Navigating the Yeast Secretory Pathway: A Review of the MFa Signal Sequence

Merkaš, M., Grujicic, N., Geier, M., Glieder, A., & Emmerstorfer-Augustin, A.

This mini-review provides a comprehensive overview of the mating factor alpha (MFa) signal sequence from Saccharomyces cerevisiae, a cornerstone for recombinant protein secretion in yeast. The paper systematically explores the historical context and molecular mechanisms of MFa-guided protein transport via the post-translational secretory pathway. Key challenges are highlighted, including endoplasmic reticulum (ER) aggregation, protein missorting, and inefficient proteolytic processing by Kex2 and Ste13, which often limit secretion efficiency and product quality. The authors summarize major innovations to overcome these bottlenecks, such as targeted mutagenesis, structural deletions within the MFa pro-region, and the development of hybrid signal sequences. The central finding is that while MFa remains a highly effective and widely used secretion leader, its performance is often protein-specific. This underscores the importance of tailored engineering strategies and continued research into more universally robust secretion systems for biotechnological and therapeutic applications.

In a recent mini-review published in Applied Microbiology and Biotechnology, researchers Magdalena Merkaš, Anita Emmerstorfer-Augustin, and colleagues provide a detailed and current overview of the mating factor alpha (MFa) signal sequence, one of the most crucial tools for producing heterologous proteins in yeast expression systems. The work, originating from a strong Austrian research consortium including the Graz University of Technology, the Austrian Centre of Industrial Biotechnology (ACIB), BioTechMed-Graz, and the company Bisy GmbH, consolidates decades of research and outlines modern strategies for optimizing this pivotal component of yeast biotechnology.

Mechanisms and secretion bottlenecks

The paper elaborates on how the MFa pre-pro-leader sequence directs target proteins into the post-translational secretion pathway in yeast. This process, while generally robust, is fraught with several well-documented bottlenecks that can severely limit protein yield and quality. The authors identify three primary challenges:

Incorrect Proteolytic Processing: The sequential cleavage of the pro-region by the Kex2 and Ste13 proteases is a critical step. Inefficient processing can lead to the secretion of proteins with unwanted N-terminal extensions (e.g., EAEA sequences), which can impair function and complicate downstream processing, a significant issue for therapeutic protein production.
ER Aggregation and Stress: The MFa pro-region itself is prone to misfolding and aggregation within the endoplasmic reticulum (ER). This can trigger the unfolded protein response (UPR), placing a metabolic burden on the cell and ultimately blocking the secretory pathway.
Protein Missorting: For some proteins, the post-translational translocation mechanism allows for premature folding in the cytosol, which can impede their entry into the ER and lead to missorting to the vacuole for degradation.

Engineering strategies and key innovations

A core focus of the review is the summary of innovative engineering approaches developed to mitigate these issues. The research highlights that there is no universal solution; rather, optimization often requires a strategy tailored to the specific recombinant protein. Key advancements discussed include:

Mutational Analysis and Truncation: Targeted point mutations and deletions within the MFa sequence have proven highly effective. For instance, the authors note studies where deleting helical regions of the pro-peptide led to a more than 50% increase in the secretion of reporter proteins like horseradish peroxidase (HRP) and Candida antarctica lipase B (CalB).
Hybrid Signal Leaders: A particularly promising strategy is the creation of chimeric signal sequences. The review highlights the success of fusing the pre-region of the Ost1 signal peptide, which facilitates co-translational translocation, with the pro-region of MFa. This hybrid leader has been shown to dramatically increase the secretion of difficult-to-express proteins by shifting the translocation mechanism and avoiding cytosolic folding issues.

Significance and future outlook

This review underscores the central role of Austrian science, driven by researchers like Merkaš and Emmerstorfer-Augustin, in advancing industrial biotechnology. Supported by Austrian funding bodies such as the FFG and the COMET Center ACIB, this work positions the national research landscape at the forefront of protein expression technology.

For the broader scientific community, the paper serves as an essential guide for troubleshooting and enhancing protein secretion in yeast. It makes clear that while MFa remains the „top choice“ for recombinant secretion, its optimization is a complex, multi-factorial problem. The future of the field, as the authors suggest, lies in combining sequence engineering with host strain engineering and leveraging high-throughput screening and machine learning to discover and design novel, more predictable, and universally applicable signal peptides. These advancements are critical for the cost-effective industrial production of biopharmaceuticals, enzymes, and other high-value proteins.