Physiological state as transferable operating criterion to improve recombinant protein production in <i>Pichia pastoris</i> through oxygen limitation

Garcia-Ortega, Xavier; Valero, Francisco; Montesinos-Seguí, José Luis

doi:10.1002/jctb.5272

Cited by 20 publications

(34 citation statements)

References 72 publications

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“…Once a suitable expression system has been chosen, the next step is to optimize culture conditions to achieve the target productivity. Factors such as temperature, pH, osmolality, specific growth rate (μ) and dissolved oxygen (DO) are critical for the efficient operation of the culture, and their influence on protein production and culture performance has been individually assessed (Baumann et al., 2008; Charoenrat et al., 2005; Dragosits et al., 2009, 2010; Garcia-Ortega et al., 2017; Heyland et al., 2010; Maurer et al., 2006). Although several studies have reviewed the relationships between protein production and growth (refer to Looser et al.…”

Section: Introductionmentioning

confidence: 99%

Contextualized genome-scale model unveils high-order metabolic effects of the specific growth rate and oxygenation level in recombinant Pichia pastoris

Torres

Saa

Albiol

et al. 2019

Metabolic Engineering Communications

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Pichia pastoris is recognized as a biotechnological workhorse for recombinant protein expression. The metabolic performance of this microorganism depends on genetic makeup and culture conditions, amongst which the specific growth rate and oxygenation level are critical. Despite their importance, only their individual effects have been assessed so far, and thus their combined effects and metabolic consequences still remain to be elucidated. In this work, we present a comprehensive framework for revealing high-order (i.e., individual and combined) metabolic effects of the above parameters in glucose-limited continuous cultures of P. pastoris, using thaumatin production as a case study. Specifically, we employed a rational experimental design to calculate statistically significant metabolic effects from multiple chemostat data, which were later contextualized using a refined and highly predictive genome-scale metabolic model of this yeast under the simulated conditions. Our results revealed a negative effect of the oxygenation on the specific product formation rate (thaumatin), and a positive effect on the biomass yield. Notably, we identified a novel positive combined effect of both the specific growth rate and oxygenation level on the specific product formation rate. Finally, model predictions indicated an opposite relationship between the oxygenation level and the growth-associated maintenance energy (GAME) requirement, suggesting a linear GAME decrease of 0.56 mmol ATP/gDCW per each 1% increase in oxygenation level, which translated into a 44% higher metabolic cost under low oxygenation compared to high oxygenation. Overall, this work provides a systematic framework for mapping high-order metabolic effects of different culture parameters on the performance of a microbial cell factory. Particularly in this case, it provided valuable insights about optimal operational conditions for protein production in P. pastoris.

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Section: Introductionmentioning

confidence: 99%

Contextualized genome-scale model unveils high-order metabolic effects of the specific growth rate and oxygenation level in recombinant Pichia pastoris

Torres

Saa

Albiol

et al. 2019

Metabolic Engineering Communications

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“…The concentrations of the different carbon sources used in the batch (glycerol) and fed‐batch tests (glucose), and of potential fermentation by‐products (arabitol or ethanol), were all determined by HPLC. The column and procedure used for this purpose are described elsewhere (Garcia‐Ortega et al , ). RSD was always < 1%.…”

Section: Methodsmentioning

confidence: 99%

Rationale‐based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffii (Pichia pastoris)

Nieto-Taype

Garrigós-Martínez

Sánchez-Farrando

et al. 2019

Microbial Biotechnology

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Summary Its features as a microbial and eukaryotic organism have turned Komagataella phaffii (Pichia pastoris) into an emerging cell factory for recombinant protein production (RPP). As a key step of the bioprocess development, this work aimed to demonstrate the importance of tailor designing the cultivation strategy according to the production kinetics of the cell factory. For this purpose, K. phaffii clones constitutively expressing (PGAP) Candida rugosa lipase 1 (Crl1) with different gene dosage were used as models in continuous and fed‐batch cultures. Production parameters were much greater with a multicopy clone (MCC) than with the single‐copy clone (SCC). Regarding production kinetics, the specific product generation rate (qP) increased linearly with increasing specific growth rate (µ) in SCC; by contrast, qP exhibited saturation in MCC. A transcriptional analysis in chemostat cultures suggested the presence of eventual post‐transcriptional bottlenecks in MCC. After the strain characterization, in order to fulfil overall development of the bioprocess, the performance of both clones was also evaluated in fed‐batch mode. Strikingly, different optimal strategies were determined for both models due to the different production kinetic patterns observed as a trade‐off for product titre, yields and productivity. The combined effect of gene dosage and adequate µ enables rational process development with a view to optimize K. phaffii RPP bioprocesses.

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“…The concentration of the different carbon sources used in the batch (glycerol), and chemostat and fed-batch cultivations (methanol), and the potential fermentation byproducts, were all determined by HPLC. The column and program used are described elsewhere [48]. RSD was invariably less than 1%.…”

Section: Methodsmentioning

confidence: 99%

Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris (Komagataella phaffii) PAOX1-driven recombinant producer strains with different target gene dosage

et al. 2019

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BackgroundThe PAOX1-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). Despite relevant recent advances in regulation of the methanol utilization (MUT) pathway have been made, the role of specific growth rate (µ) in AOX1 regulation remains unknown, and therefore, its impact on protein production kinetics is still unclear.ResultsThe influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two PAOX1-driven Candida rugosa lipase 1 (Crl1) producer clones. Specifically, a clone integrating a single expression cassette of CRL1 was compared with one containing three cassettes over broad dilution rate and µ ranges in both chemostat and fed-batch cultivations. Chemostat cultivations allowed to establish the impact of µ on the MUT-related MIT1 pool which leads to a bell-shaped relationship between µ and PAOX1-driven gene expression, influencing directly Crl1 production kinetics. Also, chemostat and fed-batch cultivations exposed the favorable effects of increasing the CRL1 gene dosage (up to 2.4 fold in qp) on Crl1 production with no significant detrimental effects on physiological capabilities.ConclusionsPAOX1-driven gene expression and Crl1 production kinetics in P. pastoris were successfully correlated with µ. In fact, µ governs MUT-related MIT1 amount that triggers PAOX1-driven gene expression—heterologous genes included—, thus directly influencing the production kinetics of recombinant protein.

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Physiological state as transferable operating criterion to improve recombinant protein production in Pichia pastoris through oxygen limitation

Abstract: BACKGROUND: The yeast Pichia pastoris is widely used as a production platform for

Cited by 20 publications

References 72 publications

Contextualized genome-scale model unveils high-order metabolic effects of the specific growth rate and oxygenation level in recombinant Pichia pastoris

Contextualized genome-scale model unveils high-order metabolic effects of the specific growth rate and oxygenation level in recombinant Pichia pastoris

Rationale‐based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffii (Pichia pastoris)

Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris (Komagataella phaffii) PAOX1-driven recombinant producer strains with different target gene dosage

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