Yeast Suspension Culture

Mattanovich, Diethard; Jungo, Carmen; Wenger, Jana; Dabros, Michal; Maurer, Michael

doi:10.1002/9783527683321.ch02

Cited by 8 publications

(5 citation statements)

References 31 publications

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“…While pAOX1 offers strong inducible expression with methanol – thereby enabling uncoupling fast growth from production –, pGAP provides comparable constitutive expression (Peña et al., 2018). P. pastoris cultures incur in high oxygen consumption and heat production during methanol oxidation, and hence, its use poses major challenges for large-scale protein production (Mattanovich et al., 2014). Once a suitable expression system has been chosen, the next step is to optimize culture conditions to achieve the target productivity.…”

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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“…More importantly still, the impact of growth rate controlled fed-batch cultures on the productivity of the strains and on the quality of the product deserves a thorough investigation. This may be of particular interest in controlling multiple-phase fed-batch processes such as the recombinant protein production strategy with Pichia pastoris reported by Mattanovich et al [1]. On-line monitoring of medium metabolites with, for example, FTIR could further enhance the control strategy, provided that the measurements are done above the limit of detection.…”

Section: Discussionmentioning

confidence: 99%

“…Many strains of the primary hosts used in the industrial production of recombinant protein, bioproducts or biofuels, including Escherichia coli and Saccharomyces cerevisiae, undergo the Crabtree effect and produce various overflow metabolites. The restricted respiratory capacity of Crabtree-positive strains and the resulting overflow metabolism can have significant implications in terms of inhibited growth, decreased productivity, and suboptimal product quality [1][2][3][4][5][6].…”

Section: Introductionmentioning

confidence: 99%

“…Overcoming the drawbacks related to the Crabtree effect usually involves the implementation of fed-batch cultures, where either the carbon substrate concentration [6,7], the undesired overflow metabolite itself [8,9], or the specific growth rate [3][4][5]10] are maintained below a certain critical value. For this reason, among others, fed-batch cultures have become an important and widely applied production strategy in bioprocessing [1,11]. The major challenge of controlled fed-batch cultivations lies in selecting a suitable on-line monitoring technique for the appropriate control variable and in developing an effective control strategy with sufficient simplicity and robustness to render industrial implementation feasible [11].…”

Section: Introductionmentioning

confidence: 99%

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Preventing Overflow Metabolism in Crabtree-Positive Microorganisms through On-Line Monitoring and Control of Fed-Batch Fermentations

2018

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At specific growth rates above a particular critical value, Crabtree-positive microorganisms exceed their respiratory capacity and enter diauxic growth metabolism. Excess substrate is converted reductively to an overflow metabolite, resulting in decreased biomass yield and productivity. To prevent this scenario, the cells can be cultivated in a fed-batch mode at a growth rate maintained below the critical value, µcrit. This approach entails two major challenges: accurately estimating the current specific growth rate and controlling it successfully over the course of the fermentation. In this work, the specific growth rate of S. cerevisiae and E. coli was estimated from enhanced on-line biomass concentration measurements obtained with dielectric spectroscopy and turbidity. A feedforward-feedback control scheme was implemented to maintain the specific growth rate at a setpoint below µcrit, while on-line FTIR measurements provided the early detection of the overflow metabolites. The proposed approach is in line with the principles of Bioprocess Analytical Technology (BioPAT), and provides a means to increase the productivity of Crabtree-positive microorganisms.

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“…However, utilization of methanol as an inducer has some disadvantages, as it is flammable and toxic. Furthermore, alcohol oxidase requires oxygen as an electron acceptor, which leads to a high oxygen demand and heat generation in methanol-based industrial production processes ( 29 , 30 ). Therefore, much effort was put into establishing an array of various promoters that are methanol-independent.…”

Section: Introductionmentioning

confidence: 99%

Systematic sequence engineering enhances the induction strength of the glucose-regulated GTH1 promoter of Komagataella phaffii

Flores-Villegas,

Rebnegger,

Kowarz

et al. 2023

Nucleic Acids Research

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The promoter of the high-affinity glucose transporter Gth1 (PGTH1) is tightly repressed on glucose and glycerol surplus, and strongly induced in glucose-limitation, thus enabling regulated methanol-free production processes in the yeast production host Komagataella phaffii. To further improve this promoter, an intertwined approach of nucleotide diversification through random and rational engineering was pursued. Random mutagenesis and fluorescence activated cell sorting of PGTH1 yielded five variants with enhanced induction strength. Reverse engineering of individual point mutations found in the improved variants identified two single point mutations with synergistic action. Sequential deletions revealed the key promoter segments for induction and repression properties, respectively. Combination of the single point mutations and the amplification of key promoter segments led to a library of novel promoter variants with up to 3-fold higher activity. Unexpectedly, the effect of gaining or losing a certain transcription factor binding site (TFBS) was highly dependent on its context within the promoter. Finally, the applicability of the novel promoter variants for biotechnological production was proven for the secretion of different recombinant model proteins in fed batch cultivation, where they clearly outperformed their ancestors. In addition to advancing the toolbox for recombinant protein production and metabolic engineering of K. phaffii, we discovered single nucleotide positions and correspondingly affected TFBS that distinguish between glycerol- and glucose-mediated repression of the native promoter.

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Yeast Suspension Culture

Cited by 8 publications

References 31 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

Preventing Overflow Metabolism in Crabtree-Positive Microorganisms through On-Line Monitoring and Control of Fed-Batch Fermentations

Systematic sequence engineering enhances the induction strength of the glucose-regulated GTH1 promoter of Komagataella phaffii

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