About This Project
Artificial methane monooxygenases (MMOs) using Cu and Fe metal coordination centers within coiled-coil tetratricopeptide repeat (CTPR) scaffolds offer a promising approach for methane oxidation. By engineering CTPRs to host Cu and Fe active sites, these artificial enzymes mimic natural MMOs, enabling selective CH₄-to-CH₃OH conversion. This strategy provides stability, tunability, and catalytic efficiency, advancing biocatalysis for sustainable fuel production and methane mitigation
Ask the Scientists
Join The DiscussionMotivating Factor
Methane (CH₄) is a potent greenhouse gas and a valuable feedstock for chemical synthesis. However, its selective oxidation to methanol (CH₃OH) remains a significant challenge due to the high C–H bond strength (104 kcal/mol). Natural methane monooxygenases (MMOs), including soluble MMO (sMMO) and particulate MMO (pMMO), catalyze this transformation efficiently under mild conditions. sMMO is a diiron-containing enzyme that activates dioxygen to form a highly reactive intermediate (Intermediate Q), responsible for methane oxidation [Schulz et al. 2021]. pMMO, on the other hand, is a copper-dependent enzyme, with recent studies suggesting a dicopper active site in the pmoB subunit [Balasubramanian et al. 2010]. Despite their efficiency, MMOs pose challenges for industrial application due to their complex structures and difficulty in large-scale production. The development of artificial methane monooxygenases (AMMOs) aims to mimic these enzymatic processes for industrial applications.
Specific Bottleneck
Several bottlenecks still challenge the development of efficient AMMOs. Many bioinspired systems exhibit low stability, requiring further optimization to improve durability and catalytic efficiency. The transition from laboratory-scale research to industrial applications is hindered by high costs, scalability issues, and efficiency constraints. Additionally, controlling oxidation to maximize methanol yield while preventing overoxidation to CO₂ remains a critical hurdle. Overcoming these challenges will require a multidisciplinary approach, integrating computational modeling, synthetic biology, and materials science to design more robust and scalable AMMOs. Further advancements in protein engineering and microfluidics screening could also accelerate the discovery of improved variants with enhanced catalytic properties.
Actionable Goals
Goal 1 (G1): Design and engineer artificial methane monooxygenases (AMMOs) with specific amino acid residues for Fe and Cu coordination, inspired by the structures of sMMO and pMMO, while improving stability and catalytic efficiency.
Goal 2 (G2): Incorporate Fe and Cu centers into sMMO- and pMMO-like proteins, ensuring stable metal coordination and preventing structural degradation.
Goal 3 (G3): Develop artificial enzyme variants with MMO-like activity, optimizing methanol yields while minimizing overoxidation to CO₂.
Goal 4 (G4): Create an artificial enzyme library to enhance catalytic activity through iterative design, screening, and optimization for improved performance and scalability.
Budget
A significant portion of the workforce will be dedicated to expressing, purifying, and coordinating the CTPR protein, which is crucial for the success of the project. As such, we will need to allocate part of the salary for a technician to assist with these tasks. In addition, we will require consumables for protein purification, metal coordination, library preparation, and the initial microfluidic experiments. Furthermore, we will need to outsource the gas chromatography experiments for methane oxidation activity analysis, as well as the photolithography for the microfluidic chip fabrication. These resources are essential for ensuring the smooth progression and success of the project.
Meet the Team
Team Bio
The Molecular Nanobiotechnology Lab, directed by Aitziber L. Cortajarena, focuses on developing functional nanostructures and bioinspired materials for nanobiotechnology and nanomedicine. The research includes protein scaffolds for nanofabrication, hybrid protein-nanomaterial systems for biomedical and technological applications, designed proteins for biomolecular electronics, protein-nanomaterial systems for biocatalysis and biosensing, and in vitro diagnostic tests for antibody detection.
Aitor Manteca
Aitor Manteca is a Research Associate at the Biomolecular Nanotechnology Lab, CIC biomaGUNE, since 2021, funded by the Gipuzkoa Fellow Grant. He has authored 8 peer-reviewed articles (as first, second, or corresponding author) and contributed to the creation of two start-ups. Dr. Manteca has delivered over 10 keynote, invited, and selected talks at international events. He holds dual B.Eng degrees in Industrial Chemistry and Material Science, with research stays in The Netherlands and Spain. In 2009, he earned the Iñaki Goenaga scholarship to study magnetoresistive properties, resulting in a first-author publication in Biosensors and Bioelectronics. He later worked in R&D at ITW-Krafft, integrating nanomaterials into polymers and completing an M.Eng in Renewable Materials. From 2013–2017, he pursued a PhD at CIC nanoGUNE, studying titin's nanoscale properties under Raul Pérez-Jimenez and David de Sancho. His research yielded a European PhD with Cum Laude, publications in JBC and Nature Structural & Molecular Biology, and the spin-off Evolgene Genomics. A secondment in Germany, funded by EBSA, expanded his expertise in molecular cardiology. His postdoctoral research (2017–2021) with Dr. Axel Innis (INSERM) and Prof. JC Baret (CNRS) focused on peptide-based bacterial ribosome inhibitors using droplet microfluidics, supported by an EMBO Grant. He published in ACS Synthetic Biology and Biochemistry. At CIC biomaGUNE, Dr. Manteca has secured over €500,000 in funding for protein engineering, quantum-inspired algorithms, and immunotherapy. His collaborations include CRG, NavarraBiomed, CNRS, and Pasteur Institute, alongside industrial partners like ACX Instruments and Multiverse Computing. He co-leads outreach programs such as Pint of Science and Biophysics Day. He has supervised and advised multiple PhD and graduate students and developed innovative technologies, including a droplet sorter and quantum-based epitope analysis tools, with patents underway.
Liliana R. Teixeira
Dr. Liliana Teixeira is a Postdoctoral Researcher in the Biomolecular Nanotechnology group at CIC biomaGUNE, under the supervision of Prof. Dr. Aitziber López Cortajarena with a Bikaintek grant. Her work focuses on the development and screening of protein libraries.
Dr. Teixeira has a bachelor's and master's in Biotechnology from the University of Aveiro, with a research stay at the University of Ghent. She obtained her PhD thesis at NOVA University in Lisbon, under the supervision of Prof. Dr. Carlos A. Salgueiro in the area of biochemistry and biophysics. She carried out her PhD thesis on the mechanisms of electricity bioproduction, and did a secondment under the supervision of Prof. Dr. Marta Bruix at IQF-CSIC (Madrid).
Subsequently, she worked as a postdoctoral researcher at UTSW Medical Center, under the supervision of Dr. Goldsmith. Her project focused on the biochemical and biophysical study of a family of proteins involved in the process of breast cancer metastasis.
Dr. Teixeira has published 7 articles in international journals and has presented her work in several national and international conferences.
Eugenio Roubieu
Eugenio Roubieu is a PhD researcher at CIC biomaGUNE's Biomolecular Nanotechnology group conducted research under the guidance of Prof. Dr. Aitziber L. Cortajarena, with funding from the European Project iSenseDNA, specializing in advanced protein engineering methodologies.
Eugenio Roubieu holds a Bachelor's degree in Biotechnology from the University of Pablo de Olavide. He pursued two distinct Master's degrees, one in Engineering and Chemistry of Biomolecules at the University Paris-Sud/Saclay, where he specialized in NANO-IR techniques under the supervision of Prof. Dr. Ariane Deniset-Besseau within the field of biophysics, and another in Medical Chemistry at the University of Cádiz. In the latter, his research focused on the chemical modification of polycaprolactone to graft antibiotics for the development of 3D-printed scaffolds.
Aitziber L. Cortajarena
Aitziber L. Cortajarena is an Ikerbasque Research Professor who leads the Biomolecular Nanotechnology Laboratory at CIC biomaGUNE, where she served as the Scientific ViceDirector in 2021, and has been the Scientific Director since 2022. She has published over 100 peer-reviewed articles in internationally recognized journals, which have been cited over 3300 times, 6 invited reviews, 2 edited books, and 5 granted patents. She has presented her work at over 50 internationally recognized conferences. Her work has been recognized by the Horizon Prize from The Royal Society of Chemistry, the Research Excellence Award from The Spanish Royal Society of Chemistry, and the Women in Science Career Award from Ikerbasque. She is Associate Editor of ACS Applied Biomaterials, ACS Publications, and Senior Editor at Protein Science, Wiley. She is the vice president of the Spanish Biophysical Society and Member of the International Protein Society Council. Her mentoring abilities are reflected in the number of researchers she has supervised: 22 Master students (10 of which international), 18 PhD students, and 14 postdoctoral researchers in the past 5 years. Many of her alumni have gone onto successful academic or industry careers. Cortajarena has obtained numerous European projects, including an ERC Consolidator Grant (ProNANO), two ERC-PoC (NIMM, NanoImaging), an ERA-CoBioTech (coordinator), four FET-Open projects, one coordinated (e-Prot, ARTIBLED, FairyLights, DeDNAed), and one EICPathfinder project (iSenseDNA), which cover from the fundamental development of protein-based tools to the validation of biomolecule-based technologies in biomedical and technological applications.
Additional Information
This project addresses two key actionable problems from the repository:
Discover or Engineer Efficient Soluble Methane Monooxygenases: By designing and producing artificial methane monooxygenases (AMMOs) based on engineered CTPR scaffolds, the project aims to create efficient, soluble catalysts for methane oxidation.
Design a De Novo Methane Monooxygenase: Through computational and synthetic biology approaches, the project seeks to design entirely new methane monooxygenase systems from scratch, enabling novel and scalable methods for methane valorization.
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