Xavier Garcia Ortega
Verified @gmail.com
Scopus Publications
- Bioprocess intensification for 3-hydroxypropionic acid production from methanol using engineered Komagataella phaffii
Giovanni Covaleda-Cortés, Silvia Àvila-Cabré, Arnau Gasset, Xavier García-Ortega, José Luis Montesinos-Seguí, Pau Ferrer, Francisco Valero
Chemical Engineering Journal Advances, 2026
Green methanol is increasingly recognized as an attractive feedstock to produce value-added biochemicals, providing a unique opportunity to positively impact sustainability. The methylotrophic yeast Komagataella phaffii has been recently engineered for 3-hydroxypropionic acid (3-HP) production from methanol. However, challenges remain in developing techno-economically viable and environmentally sustainable bioprocesses.In this study, different fed-batch operational strategies and conditions, using K. phaffii strain engineered with the synthetic β-alanine pathway to produce 3-HP, based on a preprogrammed exponential feeding of methanol to attain a constant specific growth rate or a closed-loop feedback control of methanol concentration were implemented and compared. The latter strategy resulted in better overall process performance, reaching a 3-HP concentration of 42.7 g3-HP/L in the supernatant, a yield on methanol of 0.106 g3-HP/gS and a volumetric productivity of 0.495 g3-HP/(L·h). Additionally, the use of formate as an electron donor in methanol fed-batch cultures were investigated, demonstrating improved process efficiency (final product concentration, yield on methanol and volumetric productivity increase of about 15%).Overall, the results obtained in this study highlight the potential of K. phaffii as a methanol-based platform microbial cell factory, paving the way for further bioprocess intensification for sustainable chemicals production from CO2-derived feedstocks. - Pichia pastoris Cell Disruption by High-Pressure Homogenization (HPH)
Antoni Casablancas, Xavier Garcia-Ortega, Francisco Valero
Methods in Molecular Biology, 2026 - Preface
Methods in Molecular Biology, 2026 - Pichia pastoris Continuous Cultivations for Strain Characterization and Continuous Biomanufacturing
Núria Bernat-Camps, Arnau Gasset, José Luis Montesinos-Seguí, Francisco Valero, Xavier Garcia-Ortega
Methods in Molecular Biology, 2026 - Clarification of Pichia pastoris Broth by TFF with Ceramic Membranes
Fuensanta Verdú-Navarro, Juan A. Moreno-Cid, Ana G. Del Hierro, Xavier Garcia-Ortega, Ruth Ordóñez
Methods in Molecular Biology, 2026 - Concentration and Purification of Proteins Produced by Pichia pastoris by TFF with Ceramic Membranes
Fuensanta Verdú-Navarro, Juan A. Moreno-Cid, Ana G. Del Hierro, Xavier Garcia-Ortega, Ruth Ordóñez
Methods in Molecular Biology, 2026 - Fed-Batch Operational Strategies for Efficient and Robust Pichia Pastoris Cultivations Achieving High Cell Densities
Albert Sales-Vallverdú, Miguel Angel Nieto-Taype, Arnau Gasset, José Luis Montesinos-Seguí, Francisco Valero, Xavier Garcia-Ortega
Methods in Molecular Biology, 2026 - Next-generation stress-inducible Komagataella phaffii promoter variants
Katharina Ebner, Núria Bernat-Camps, Simona Scheipel, Corina Dörner, Francisco Valero, Anton Glieder, Xavier Garcia-Ortega
Microbial Cell Factories, 2025
Background Expanding the promoter toolbox of Komagataella phaffii (K. phaffii) in terms of strength and regulatory flexibility can significantly enhance bioprocess efficiency for recombinant protein and metabolite production. The most frequently used promoters are still derived from the methanol utilization (MUT) pathway or genes of the central metabolism. However, the hazards and costs associated with methanol have prompted the search for alternative promoters, including engineered variants. A key limitation remains, many available promoters are still growth-coupled, tying production to biomass accumulation and shortening process duration. Promoters with growth-decoupled expression are therefore highly desirable. In this context, the recently described P DH promoter is of interest due to its methanol independence, strong expression, and growth-decoupled regulation. Results In order to identify potential activator sites of the P DH , a systematic semi-rational block-scanning approach was used, employing single-base and sequence block walking mutagenesis. The strength of 152 systematically generated variants was characterized using the intracellular reporter eGFP. Variants showed altered strengths and regulatory patterns with fluorescence levels spanning approximately 10–150% of the parental promoter. Subsequently, the best-performing variants were combined to multi-combination variants, which showed activities up to 250% of the parental P DH . Selected variants were also evaluated with the industrially relevant and secreted enzyme Cal B, a lipase from Candida antarctica. Lipase product titers were approx. 2-fold higher than with the parental native promoter sequence and also outperformed the typical state-of-the-art benchmark and constitutive and growth-coupled GAP promoter (P GAP ). Conclusions Creating and characterizing variants of the P DH sequence supported the elucidation of the sequence-function relationships of this promoter. In addition, the surprisingly beneficial effects of a synthetic 10 bp sequence stretch opened up opportunities for further engineering of this system and extended the toolbox of efficient vector parts for methanol-free and growth-decoupled protein production with K. phaffii . Those additional promoter sequences will also support the construction of stable engineered strains with a balanced expression of multiple genes, as needed for e.g. multienzyme pathways and synthetic biology applications. - Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection
Florian Weiss, Guillermo Requena-Moreno, Carsten Pichler, Francisco Valero, Anton Glieder, Xavier Garcia-Ortega
Microbial Cell Factories, 2024
Background Most recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors. Results In micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct. Conclusions For the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production. - Synergic kinetic and physiological control to improve the efficiency of Komagataella phaffii recombinant protein production bioprocesses
Albert Sales‐Vallverdú, Arnau Gasset, Guillermo Requena‐Moreno, Francisco Valero, José Luis Montesinos‐Seguí, Xavier Garcia‐Ortega
Microbial Biotechnology, 2024
The yeast Komagataella phaffii (Pichia pastoris) is currently considered a versatile and highly efficient host for recombinant protein production (RPP). Interestingly, the regulated application of specific stress factors as part of bioprocess engineering strategies has proven potential for increasing the production of recombinant products. This study aims to evaluate the impact of controlled oxygen‐limiting conditions on the performance of K. phaffii bioprocesses for RPP in combination with the specific growth rate (μ) in fed‐batch cultivations. In this work, Candida rugosa lipase 1 (Crl1) production, regulated by the constitutive GAP promoter, growing at different nominal μ (0.030, 0.065, 0.100 and 0.120 h−1) under both normoxic and hypoxic conditions in carbon‐limiting fed‐batch cultures is analysed. Hypoxic fermentations were controlled at a target respiratory quotient (RQ) of 1.4, with excellent performance, using an innovative automated control based on the stirring rate as the manipulated variable developed during this study. The results conclude that oxygen limitation positively affects bioprocess efficiency under all growing conditions compared. The shift from respiratory to respiro‐fermentative metabolism increases bioprocess productivity by up to twofold for the specific growth rates evaluated. Moreover, the specific product generation rate (qp) increases linearly with μ, regardless of oxygen availability. Furthermore, this hypoxic boosting effect was also observed in the production of Candida antarctica lipase B (CalB) and pro‐Rhizopus oryzae lipase (proRol), thus proving the synergic effect of kinetic and physiological stress control. Finally, the Crl1 production scale‐up was conducted successfully, confirming the strategy's scalability and the robustness of the results obtained at the bench‐scale level. - Continuous Process Verification 4.0 application in upstream: adaptiveness implementation managed by AI in the hypoxic bioprocess of the Pichia pastoris cell factory
Arnau Gasset, Joeri Van Wijngaarden, Ferran Mirabent, Albert Sales-Vallverdú, Xavier Garcia-Ortega, José Luis Montesinos-Seguí, Toni Manzano, Francisco Valero
Frontiers in Bioengineering and Biotechnology, 2024 - Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free PDH promoter
Núria Bernat-Camps, Katharina Ebner, Veronika Schusterbauer, Jasmin Elgin Fischer, Miguel Angel Nieto-Taype, Francisco Valero, Anton Glieder, Xavier Garcia-Ortega
Frontiers in Bioengineering and Biotechnology, 2023 - Innovative Bioprocess Strategies Combining Physiological Control and Strain Engineering of Pichia pastoris to Improve Recombinant Protein Production
Arnau Gasset, Xavier Garcia-Ortega, Javier Garrigós-Martínez, Francisco Valero, José Luis Montesinos-Seguí
Frontiers in Bioengineering and Biotechnology, 2022 - Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C9
Javier Garrigós-Martínez, Astrid Weninger, José Luis Montesinos-Seguí, Christian Schmid, Francisco Valero, Claudia Rinnofner, Anton Glieder, Xavier Garcia-Ortega
Microbial Cell Factories, 2021 - Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris
Javier Garrigós-Martínez, Kiira Vuoristo, Miguel Angel Nieto-Taype, Juha Tähtiharju, Jaana Uusitalo, Pauliina Tukiainen, Christian Schmid, Ilya Tolstorukov, Knut Madden, Merja Penttilä, José Luis Montesinos-Seguí, Francisco Valero, Anton Glieder, Xavier Garcia-Ortega
Microbial Cell Factories, 2021 - Continuous Cultivation as a Tool Toward the Rational Bioprocess Development With Pichia Pastoris Cell Factory
Miguel Angel Nieto-Taype, Xavier Garcia-Ortega, Joan Albiol, José Luis Montesinos-Seguí, Francisco Valero
Frontiers in Bioengineering and Biotechnology, 2020 - Rationale-based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffii (Pichia pastoris)
Miguel Angel Nieto‐Taype, Javier Garrigós‐Martínez, Marc Sánchez‐Farrando, Francisco Valero, Xavier Garcia‐Ortega, José Luis Montesinos‐Seguí
Microbial Biotechnology, 2020 - Rational development of bioprocess engineering strategies for recombinant protein production in Pichia pastoris (Komagataella phaffii) using the methanol-free GAP promoter. Where do we stand?
Xavier García-Ortega, Elena Cámara, Pau Ferrer, Joan Albiol, José Luis Montesinos-Seguí, Francisco Valero
New Biotechnology, 2019 - Continuous operation, a realistic alternative to fed-batch fermentation for the production of recombinant lipase B from Candida antarctica under the constitutive promoter PGK in Pichia pastoris
Julia de Macedo Robert, Xavier Garcia-Ortega, José Luis Montesinos-Seguí, Denise Maria Guimaraes Freire, Francisco Valero
Biochemical Engineering Journal, 2019 - Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris (Komagataella phaffii) P AOX1 -driven recombinant producer strains with different target gene dosage
Javier Garrigós-Martínez, Miguel Angel Nieto-Taype, Arnau Gasset-Franch, José Luis Montesinos-Seguí, Xavier Garcia-Ortega, Francisco Valero
Microbial Cell Factories, 2019 - Physiological state as transferable operating criterion to improve recombinant protein production in Pichia pastoris through oxygen limitation
Xavier Garcia-Ortega, Francisco Valero, José Luis Montesinos-Seguí
Journal of Chemical Technology and Biotechnology, 2017 - The effect of hypoxia on the lipidome of recombinant Pichia pastoris
Núria Adelantado, Pablo Tarazona, Karlheinz Grillitsch, Xavier García-Ortega, Sergi Monforte, Francisco Valero, Ivo Feussner, Günther Daum, Pau Ferrer
Microbial Cell Factories, 2017 - A step forward to improve recombinant protein production in Pichia pastoris: From specific growth rate effect on protein secretion to carbon-starving conditions as advanced strategy
Xavier Garcia-Ortega, Núria Adelantado, Pau Ferrer, José Luis Montesinos, Francisco Valero
Process Biochemistry, 2016 - Overall key performance indicator to optimizing operation of high-pressure homogenizers for a reliable quantification of intracellular components in Pichia pastoris
Xavier Garcia-Ortega, Cecilia Reyes, José Luis Montesinos, Francisco Valero
Frontiers in Bioengineering and Biotechnology, 2015 - Fed-batch operational strategies for recombinant Fab production with Pichia pastoris using the constitutive GAP promoter
Xavier Garcia-Ortega, Pau Ferrer, José Luis Montesinos, Francisco Valero
Biochemical Engineering Journal, 2013
