Periodic Reporting for period 1 - MENTHOL (Metabolic heterogeneity of monoclonal bacterial cells as a biotechnological tool to produce natural compounds.)
Okres sprawozdawczy: 2021-07-01 do 2023-06-30
The MSCA action MENTHOL aimed to create a new biotechnological platform for microbial production of high-value plant metabolites. The project involved the establishment of a distributed catalysis within single-species-based multifunctional bacterial populations. It was focused on producing isoprenoids such as limonene, menthol, and p-cymene, which are compounds with significant industrial relevance. Fully aligned with the sustainable development strategies promoted by the European Union, this project positively influenced microbial biotech progress.
In recent decades, synthetic biology (SynBio) has advanced microbial metabolite production. For early strategies, metabolic burden of monoclonal fermentations and complex ecological interactions within synthetic consortia are yet issues to be solved. MENTHOL innovatively addressed these by decoupling complex biosynthetic pathways into minimal functional modules, to be reassembled within metabolically differentiated isogenic populations. The outcomes of this MSCA action contributed to the emergence of a novel hypothesis: programmable metabolic heterogeneity as a strategy to promote division of labour within monoclonal bacterial populations.
Specific objectives of this MSCA action have been: (1) engineering a pre-programmable heterogeneous monoclonal population of P. putida cells through cutting-edge SynBio approaches (WP3); (2) engineering a P. putida strain overproducing isopentenyl pyrophosphate (IPP) (WP4); (3) model-based engineering of multifunctional P. putida populations overproducing limonene, menthol, and p-cymene (WP5-6). Additional goals were focused on fostering training and development of the researcher (WP1-2).
In recent decades, synthetic biology (SynBio) has advanced microbial metabolite production. For early strategies, metabolic burden of monoclonal fermentations and complex ecological interactions within synthetic consortia are yet issues to be solved. MENTHOL innovatively addressed these by decoupling complex biosynthetic pathways into minimal functional modules, to be reassembled within metabolically differentiated isogenic populations. The outcomes of this MSCA action contributed to the emergence of a novel hypothesis: programmable metabolic heterogeneity as a strategy to promote division of labour within monoclonal bacterial populations.
Specific objectives of this MSCA action have been: (1) engineering a pre-programmable heterogeneous monoclonal population of P. putida cells through cutting-edge SynBio approaches (WP3); (2) engineering a P. putida strain overproducing isopentenyl pyrophosphate (IPP) (WP4); (3) model-based engineering of multifunctional P. putida populations overproducing limonene, menthol, and p-cymene (WP5-6). Additional goals were focused on fostering training and development of the researcher (WP1-2).
Work was conducted via work packages. WP1-2 included all activities related with project management, career development, and growth independence of the fellow as research scientist. Throughout the cultivation of personal brand, the researcher not only enhance the CV, but was also able to identify strengths and focus on the type of research to lead in future projects. Working with Design-Build-Test-Learn (DBTL) cycles enhanced research skills in various domains, creating solid foundations to success in the field of SynBio and biotechnology. As featured deliverables there is the application for a national grant IF-ERC; successful participation in new project proposals, and the supervision of forthcoming Master thesis.
In WP 3-6, the research trajectory evolved from foundational steps to more ambitious outcomes.
The pivotal task of this project involved engineering synthetic pluripotent P. putida cells through the implementation of a toggle switch (TS) controlled by cell density. The scope was broadened to encompass the development of ProMetEO (from PROgrammable METabolic hetErOgeneity) a toolbox consisting of a library of 27 bistable dynamic systems that allow to split an isogenic bacteria population in two fractions expressing complementary metabolic labors. ProMetEO is available to the metabolic engineering community as a SynBio toolbox platform presented as MoClo-based Level 1 Module to offer a time-saving solution for their implementation (WP3).
Expanding the known potential of the TS, the fellow established compelling proof-of-concepts, exemplifying applications with valuable results for the future of synthetic pattern formation. This is particularly pertinent for genetic network design aimed at engineering living materials with sustainability implications.
Focused on the production of isoprenoids, a P. putida strain overproducing IPP/DMAPP was engineered and optimized (WP4). Along the way, metabolite biosensor to the high throughput testing of multiple metabolic pathways designs was necessary to select the architecture promoting higher IPP production. As this was necessary, not only for the current project, but for any endeavor involving the optimization of synthetic metabolic pathways, the scope of this task has been broadened to the development of modular biosensing devices with customizable ligand-specificity, providing a versatile platform for the current and future investigations.
Following a similar strategy, the engineering and development of four P. putida strains — MEVI (IPP overproducer), LIMON (limonene biosynthesizer), MENTHOL (menthol producer), and CYME (p-cymene biosynthesizer) — were accomplished, underlining the multiplicity of achievements within this fellowship.
Dissemination and public engagement are crucial activities for scientific development. Throughout the course of this project, the fellow was actively engaged in various activities to promote the dissemination of scientific knowledge and foster collaboration and interdisciplinary dialogue. Some of the key activities undertaken include: (1) Poster and Rapid Fire Tak at Metabolic Engineering International Conference (ME15); (2) multiple seminars organized by the National Centre of Biotechnology in Spain; (3) Tutoring at “High School - Enterprise Program” from Madrid government; (4) Speaker at National Conference on Biotechnology of Microbiomes at Universidad del Valle (Colombia); (5) Two times speaker of European Researcher’s Night in Madrid, Spain; (6) Speaker at LGTBIQ+ Figures in STEM from Los Andes University (Colombia); (7) International Day of Women and Girls in Science (Madrid, Spain).
Results of this project are reported in: 3 forthcoming papers on Q1 scientific journal; Poster and Rapid Fire Talk in Metabolic Engineering Conference 15 (2023); Poster in Scientific Conference in Autumn 2023; (4) Master Thesis supervised by the fellow.
In WP 3-6, the research trajectory evolved from foundational steps to more ambitious outcomes.
The pivotal task of this project involved engineering synthetic pluripotent P. putida cells through the implementation of a toggle switch (TS) controlled by cell density. The scope was broadened to encompass the development of ProMetEO (from PROgrammable METabolic hetErOgeneity) a toolbox consisting of a library of 27 bistable dynamic systems that allow to split an isogenic bacteria population in two fractions expressing complementary metabolic labors. ProMetEO is available to the metabolic engineering community as a SynBio toolbox platform presented as MoClo-based Level 1 Module to offer a time-saving solution for their implementation (WP3).
Expanding the known potential of the TS, the fellow established compelling proof-of-concepts, exemplifying applications with valuable results for the future of synthetic pattern formation. This is particularly pertinent for genetic network design aimed at engineering living materials with sustainability implications.
Focused on the production of isoprenoids, a P. putida strain overproducing IPP/DMAPP was engineered and optimized (WP4). Along the way, metabolite biosensor to the high throughput testing of multiple metabolic pathways designs was necessary to select the architecture promoting higher IPP production. As this was necessary, not only for the current project, but for any endeavor involving the optimization of synthetic metabolic pathways, the scope of this task has been broadened to the development of modular biosensing devices with customizable ligand-specificity, providing a versatile platform for the current and future investigations.
Following a similar strategy, the engineering and development of four P. putida strains — MEVI (IPP overproducer), LIMON (limonene biosynthesizer), MENTHOL (menthol producer), and CYME (p-cymene biosynthesizer) — were accomplished, underlining the multiplicity of achievements within this fellowship.
Dissemination and public engagement are crucial activities for scientific development. Throughout the course of this project, the fellow was actively engaged in various activities to promote the dissemination of scientific knowledge and foster collaboration and interdisciplinary dialogue. Some of the key activities undertaken include: (1) Poster and Rapid Fire Tak at Metabolic Engineering International Conference (ME15); (2) multiple seminars organized by the National Centre of Biotechnology in Spain; (3) Tutoring at “High School - Enterprise Program” from Madrid government; (4) Speaker at National Conference on Biotechnology of Microbiomes at Universidad del Valle (Colombia); (5) Two times speaker of European Researcher’s Night in Madrid, Spain; (6) Speaker at LGTBIQ+ Figures in STEM from Los Andes University (Colombia); (7) International Day of Women and Girls in Science (Madrid, Spain).
Results of this project are reported in: 3 forthcoming papers on Q1 scientific journal; Poster and Rapid Fire Talk in Metabolic Engineering Conference 15 (2023); Poster in Scientific Conference in Autumn 2023; (4) Master Thesis supervised by the fellow.
The anticipated impacts of the MSCA action have been increased and improved. In terms of scientific production, two groundbreaking SynBio tools have been developed. These tools are expected to significantly facilitate research in systems metabolic engineering, particularly in the design of synthetic metabolic pathways. As part of our commitment to knowledge sharing and collaboration, these tools will be made accessible to the wider scientific community, thereby fostering advancements in biotechnology and metabolic engineering. The results achieved through the exploration of the potential of ProMetEO have led to the emergence of novel and more advanced projects and initiatives, that have successfully secured funding and are currently underway within the Systems Biotechnology Research Group (SBG). MENTHOL results are valuable for the future of synthetic pattern formation, for example, in the context of designing genetic networks for the bottom-up engineering of living materials for sustainability. Likewise, the progresses in the optimization of synthetic pathways to produce isoprenoids have laid important foundations for a new line of research within the group.
Outcomes and achievements presented here are aligned with the sustainable development strategies promoted by the European Union, yielding solid positive impacts in the advancement of microbial biotechnology platforms and their integration to bioeconomy.
Outcomes and achievements presented here are aligned with the sustainable development strategies promoted by the European Union, yielding solid positive impacts in the advancement of microbial biotechnology platforms and their integration to bioeconomy.