Flo5-1 and Nrg1 are involved in reversible pH-dependent flocculation in Komagataella phaffii.

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.