Downregulation by organic nitrogen of <scp><i>AOX1</i></scp> promoter used for controlled expression of foreign genes in the yeast <scp><i>Pichia pastoris</i></scp>

Velastegui, Edgar; Theron, Chrispian W.; Berríos, Julio; Fickers, Patrick

doi:10.1002/yea.3381

Cited by 10 publications

(8 citation statements)

References 27 publications

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“…In Pichia pastoris, it has been shown that the choice of N source can regulate the activity of genes involved in methanol assimilation (Rumjantsev et al, 2014). For example, the activity of the AOX1 promoter in P. pastoris was reported to be reduced in the presence of free amino acids in the cultivation medium (Velastegui et al, 2019). The expression of the LDH in this study is regulated by the MOX promoter, which is O. polymorpha's native homolog to the AOX1 promoter.…”

Section: Discussionmentioning

confidence: 59%

Engineering the methylotrophic yeast Ogataea polymorpha for lactate production from methanol

Wefelmeier,

Schmitz,

Haut

et al. 2023

Front. Bioeng. Biotechnol.

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Introduction: Lactate has gained increasing attention as a platform chemical, particularly for the production of the bioplastic poly-lactic acid (PLA). While current microbial lactate production processes primarily rely on the use of sugars as carbon sources, it is possible to envision a future where lactate can be produced from sustainable, non-food substrates. Methanol could be such a potential substrate, as it can be produced by (electro)chemical hydrogenation from CO2.Methods: In this study, the use of the methylotrophic yeast Ogataea polymorpha as a host organism for lactate production from methanol was explored. To enable lactate production in Ogataea polymorpha, four different lactate dehydrogenases were expressed under the control of the methanol-inducible MOX promoter. The L-lactate dehydrogenase of Lactobacillus helveticus performed well in the yeast, and the lactate production of this engineered strain could additionally be improved by conducting methanol fed-batch experiments in shake flasks. Further, the impact of different nitrogen sources and the resulting pH levels on production was examined more closely. In order to increase methanol assimilation of the lactate-producing strain, an adaptive laboratory evolution experiment was performed.Results and Discussion: The growth rate of the lactate-producing strain on methanol was increased by 55%, while at the same time lactate production was preserved. The highest lactate titer of 3.8 g/L in this study was obtained by cultivating this evolved strain in a methanol fed-batch experiment in shake flasks with urea as nitrogen source. This study provides a proof of principle that Ogataea polymorpha is a suitable host organism for the production of lactate using methanol as carbon source. In addition, it offers guidance for the engineering of methylotrophic organisms that produce platform chemicals from CO2-derived substrates. With reduced land use, this technology will promote the development of a sustainable industrial biotechnology in the future.

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Section: Discussionmentioning

confidence: 59%

Engineering the methylotrophic yeast Ogataea polymorpha for lactate production from methanol

Wefelmeier,

Schmitz,

Haut

et al. 2023

Front. Bioeng. Biotechnol.

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“…Specifically, addition of proline, glutamic acid, and casamino acids was reported to downregulate expression from the commonly used AOX1 promoter. 31,32 This indicates that intracellular amino acid concentrations may also trigger several other metabolic processes within a cell which influence protein synthesis. Our metabolomics data could display a direct impact of recombinant protein production on intracellular amino acid levels.…”

Section: Discussionmentioning

confidence: 99%

“…Most amino acids are known as rather poor nitrogen sources and thus supplementation may have a negative influence on protein synthesis. Specifically, addition of proline, glutamic acid, and casamino acids was reported to downregulate expression from the commonly used AOX1 promoter 31,32 . This indicates that intracellular amino acid concentrations may also trigger several other metabolic processes within a cell which influence protein synthesis.…”

Section: Discussionmentioning

confidence: 99%

Customizing amino acid metabolism of Pichia pastoris for recombinant protein production

Rußmayer,

Buchetics,

Mattanovich

et al. 2023

Biotechnology Journal

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Amino acids are the building blocks of proteins. In this respect, a reciprocal effect of recombinant protein production on amino acid biosynthesis as well as the impact of the availability of free amino acids on protein production can be anticipated. In this study, the impact of engineering the amino acid metabolism on the production of recombinant proteins was investigated in the yeast Pichia pastoris (syn Komagataella phaffii). Based on comprehensive systems‐level analyses of the metabolomes and transcriptomes of different P. pastoris strains secreting antibody fragments, cell engineering targets were selected. Our working hypothesis that increasing intracellular amino acid levels could help unburden cellular metabolism and improve recombinant protein production was examined by constitutive overexpression of genes related to amino acid metabolism. In addition to twelve genes involved in specific amino acid biosynthetic pathways, the transcription factor GCN4 responsible for regulation of amino acid biosynthetic genes was overexpressed. The production of the used model protein, a secreted carboxylesterase (CES) from Sphingopyxis macrogoltabida, was increased by overexpression of pathway genes for alanine and for aromatic amino acids, and most pronounced, when overexpressing the regulator GCN4. The analysis of intracellular amino acid levels of selected clones indicated a direct linkage of improved recombinant protein production to the increased availability of intracellular amino acids. Finally, fed batch cultures showed that overexpression of GCN4 increased CES titers 2.6‐fold, while the positive effect of other amino acid synthesis genes could not be transferred from screening to bioreactor cultures.This article is protected by copyright. All rights reserved

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“…Beside this, nitrogen sources such as casamino acid have also been reported to regulate methanol metabolism (Velastegui et al . 2019 ). Indeed, expression of the genes AOX1 , DAS1 and FLD1 is reduced in the presence of 0.1% of casamino acid.…”

Section: Nitrogen Metabolismmentioning

confidence: 99%

What makes Komagataella phaffii non-conventional?

Ata

Ergün

Fickers

et al. 2021

FEMS Yeast Research

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The important industrial protein production host Komagataella phaffii (syn Pichia pastoris) is classified as a non-conventional yeast. But what exactly makes K. phaffii non-conventional? In this review we set out to address the main differences to the ‘conventional’ yeast Saccharomyces cerevisiae, but also pinpoint differences to other non-conventional yeasts used in biotechnology. Apart from its methylotrophic lifestyle, K. phaffii is a Crabtree-negative yeast species. But even within the methylotrophs, K. phaffii possesses distinct regulatory features such as glycerol-repression of the methanol-utilization pathway or the lack of nitrate assimilation. Rewiring of the transcriptional networks regulating carbon (and nitrogen) source utilization clearly contributes to our understanding of genetic events occurring during evolution of yeast species. The mechanisms of mating-type switching and the triggers of morphogenic phenotypes represent further examples for how K. phaffii is distinguished from the model yeast S. cerevisiae. With respect to heterologous protein production, K. phaffii features high secretory capacity but secretes only low amounts of endogenous proteins. Different to S. cerevisiae, the Golgi apparatus of K. phaffii is stacked like in mammals. While it is tempting to speculate that Golgi architecture is correlated to the high secretion levels or the different N-glycan structures observed in K. phaffii, there is recent evidence against this. We conclude that K. phaffii is a yeast with unique features that has a lot of potential to explore both fundamental research questions and industrial applications.

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Downregulation by organic nitrogen of AOX1 promoter used for controlled expression of foreign genes in the yeast Pichia pastoris

Cited by 10 publications

References 27 publications

Engineering the methylotrophic yeast Ogataea polymorpha for lactate production from methanol

Engineering the methylotrophic yeast Ogataea polymorpha for lactate production from methanol

Customizing amino acid metabolism of Pichia pastoris for recombinant protein production

What makes Komagataella phaffii non-conventional?

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