GSE131106
Pseudohyphal differentiation in Komagataella phaffii: investigating the FLO gene family
The protein production host Komagataella phaffii has possess the ability to differentiate into pseudohyphal form when cultivated at slow growth rates (µ=0.05 h-1) in glucose-limited chemostats. In this study, we investigated the K. phaffii FLO gene family in the context of pseudohyphae formation. Transcriptional analysis helped us identify 3 possible responsible genes, FLO11, FLO400 and FLO5-1, all of which are under control of Flo8, a transcription factor whose disruption prevents pseudohyphae formation. Knocking out FLO11 revealed that this is not the sole protein responsible for this phenotype. Strikingly, the expression of FLO400 and FLO5-1 was negatively correlated with pseudohyphae formation, and shown to be under epigenetic control by FAIRE-Seq analysis. Knock outs of these two genes completely inhibited the appearance of pseudohyphal cells and prevented the expression of FLO11. Even though the    mechanism is unclear at present, we propose that in K. phaffii Flo400 and/or Flo5-1 act as upstream signals that lead to the induction of FLO11 expression upon severe glucose limitation in chemostats at slow growth rate, and that the expression of FLO400 and FLO5-1 is controlled by epigenetic silencing, which acts independently from the general activation of FLO gene expression by the transcriptional regulator Flo8. Overall design: Comparison of transcript levels of FLO genes of Komagataella phaffii (wild-type strain) when cultivated in a glucose-limited chemostat at fast (µ=0.1 h-1), slow (µ=0.05 h-1) and then switched back to fast (µ=0.1 h-1) growth rates. Samples were taken after 5 residence times at each growth rate, in triplicates. A similar cultivation was carried out for the K. phaffii strain with disrupted FLO8 gene to identify the FLO genes under the control of the Flo8 transcription factor.  In this case samples were taken at the fast (µ=0.1 h-1) and the slow (µ=0.05 h-1) growth rates.