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


Morbidostat for heterotic computation of (bio)chemical problems

This is about programming evolution by switching between certain combinations of nutrientssubstrates along with a simple deterministic model to compute evolution heterotically This system is an automated continuousculture 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 24dinitrotoluene 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.

Automated generation of metalloenzymes and information capture through a fitness function

We will take the reaction invented chemically (ie CO2 capture with a metalloenzyme) and and run it in the presence of cells using a chemical droplet robot in which the biological system is programmed to generate peptides that can chelate metal ions to generate ArMs that can be symbiotically selected by the system. This links process engineering to the fitness functions and allows the systems to be linked artificially using such a chem-o-biological robot. The link is software and spectroscopy with chemical feedback. The chemicals can acts as mutagens, and also act as selection agents on the peptides expressed.

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 userfriendly webpage for Communication and Dissemination of Projects 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 smallscale and largescale peribolism Meso and largescale models of ecosystems incorporating the NTN reactions set by MADONNA will be produced Our ecosystem description incorporates both standard habitats as well as the highenergy fuelpowered counterparts that are defined by large urban centres and industry By incorporating peribolism we will transform these type of wastesink components into new largescale 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

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Large-scale DNA-based phenotypic recording and deep learning enable highly accurate sequence-function mapping

Author(s): Simon Höllerer, Laetitia Papaxanthos, Anja Cathrin Gumpinger, Katrin Fischer, Christian Beisel, Karsten Borgwardt, Yaakov Benenson, Markus Jeschek
Published in: Nature Communications, 11/1, 2020, ISSN 2041-1723
Publisher: Nature Publishing Group
DOI: 10.1038/s41467-020-17222-4

The Quest for Xenobiotic Enzymes: From New Enzymes for Chemistry to a Novel Chemistry of Life

Author(s): Tobias Vornholt, Markus Jeschek
Published in: ChemBioChem, 21/16, 2020, Page(s) 2241-2249, ISSN 1439-4227
Publisher: John Wiley & Sons Ltd.
DOI: 10.1002/cbic.202000121

Xenobiology: A Journey towards Parallel Life Forms

Author(s): Nediljko Budisa, Vladimir Kubyshkin, Markus Schmidt
Published in: ChemBioChem, 21/16, 2020, Page(s) 2228-2231, ISSN 1439-4227
Publisher: John Wiley & Sons Ltd.
DOI: 10.1002/cbic.202000141

Synthetic soil crusts against green-desert transitions: a spatial model

Author(s): Blai Vidiella, Josep Sardanyés, Ricard V. Solé
Published in: Royal Society Open Science, 7/8, 2020, Page(s) 200161, ISSN 2054-5703
Publisher: The Royal Society
DOI: 10.1098/rsos.200161

The Wsp intermembrane complex mediates metabolic control of the swim‐attach decision of Pseudomonas putida

Author(s): Ángeles Hueso‐Gil, Belén Calles, Víctor Lorenzo
Published in: Environmental Microbiology, 22/8, 2020, Page(s) 3535-3547, ISSN 1462-2912
Publisher: Blackwell Publishing Inc.
DOI: 10.1111/1462-2920.15126

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

Author(s): Víctor de Lorenzo
Published in: Life Sciences, Society and Policy, 14/1, 2018, ISSN 2195-7819
Publisher: Springer
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, 7/11, 2018, Page(s) 2686-2697, ISSN 2161-5063
Publisher: American Chemical Society
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, 9/4, 2018, ISSN 2150-7511
Publisher: ASM
DOI: 10.1128/mbio.01512-18

Improved Thermotolerance of Genome-Reduced Pseudomonas putida EM42 Enables Effective Functioning of the P L / c I857 System

Author(s): Tomás Aparicio, Víctor de Lorenzo, Esteban Martínez-García
Published in: Biotechnology Journal, 14/1, 2019, Page(s) 1800483, ISSN 1860-6768
Publisher: Wiley - VCH Verlag GmbH & CO. KGaA
DOI: 10.1002/biot.201800483

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, 21/1, 2019, Page(s) 314-326, ISSN 1462-2912
Publisher: Blackwell Publishing Inc.
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, 5/7, 2018, Page(s) 180121, ISSN 2054-5703
Publisher: Royal Society
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, 109/3, 2018, Page(s) 273-277, ISSN 0950-382X
Publisher: Blackwell Publishing Inc.
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, 48, 2018, Page(s) 94-108, ISSN 1096-7176
Publisher: Academic Press
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, 50, 2018, Page(s) 142-155, ISSN 1096-7176
Publisher: Academic Press
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, 15/143, 2018, Page(s) 20180083, ISSN 1742-5689
Publisher: The Royal Society
DOI: 10.1098/rsif.2018.0083

CRISPR /Cas9‐enhanced ss DNA recombineering for Pseudomonas putida

Author(s): Tomás Aparicio, Víctor Lorenzo, Esteban Martínez‐García
Published in: Microbial Biotechnology, 12/5, 2019, Page(s) 1076-1089, ISSN 1751-7915
Publisher: John Wiley & Sons
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, 48/D1, 2019, Page(s) D1164-D1170, ISSN 0305-1048
Publisher: Oxford University Press
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, 13/1, 2020, Page(s) 263-273, ISSN 1751-7915
Publisher: Society for Applied Microbiology
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, 9/10, 2019, ISSN 2331-8325
Publisher: Bio-Protocol
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, 9/1, 2019, Page(s) 104-114, ISSN 2161-5063
Publisher: American Chemical Society
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, 15/12, 2019, ISSN 1744-4292
Publisher: Nature Publishing Group
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, 22/1, 2019, Page(s) 45-58, ISSN 1462-2912
Publisher: Blackwell Publishing Inc.
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, 10/2, 2020, Page(s) 14, ISSN 2075-1729
Publisher: MDPI
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, 23/3, 2020, Page(s) 100946, ISSN 2589-0042
Publisher: Elsevier
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, 54, 2019, Page(s) 200-211, ISSN 1096-7176
Publisher: Academic Press
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, 59, 2019, Page(s) 111-121, ISSN 0958-1669
Publisher: Elsevier BV
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, 21/5, 2019, Page(s) 1645-1658, ISSN 1462-2912
Publisher: Blackwell Publishing Inc.
DOI: 10.1111/1462-2920.14544

A metric space for semantic containment: Towards the implementation of genetic firewalls

Author(s): Markus Schmidt
Published in: Biosystems, 185, 2019, Page(s) 104015, ISSN 0303-2647
Publisher: Elsevier BV
DOI: 10.1016/j.biosystems.2019.104015

Global ecosystem thresholds driven by aridity

Author(s): Miguel Berdugo, Manuel Delgado-Baquerizo, Santiago Soliveres, Rocío Hernández-Clemente, Yanchuang Zhao, Juan J. Gaitán, Nicolas Gross, Hugo Saiz, Vincent Maire, Anika Lehmann, Matthias C. Rillig, Ricard V. Solé, Fernando T. Maestre
Published in: Science, 367/6479, 2020, Page(s) 787-790, ISSN 0036-8075
Publisher: American Association for the Advancement of Science
DOI: 10.1126/science.aay5958

Emergence of Function and Selection from Recursively Programmed Polymerisation Reactions in Mineral Environments

Author(s): Doran, David; Abul-Haija, Yousef M.; Cronin, Leroy
Published in: Angewandte Chemie (International Ed. in English), 1, 2019
Publisher: American Chemical Society (ACS), Chinese Chemical Society, Chemical Society of Japan, German Chemical Society (GDCh) and the Royal Society of Chemistry
DOI: 10.26434/chemrxiv.7712297.v1

Synthetic soil crusts against green-desert transitions: a spatial model

Author(s): Blai Vidiella, Josep Sardanyés, Ricard V. Solé
Published in: bioRxiv, 2019
Publisher: Cold Spring Harbor Laboratory
DOI: 10.1101/838631

Systematic Engineering of Artificial Metalloenzymes for New-to-Nature Reactions

Author(s): Tobias Vornholt,Fadri Christoffel, Michela M. Pellizzoni, Sven Panke, Thomas R. Ward, Markus Jeschek
Published in: bioRxiv, 2020
Publisher: Cold Spring Harbor Laboratory
DOI: 10.1101/2020.07.15.204206

Alternative Biofacts – Life as we don’t (yet) know it

Author(s): Markus Schmidt, Nediljko Budisa
Published in: Art as We Don’t Know It, 2020, Page(s) 20-36, ISBN 978-952-60-8823-5
Publisher: Aalto University Press

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, ISBN 978-3-319-44535-9
Publisher: Springer International Publishing
DOI: 10.1007/978-3-319-44535-9_25-1