Is heterogeneity in large-scale bioreactors a real problem in recombinant protein synthesis by Pichia pastoris?

Velastegui, Edgar; Quezada, Johan; Guerrero, Karlo; Altamirano, Claudia; Martínez, Juan A.; Berríos, Julio; Fickers, Patrick

doi:10.1007/s00253-023-12434-2

Cited by 3 publications

(1 citation statement)

References 42 publications

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“…In contrary, the cultivation in the TCB showed that cell growth was remarkably affected as the maximum achieved biomass concentration dropped to 26.8 g L −1 , which means a loss of 21% compared to the cultivation in a stirred-tank bioreactor. One possible reason for the lower cell growth is the shift of carbon fluxes in the metabolism of the cells as a response to dynamic environments, which was also reported by Velastegui et al [ 38 ]. In the here conducted L-phenylalanine production processes, carbon dioxide covered 25% more of the integral carbon balance, especially during the initial biomass production phase compared to the cultivation in the STR.…”

Section: Discussionmentioning

confidence: 63%

Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor

Hoang,

Polte,

Frantzmann

et al. 2023

Microb Cell Fact

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Background The omnipresence of population heterogeneity in industrial bioprocesses originates from prevailing dynamic bioprocess conditions, which promote differences in the expression of cellular characteristics. Despite the awareness, the concrete consequences of this phenomenon remain poorly understood. Results Therefore, for the first time, a L-phenylalanine overproducing Escherichia coli quadruple reporter strain was established for monitoring of general stress response, growth behavior, oxygen limitation and product formation of single cells based on mTagBFP2, mEmerald, CyOFP1, and mCardinal2 expression measured by flow cytometry. This strain was applied for the fed-batch production of L-phenylalanine from glycerol and ammonia in a stirred-tank bioreactor at homogeneous conditions compared to the same process in a novel two-compartment bioreactor. This two-compartment bioreactor consists of a stirred-tank bioreactor with an initial volume of 0.9 L (homogeneous zone) with a coiled flow inverter with a fixed working volume of 0.45 L as a bypass (limitation zone) operated at a mean hydraulic residence time of 102 s. The product formation was similar in both bioreactor setups with maximum L-phenylalanine concentrations of 21.1 ± 0.6 g L−1 demonstrating the consistency of this study’s microbial L-phenylalanine production. However, cell growth was vulnerable to repetitive exposure to the dynamically changing conditions in the two-compartment bioreactor with maximum biomass yields reduced by 21%. The functionality of reporter molecules was approved in the stirred-tank bioreactor cultivation, in which expressed fluorescence levels of all four markers were in accordance with respective process state variables. Additional evaluation of the distributions on single-cell level revealed the presence of population heterogeneity in both bioprocesses. Especially for the marker of the general stress response and the product formation, the corresponding histograms were characterized by bimodal shapes and broad distributions. These phenomena were pronounced particularly at the beginning and the end of the fed-batch process. Conclusions The here shown findings confirm multiple reporter strains to be a noninvasive tool for monitoring cellular characteristics and identifying potential subpopulations in bioprocesses. In combination with experiments in scale-down setups, these can be utilized for a better physiological understanding of bioprocesses and support future scale-up procedures.

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Section: Discussionmentioning

confidence: 63%

Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor

Hoang,

Polte,

Frantzmann

et al. 2023

Microb Cell Fact

View full text Add to dashboard Cite

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Co-feeding strategy alleviates hypoxic stress in large-scale bioreactor for optimal production of secretory recombinant proteins in Pichia pastoris

Velastegui

Quezada

Altamirano

et al. 2023

Journal of Biotechnology

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Real-time monitoring of recombinant GFP expression in single-cell Komagataella phaffii through filamentous pelletization technology

Zheng,

Cong,

Zhou

et al. 2023

Reviews in Analytical Chemistry

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Komagataella phaffii is an important cell factory for recombinant protein production under methanol induction. Tracking of recombinant protein expression by single K. phaffii cell is a hot topic to identify the variation of expression level in submerge cultivation system. In this study, a platform system harnessing Aspergillus niger pellets for K. phaffii cell immobilization in fabricated glass plate was developed to measure the green fluorescent protein (GFP) expression of single cell using fluorescence microscope. This system was optimized through pellets preparation, K. phaffii cell absorption, inducer comparison, methanol concentration, flow velocity of medium, and obtaining a successful platform to track GFP expression of single K. phaffii cell. This system provided an on-line analytical technology to discover the heterogeneous capability of various K. phaffii cells.

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Is heterogeneity in large-scale bioreactors a real problem in recombinant protein synthesis by Pichia pastoris?

Cited by 3 publications

References 42 publications

Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor

Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor

Co-feeding strategy alleviates hypoxic stress in large-scale bioreactor for optimal production of secretory recombinant proteins in Pichia pastoris

Real-time monitoring of recombinant GFP expression in single-cell Komagataella phaffii through filamentous pelletization technology

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