Pseudohyphal differentiation in Komagataella phaffii: investigating the FLO gene family

Abstract

Many yeasts differentiate into multicellular phenotypes in adverse environmental conditions. Here, we investigate pseudohyphal growth in Komagataella phaffii and the involvement of the flocculin (FLO) gene family in its regulation. The K. phaffii FLO family consists of 13 members, and the conditions inducing pseudohyphal growth are different from Saccharomyces cerevisiae. So far, this phenotype was only observed when K. phaffii was cultivated at slow growth rates in glucose-limited chemostats, but not upon nitrogen starvation or the presence of fusel alcohols. Transcriptional analysis identified that FLO11, FLO400 and FLO5-1 are involved in the phenotype, all being controlled by the transcriptional regulator Flo8. The three genes exhibit a complex mechanism of expression and repression during transition from yeast to pseudohyphal form. Unlike in S. cerevisiae, deletion of FLO11 does not completely prevent the phenotype. In contrast, deletion of FLO400 or FLO5-1 prevents pseudohyphae formation, and hampers FLO11 expression. FAIRE-Seq data shows that the expression and repression of FLO400 and FLO5-1 are correlated to open or closed chromatin regions upstream of these genes, respectively. Our findings indicate that K. phaffii Flo400 and/or Flo5-1 act as upstream signals that lead to the induction of FLO11 upon glucose limitation in chemostats at slow growth and chromatin modulation is involved in the regulation of their expression.

Keywords: FLO genes; Komagataella phaffii; Pichia pastoris; FAIRE-Seq; epigenetics; pseudohyphal growth; specific growth rate.

Figure 1.

Schematic representation of the structures of the K. phaffii FLO family proteins. (A)Line diagrams representing the Flo proteins with their predicted N-terminal signal peptides, identified conserved domains and GPI anchor. (B) Representation of Flo8, master transcription regulator controlling several FLO genes harboring an N-terminal Lis homology (LisH) domain, a putative single‐stranded DNA‐binding protein (SSDP) domain and a nuclear localization signal (NLS).

Figure 2.

Location of the FLO genes on the chromosomes of K. phaffii CBS7435. // indicates the localization of the centromeres.

Figure 3.

Cultivation at low dilution rate (corresponding to µ = 0.05 h⁻¹) in glucose-limited chemostat triggers pseudohyphal growth in K. phaffii, which seems to be connected to the expression of FLO11. (A) Growth rate profile of the glucose-limited chemostat experiments, sampling time points (S1–S5) and cell morphology of the wild-type and the flo8Δ strains.(B) The plot shows the transcript level of FLO11 in the wild-type and the flo8Δ strains, measured by qRT-PCR, relative to the reference sample S1 in the wild type.

Figure 4.

Microscope pictures of both the wild-type and the flo11Δ strains at different sampling time points. The graph shows the percentage of pseudohyphal cells in the population for both the wild-type and the flo11Δ strains (n = 3).

Figure 5.

qRT-PCR data showing expression pattern of several FLO genes relative to the reference sample (wild type in S1). Asterisks above the bars denote statistical significance of gene expression levels in comparison with the reference sample (P-values calculated by Student'st-test; **P-value < 0.01 and ***P-value < 0.001).

Figure 6.

Open chromatin regions identified in the proximity of FLO genes by FAIRE-Seq analysis. Screenshot from IGB showing differential peaks upstream of FLO5-1 and FLO400 (highlighted in yellow); such statistically differential peaks are not observed in proximity of FLO11. ACT1 is included here as control where an upstream open region is detected in all the three samples.

Figure 7.

FLO5-1 and FLO400 are major players involved in the regulation of pseudohyphal growth. (A)Microscope pictures of flo5-1Δ and flo400Δ strains in both wild-type and flo11Δ backgrounds at sampling time points S1, S3 and S5. (B) qRT-PCR data for FLO11 expression in the flo400Δ (right) and flo5-1Δ (left) strains relative to the reference sample at S1.

Figure 8.

Scheme representing the proposed interplay of Flo8, Flo400, Flo5-1 and Flo11 in the cascade leading to pseudohyphal growth in K. phaffii.