Pau Ferrer | Universitat Autònoma de Barcelona (original) (raw)
Papers by Pau Ferrer
Biotech, Jan 6, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Journal of Bacteriology, 1996
The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA ... more The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA (betaglIIA) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined. Sequence comparison shows that the betaglIIA enzyme has over 80% identity to the betaglII isoenzyme, an endo-beta-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium. The betaglIIA enzyme lacks a glucan- or mannan-binding domain, such as those observed in beta-1,3-glucanases and proteases having high yeast/fungus-lytic activity. It can be included in the glycosyl hydrolase family 16. Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that betaglIIA has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca. 4.0).
Microbial Cell Factories
Background Methanol is increasingly gaining attraction as renewable carbon source to produce spec... more Background Methanol is increasingly gaining attraction as renewable carbon source to produce specialty and commodity chemicals, as it can be generated from renewable sources such as carbon dioxide (CO2). In this context, native methylotrophs such as the yeast Komagataella phaffii (syn Pichia pastoris) are potentially attractive cell factories to produce a wide range of products from this highly reduced substrate. However, studies addressing the potential of this yeast to produce bulk chemicals from methanol are still scarce. 3-Hydroxypropionic acid (3-HP) is a platform chemical which can be converted into acrylic acid and other commodity chemicals and biopolymers. 3-HP can be naturally produced by several bacteria through different metabolic pathways. Results In this study, production of 3-HP via the synthetic β-alanine pathway has been established in K. phaffii for the first time by expressing three heterologous genes, namely panD from Tribolium castaneum, yhxA from Bacillus cereus...
Wiley-VCH Verlag GmbH & Co. KGaA eBooks, Feb 26, 2016
We report a genetic approach for the overproduction of a Rhizopus oryzae lipase (ROL) expressed i... more We report a genetic approach for the overproduction of a Rhizopus oryzae lipase (ROL) expressed in Pichia pastoris under the alcohol oxidase I promoter, P AOXI. It is based on the development of a stable P. pastoris strain containing multiple copies of the heterologous gene, ROL. This strain was obtained after transforming P.pastoris with the expression vector pPICZaA-ROL, and subsequent selection of strains containing multiple vector insertion events in the presence of higher Zeocin concentrations. The final ROL multicopy strains showed higher lipolytic activity and were isogenic with regard to the single copy ROL strain genetic background (i.e., His+, Mut), being therefore suitable for high density cultures using a defined synthetic medium.
New Biotechnology, 2021
Pichia pastoris (Komagataella spp.) has become one of the most important host organisms for produ... more Pichia pastoris (Komagataella spp.) has become one of the most important host organisms for production of heterologous proteins of biotechnological interest, many of them extracellular. The protein secretion pathway has been recognized as a limiting process in which many roadblocks have been pinpointed. Recently, we have identified a bottleneck at the ER translocation level. In earlier exploratory studies, this limitation could be largely overcome by using an improved chimeric secretion signal to drive proteins through the co-translational translocation pathway. Here, we have further tested at bioreactor scale the improved secretion signal consisting of the pre-Ost1 signal sequence, which drives proteins through co-translational translocation, followed by the pro region from the secretion signal of the Saccharomyces cerevisiae α-factor mating pheromone. For comparison, the commonly used full-length α-factor secretion signal, which drives proteins through post-translational translocation, was tested. These two secretion signals were fused to three different model proteins: the tetrameric red fluorescent protein E2-Crimson, which can be used to visualize roadblocks in the secretory pathway; the lipase 2 from Bacillus thermocatenulatus (BTL2); and the Rhizopus oryzae lipase (ROL). All strains were tested in batch cultivation to study the different growth parameters obtained. The strains carrying the improved secretion signal showed increased final production of the proteins of interest. Interestingly, they were able to grow at significantly higher maximum specific growth rates than their counterparts carrying the conventional secretion signal. These results were corroborated in a 5L fed-batch cultivation, where the final product concentration and volumetric productivity were also shown to be improved.
Microbial Cell Factories, Oct 12, 2018
Background: Proteins can be secreted from a host organism with the aid of N-terminal secretion si... more Background: Proteins can be secreted from a host organism with the aid of N-terminal secretion signals. The budding yeast Pichia pastoris (Komagataella sp.) is widely employed to secrete proteins of academic and industrial interest. For this yeast, the most commonly used secretion signal is the N-terminal portion of pre-pro-α-factor from Saccharomyces cerevisiae. However, this secretion signal promotes posttranslational translocation into the endoplasmic reticulum (ER), so proteins that can fold in the cytosol may be inefficiently translocated and thus poorly secreted. In addition, if a protein self-associates, the α-factor pro region can potentially cause aggregation, thereby hampering export from the ER. This study addresses both limitations of the pre-pro-α-factor secretion signal. Results: We engineered a hybrid secretion signal consisting of the S. cerevisiae Ost1 signal sequence, which promotes cotranslational translocation into the ER, followed by the α-factor pro region. Secretion and intracellular localization were assessed using as a model protein the tetrameric red fluorescent protein E2-Crimson. When paired with the α-factor pro region, the Ost1 signal sequence yielded much more efficient secretion than the α-factor signal sequence. Moreover, an allelic variant of the α-factor pro region reduced aggregation of the E2-Crimson construct in the ER. The resulting improved secretion signal enhanced secretion of E2-Crimson up to 20-fold compared to the levels obtained with the original α-factor secretion signal. Similar findings were obtained with the lipase BTL2, which exhibited 10-fold enhanced secretion with the improved secretion signal. Conclusions: The improved secretion signal confers dramatic benefits for the secretion of certain proteins from P. pastoris. These benefits are likely to be most evident for proteins that can fold in the cytosol and for oligomeric proteins.
Dipòsit Digital de Documents de la UAB, Nov 7, 2022
Dipòsit Digital de Documents de la UAB
Dipòsit Digital de Documents de la UAB
Dipòsit Digital de Documents de la UAB
Dataset corresponding to the NAD kinase redox engineering study in P.pastoris referenced below. I... more Dataset corresponding to the NAD kinase redox engineering study in P.pastoris referenced below. Includes cultivation experimental data, droplet digital PCR data and computer simulation results in different files in Microsoft Excel 2013 Format
New Biotechnology, 2019
Rational development of bioprocess engineering strategies for recombinant protein production in P... more Rational development of bioprocess engineering strategies for recombinant protein production in Pichia pastoris (Komagataella phaffi) using the methanol free GAP promoter. Where do we stand?
A thorough description of transcriptome is one of the foundations for understanding the cell as a... more A thorough description of transcriptome is one of the foundations for understanding the cell as a molecular system. Toward this goal, we have launched three projects, namely full-length cDNA analysis, absolute quantification of mRNAs, and enforced activation of transcription factors. In the full-length cDNA analysis, we have read two cDNA libraries, one from S288C strain exponentially growing in the SD medium and the other from SK1 strain at meiotic stages. Both libraries were constructed using a vectorcapping method, which allows one to precisely map transcription start sites based on a cap-dependent nucleotide addition phenomenon. Consequently, we identified 11,519 transcription start sites for 3,638 annotated genes. The analysis also revealed 50 novel introns including those affecting ORF annotations and those spliced alternatively, 576 novel transcripts derived from intergenic regions, and antisense transcripts for, at least, 665 annotated genes. Thus, the budding yeast transcriptome is much more complex than previously thought. It may provide a tractable model system to reveal functions of noncoding RNAs found in higher eukaryotes, and the full-length cDNA clones would serve as an invaluable resource for such studies.
Journal of Industrial Microbiology & Biotechnology, Jun 6, 2022
To successfully design expression systems for industrial biotechnology and biopharmaceutical appl... more To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, efficient synthesis of the desired product and the use of selection markers acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of IPTG-inducible protein expression systems-harboring different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA)-in high-cell density cultures to evaluate their suitability in bioprocess conditions that resemble industrial settings. Results revealed that the first generation of engineered strain showed a 50% reduction in the production of the model recombinant protein fuculose-1-phosphate aldolase (FucA) compared to the reference system from QIAGEN. The over-transcription of glyA was found to be a major factor responsible for the metabolic burden. The second-and third-generation of expression systems presented an increase in FucA production and advantageous features. In particular, the third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and, is capable to produce FucA at similar levels compared to the original commercial expression system. These new tools open new avenues for high-yield and robust expression of recombinant proteins in E. coli.
Pichia pastoris has been recognized as an effective host for recombinant protein production. In t... more Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary 13 C metabolic flux analysis (INST 13 C-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 CO 2 production rates and slightly reduced biomass yield suggest an increased energy demand for the producing strain. This observation is further confirmed by 13 C-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.
Aids Reviews, 2019
Virus-like particles (VLPs) are a type of subunit vaccine which resembles viruses but do not cont... more Virus-like particles (VLPs) are a type of subunit vaccine which resembles viruses but do not contain any genetic material so that they are not infectious. VLPs maintain the same antigenic conformation to the original virus, and they could be a better vaccine candidate than live-attenuated and inactivated vaccines. In addition, compared to other subunit vaccines such as soluble protein, VLPs can stimulate both innate and adaptive immune responses effectively and safely against several pathogens by the closer morphology to its native virus. They have already been licensed as vaccines against Hepatitis B virus, human papillomavirus (HPV), and several veterinary diseases. Moreover, it has been investigated to prevent other viral infections including HIV. While HIV VLP-based vaccines have been studied over 35 years, none of them has been successful enough to reach even Phase III clinical trials. In this review, we summarize: (i) general features of VLPs; (ii) epidemiological data and current status of vaccine research and development on HPV and HIV; and (iii) previous studies held on HPV VLPs, HIV VLPs, and chimeric HPV/HIV VLPs including production methods and different animal immunization assays. Furthermore, we review present state of human clinical trials with VLPs and consider the potential to develop a successful preventive HIV vaccine using HPV VLP models. Finally, we discuss the benefits, limitations, and challenges of developing chimeric VLPbased HPV/HIV vaccines with recent findings, critical issues to improve VLP-based vaccines, and hot topics for the next 5 years to join the global effort to fight against these two pathogens.
Background Production of 3-hydroxypropionic acid (3-HP) through the malonyl-CoA pathway has yield... more Background Production of 3-hydroxypropionic acid (3-HP) through the malonyl-CoA pathway has yielded promising results in Pichia pastoris (Komagataella phaffii), demonstrating the potential of this cell factory to produce this platform chemical and other acetyl-CoA-derived products using glycerol as a carbon source. However, further metabolic engineering of the original P. pastoris 3-HP-producing strains resulted in unexpected outcomes, e.g. significantly lower product yield and/or growth rate. To gain understanding on the metabolic constraints underlying these observations, the fluxome of ten 3-HP-producing P. pastoris strains has been characterized using a high throughput 13C-metabolic flux analysis platform. Results Results indicate that the expression of the NADH kinase leads to a reduction in the fluxes of the pentose phosphate pathway reactions. Moreover, an increase in the pentose phosphate pathway fluxes was observed when the cytosolic acetyl-CoA pathway was overexpressed. Re...
Biotech, Jan 6, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Journal of Bacteriology, 1996
The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA ... more The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA (betaglIIA) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined. Sequence comparison shows that the betaglIIA enzyme has over 80% identity to the betaglII isoenzyme, an endo-beta-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium. The betaglIIA enzyme lacks a glucan- or mannan-binding domain, such as those observed in beta-1,3-glucanases and proteases having high yeast/fungus-lytic activity. It can be included in the glycosyl hydrolase family 16. Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that betaglIIA has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca. 4.0).
Microbial Cell Factories
Background Methanol is increasingly gaining attraction as renewable carbon source to produce spec... more Background Methanol is increasingly gaining attraction as renewable carbon source to produce specialty and commodity chemicals, as it can be generated from renewable sources such as carbon dioxide (CO2). In this context, native methylotrophs such as the yeast Komagataella phaffii (syn Pichia pastoris) are potentially attractive cell factories to produce a wide range of products from this highly reduced substrate. However, studies addressing the potential of this yeast to produce bulk chemicals from methanol are still scarce. 3-Hydroxypropionic acid (3-HP) is a platform chemical which can be converted into acrylic acid and other commodity chemicals and biopolymers. 3-HP can be naturally produced by several bacteria through different metabolic pathways. Results In this study, production of 3-HP via the synthetic β-alanine pathway has been established in K. phaffii for the first time by expressing three heterologous genes, namely panD from Tribolium castaneum, yhxA from Bacillus cereus...
Wiley-VCH Verlag GmbH & Co. KGaA eBooks, Feb 26, 2016
We report a genetic approach for the overproduction of a Rhizopus oryzae lipase (ROL) expressed i... more We report a genetic approach for the overproduction of a Rhizopus oryzae lipase (ROL) expressed in Pichia pastoris under the alcohol oxidase I promoter, P AOXI. It is based on the development of a stable P. pastoris strain containing multiple copies of the heterologous gene, ROL. This strain was obtained after transforming P.pastoris with the expression vector pPICZaA-ROL, and subsequent selection of strains containing multiple vector insertion events in the presence of higher Zeocin concentrations. The final ROL multicopy strains showed higher lipolytic activity and were isogenic with regard to the single copy ROL strain genetic background (i.e., His+, Mut), being therefore suitable for high density cultures using a defined synthetic medium.
New Biotechnology, 2021
Pichia pastoris (Komagataella spp.) has become one of the most important host organisms for produ... more Pichia pastoris (Komagataella spp.) has become one of the most important host organisms for production of heterologous proteins of biotechnological interest, many of them extracellular. The protein secretion pathway has been recognized as a limiting process in which many roadblocks have been pinpointed. Recently, we have identified a bottleneck at the ER translocation level. In earlier exploratory studies, this limitation could be largely overcome by using an improved chimeric secretion signal to drive proteins through the co-translational translocation pathway. Here, we have further tested at bioreactor scale the improved secretion signal consisting of the pre-Ost1 signal sequence, which drives proteins through co-translational translocation, followed by the pro region from the secretion signal of the Saccharomyces cerevisiae α-factor mating pheromone. For comparison, the commonly used full-length α-factor secretion signal, which drives proteins through post-translational translocation, was tested. These two secretion signals were fused to three different model proteins: the tetrameric red fluorescent protein E2-Crimson, which can be used to visualize roadblocks in the secretory pathway; the lipase 2 from Bacillus thermocatenulatus (BTL2); and the Rhizopus oryzae lipase (ROL). All strains were tested in batch cultivation to study the different growth parameters obtained. The strains carrying the improved secretion signal showed increased final production of the proteins of interest. Interestingly, they were able to grow at significantly higher maximum specific growth rates than their counterparts carrying the conventional secretion signal. These results were corroborated in a 5L fed-batch cultivation, where the final product concentration and volumetric productivity were also shown to be improved.
Microbial Cell Factories, Oct 12, 2018
Background: Proteins can be secreted from a host organism with the aid of N-terminal secretion si... more Background: Proteins can be secreted from a host organism with the aid of N-terminal secretion signals. The budding yeast Pichia pastoris (Komagataella sp.) is widely employed to secrete proteins of academic and industrial interest. For this yeast, the most commonly used secretion signal is the N-terminal portion of pre-pro-α-factor from Saccharomyces cerevisiae. However, this secretion signal promotes posttranslational translocation into the endoplasmic reticulum (ER), so proteins that can fold in the cytosol may be inefficiently translocated and thus poorly secreted. In addition, if a protein self-associates, the α-factor pro region can potentially cause aggregation, thereby hampering export from the ER. This study addresses both limitations of the pre-pro-α-factor secretion signal. Results: We engineered a hybrid secretion signal consisting of the S. cerevisiae Ost1 signal sequence, which promotes cotranslational translocation into the ER, followed by the α-factor pro region. Secretion and intracellular localization were assessed using as a model protein the tetrameric red fluorescent protein E2-Crimson. When paired with the α-factor pro region, the Ost1 signal sequence yielded much more efficient secretion than the α-factor signal sequence. Moreover, an allelic variant of the α-factor pro region reduced aggregation of the E2-Crimson construct in the ER. The resulting improved secretion signal enhanced secretion of E2-Crimson up to 20-fold compared to the levels obtained with the original α-factor secretion signal. Similar findings were obtained with the lipase BTL2, which exhibited 10-fold enhanced secretion with the improved secretion signal. Conclusions: The improved secretion signal confers dramatic benefits for the secretion of certain proteins from P. pastoris. These benefits are likely to be most evident for proteins that can fold in the cytosol and for oligomeric proteins.
Dipòsit Digital de Documents de la UAB, Nov 7, 2022
Dipòsit Digital de Documents de la UAB
Dipòsit Digital de Documents de la UAB
Dipòsit Digital de Documents de la UAB
Dataset corresponding to the NAD kinase redox engineering study in P.pastoris referenced below. I... more Dataset corresponding to the NAD kinase redox engineering study in P.pastoris referenced below. Includes cultivation experimental data, droplet digital PCR data and computer simulation results in different files in Microsoft Excel 2013 Format
New Biotechnology, 2019
Rational development of bioprocess engineering strategies for recombinant protein production in P... more Rational development of bioprocess engineering strategies for recombinant protein production in Pichia pastoris (Komagataella phaffi) using the methanol free GAP promoter. Where do we stand?
A thorough description of transcriptome is one of the foundations for understanding the cell as a... more A thorough description of transcriptome is one of the foundations for understanding the cell as a molecular system. Toward this goal, we have launched three projects, namely full-length cDNA analysis, absolute quantification of mRNAs, and enforced activation of transcription factors. In the full-length cDNA analysis, we have read two cDNA libraries, one from S288C strain exponentially growing in the SD medium and the other from SK1 strain at meiotic stages. Both libraries were constructed using a vectorcapping method, which allows one to precisely map transcription start sites based on a cap-dependent nucleotide addition phenomenon. Consequently, we identified 11,519 transcription start sites for 3,638 annotated genes. The analysis also revealed 50 novel introns including those affecting ORF annotations and those spliced alternatively, 576 novel transcripts derived from intergenic regions, and antisense transcripts for, at least, 665 annotated genes. Thus, the budding yeast transcriptome is much more complex than previously thought. It may provide a tractable model system to reveal functions of noncoding RNAs found in higher eukaryotes, and the full-length cDNA clones would serve as an invaluable resource for such studies.
Journal of Industrial Microbiology & Biotechnology, Jun 6, 2022
To successfully design expression systems for industrial biotechnology and biopharmaceutical appl... more To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, efficient synthesis of the desired product and the use of selection markers acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of IPTG-inducible protein expression systems-harboring different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA)-in high-cell density cultures to evaluate their suitability in bioprocess conditions that resemble industrial settings. Results revealed that the first generation of engineered strain showed a 50% reduction in the production of the model recombinant protein fuculose-1-phosphate aldolase (FucA) compared to the reference system from QIAGEN. The over-transcription of glyA was found to be a major factor responsible for the metabolic burden. The second-and third-generation of expression systems presented an increase in FucA production and advantageous features. In particular, the third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and, is capable to produce FucA at similar levels compared to the original commercial expression system. These new tools open new avenues for high-yield and robust expression of recombinant proteins in E. coli.
Pichia pastoris has been recognized as an effective host for recombinant protein production. In t... more Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary 13 C metabolic flux analysis (INST 13 C-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 CO 2 production rates and slightly reduced biomass yield suggest an increased energy demand for the producing strain. This observation is further confirmed by 13 C-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.
Aids Reviews, 2019
Virus-like particles (VLPs) are a type of subunit vaccine which resembles viruses but do not cont... more Virus-like particles (VLPs) are a type of subunit vaccine which resembles viruses but do not contain any genetic material so that they are not infectious. VLPs maintain the same antigenic conformation to the original virus, and they could be a better vaccine candidate than live-attenuated and inactivated vaccines. In addition, compared to other subunit vaccines such as soluble protein, VLPs can stimulate both innate and adaptive immune responses effectively and safely against several pathogens by the closer morphology to its native virus. They have already been licensed as vaccines against Hepatitis B virus, human papillomavirus (HPV), and several veterinary diseases. Moreover, it has been investigated to prevent other viral infections including HIV. While HIV VLP-based vaccines have been studied over 35 years, none of them has been successful enough to reach even Phase III clinical trials. In this review, we summarize: (i) general features of VLPs; (ii) epidemiological data and current status of vaccine research and development on HPV and HIV; and (iii) previous studies held on HPV VLPs, HIV VLPs, and chimeric HPV/HIV VLPs including production methods and different animal immunization assays. Furthermore, we review present state of human clinical trials with VLPs and consider the potential to develop a successful preventive HIV vaccine using HPV VLP models. Finally, we discuss the benefits, limitations, and challenges of developing chimeric VLPbased HPV/HIV vaccines with recent findings, critical issues to improve VLP-based vaccines, and hot topics for the next 5 years to join the global effort to fight against these two pathogens.
Background Production of 3-hydroxypropionic acid (3-HP) through the malonyl-CoA pathway has yield... more Background Production of 3-hydroxypropionic acid (3-HP) through the malonyl-CoA pathway has yielded promising results in Pichia pastoris (Komagataella phaffii), demonstrating the potential of this cell factory to produce this platform chemical and other acetyl-CoA-derived products using glycerol as a carbon source. However, further metabolic engineering of the original P. pastoris 3-HP-producing strains resulted in unexpected outcomes, e.g. significantly lower product yield and/or growth rate. To gain understanding on the metabolic constraints underlying these observations, the fluxome of ten 3-HP-producing P. pastoris strains has been characterized using a high throughput 13C-metabolic flux analysis platform. Results Results indicate that the expression of the NADH kinase leads to a reduction in the fluxes of the pentose phosphate pathway reactions. Moreover, an increase in the pentose phosphate pathway fluxes was observed when the cytosolic acetyl-CoA pathway was overexpressed. Re...
Metabolic flux analysis based on tracing patterns of stable isotopes, particularly 13C, comprises... more Metabolic flux analysis based on tracing patterns of stable isotopes, particularly 13C, comprises a set of methodologies to experimentally quantify intracellular biochemical reaction rates, i.e., to measure carbon flux distributions through a metabolic network. This allows quantifying the response of a metabolic network to an environmental or genetic perturbation (i.e., the metabolic phenotype). Here, we describe a protocol based on growing yeast on a 13C-labelled substrate and subsequent NMR detection of 13C-patterns in proteinogenic amino acids. To calculate metabolic fluxes, we describe two complementary mathematical approaches using available software; namely, an approach based on the estimation of local ratios in network nodes, and a method based on a global iterative fitting approach. Furthermore, we consider specificities of these protocols for their application to the yeast Pichia pastoris growing on multicarbon substrates other than glucose (glycerol), as well as the case when methanol is used as co-substrate in combination with glucose or glycerol.
Overexpression of a foreign protein may negatively affect several cell growth parameters, as we... more Overexpression of a foreign protein may negatively affect several cell growth parameters, as well as cause cellular stress. Central (or core) metabolism plays a crucial role since it supplies energy, reduction equivalents, and precursor molecules for the recombinant product, cell’s maintenance, and growth needs. However, the number of quantitative physiology studies of the impact of recombinant protein production on the central metabolic pathways of yeast cell factories has been traditionally rather limited, thereby hampering the application of rational strain engineering strategies targeting central metabolism.
The development and application of quantitative physiology and modelling tools and methodologies is allowing for a systems-level understanding of the effect of bioprocess parameters such as growth rate, temperature, oxygen availability, and substrate(s) choice on metabolism, and its subsequent interactions with recombinant protein synthesis, folding, and secretion.
Here, we review the recent developments and applications of 13C-based metabolic flux analysis (13C-MFA) of Pichia pastoris and the gained understanding of the metabolic behavior of this yeast in recombinant protein production bioprocesses. We also discuss the potential of multilevel studies integrating 13C-MFA with other omics analyses, as well as future perspectives on the metabolic modelling approaches to study and design metabolic engineering strategies for improved protein production.