In <i>Pichia pastoris</i>, growth rate regulates protein synthesis and secretion, mating and stress response

Rebnegger, Corinna; Graf, Alexandra B.; Valli, Minoska; Steiger, Matthias G.; Gasser, Brigitte; Maurer, Michael; Mattanovich, Diethard

doi:10.1002/biot.201300334

Cited by 89 publications

(145 citation statements)

References 52 publications

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“…For serum albumin, opposite relationships were observed in cultures with methanol or glucose. AOX1-controlled production with methanol as a substrate showed negative growth-related product formation (Kobayashi et al, 2000) whereas for glucose and the GAP-promoter, positive growth-related product formation was observed (Rebnegger et al, 2014).…”

Section: Production Kineticsmentioning

confidence: 99%

“…A few examples of recent experimental studies show and quantify effects of culture conditions such as specific growth rate, substrate composition, temperature and oxygen supply on correct and sufficient post-translational protein modifications (Ha et al, 2011a, Hesketh et al, 2013, Jacobs et al, 2010, Rebnegger et al, 2014, Schenk et al, 2008, Seman et al, 2014, Wu et al, 2011. Examples of complex proteins of high scientific and commercial relevance are human membrane proteins.…”

Section: Product Qualitymentioning

confidence: 99%

“…Extensive lists of proteins that have been successfully produced with P. pastoris highlight the increasing future relevance of this microbial production system [reviewed in (Ahmad et al, 2014, Calik et al, 2015, Cereghino and Cregg, 2000, Li et al, 2007, Macauley-Patrick et al, 2005]. Due to heightened interest in its application, new genetic tools for P. pastoris have been developed (Ahmad et al, 2014, Delic et al, 2014, and thus the availability of new strains has intensified the need for a systematic design of cultivation and production processes with this yeast (Buchetics et al, 2011, Potgieter et al, 2010, Rebnegger et al, 2014.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Cultivation strategies to enhance productivity of Pichia pastoris: A review

Looser

Brühlmann

Bumbak

et al. 2015

Biotechnology Advances

285

268

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Pichia pastoris, a methylotrophic yeast, is an established system for the production of heterologous proteins, particularly biopharmaceuticals and industrial enzymes. To maximise and optimise the production of recombinant products, recent molecular research has focused on numerous issues including the design of expression vectors, optimisation of gene copy number, co-expression of secretory proteins such as chaperones, engineering of glycosylation and secretory pathways, etc. However, the physiological effects of different cultivation strategies are often difficult to separate from the molecular effects of the gene construct (e.g., cellular stress through over-expression or incorrect post-translational processing). Hence, overall system optimisation is difficult, even though it is urgently required in order to describe and understand the behaviour of new molecular constructs. This review focuses on particular aspects of recombinant protein production related to variations in biomass growth and their implications for strain design and screening, as well as on the concept of rational comparisons between cultivation systems for the development of specific production processes in bioreactors. The relationship between specific formation rates of secreted recombinant proteins, qp, and specific growth rates, μ, has been analysed in a conceptual attempt to compare different systems, particularly those based on AOX1/methanol and GAP/glucose, and this has now evolved into a pivotal concept for bioprocess engineering of P. pastoris.

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Section: Production Kineticsmentioning

confidence: 99%

Section: Product Qualitymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cultivation strategies to enhance productivity of Pichia pastoris: A review

Looser

Brühlmann

Bumbak

et al. 2015

Biotechnology Advances

285

268

View full text Add to dashboard Cite

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“…The same promoter and substrate combination tended to have a greater influence on product formation kinetics than similar categories of recombinant products (Looser et al 2015). Specific rates of P GAP -controlled protein production with glucose as a substrate typically increase with specific growth rates close to μ max (Buchetics et al 2011; Maurer et al 2006; Rebnegger et al 2014). The opposite is often found for P AOX1 -controlled high-level recombinant protein formation induced by methanol, where maximum specific secretion rates were observed at a μ considerably below μ max (Kobayashi et al 2000; Potgieter et al 2010; Wu et al 2010).…”

Section: Introductionmentioning

confidence: 99%

Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter

Looser

Lüthy

Straumann

et al. 2017

Appl Microbiol Biotechnol

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As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting Candida antarctica lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (q p) and specific growth rate (μ). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source. CALB was not synthesised during batch cultivation in excess glycerol (>10 g l−1) and at a growth rate close to μ max (0.15 h−1). Between 0.017 and 0.115 h−1 in glycerol-limited fedbatch cultures, basal levels of q p > 0.4 mg g−1 h−1 CALB were reached, independent of the μ at which the culture grew. At μ > 0.04 h−1, an elevated q p occurred temporarily during the first 20 h after changing to fedbatch mode and decreased thereafter to basal. In order to accelerate the determination of the q p(μ) relationship (kinetics of product formation), the entire μ range was covered in a single fedbatch experiment. By linearly increasing and decreasing glycerol addition rates, μ values were repeatedly shifted from 0.004 to 0.074 h−1 and vice versa. Changes in q p were related to changes in μ. A rough estimation of μ range suitable for production was possible in a single fedbatch, thus significantly reducing the experimental input over previous approaches comprising several experiments.

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“…When lactose utilization rate gets higher (as in the CreA loss-of-function mutants), ST production will not occur before its complete exhaustion, just like on d -glucose. While we cannot yet link our observations to a defined molecular step, it is to be noted that in Pichia pastoris , carbon source-responsive genes and transcription factors are upregulated at low growth rates [41]. Recent evidence also suggests that fungal secondary metabolite gene clusters are differentially regulated by the various sugars available [34,42].…”

Section: Discussionmentioning

confidence: 98%

Growth-Phase Sterigmatocystin Formation on Lactose Is Mediated via Low Specific Growth Rates in Aspergillus nidulans

Németh

Molnár

Fejes

et al. 2016

Toxins

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Seed contamination with polyketide mycotoxins such as sterigmatocystin (ST) produced by Aspergilli is a worldwide issue. The ST biosynthetic pathway is well-characterized in A. nidulans, but regulatory aspects related to the carbon source are still enigmatic. This is particularly true for lactose, inasmuch as some ST production mutant strains still synthesize ST on lactose but not on other carbon substrates. Here, kinetic data revealed that on d-glucose, ST forms only after the sugar is depleted from the medium, while on lactose, ST appears when most of the carbon source is still available. Biomass-specified ST production on lactose was significantly higher than on d-glucose, suggesting that ST formation may either be mediated by a carbon catabolite regulatory mechanism, or induced by low specific growth rates attainable on lactose. These hypotheses were tested by d-glucose limited chemostat-type continuous fermentations. No ST formed at a high growth rate, while a low growth rate led to the formation of 0.4 mg·L−1 ST. Similar results were obtained with a CreA mutant strain. We concluded that low specific growth rates may be the primary cause of mid-growth ST formation on lactose in A. nidulans, and that carbon utilization rates likely play a general regulatory role during biosynthesis.

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In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response

Cited by 89 publications

References 52 publications

Cultivation strategies to enhance productivity of Pichia pastoris: A review

Cultivation strategies to enhance productivity of Pichia pastoris: A review

Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter

Growth-Phase Sterigmatocystin Formation on Lactose Is Mediated via Low Specific Growth Rates in Aspergillus nidulans

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