Brigitte Gasser | University of Natural Resources and Life Sciences, Vienna (BOKU) (original) (raw)

Papers by Brigitte Gasser

Trends in Biotechnology, 2021

The biopharmaceutical market is growing faster than ever, with two production systems competing f... more The biopharmaceutical market is growing faster than ever, with two production systems competing for market dominance: mammalian cells and microorganisms. In recent years, based on the rise of antibody-based therapies, new biotherapeutic approvals have favored mammalian hosts. However, not only has extensive research elevated our understanding of microbes to new levels, but emerging therapeutic molecules also facilitate their use; thus, is it time for microbes to fight back? In this review, we answer this timely question by cross-comparing four microbial production hosts and examining the innovations made to both their secretion and post-translational modification (PTM) capabilities. Furthermore, we discuss the impact of tools, such as omics and systems biology, as well as alternative production systems and emerging biotherapeutics. Small cells in an expanding market Recombinant proteins have dramatically changed our lives, and their market size and impact are projected to keep expanding i. Despite the widespread use of recombinant proteins in many industrial sectors, one of the main driving forces for continuous market expansion are biopharmaceuticals, the fastest growing group in the pharmaceutical industry [1]. This has triggered the development of a large spectrum of industrial expression platforms for their production, including both microbial and mammalian cell hosts [2]. The trend in recent years has seen mammalian cell lines increasingly outcompete their microbial counterparts (Figure 1A). From 2014 to mid-2018, more than 87% of the genuinely new biopharmaceutical active ingredients that were released to the market were proteins [3]. Of these, 84% were expressed in mammalian expression systems, with Chinese hamster ovary (CHO) (see Glossary) cell-based systems being the most widely used (Box 1). This surge in the biotherapeutics sector can be explained mainly by the increasing dominance of monoclonal antibodies (mAbs), which require humanized PTMs [3]. Highlights Microbial production systems for biopharmaceutics have been outcompeted by mammalian systems in recent years, mostly due to the increased demand for antibody-based products. In most other categories, microbes have maintained their dominant position in bioproduction. The post-translational modification capabilities of Escherichia coli have been significantly improved, specifically regarding disulfide bond formation. Recent developments of Bacillus subtilis platforms hold promise for biopharmaceutical protein production. The toolbox for protein glycosylation and secretion in Saccharomyces cerevisiae and Pichia pastoris has been significantly improved. Omics and systems biology tools are significantly enhancing our understanding of cellular processes. Antibody mimetics are promising biotherapeutics that are likely to increase the demand for microbial biopharmaceutical production platforms.

Genetics and Biotechnology, 2020

Brigitte Gasser, BOKU University of Natural Resources and Life Sciences ; Austrian Centre of Indu... more Brigitte Gasser, BOKU University of Natural Resources and Life Sciences ; Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria brigitte.gasser@boku.ac.at Lukas Marsalek, BOKU University of Natural Resources and Life Sciences ; Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria Verena Puxbaum, BOKU University of Natural Resources and Life Sciences ; Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria Clemens Gruber-Grünwald, BOKU University of Natural Resources and Life Sciences ; Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria Friedrich Altmann, BOKU University of Natural Resources and Life Sciences ; Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria Diethard Mattanovich, BOKU University of Natural Resources and Life Sciences ; Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria

BOOK Pichia 2016 April 3-6, 2016  Antalya, Turkey www.pichia2016.com 2 Our Sponsors Pichia 2016 ... more BOOK Pichia 2016 April 3-6, 2016  Antalya, Turkey www.pichia2016.com 2 Our Sponsors Pichia 2016 April 3-6, 2016  Antalya, Turkey www.pichia2016.com 3 Resort and Conference Map Pichia 2016 April 3-6, 2016  Antalya, Turkey www.pichia2016.com 4 Table of

Background: It has become evident that host cells react to recombinant protein production with a ... more Background: It has become evident that host cells react to recombinant protein production with a variety of metabolic and intrinsic stresses such as the unfolded protein response (UPR) pathway. Additionally, environmental conditions such as growth temperature may have a strong impact on cell physiology and specific productivity. However, there is little information about the molecular reactions of the host cells on a genomic level, especially in context to recombinant protein secretion. For the first time, we monitored transcriptional regulation of a subset of marker genes in the common production host Pichia pastoris to gain insights into the general physiological status of the cells under protein production conditions, with the main focus on secretion stress related genes. Results: Overexpression of the UPR activating transcription factor Hac1p was employed to identify UPR target genes in P. pastoris and the responses were compared to those known for Saccharomyces cerevisiae. Most of the folding/secretion related genes showed similar regulation patterns in both yeasts, whereas genes associated with the general stress response were differentially regulated. Secretion of an antibody Fab fragment led to induction of UPR target genes in P. pastoris, however not to the same magnitude as Hac1p overproduction. Overexpression of S. cerevisiae protein disulfide isomerase (PDI1) enhances Fab secretion rates 1.9 fold, but did not relief UPR stress. Reduction of cultivation temperature from 25°C to 20°C led to a 1.4-fold increase of specific product secretion rate in chemostat cultivations, although the transcriptional levels of the product genes (Fab light and heavy chain) were significantly reduced at the lower temperature. A subset of folding related genes appeared to be down-regulated at the reduced temperature, whereas transcription of components of the ER associated degradation and the secretory transport was enhanced. Conclusion: Monitoring of genomic regulation of marker genes with the transcriptional profiling method TRAC in P. pastoris revealed similarities and discrepancies of the responses compared to S. cerevisiae. Thus our results emphasize the importance to analyse the individual hosts under real production conditions instead of drawing conclusions from model organisms. Cultivation temperature has a significant influence on specific productivity that cannot be related just to thermodynamic effects, but strongly impacts the regulation of specific genes.

Biotechnology Journal, 2020

The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of hete... more The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of heterologous proteins. While N‐linked protein glycosylation is well characterized in P. pastoris there is less knowledge of the patterns of O‐glycosylation. O‐glycans produced by P. pastoris consist of short linear mannose chains, which in the case of recombinant biopharmaceuticals can trigger an immune response in humans. This study aims to reveal the influence of different cultivation strategies on O‐mannosylation profiles in P. pastoris. Sixteen different model proteins, produced by different P. pastoris strains, are analyzed for their O‐glycosylation profile. Based on the obtained data, human serum albumin (HSA) is chosen to be produced in fast and slow growth fed batch fermentations by using common promoters, PGAP and PAOX1. After purification and protein digestion, glycopeptides are analyzed by LC/ESI‐MS. In the samples expressed with PGAP it is found that the degree of glycosylation is slightly higher when a slow growth rate is used, regardless of the efficiency of the producing strain. The highest glycosylation intensity is observed in HSA produced with PAOX1. The results indicate that the O‐glycosylation level is markedly higher when the protein is produced in a methanol‐based expression system.

Microbial Cell Factories, 2017

Background: Industrial processes for recombinant protein production challenge production hosts, s... more Background: Industrial processes for recombinant protein production challenge production hosts, such as the yeast Pichia pastoris, on multiple levels. During a common P. pastoris fed-batch process, cells experience strong adaptations to different metabolic states or suffer from environmental stresses due to high cell density cultivation. Additionally, recombinant protein production and nutrient limitations are challenging in these processes. Results: Pichia pastoris producing porcine carboxypeptidase B (CpB) was cultivated in glucose or methanol-limited fed-batch mode, and the cellular response was analyzed using microarrays. Thereby, strong transcriptional regulations in transport-, regulatory-and metabolic processes connected to sulfur, phosphorus and nitrogen metabolism became obvious. The induction of these genes was observed in both glucose-and methanol-limited fed batch cultivations, but were stronger in the latter condition. As the transcriptional pattern was indicative for nutrient limitations, we performed fed-batch cultivations where we added the respective nutrients and compared them to non-supplemented cultures regarding cell growth, productivity and expression levels of selected biomarker genes. In the non-supplemented reference cultures we observed a strong increase in transcript levels of up to 89-fold for phosphorus limitation marker genes in the late fed-batch phase. Transcript levels of sulfur limitation marker genes were up to 35-fold increased. By addition of (NH 4) 2 SO 4 or (NH 4) 2 HPO 4 , respectively, we were able to suppress the transcriptional response of the marker genes to levels initially observed at the start of the fed batch. Additionally, supplementation had also a positive impact on biomass generation and recombinant protein production. Supplementation with (NH 4) 2 SO 4 led to 5% increase in biomass and 52% higher CpB activity in the supernatant, compared to the non-supplemented reference cultivations. In (NH 4) 2 HPO 4 supplemented cultures 9% higher biomass concentrations and 60% more CpB activity were reached. Conclusions: Transcriptional analysis of P. pastoris fed-batch cultivations led to the identification of nutrient limitations in the later phases, and respective biomarker genes for indication of limitations. Supplementation of the cultivation media with those nutrients eliminated the limitations on the transcriptional level, and was also shown to enhance productivity of a recombinant protein. The biomarker genes are versatily applicable to media and process optimization approaches, where tailor-made solutions are envisioned.

Industrial Biotechnology, 2016

FEMS Yeast Research, 2016

Improved gene annotation and functional prediction of Pichia pastoris CBS7435 based on RNAseq, pr... more Improved gene annotation and functional prediction of Pichia pastoris CBS7435 based on RNAseq, proteomics and manual curation is presented in the database and genome browser available at www.pichiagenome.org.

Applied Microbiology and Biotechnology, 2016

Production of heterologous proteins in Pichia pastoris (syn. Komagataella sp.) has been shown to ... more Production of heterologous proteins in Pichia pastoris (syn. Komagataella sp.) has been shown to exert a metabolic burden on the host metabolism. This burden is associated with metabolite drain, which redirects nucleotides and amino acids from primary metabolism. On the other hand, recombinant protein production affects energy and redox homeostasis of the host cell. In a previous study, we have demonstrated that overexpression of single genes of the oxidative pentose phosphate pathway (PPP) had a positive influence on recombinant production of cytosolic human superoxide dismutase (hSOD). In this study, different combinations of these genes belonging to the oxidative PPP were generated and analyzed. Thereby, a 3.8-fold increase of hSOD production was detected when glucose-6-phosphate dehydrogenase (ZWF1) and 6-gluconolactonase (SOL3) were simultaneously overexpressed, while the combinations of other genes from PPP had no positive effect on protein production. By measuring isotopologue patterns of 13 C-labelled metabolites, we could detect an upshift in the flux ratio of PPP to glycolysis upon ZWF1 and SOL3 co-overexpression, as well as increased levels of 6-phosphogluconate. The substantial improvement of hSOD production by ZWF1 and SOL3 co-overexpression appeared to be connected to an increase in PPP flux. In conclusion, we show that overexpression of SOL3 together with ZWF1 enhanced both the PPP flux ratio and hSOD accumulation, providing evidence that in P. pastoris Sol3 limits the flux through PPP and recombinant protein production.

BMC Genomics, 2015

Background: The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heter... more Background: The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. Results: We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol-or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. Conclusions: P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength.

Microbial Cell Factories, 2006

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Microbial Cell Factories, 2008

Different species of microorganisms including yeasts, filamentous fungi and bacteria have been us... more Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell fa...

Biotechnology Progress, 2010

in Wiley Online Library (wileyonlinelibrary.com). Microorganisms encounter diverse stress conditi... more in Wiley Online Library (wileyonlinelibrary.com). Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes. V