Quantitative Metabolomics and Instationary 13C-Metabolic Flux Analysis Reveals Impact of Recombinant Protein Production on Trehalose and Energy Metabolism in Pichia pastoris

Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary 13C metabolic flux analysis (INST 13C-MFA) using GC-MS and LC-MS/MS to evaluate the potential impact of the production of a Rhizopus oryzae lipase (Rol) on P. pastoris central carbon metabolism. Higher oxygen uptake and CO2 production rates and slightly reduced biomass yield suggest an increased energy demand for the producing strain. This observation is further confirmed by 13C-based metabolic flux analysis. In particular, the flux through the methanol oxidation pathway and the TCA cycle was increased in the Rol-producing strain compared to the reference strain. Next to changes in the flux distribution, significant variations in intracellular metabolite concentrations were observed. Most notably, the pools of trehalose, which is related to cellular stress response, and xylose, which is linked to methanol assimilation, were significantly increased in the recombinant strain

Recent advances in Pichia pastoris as host for heterologous expression system for lipases : a review

The production of heterologous lipases is one of the most promising strategies to increase the productivity of the bioprocesses and to reduce costs, with the final objective that more industrial lipase applications could be implemented. In this chapter, an overview of the new success in synthetic biology, with traditional molecular genetic techniques and bioprocess engineering in the last 5 years in the cell factory Pichia pastoris, the most promising host system for heterologous lipase production, is presented. The goals get on heterologous Candida antarctica, Rhizopus oryzae, and Candida rugosa lipases, three of the most common lipases used in biocatalysis, are showed. Finally, new cell factories producing heterologous lipases are presented

Detection and elimination of cellular bottlenecks in protein-producing yeasts

Yeasts are efficient cell factories and are commonly used for the production of recombinant proteins for biopharmaceutical and industrial purposes. For such products high levels of correctly folded proteins are needed, which sometimes requires improvement and engineering of the expression system. The article summarizes major breakthroughs that led to the efficient use of yeasts as production platforms and reviews bottlenecks occurring during protein production. Special focus is given to the metabolic impact of protein production. Furthermore, strategies that were shown to enhance secretion of recombinant proteins in different yeast species are presented

Improving functional annotation for industrial microbes: a case study with Pichia pastoris.

The research communities studying microbial model organisms, such as Escherichia coli or Saccharomyces cerevisiae, are well served by model organism databases that have extensive functional annotation. However, this is not true of many industrial microbes that are used widely in biotechnology. In this Opinion piece, we use Pichia (Komagataella) pastoris to illustrate the limitations of the available annotation. We consider the resources that can be implemented in the short term both to improve Gene Ontology (GO) annotation coverage based on annotation transfer, and to establish curation pipelines for the literature corpus of this organism.We gratefully acknowledge funding from the Wellcome Trust (PomBase and Canto; WT090548MA to SGO), and the EU 7th Framework Programme (BIOLEDGE Contract No: 289126 to SGO).This is the published version distributed under a Creative Commons Attribution License 2.0, which can also be found on the publisher's website at: http://www.sciencedirect.com/science/article/pii/S0167779914001061

Systems-level organization of yeast methylotrophic lifestyle

BACKGROUND: Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date. RESULTS: In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation. CONCLUSIONS: Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-015-0186-5) contains supplementary material, which is available to authorized users

Continuous Cultivation as a Tool Toward the Rational Bioprocess Development With Pichia Pastoris Cell Factory

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is currently considered one of the most promising hosts for recombinant protein production (RPP) and metabolites due to the availability of several tools to efficiently regulate the recombinant expression, its ability to perform eukaryotic post-translational modifications and to secrete the product in the extracellular media. The challenge of improving the bioprocess efficiency can be faced from two main approaches: the strain engineering, which includes enhancements in the recombinant expression regulation as well as overcoming potential cell capacity bottlenecks; and the bioprocess engineering, focused on the development of rational-based efficient operational strategies. Understanding the effect of strain and operational improvements in bioprocess efficiency requires to attain a robust knowledge about the metabolic and physiological changes triggered into the cells. For this purpose, a number of studies have revealed chemostat cultures to provide a robust tool for accurate, reliable, and reproducible bioprocess characterization. It should involve the determination of key specific rates, productivities, and yields for different C and N sources, as well as optimizing media formulation and operating conditions. Furthermore, studies along the different levels of systems biology are usually performed also in chemostat cultures. Transcriptomic, proteomic and metabolic flux analysis, using different techniques like differential target gene expression, protein description and 13 C-based metabolic flux analysis, are widely described as valued examples in the literature. In this scenario, the main advantage of a continuous operation relies on the quality of the homogeneous samples obtained under steady-state conditions, where both the metabolic and physiological status of the cells remain unaltered in an all-encompassing picture of the cell environment. This contribution aims to provide the state of the art of the different approaches that allow the design of rational strain and bioprocess engineering improvements in Pichia pastoris toward optimizing bioprocesses based on the results obtained in chemostat cultures. Interestingly, continuous cultivation is also currently emerging as an alternative operational mode in industrial biotechnology for implementing continuous process operations

Validation of a FBA model for Pichia pastoris in chemostat cultures

Background: Constraint-based metabolic models and flux balance analysis (FBA) have been extensively used in the last years to investigate the behavior of cells and also as basis for different industrial applications. In this context, this work provides a validation of a small-sized FBA model of the yeast Pichia pastoris. Our main objective is testing how accurate is the hypothesis of maximum growth to predict the behavior of P. pastoris in a range of experimental environments. Results: A constraint-based model of P. pastoris was previously validated using metabolic flux analysis (MFA). In this paper we have verified the model ability to predict the cells behavior in different conditions without introducing measurements, experimental parameters, or any additional constraint, just by assuming that cells will make the best use of the available resources to maximize its growth. In particular, we have tested FBA model ability to: (a) predict growth yields over single substrates (glucose, glycerol, and methanol); (b) predict growth rate, substrate uptakes, respiration rates, and by-product formation in scenarios where different substrates are available (glucose, glycerol, methanol, or mixes of methanol and glycerol); (c) predict the different behaviors of P. pastoris cultures in aerobic and hypoxic conditions for each single substrate. In every case, experimental data from literature are used as validation. Conclusions: We conclude that our predictions based on growth maximisation are reasonably accurate, but still far from perfect. The deviations are significant in scenarios where P. pastoris grows on methanol, suggesting that the hypothesis of maximum growth could be not dominating in these situations. However, predictions are much better when glycerol or glucose are used as substrates. 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Systematic investigation into the clonal variability of the non-conventional yeast Pichia pastoris

Schwarzhans JP. Systematic investigation into the clonal variability of the non-conventional yeast Pichia pastoris. Bielefeld: Universität Bielefeld; 2017.The non-conventional methylotrophic yeast *Pichia pastoris* has become a firmly established host for recombinant protein production in both the industry and academia. High product titers, an efficient secretory machinery and the ability to express complex proteins from bacterial to human origin have given *P. pastoris* an advantage over many other host systems. In recent years, its aptitude for foreign gene expression has also been applied in a rising number of metabolic engineering studies. However, scientists trying to create the *P. pastoris* strain optimal for their application are faced with a challenge. The high clonal variability results in clones from one transformation exhibiting wildly different expression levels, no detectable expression at all or altered growth behaviors. In consequence, a laborious screening process has to be applied to identify the desired strain from among hundreds or thousands of clones. Surprisingly, only few studies tried to analyze clonal variability in *P. pastoris* so far. Although the connections between gene dosage and product titers have been investigated thoroughly, the underlying causes and mechanisms of clonal variability remained unknown. In this project, we present the first systematic investigation into the clonal variability of *P. pastoris*, the discovered genetic events and their impact on both recombinant protein production and growth behavior. By applying well-established standard methods for *P. pastoris* experiments, we aimed to provide relevant results and insights for other scientists working with this yeast. A library of 845 strains, transformed with an easy to detect reporter protein, was characterized for classic properties including colony morphology, gene dosage and productivity. Thereby, we analyzed a significantly larger clone library than previous *P. pastoris* publications, exceeding their size ca. 20 to 100 fold. Based on the characterization data, 31 strains with very peculiar features were selected for whole genome sequencing. Enabled by a combination of characterization and genome sequencing data, we discovered novel connections between integration event and strain properties. A clear correlation between cassette-to-cassette orientation and productivity was found. Additionally, a surprising ratio between the different orientation forms suggested the existence of two competing integration mechanisms that excluded each other. We also observed a rather high occurrence of false-positive clones containing the same integration event. Our combinatorial approach enabled us to identify a surplus homologous sequence inside the expression cassette as the likely cause for this secondary integration event. The theory was validated by optimization of the expression cassette and subsequent elimination of the undesired integration event. Besides productivity related effects, we also analyzed strains that displayed a marked change in their colony morphology. Multiple new non-canonical integration events were discovered in them. Off-target gene disruptions could be correlated with the change in colony morphology. Particularly, the relocation of the knock-out target to a different chromosome and the subsequent gene disruption provided important insights for genetic engineering studies. In a number of clones we found *E. coli* DNA from the plasmid host, which had co-integrated in fusion with the expression cassette. Moreover, qRT-PCR experiments confirmed the transcriptional activity of the *E. coli* genes in *P. pastoris*. Strikingly, the clonal variability also resulted in the creation of a novel genetic tool for recombinant protein production in *P. pastoris*. In one strain with exceptionally good productivity features, the creation of a circular plasmid consisting of the expression cassette and mitochondrial DNA was found. We could validate its replicative capabilities and successfully applied it for transformation of both *P. pastoris* and *Saccharomyces cerevisiae*. In *P. pastoris*, newly created pMito clones exhibited a highly uniform expression level that significantly exceeded a reference strain with a single copy of the expression cassette in its genome by up to fourfold. Taken together, our project provides scientists working with *P. pastoris* with important references for studies both focused on recombinant protein production as well as genetic or metabolic engineering. Thereby, we aim to promote further development of this yeast and aid in the implementation of more complex genetic engineering strategies. Ways to reduce the frequency of low-producer strains enable streamlined screening procedures for high producer strains. Simultaneously, the documentation of off-target integration events helps to devise strategies that prevent their occurrence or highlight events that should be assayed for in constructed strains. Lastly, the novel episomal vector we discovered displayed great potential, especially for protein engineering studies in which a great number of different target variants need to be assayed