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Microbial deployment of new-to-nature chemistries for refactoring the barriers between living and non-living matter

Deliverables

Morbidostat for heterotic computation of (bio)chemical problems

This is about programming evolution by switching between certain combinations of nutrients/substrates along with a simple deterministic model to compute evolution heterotically. This system is an automated continuous-culture device that controls substrate concentration to maintain constant growth inhibition. The device runs at a certain interval and adds either fresh medium with a high substrate concentration (2,4-dinitrotoluene, DNT as the target molecule in our case) or without it. By giving it pulses of DNT and letting it recover we expect that the ROS produced by faulty reactions by suboptimal dioxygenases will diversify the corresponding DNA sequences until a biochemical solution is found.

Genome edited strains of E. coli and P. putida entirely cyborgized for hosting NTN reactions

The genomes of the two bacterial species adopted in this Project (E. colo and Pseudomonas putida) will be [i] erased of instability determinants and other genes encoding functions considered to be irrelevant for the sake of MADONNA and [ii] endowed with new traits to make them withstand the working conditions of the reactions of interest.

Microdroplet-based environmental simulator

This will be based on a fully autonomous chemical robot system that is able to generate random physicochemical inputs in microdroplet scale. The droplet behaviours will then be analysed, and ranked using a fitness function. This process is continued through iterated generations. This allows inspecting how the genotype is modulated through an envirotype to express the phenotype of interest.

Proposition of containment needs for microbial agents bearing peribolic reactions

Modeling approaches will be adopted to inspect the pluses and minuses of dealing with live —but heavily refactored microbial agents that could propagate through the Biosphere. This will help to determine the needs of containment of the agents at stake. This lower-scale models will help to understand the resilience and optimal yield conditions of designed microorganisms and microbial consortia and create in silico tests of the proposed peribolic reactions.

Detailed report a genetically optimized artificial carboxylase based on evolved Sav isoforms

The reaction conditions that accompany desired carboxylase recations are often different from physiological conditions (defined as pH of approximately 7, aqueous environments, and ambient temperatures) and turnover numbers very low. In order to make such reaction sustainable and to ultimately recruit such reactions into biology, the protein scaffold has to be evolved. Therefore, we will develope an strategy to implement an enzyme engineering trajectory towards a highly efficient Sav-Pd-catalyst for the production of acrylic acid.

Responsible innovation. Report of ethics, safety and security tasks

Reconciliation of geochemical, biological and industrial/human induced global cycles will not only provide a sustainable way of producing and consuming, but will inevitably change the characteristic of the three cycles, diffusing and enlarging the limits of human intervention on the planet. In this report we will ask: what rationale (world view) is behind the projects aim and goal, what other rationales co-exist, which solutions do they provide? What are the ethical dimensions of going forward with the project versus continuing with the unsustainable business-as-usual.

Setup of a Project website and Logo

This Deliverable implies the elaboration of an user-friendly webpage for Communication and Dissemination of Project's activities as well as for internal communication among MADONNA members

Data management plan

This will consist of a document specifying the formats to be followed by the partners for storing the data of interest in a joint repository as well as formal languages for description of actions and constructs

An integrated computational frame for merging NTN reactions to extant metabolism

This is about application of complex networks theory to model new metabolic loops brought about by small-scale and large-scale peribolism. Meso- and large-scale models of ecosystems incorporating the NTN reactions set by MADONNA will be produced. Our ecosystem description incorporates both standard habitats as well as the high-energy, fuel-powered counterparts that are defined by large urban centres and industry. By incorporating peribolism, we will transform these type of waste/sink components into new large-scale chemical processes thus deeply changing the global dynamics. To this goal we will develop a set of mathematical and computational tools that provide a rationale for the expected responses of this global metabolism on different scales.

Publications

Evolutionary tinkering vs. rational engineering in the times of synthetic biology

Author(s): Víctor de Lorenzo
Published in: Life Sciences, Society and Policy, Issue 14/1, 2018, ISSN 2195-7819
DOI: 10.1186/s40504-018-0086-x

A Post-translational Metabolic Switch Enables Complete Decoupling of Bacterial Growth from Biopolymer Production in Engineered Escherichia coli

Author(s): Gonzalo Durante-Rodríguez, Víctor de Lorenzo, Pablo I. Nikel
Published in: ACS Synthetic Biology, Issue 7/11, 2018, Page(s) 2686-2697, ISSN 2161-5063
DOI: 10.1021/acssynbio.8b00345

The Metabolic Redox Regime of Pseudomonas putida Tunes Its Evolvability toward Novel Xenobiotic Substrates

Author(s): Özlem Akkaya, Danilo R. Pérez-Pantoja, Belén Calles, Pablo I. Nikel, Víctor de Lorenzo
Published in: mBio, Issue 9/4, 2018, ISSN 2150-7511
DOI: 10.1128/mbio.01512-18

Evolving metabolism of 2,4-dinitrotoluene triggers SOS-independent diversification of host cells

Author(s): Özlem Akkaya, Pablo I. Nikel, Danilo Pérez-Pantoja, Víctor de Lorenzo
Published in: Environmental Microbiology, Issue 21/1, 2019, Page(s) 314-326, ISSN 1462-2912
DOI: 10.1111/1462-2920.14459

Population dynamics of synthetic terraformation motifs

Author(s): Ricard V. Solé, Raúl Montañez, Salva Duran-Nebreda, Daniel Rodriguez-Amor, Blai Vidiella, Josep Sardanyés
Published in: Royal Society Open Science, Issue 5/7, 2018, Page(s) 180121, ISSN 2054-5703
DOI: 10.1098/rsos.180121

The imbroglio of the physiological Cra effector clarified at last

Author(s): Max Chavarría, Víctor de Lorenzo
Published in: Molecular Microbiology, Issue 109/3, 2018, Page(s) 273-277, ISSN 0950-382X
DOI: 10.1111/mmi.14080

Refactoring the upper sugar metabolism of Pseudomonas putida for co-utilization of cellobiose, xylose, and glucose

Author(s): Pavel Dvořák, Víctor de Lorenzo
Published in: Metabolic Engineering, Issue 48, 2018, Page(s) 94-108, ISSN 1096-7176
DOI: 10.1016/j.ymben.2018.05.019

Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism

Author(s): Pablo I. Nikel, Víctor de Lorenzo
Published in: Metabolic Engineering, Issue 50, 2018, Page(s) 142-155, ISSN 1096-7176
DOI: 10.1016/j.ymben.2018.05.005

Exploiting delayed transitions to sustain semiarid ecosystems after catastrophic shifts

Author(s): Blai Vidiella, Josep Sardanyés, Ricard Solé
Published in: Journal of The Royal Society Interface, Issue 15/143, 2018, Page(s) 20180083, ISSN 1742-5689
DOI: 10.1098/rsif.2018.0083

CRISPR /Cas9‐enhanced ss DNA recombineering for Pseudomonas putida


Published in: ISSN 1751-7915
DOI: 10.1111/1751-7915.13453

SEVA 3.0: an update of the Standard European Vector Architecture for enabling portability of genetic constructs among diverse bacterial hosts

Author(s): Esteban Martínez-García, Angel Goñi-Moreno, Bryan Bartley, James McLaughlin, Lucas Sánchez-Sampedro, Héctor Pascual del Pozo, Clara Prieto Hernández, Ada Serena Marletta, Davide De Lucrezia, Guzmán Sánchez-Fernández, Sofía Fraile, Víctor de Lorenzo
Published in: Nucleic Acids Research, Issue 48/D1, 2019, Page(s) D1164-D1170, ISSN 0305-1048
DOI: 10.1093/nar/gkz1024

Gross transcriptomic analysis of Pseudomonas putida for diagnosing environmental shifts

Author(s): Ángeles Hueso‐Gil, Belén Calles, George A. O'Toole, Víctor Lorenzo
Published in: Microbial Biotechnology, Issue 13/1, 2020, Page(s) 263-273, ISSN 1751-7915
DOI: 10.1111/1751-7915.13404

Assembly of a Custom-made Device to Study Spreading Patterns of Pseudomonas putida Biofilms

Author(s): David Espeso, Esteban Martínez-García, Víctor Lorenzo
Published in: BIO-PROTOCOL, Issue 9/10, 2019, ISSN 2331-8325
DOI: 10.21769/bioprotoc.3238

Multiple-Site Diversification of Regulatory Sequences Enables Interspecies Operability of Genetic Devices

Author(s): Angeles Hueso-Gil, Ákos Nyerges, Csaba Pál, Belén Calles, Víctor de Lorenzo
Published in: ACS Synthetic Biology, Issue 9/1, 2019, Page(s) 104-114, ISSN 2161-5063
DOI: 10.1021/acssynbio.9b00375

Digitalizing heterologous gene expression in Gram‐negative bacteria with a portable ON/OFF module

Author(s): Belén Calles, Ángel Goñi‐Moreno, Víctor Lorenzo
Published in: Molecular Systems Biology, Issue 15/12, 2019, ISSN 1744-4292
DOI: 10.15252/msb.20188777

Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene

Author(s): Tomas Aparicio, Akos Nyerges, István Nagy, Csaba Pal, Esteban Martínez‐García, Víctor Lorenzo
Published in: Environmental Microbiology, Issue 22/1, 2019, Page(s) 45-58, ISSN 1462-2912
DOI: 10.1111/1462-2920.14814

Synthetic Biology for Terraformation Lessons from Mars, Earth, and the Microbiome

Author(s): Nuria Conde-Pueyo, Blai Vidiella, Josep Sardanyés, Miguel Berdugo, Fernando T. Maestre, Victor de Lorenzo, Ricard Solé
Published in: Life, Issue 10/2, 2020, Page(s) 14, ISSN 2075-1729
DOI: 10.3390/life10020014

High-Efficiency Multi-site Genomic Editing of Pseudomonas putida through Thermoinducible ssDNA Recombineering

Author(s): Tomas Aparicio, Akos Nyerges, Esteban Martínez-García, Víctor de Lorenzo
Published in: iScience, Issue 23/3, 2020, Page(s) 100946, ISSN 2589-0042
DOI: 10.1016/j.isci.2020.100946

Functional implementation of a linear glycolysis for sugar catabolism in Pseudomonas putida

Author(s): Alberto Sánchez-Pascuala, Lorena Fernández-Cabezón, Víctor de Lorenzo, Pablo I. Nikel
Published in: Metabolic Engineering, Issue 54, 2019, Page(s) 200-211, ISSN 1096-7176
DOI: 10.1016/j.ymben.2019.04.005

Pseudomonas putida in the quest of programmable chemistry

Author(s): Esteban Martínez-García, Víctor de Lorenzo
Published in: Current Opinion in Biotechnology, Issue 59, 2019, Page(s) 111-121, ISSN 0958-1669
DOI: 10.1016/j.copbio.2019.03.012

Spatial organization of the gene expression hardware in Pseudomonas putida

Author(s): Juhyun Kim, Angel Goñi‐Moreno, Belén Calles, Víctor de Lorenzo
Published in: Environmental Microbiology, Issue 21/5, 2019, Page(s) 1645-1658, ISSN 1462-2912
DOI: 10.1111/1462-2920.14544

Genomic Responses of Pseudomonas putida to Aromatic Hydrocarbons

Author(s): Víctor de Lorenzo, Hiren Joshi
Published in: Consequences of Microbial Interactions with Hydrocarbons, Oils, and Lipids: Biodegradation and Bioremediation, 2019, Page(s) 1-15
DOI: 10.1007/978-3-319-44535-9_25-1

Datasets