The secretome of <i>Pichia pastoris</i> in fed‐batch cultivations is largely independent of the carbon source but changes quantitatively over cultivation time

Burgard, Jonas; Grünwald‐Gruber, Clemens; Altmann, Friedrich; Zanghellini, Jürgen; Valli, Minoska; Mattanovich, Diethard; Gasser, Brigitte

doi:10.1111/1751-7915.13499

Cited by 16 publications

(14 citation statements)

References 46 publications

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“…[ 27,28 ] Typically, a relatively small amount of endogenous protein is present in the culture supernatant, facilitating down‐stream processing tremendously. [ 29 ] On top of that, P. pastoris can rapidly grow to very high cell densities, allowing large‐scale production for industrial applications. [ 30,31 ]…”

Section: Introductionmentioning

confidence: 99%

Secretion of Pseudomonas aeruginosa Lipoxygenase by Pichia pastoris upon Glycerol Feed

Hashem

Stolterfoht

Rinnofner

et al. 2020

Biotechnology Journal

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Pseudomonas aeruginosa lipoxygenase (PaLOX) catalyzes the peroxidation of unsaturated fatty acids. Not only linoleic acid, but also linolenic acid and oleic acid are oxidized. The natural host secretes PaLOX into the periplasmic space. Herein, the aim is to secrete PaLOX to the culture supernatant of Pichia pastoris. Since protein background in the culture supernatant is typically rather low, this strategy allows for almost pure production of PaLOX applicable for the valorization of renewable fatty acids, for example for the production of green leaf volatiles. Using the CAT1 promoter system and the well‐established α‐factor signal sequence for secretion, methanol‐ and glycerol‐induced secretion are compared and the latter shows four times more LOX activity in the culture supernatant under methanol‐free conditions. In addition, secreted PaLOX is purified and the specific activity with enzyme in culture supernatant is compared. Notably, the predominant specific activity is achieved for enzyme in culture supernatant ‐ 11.6 U mg−1 ‐ reaching five times higher specific activity than purified PaLOX.

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Section: Introductionmentioning

confidence: 99%

Secretion of Pseudomonas aeruginosa Lipoxygenase by Pichia pastoris upon Glycerol Feed

Hashem

Stolterfoht

Rinnofner

et al. 2020

Biotechnology Journal

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“…Interestingly, during secretome analysis the Flo5-1 protein was found in the supernatants of K. phaffii cells in the later samples of glucose- or methanol-limited fed batch cultivations, where cells tend to adhere to the reactor surface as well (Burgard et al . 2020 ), indicating that a mechanism of cellular release might also act in K. phaffii .…”

Section: Discussionmentioning

confidence: 99%

Pseudohyphal differentiation inKomagataella phaffii: investigating theFLOgene family

Rebnegger

Moser

et al. 2020

FEMS Yeast Research

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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.

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“…The most recent study carried out by Burgard et al . ( 2020 ) investigated how the secretome of K. phaffii changes throughout a typical recombinant protein production process and how the choice of carbon source (glucose or glycerol/methanol) affects native protein secretion. In total 51 proteins were identified, concordant with previous observations.…”

Section: Protein Secretionmentioning

confidence: 99%

What makes Komagataella phaffii non-conventional?

Ata

Ergün

Fickers

et al. 2021

FEMS Yeast Research

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The important industrial protein production host Komagataella phaffii (syn Pichia pastoris) is classified as a non-conventional yeast. But what exactly makes K. phaffii non-conventional? In this review we set out to address the main differences to the ‘conventional’ yeast Saccharomyces cerevisiae, but also pinpoint differences to other non-conventional yeasts used in biotechnology. Apart from its methylotrophic lifestyle, K. phaffii is a Crabtree-negative yeast species. But even within the methylotrophs, K. phaffii possesses distinct regulatory features such as glycerol-repression of the methanol-utilization pathway or the lack of nitrate assimilation. Rewiring of the transcriptional networks regulating carbon (and nitrogen) source utilization clearly contributes to our understanding of genetic events occurring during evolution of yeast species. The mechanisms of mating-type switching and the triggers of morphogenic phenotypes represent further examples for how K. phaffii is distinguished from the model yeast S. cerevisiae. With respect to heterologous protein production, K. phaffii features high secretory capacity but secretes only low amounts of endogenous proteins. Different to S. cerevisiae, the Golgi apparatus of K. phaffii is stacked like in mammals. While it is tempting to speculate that Golgi architecture is correlated to the high secretion levels or the different N-glycan structures observed in K. phaffii, there is recent evidence against this. We conclude that K. phaffii is a yeast with unique features that has a lot of potential to explore both fundamental research questions and industrial applications.

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The secretome of Pichia pastoris in fed‐batch cultivations is largely independent of the carbon source but changes quantitatively over cultivation time

Cited by 16 publications

References 46 publications

Secretion of Pseudomonas aeruginosa Lipoxygenase by Pichia pastoris upon Glycerol Feed

Secretion of Pseudomonas aeruginosa Lipoxygenase by Pichia pastoris upon Glycerol Feed

Pseudohyphal differentiation inKomagataella phaffii: investigating theFLOgene family

What makes Komagataella phaffii non-conventional?

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