Pseudohyphal differentiation in Komagataella phaffii: investigating the FLO gene family.

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.

Identification and characterization of the Komagataella phaffii mating pheromone genes.

The methylotrophic yeast Komagataella phaffii (Pichia pastoris) is a haploid yeast that is able to form diploid cells by mating once nitrogen becomes limiting. Activation of the mating response requires the secretion of a- and α-factor pheromones, which bind to G-protein coupled receptors on cells of opposite mating type. In K. phaffii, the genes coding for the α-factor (MFα), the pheromone surface receptors and the conserved a-factor biogenesis pathway have been annotated previously. Initial homology-based search failed to identify potential a-factor genes (MFA). By using transcriptome data of heterothallic strains under mating conditions, we found two K. phaffiia-factor genes. Deletion of both MFA genes prevented mating of a-type cells. MFA single mutants were still able to mate and activate the mating response pathway in α-type cells. A reporter assay was used to confirm the biological activity of synthetic a- and α-factor peptides. The identification of the a-factor genes enabled the first characterization of the role and regulation of the mating pheromone genes and the response of K. phaffii to synthetic pheromones and will help to gain a better understanding of the mating behavior of K. phaffii.

The adaptive landscape of wildtype and glycosylation-deficient populations of the industrial yeast Pichia pastoris.

BACKGROUND: The effects of long-term environmental adaptation and the implications of major cellular malfunctions are still poorly understood for non-model but biotechnologically relevant species. In this study we performed a large-scale laboratory evolution experiment with 48 populations of the yeast Pichia pastoris in order to establish a general adaptive landscape upon long-term selection in several glucose-based growth environments. As a model for a cellular malfunction the implications of OCH1 mannosyltransferase knockout-mediated glycosylation-deficiency were analyzed. RESULTS: In-depth growth profiling of evolved populations revealed several instances of genotype-dependent growth trade-off/cross-benefit correlations in non-evolutionary growth conditions. On the genome level a high degree of mutational convergence was observed among independent populations. Environment-dependent mutational hotspots were related to osmotic stress-, Rim - and cAMP signaling pathways. In agreement with the observed growth phenotypes, our data also suggest diverging compensatory mutations in glycosylation-deficient populations. High osmolarity glycerol (HOG) pathway loss-of-functions mutations, including genes such as SSK2 and SSK4, represented a major adaptive strategy during environmental adaptation. However, genotype-specific HOG-related mutations were predominantly observed in opposing environmental conditions. Surprisingly, such mutations emerged during salt stress adaptation in OCH1 knockout populations and led to growth trade-offs in non-adaptive conditions that were distinct from wildtype HOG-mutants. Further environment-dependent mutations were identified for a hitherto uncharacterized species-specific Gal4-like transcriptional regulator involved in environmental sensing. CONCLUSION: We show that metabolic constraints such as glycosylation-deficiency can contribute to evolution on the molecular level, even in non-diverging growth environments. Our dataset suggests universal adaptive mechanisms involving cellular stress response and cAMP/PKA signaling but also the existence of highly species-specific strategies involving unique transcriptional regulators, improving our biological understanding of distinct Ascomycetes species.

Implications of evolutionary engineering for growth and recombinant protein production in methanol-based growth media in the yeast Pichia pastoris.

BACKGROUND: Pichia pastoris is a widely used eukaryotic expression host for recombinant protein production. Adaptive laboratory evolution (ALE) has been applied in a wide range of studies in order to improve strains for biotechnological purposes. In this context, the impact of long-term carbon source adaptation in P. pastoris has not been addressed so far. Thus, we performed a pilot experiment in order to analyze the applicability and potential benefits of ALE towards improved growth and recombinant protein production in P. pastoris. RESULTS: Adaptation towards growth on methanol was performed in replicate cultures in rich and minimal growth medium for 250 generations. Increased growth rates on these growth media were observed at the population and single clone level. Evolved populations showed various degrees of growth advantages and trade-offs in non-evolutionary growth conditions. Genome resequencing revealed a wide variety of potential genetic targets associated with improved growth performance on methanol-based growth media. Alcohol oxidase represented a mutational hotspot since four out of seven evolved P. pastoris clones harbored mutations in this gene, resulting in decreased Aox activity, despite increased growth rates. Selected clones displayed strain-dependent variations for AOX-promoter based recombinant protein expression yield. One particularly interesting clone showed increased product titers ranging from a 2.5-fold increase in shake flask batch culture to a 1.8-fold increase during fed batch cultivation. CONCLUSIONS: Our data indicate a complex correlation of carbon source, growth context and recombinant protein production. While similar experiments have already shown their potential in other biotechnological areas where microbes were evolutionary engineered for improved stress resistance and growth, the current dataset encourages the analysis of the potential of ALE for improved protein production in P. pastoris on a broader scale.