Industrial Microorganisms:<i>Pichia pastoris</i>

Mattanovich, Diethard; Sauer, Michael; Gasser, Brigitte

doi:10.1002/9783527807796.ch19

Cited by 11 publications

(8 citation statements)

References 165 publications

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“…Extensive studies have been carried out to find suitable production systems for high-level expression of recombinant proteins ( Kita et al, 2016 ; Rosano et al, 2019 ; Rozov and Deineko, 2019 ; Ahmad et al, 2020 ). The yeast Pichia pastoris ( Komagataella phaffii ) has become one of the most widely used expression systems to express several hundreds of proteins from viruses, bacteria, fungi, animals, plants, and humans ( Mattanovich et al, 2017 ; Liu et al, 2019a ). As a methylotrophic yeast with eukaryotic machinery, P. pastoris can grow on the only carbon source of methanol.…”

Section: Introductionmentioning

confidence: 99%

Development of a New High-Cell Density Fermentation Strategy for Enhanced Production of a Fungus β-Glucosidase in Pichia pastoris

et al. 2020

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Section: Introductionmentioning

confidence: 99%

Development of a New High-Cell Density Fermentation Strategy for Enhanced Production of a Fungus β-Glucosidase in Pichia pastoris

et al. 2020

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“…Indeed, a previous study identified several metabolic engineering targets for improving recombinant protein production using a genome-scale metabolic model (11). Interestingly, about the 50% of the identified targets pointed toward NADPH supply reactions (15).…”

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confidence: 99%

Redox Engineering by Ectopic Overexpression of NADH Kinase in Recombinant Pichia pastoris ( Komagataella phaffii ): Impact on Cell Physiology and Recombinant Production of Secreted Proteins

Tomàs-Gamisans

Andrade

Maresca

et al. 2020

Appl Environ Microbiol

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High-level expression and secretion of heterologous proteins in yeast cause an increased energy demand, which may result in altered metabolic flux distributions. Moreover, recombinant protein overproduction often results in endoplasmic reticulum (ER) stress and oxidative stress, causing deviations from the optimal NAD(P)H regeneration balance. In this context, overexpression of genes encoding enzymes catalyzing endogenous NADPH-producing reactions, such as the oxidative branch of the pentose phosphate pathway, has been previously shown to improve protein production in Pichia pastoris (syn. Komagataella spp.). In this study, we evaluate the overexpression of the Saccharomyces cerevisiae POS5-encoded NADH kinase in a recombinant P. pastoris strain as an alternative approach to overcome such redox constraints. Specifically, POS5 was cooverexpressed in a strain secreting an antibody fragment, either by directing Pos5 to the cytosol or to the mitochondria. The physiology of the resulting strains was evaluated in continuous cultivations with glycerol or glucose as the sole carbon source, as well as under hypoxia (on glucose). Cytosolic targeting of Pos5 NADH kinase resulted in lower biomass-substrate yields but allowed for a 2-fold increase in product specific productivity. In contrast, Pos5 NADH kinase targeting to the mitochondria did not affect growth physiology and recombinant protein production significantly. Growth physiological parameters were in silico evaluated using the recent upgraded version (v3.0) of the P. pastoris consensus genome-scale metabolic model iMT1026, providing insights on the impact of POS5 overexpression on metabolic flux distributions. IMPORTANCE Recombinant protein overproduction often results in oxidative stress, causing deviations from the optimal redox cofactor regeneration balance. This becomes one of the limiting factors in obtaining high levels of heterologous protein production. Overexpression of redox-affecting enzymes has been explored in other organisms, such as Saccharomyces cerevisiae, as a means to fine tune the cofactor regeneration balance in order to obtain higher protein titers. In the present work, this strategy is explored in P. pastoris. In particular, one NADH kinase enzyme from S. cerevisiae (Pos5) is used, either in the cytosol or in mitochondria of P. pastoris, and its impact on the production of a model protein (antibody fragment) is evaluated. A significant improvement in the production of the model protein is observed when the kinase is directed to the cytosol. These results are significant in the field of heterologous protein production in general and in particular in the development of improved metabolic engineering strategies for P. pastoris.

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“…Genes for the orthogonal pair of amber suppressor tRNA (tRNA CUA ) and pAzF-specific RS (RS pAzF ) were derived from E. coli tyrosyl-tRNA CUA and tyrosyl-RS, as reported previously [ 18 , 20 , 21 ]. Methanol-inducible promoters of varying strength were chosen for each gene based on the knowledge of the methanol utilization pathway in P. pastoris [ 15 , 22 ]. Enhanced green fluorescent protein (eGFP) was used as a reporter protein for pAzF incorporation in small-scale screenings and detection was done by flow cytometry.…”

Section: Resultsmentioning

confidence: 99%

“…Since P. pastoris has genes for methanol utilization expressed under tightly controllable methanol-inducible promoters, the choice of promoters for expression of all heterologous genes in this work was based on methanol metabolism [ 15 ]. The gene for the tRNA CUA was derived from E. coli tyrosyl-tRNA, as described previously [ 21 ].…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, P. pastoris , unlike Saccharomyces cerevisiae , has a Crabtree-negative phenotype [ 13 , 14 ], facilitating its cultivation at high cell densities to obtain higher product titers. Due to its methylotrophic metabolism and genes tightly regulated by methanol, it is commonly used for the inducible production of large amounts of heterologous proteins [ 15 ]. In addition, P. pastoris secretes very few endogenous proteins in the supernatant, which reduces the effort to purify the secreted product.…”

Section: Introductionmentioning

confidence: 99%

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From strain engineering to process development: monoclonal antibody production with an unnatural amino acid in Pichia pastoris

et al. 2022

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Background Expansion of the genetic code is a frequently employed approach for the modification of recombinant protein properties. It involves reassignment of a codon to another, e.g., unnatural, amino acid and requires the action of a pair of orthogonal tRNA and aminoacyl tRNA synthetase modified to recognize only the desired amino acid. This approach was applied for the production of trastuzumab IgG carrying p-azido-l-phenylalanine (pAzF) in the industrial yeast Pichia pastoris. Combining the knowledge of protein folding and secretion with bioreactor cultivations, the aim of the work was to make the production of monoclonal antibodies with an expanded genetic code cost-effective on a laboratory scale. Results Co-translational transport of proteins into the endoplasmic reticulum through secretion signal prepeptide change and overexpression of lumenal chaperones Kar2p and Lhs1p improved the production of trastuzumab IgG and its Fab fragment with incorporated pAzF. In the case of Fab, a knockout of vacuolar targeting for protein degradation further increased protein yield. Fed-batch bioreactor cultivations of engineered P. pastoris strains increased IgG and IgGpAzF productivity by around 50- and 20-fold compared to screenings, yielding up to 238 mg L−1 and 15 mg L−1 of fully assembled tetrameric protein, respectively. Successful site-specific incorporation of pAzF was confirmed by mass spectrometry. Conclusions Pichia pastoris was successfully employed for cost-effective laboratory-scale production of a monoclonal antibody with an unnatural amino acid. Applying the results of this work in glycoengineered strains, and taking further steps in process development opens great possibilities for utilizing P. pastoris in the development of antibodies for subsequent conjugations with, e.g., bioactive payloads.

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Industrial Microorganisms:Pichia pastoris

Cited by 11 publications

References 165 publications

Development of a New High-Cell Density Fermentation Strategy for Enhanced Production of a Fungus β-Glucosidase in Pichia pastoris

Development of a New High-Cell Density Fermentation Strategy for Enhanced Production of a Fungus β-Glucosidase in Pichia pastoris

Redox Engineering by Ectopic Overexpression of NADH Kinase in Recombinant Pichia pastoris ( Komagataella phaffii ): Impact on Cell Physiology and Recombinant Production of Secreted Proteins

From strain engineering to process development: monoclonal antibody production with an unnatural amino acid in Pichia pastoris

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