De Sonakshi, Stadlmayr Gerhard, Rebnegger Corinna, Mattanovich Diethard, Gasser Brigitte
Austrian Centre of Industrial Biotechnology (acib), Vienna, Austria.
Department of Biotechnology and Food Science, Institute of Microbiology and Microbial Biotechnology, BOKU University, Vienna, Austria.
Appl Microbiol Biotechnol. 2025 Aug 4;109(1):178. doi: 10.1007/s00253-025-13562-7.
The non-conventional yeast Komagataella phaffii (syn Pichia pastoris) is a well-established host for biotechnological production processes, especially for recombinant protein production. Such processes are mostly run at neutral or slightly acid pH values between pH 5.0 and 6.5, but K. phaffii can grow also at lower or higher pH. Strikingly, we found that K. phaffii displays pH-dependent flocculation at pH 4, which is reversible when the cells are shifted to higher or lower pH. Six members of the flocculin (FLO) gene family were differentially regulated at pH 4.0. Of these, Flo5-1 was revealed to be crucially involved in the pH-triggered flocculation behavior, as cells lacking this flocculin (flo5-1Δ) settled at a much faster rate in the sedimentation assays. The transcriptional regulator Nrg1 was identified to negatively regulate this process, and cells overexpressing NRG1 do not show the pH-dependent flocculation phenotype. In contrast to the model yeast Saccharomyces cerevisiae, neither the flocculin Flo11 nor the transcriptional activator Flo8 are involved in pH-dependent flocculation, once again highlighting the importance of studying transcriptional regulation mechanisms in non-conventional yeasts. KEY POINTS: • Komagataella phaffii shows flocculation at pH 4, which is reversible at other pH. • Six FLO genes are differentially expressed at low pH; flo5-1Δ flocculates stronger. • K. phaffii Nrg1, but not Flo8, is involved in regulating pH-dependent flocculation.
非常规酵母毕赤酵母（Komagataella phaffii，曾用名巴斯德毕赤酵母Pichia pastoris）是生物技术生产过程中成熟的宿主，尤其适用于重组蛋白的生产。此类生产过程大多在pH 5.0至6.5的中性或微酸性pH值下进行，但毕赤酵母也能在更低或更高的pH值下生长。令人惊讶的是，我们发现毕赤酵母在pH 4时表现出pH依赖性絮凝，当细胞转移到更高或更低的pH值时，这种絮凝是可逆的。絮凝素（FLO）基因家族的六个成员在pH 4.0时受到不同程度的调控。其中，Flo5-1被发现与pH触发的絮凝行为密切相关，因为缺乏这种絮凝素的细胞（flo5-1Δ）在沉降试验中沉降速度要快得多。转录调节因子Nrg1被确定对这一过程起负调控作用，过表达NRG1的细胞不表现出pH依赖性絮凝表型。与模式酵母酿酒酵母不同，絮凝素Flo11和转录激活因子Flo8均不参与pH依赖性絮凝，这再次凸显了研究非常规酵母转录调控机制的重要性。要点：• 毕赤酵母在pH 4时表现出絮凝，在其他pH值下可逆。• 六个FLO基因在低pH下差异表达；flo5-1Δ絮凝更强。• 毕赤酵母的Nrg1而非Flo8参与调节pH依赖性絮凝。
