Integrating metabolic modeling and population heterogeneity analysis into optimizing recombinant protein production by Komagataella (Pichia) pastoris

Theron, Chrispian W.; Berríos, Julio; Delvigne, Frank; Fickers, Patrick

doi:10.1007/s00253-017-8612-y

Cited by 34 publications

(15 citation statements)

References 146 publications

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“…The methanol metabolism is rather inefficient as the first step of methanol oxidation by alcohol oxidase requires a direct electron transfer from methanol to oxygen and therefore increases the oxygen demand and generates excessive heat as well as oxidative stress (Couderc & Baratti, 1980). Compared with other microbial expression systems, this is the main constraint to consider when upscaling a P. pastoris process (Krainer et al, 2012; Theron, Berrios, Delvigne, & Fickers, 2018). Deletion of the AOX1 and AOX2 genes obstructs the methanol metabolism, effectively creating a methanol utilization negative phenotype (Mut − ) which is not able to grow on methanol as a sole carbon and energy source (Cregg et al, 1989; Sreekrishna et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

Zavec

Gasser

Mattanovich

2020

Biotech & Bioengineering

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The methanol utilization (Mut) phenotype in the yeast Pichia pastoris (syn. Komagataella spp.) is defined by the deletion of the genes AOX1 and AOX2. The Mut− phenotype cannot grow on methanol as a single carbon source. We assessed the Mut− phenotype for secreted recombinant protein production. The methanol inducible AOX1 promoter (PAOX1) was active in the Mut− phenotype and showed adequate eGFP fluorescence levels and protein yields (YP/X) in small‐scale screenings. Different bioreactor cultivation scenarios with methanol excess concentrations were tested using PAOX1HSA and PAOX1vHH expression constructs. Scenario B comprising a glucose‐methanol phase and a 72‐hr‐long methanol only phase was the best performing, producing 531 mg/L HSA and 1631 mg/L vHH. 61% of the HSA was produced in the methanol only phase where no biomass growth was observed, representing a special case of growth independent production. By using the Mut− phenotype, the oxygen demand, heat output, and specific methanol uptake (qmethanol) in the methanol phase were reduced by more than 80% compared with the MutS phenotype. The highlighted improved process parameters coupled with growth independent protein production are overlooked benefits of the Mut− strain for current and future applications in the field of recombinant protein production.

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Section: Introductionmentioning

confidence: 99%

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

Zavec

Gasser

Mattanovich

2020

Biotech & Bioengineering

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“…Intracellular protein accumulation and/or degradation, together with the existence of a non-secreting phenotype results in decreased productivity of a heterologous protein. Though an important aspect of productivity and approach to a strain's optimization, little attention is typically paid to intracellular product fluxes, stress responses (UPR, ERAD), strain heterogeneity in terms of growth and production/secretion, and cell physiology in microbial cultivation processes under industrially relevant cultivation conditions (Theron et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris

et al. 2019

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Pichia pastoris ( Komagataella sp .) is broadly used for the production of secreted recombinant proteins. Due to the high rate of protein production, incorrectly folded proteins may accumulate in the endoplasmic reticulum (ER). To restore their proper folding, the cell triggers the unfolded protein response (UPR); however, if the proteins cannot be repaired, they are degraded, which impairs process productivity. Moreover, a non-producing/non-secreting subpopulation of cells might occur, which also decreases overall productivity. Therefore, an in depth understanding of intracellular protein fluxes and population heterogeneity is needed to improve productivity. Under industrially relevant cultivation conditions in bioreactors, we cultured P. pastoris strains producing three different recombinant proteins: penicillin G acylase from Escherichia coli ( Ec PGA), lipase B from Candida antarctica ( Ca LB) and xylanase A from Thermomyces lanuginosus ( Tl XynA). Extracellular and intracellular product concentrations were determined, along with flow cytometry-based single-cell measurements of cell viability and the up-regulation of UPR. The cell population was distributed into four clusters, two of which were viable cells with no UPR up-regulation, differing in cell size and complexity. The other two clusters were cells with impaired viability, and cells with up-regulated UPR. Over the time course of cultivation, the distribution of the population into these four clusters changed. After 30 h of production, 60% of the cells producing Ec PGA, which accumulated in the cells (50–70% of the product), had up-regulated UPR, but only 13% of the cells had impaired viability. A higher proportion of cells with decreased viability was observed in strains producing Ca LB (20%) and Tl XynA (27%). The proportion of cells with up-regulated UPR in Ca LB-producing (35%) and Tl XynA-producing (30%) strains was lower in comparison to the Ec PGA-producing strain, and a smaller proportion of Ca LB and Tl XynA (<10%) accumulated in the cells. These data provide an insight into the development of heterogeneity in a recombinant P. pastoris population during a biotechnological process. A deeper understanding of the relationship between protein production/secretion and the regulation of the UPR might be utilized in bioprocess control and optimization with respect to secretion and population heterogeneity.

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“…Pichia pastoris , reclassified as Komagataella phaffi [ 7 ], has become a successful host organism for production of recombinant proteins due to the benefits of high cell density cultivation, the simplicity of genetic manipulation, growth on inexpensive media, efficient secretory capabilities with a low level of endogenous protein secretion, the strong and tightly inducible AOX1 promoter and the ability of post-translational modifications to proteins [ 8 , 9 ]. Currently, over 1000 proteins have been produced in P. pastoris [ 10 ]. In our previous studies, we heterologously expressed two furin substrates using the DNA sequences encoding the mature peptides in P. pastoris .…”

Section: Introductionmentioning

confidence: 99%

Investigation on the processing and improving the cleavage efficiency of furin cleavage sites in Pichia pastoris

Huang

Long

et al. 2018

Microb Cell Fact

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BackgroundProprotein convertase furin is responsible for the processing of a wide variety of precursors consisted of signal peptide, propeptide and mature peptide in mammal. Many precursors processed by furin have important physiological functions and can be recombinantly expressed in Pichia pastoris expression system for research, pharmaceutical and vaccine applications. However, it is not clear whether the furin cleavage sites between the propeptide and mature peptide can be properly processed in P. pastoris, bringing uncertainty for proper expression of the coding DNA sequences of furin precursors containing the propeptides and mature peptides.ResultsIn this study, we evaluated the ability of P. pastoris to process furin cleavage sites and how to improve the cleavage efficiencies of furin cleavage sites in P. pastoris. The results showed that P. pastoris can process furin cleavage sites but the cleavage efficiencies are not high. Arg residue at position P1 or P4 in furin cleavage sites significantly affect cleavage efficiency in P. pastoris. Kex2 protease, but not YPS1, in P. pastoris is responsible for processing furin cleavage sites. Heterologous expression of furin or overexpression of Kex2 in P. pastoris effectively increased cleavage efficiencies of furin cleavage sites.ConclusionsOur investigation on the processing of furin cleavage sites provides important information for recombinant expression of furin precursors in P. pastoris. Furin or Kex2 overexpressing strains may be good choices for expressing precursors processed by furin in P. pastoris.

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Integrating metabolic modeling and population heterogeneity analysis into optimizing recombinant protein production by Komagataella (Pichia) pastoris

Cited by 34 publications

References 146 publications

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris

Investigation on the processing and improving the cleavage efficiency of furin cleavage sites in Pichia pastoris

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