The pH Switch: Unraveling Yeast Flocculation for Enhanced Bioproduction

De, S., Stadlmayr, G., Rebnegger, C., Mattanovich, D., & Gasser, B.

Komagataella phaffii is a vital yeast in biotechnology, particularly for producing proteins. Understanding its cellular behavior is crucial for efficient industrial processes. This study reveals that K. phaffii surprisingly forms reversible cell clumps, known as flocs, when exposed to mildly acidic conditions around pH 4. Researchers pinpointed key genetic components responsible for this phenomenon: a specific flocculin gene, Flo5-1, which appears to control rather than promote clumping, and a regulatory protein, Nrg1, that negatively influences flocculation. This unique mechanism differs from that in traditional model yeasts. These findings offer important insights into how K. phaffii responds to its environment, providing a foundation for developing better strategies to manage cell separation in bioproduction, ultimately enhancing efficiency and sustainability in various biotechnological applications.

The yeast Komagataella phaffii (Pichia pastoris) is crucial in modern biotechnology, especially for producing proteins. Controlling yeast cell behavior is vital for efficient industrial processes. One key behavior is flocculation – where cells clump together and settle. When controlled, this can significantly simplify product separation, reducing costs. Researchers from leading Austrian institutions, including the Austrian Centre of Industrial Biotechnology (acib) and BOKU University Vienna, have made a significant discovery about K. phaffii‘s flocculation. Published in Applied Microbiology and Biotechnology, their study uncovers a fascinating pH-dependent clumping mechanism with major industrial implications.

A pH-controlled clumping switch

K. phaffii cells clump most effectively at a mildly acidic pH of 4.0. This clumping is reversible: changing the pH disperses flocs. Calcium ions accelerated clumping, indicating flocculin involvement. This precise pH-responsive clumping offers a new, simple control mechanism.

Surprising gene roles and unique mechanisms

The study investigated the genetic factors behind this phenomenon. Six flocculin (FLO) genes showed altered activity at low pH. The gene Flo5-1 was particularly surprising: its deletion actually increased flocculation, suggesting it normally inhibits clumping or acts as a sensing protein.

Crucially, the study identified Nrg1, a transcriptional repressor, as a key player. Nrg1 was found to negatively regulate pH-dependent flocculation; overexpressing Nrg1 significantly reduced clumping. Nrg1 acts as a powerful genetic switch. Unlike Saccharomyces cerevisiae, K. phaffii‘s Flo8, a key regulator in other yeasts, had no role in this process. This highlights the need to study non-conventional yeasts directly, as their biological pathways can differ.

Revolutionizing bioprocesses and austrian innovation

These findings have substantial implications for biotechnology. Current cell separation methods are often costly and complex. Inducing or preventing flocculation via simple pH adjustments or genetic engineering of Flo5-1/Nrg1 offers a cost-effective, sustainable alternative for biomass separation. This means reduced manufacturing costs for biopharmaceuticals, more efficient processes due to minimized energy and resource consumption, and greater control for optimizing industrial bioreactors. This research advances yeast biology and provides new tools for tailor-made K. phaffii strains. Nrg1’s role opens new avenues for understanding K. phaffii’s multicellular structures under stress. This groundbreaking work was primarily conducted by leading Austrian research institutions, funded by Austrian programs and ministries including the Austrian Federal Ministry of Economy, Energy and Tourism (BMWET), the Austrian Federal Ministry of Innovation, Mobility and Infrastructure (BMIMI), the Austrian Science Fund (FWF), and the COMET Funding Program (FFG). This national support underscores Austria’s dedication to cutting-edge biotechnology, strengthening its global position and contributing to a sustainable bioeconomy.