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

Aparicio, T., Nyerges, Martínez-García E. and de Lorenzo, V. (2020) High-efficiency multi-site genomic editing (HEMSE) of Pseudomonas putida through thermoinducible ssDNA recombineering. iScience   DOI: 10.1016/j.isci.2020.100946 (In Press)

Single-stranded DNA recombineering is a useful tool for genome editing used mainly in enterobacteria strains such as E.coli. It is based on the presence of synthetic DNA strains and a DNA exchange mechanism called the Red system. Specifically, this system relies on one of its components, the Red-β protein – a recombinase protein that is able to include the synthetic DNA fragments into the DNA replication process and therefore change the DNA sequence. However, the implementation of this system in other non-enteric species is limited by the efficiency of the Red recombinase (which lowers drastically) and the action of repair systems that fix targeted mutations.

In this paper, we have developed a new method to use the Red recombineering system in Pseudomonas putida, a valuable non-enteric bacteria in industrial and biotechnological research. Our approach combines the expression of a Red-like protein and a reversible system to inhibit mismatch repair during a limited time. By applying multiple cycles of recombinase production and DNA transformation, high-fidelity recombination frequencies could be achieved. Thus, this method opens a new genome editing possibility for this bacteria and expands the single-stranded DNA recombineering system functionalities to other species.

An autonomous chemical robot discovers the rules of inorganic coordination chemistry without prior knowledge

Porwol, L., D. Kowalski, A. Henson, D. -L. Long, N. L. Bell, L. Cronin  (2020) An autonomous chemical robot discovers the rules of supramolecular chemistry without prior knowledge, Angew Chem Int Ed https://doi.org/10.1002/anie.202000329

Finding new chemical assemblies is challenging due to the vast range of combinations possible and the difficulty of their prediction. Moreover, it is common to perform these reactions with an outcome in mind or using sub-optimal conditions, limiting the extent of discovery. Attempts to explore the chemical space including optimal and non-optimal synthesis conditions to look for unpredicted outcomes carry a high cost in terms of time and resources. However, automation tools and algorithms can overcome these limitations.

In this paper, we have developed an autonomous discovery platform that is able to identify ligands and perform binding reactions faster than previous approaches (2h vs. 20h). This robot has been tested in a chemical space of over 109, and has already made relevant discoveries including a range of new molecules in the family of 1-benzyl-(1,2,3-triazol-4-yl)-N-alkyl-(2-pyridinemethanimine) ligands and four new complexes of Fe and Co.

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

Conde-Pueyo, N., Vidiella, B.,  Sardanyés J., Berdugo, M. Maestre, F., de Lorenzo V. and Sole, R. (2020) Synthetic biology for terraformation: lessons from Mars, Earth and the microbiome  Life 10: 14 (2020)

Synthetic biology has been widely used in different types of organisms, usually with some industrial application in mind. However, it has the potential of working in a much larger scale. Here, we discuss the possible applications of synthetic biology in the field of modifying ecosystems and ecological communities. The tools that synthetic biology provides could be used to prevent the loss of endangered areas or to minimize the consequences of climate change.

The core idea is the engineering of designed organisms able to counterbalance the impact of global warming and its associated tipping points. These organisms would act on key processes such as the reduction of greenhouse gases, removal of undesirable waste or ensuring habitat persistence. This concept is referred to as terraformation, and it is suggested here to include all scales for context: from the microbiome to the biosphere. Terraformation could not only be applied on Earth, but also to other planets such as Mars, where early studies have already been performed.

Xenobiology: A Journey towards Parallel Life Forms

Budisa N., Kubyshkin V., Schmidt M. (2020) Xenobiology: a journey towards parallel life forms. ChemBioChem doi.org/10.1002/cbic.202000141

There is a vast range of life forms living in our planet, from the most delicate species to extremophiles living in the deepest points of the ocean or above the atmosphere. But despite this tremendous diversity, all life forms share a very similar information-processing scheme and chemistry.

Xenobiology is the science that studies other possible basic workings of life, as in other ways to process information and use matter and energy. In this field, researchers aim to design or describe novel biological procedures or molecules that could have existed naturally but have not yet been found. In this research, we discuss the basics of xenobiology and how it could lead to the creation of different life forms, taxonomies or even ecosystems.


  • Kim, J., Goñi-Moreno, A., Calles, B., and de Lorenzo, V. (2019). Spatial organization of the gene expression hardware in Pseudomonas putida. Environ Microbiol (In Press) doi: 10.1111/1462-2920.14544

  • Amann RI et al (2019) Toward unrestricted use of public genomic data. Science 363: 350-352. doi 10.1126/science.aaw1280

  • Grégory Boël, G., Danot, O., de Lorenzo, V., Danchin, A.(2019) Omnipresent Maxwell’s demons orchestrate information management in living cells. Microb Biotech . 12 (2):210-242. doi: 10.1111/1751-7915.13378.

  • Aparicio, T., de Lorenzo V., Martínez-García, E.(2018) A broad host range plasmid-based roadmap for ssDNA-based recombineering in Gram-negative bacteria. Methods Mol Biol (In Press)

  • Aparicio, T., de Lorenzo V., Martínez-García, E.(2018) Improved thermotolerance of genome-reduced Pseudomonas putida EM42 enables effective functioning of the PL/cI857 system. Biotech J . 14(1):1800483 DOI: 10.1002/biot.201800483

  • de Lorenzo V., Couto, J.(2018) The important vs. the exciting: reining contradictions in contemporary biotechnology. Microb Biotech. 12(1): 32–34. doi: 10.1111/1751-7915.13348

  • Akkaya, Ö., Nikel, P.I., Pérez-Pantoja, D. and de Lorenzo, V.(2018) Evolving metabolism of 2,4-dinitrotoluene triggers SOS-independent diversification of host cells. Env Microbiol. 21(1):314-326 doi: 10.1111/1462-2920.14459.

  • Durante-Rodríguez, G., de Lorenzo, V. and Nikel, P.I. (2018) A post-translational metabolic switch enables complete decoupling of bacterial growth from biopolymer production in engineered Escherichia coli. ACS Synth Biol. 7(11):2686–2697  DOI 10.1021/acssynbio.8b00345

  • Pérez-Pantoja, D., Kim, J., Platero, R. and de Lorenzo, V. (2018) The interplay of EIIANtr with C-source regulation of the Pu promoter of Pseudomonas putida mt-2. Env Microbiol. 20(12):4555-4566. doi: 10.1111/1462-2920.14410.

  • Rodríguez Espeso, D., Martínez-García, E., Carpio, A., and de Lorenzo, V. (2018)Dynamics of Pseudomonas putida biofilms in an upscale experimental. framework. J Ind Microbiol Biotech. 45 (10):899–911 doi.org/10.1007/s10295-018-2070-0

  • Chavarría, M. and de Lorenzo, V. (2018) The imbroglio of the physiological Cra effector clarified at last. Mol Microbiol. 109(3):273-277. doi: 10.1111/mmi.14080.

  • de Lorenzo, V. (2018) Evolutionary tinkering vs. rational engineering in the times of Synthetic Biology. Life Sciences, Society & Policy 14(1):18. doi: 10.1186/s40504-018-0086-x

  • Akkaya, O., Pérez-Pantoja2, D., Calles, B., Nikel, P.I. and de Lorenzo, V.(2018) The metabolic redox regime of Pseudomonas putida tunes its evolvability towards novel xenobiotic substrates. mBio 9 (4):e01512-18 DOI: 10.1128/mBio.01512-18

  • Dvorak, P. and de Lorenzo, V. (2018). Refactoring the upper sugar metabolism of Pseudomonas putida for co-utilization of cellobiose, xylose, and glucose. Metab Eng. 48:e01512-18 94-108. doi: 10.1016/j.ymben.2018.05.019

  • O’Day, E., Hosta-Rigau, H., Oyarzún, D.A., Okano, H., de Lorenzo, V., von Kameke, C., Alsafar, H., Cao, C., Chen G.Q., Ji, W., Roberts R.J. Ronaghi, M., Yeung, K., Zhang, F. and Lee, S.Y. . (2018) Are we there yet? How and when specific biotechnologies will improve human health. Biotech J. 14(1):e1800195. doi: 10.1002/biot.201800195

  • Nikel P.I. and de Lorenzo V . (2018)Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism. Metab Eng. 50:142-155. doi: 10.1016/j.ymben.2018.05.005

  • Sánchez-Pascuala, A., Nikel, P.I. and de Lorenzo, V. .(2018) Re-factoring glycolytic genes for targeted engineering of catabolism in Gram-negative bacteria. Methods Mol Biol. 772.(:3-24. doi: 10.1007/978-1-4939-7795-6_1.
  • Goñi-Moreno, A. and de Lorenzo, V. .(2018) Bio-algorithmic workflows for standardized synthetic biology constructs. Methods Mol Biol. 1772:363-372. doi: 10.1007/978-1-4939-7795-6_20.

  • de Lorenzo, V..(2018) Environmental microbiology to the rescue of planet Earth. Environ Microbiol. 20(6):1910-16 DOI: 10.1111/1462-2920.14105

  • Nikel, P.I. and de Lorenzo V. .(2018) Assessing carbon source-dependent phenotypic variability in Pseudomonas putida. Methods Mol Biol 1745:287-301. doi: 10.1007/978-1-4939-7680-5_16.

  • de Lorenzo, V., et al. (2018) The power of synthetic biology for bioproduction, remediation and pollution control. EMBO Reports 19:e45658. doi: 10.15252/embr.201745658.

  • Ricaurte DE, Martínez-García E, Nyerges Á, Pál C, de Lorenzo V, Aparicio T.A. (2018) A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities. Microb Biotechnol. 11(1):176-188. doi: 10.1111/1751-7915.12846

  • Jonathan Grizou, Laurie J. Points, Abhishek Sharma and Leroy Cronin Discovery of Novelty in Robotically Constructed Self-Propelling Droplets Using a Curiosity Algorithm (submitted)

  • Andrew J. Surman, Marc Rodriguez Garcia, Yousef M. Abul-Haija, Geoffrey J. T. Cooper, Piotr S. Gromski, Rebecca Turk-MacLeod, Margaret Mullin, Cole Mathis, Sara I. Walker, and Leroy Cronin (2019). Environmental control programs the emergence of functional ensembles from unconstrained chemical reaction networks. Proc Natl Acad Sci.USA (In Press)
  • Soichiro Tsuda, Lewis A. Fraser, Salah Sharabi, Mohammed Hezwani, Andrew B. Kinghorn, Shaolin Liang, Gillian Douce, Julian A. Tanner and Leroy Cronin. A Portable 3D-printed Platform for Point-of-care Diagnosis of Clostridium difficile Infection and Malaria (submitted)

  • Solé RV, Montanez R, Duran-Nebreda S, Rodriguez-Amor D, Vidiella B, Sardanyés J. (2018) Population dynamics of synthetic Terraformation motifs. Royal Society open science. 25(7):180121. doi: 10.1098/rsos.180121

  • Vidiella B, Sardanyés J, Solé R. (2018) Exploiting delayed transitions to sustain semiarid ecosystems after catastrophic shifts. Journal of The Royal Society Interface. 15(143):20180083. doi.org/10.1098/rsif.2018.0083

  • Schmidt M, Budisa N. 2019. Alternative Biofacts: Life as we don’t (yet) know it. Chapter in: Berger E, Mäki-Reinikka K, O’Reilly, K, Sederholm H. Finish Bioart Society – 10th anniversary publication. Aalto ARTS Books