such as insulin or glucagon-like peptide-1 (GLP-1) analogs. Contrastingly, proteins of higher molecular weight, foremost antibodies, did not reach the market due, among other factors, to limiting productivity. Here we addressed the loss of product to protein degradation through a combination of genetic engineering of the host and medium optimization. We screened target genes that either directly or indirectly can lead to proteolytic degradation. We identified four deletions that are beneficial for expression: PEP1 and VPS30, which both can channel proteins to the vacuole for degradation; MON2, which can lead to the re-uptake of secreted proteins; and ALG3, which can affect the permeability of the cell wall. In parallel, we developed a small-scale fed-batch cultivation system for 24-well deep well plate cultivations and using an amino acid-rich medium. To stabilize secreted proteins, we screened chemical chaperones and osmolytes. We fortified the medium with arginine, 4-phenylbutyrate (4-PBA), and Tween-20. Using the engineered yeast strain, which features VPS30, PEP1, and ALG3 deletions, and the small-scale fed-batch system, we obtained 2.5 µg/mL of a secreted chimeric fusion of a nanobody to the crystallizable fragment (Fc) of a human immunoglobulin. Instrumental to the increase in the final titer were the reduced losses. This was achieved by a combination of complementary measures: improving diffusion through the cell wall, achieved through genetic engineering, and reducing losses to proteolytic degradation through medium optimization and genetic engineering. Moreover, we showed that the engineered strain and cultivation set-up are suitable for the production of different antibodies.

• Chemical chaperones and amino acid-rich medium increased secreted protein titers.

• Medium and host engineering are instrumental for improving productivity.

• Small-scale cultivation system enables production levels suitable for characterization.

The online version contains supplementary material available at 10.1007/s00253-025-13700-1.