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Synthetic biology is not only a contemporary reformulation of the recombinant DNA technologies of the last 30 years, combined with descriptive language imported from electrical and industrial engineering. It is also a new way to interpret living systems and a statement of intent for the use and reprogramming of biological objects for human benefit. In this context, the notion of designer biology is often presented as opposed to natural selection following the powerful rationale formulated by François Jacob on evolution-as-tinkering. The onset of synthetic biology opens a different perspective by leaving aside the question about the evolutionary origin of biological phenomena and focusing instead on the relational logic and the material properties of the corresponding components that make biological system work as they do. Once a functional challenge arises, the solution space for the problem is not homogeneous but it has attractors that can be accessed either through random exploration (as evolution does) or rational design (as engineers do). Although these two paths (i.e. evolution and engineering) are essentially different, they can lead to solutions to specific mechanistic bottlenecks that frequently coincide or converge—and one can easily help to understand and improve the other. Alas, productive discussions on these matters are often contaminated by ideological preconceptions that prevent adoption of the engineering metaphor to understand and ultimately reshape living systems—as ambitioned by synthetic biology. Yet, some possible ways to overcome the impasse are feasible. In parallel to Monod's evolutionary paradox of teleo-logy (finality/purpose) vs. teleo-nomy (appearance of finality/purpose), a mechanistic paradox could be entertained between techno-logy (rational engineering) vs techno-nomy (appearance of rational engineering), all for the sake of understanding the relational logic that enables live systems to function as physico-chemical entities in time and space. This article thus proposes a radical vision of synthetic biology through the lens of the engineering metaphor. ; Research in Author's Laboratory is funded by Projects HELIOS (BIO2015–66960-C3–2-R; MINECO/FEDER) and contracts of the European Union ARISYS (ERC-2012-ADG-322797), EmPowerPutida (EU-H2020-BIOTEC-2014-2015-6335536), MADONNA (H2020-FET-OPEN-RIA-2017-1 (766975) as well as the InGEMICS-CM (B2017/BMD-3691) contract of the Comunidad de Madrid (FSE, FECER).

10 páginas, 2 figuras, 5 tablas.-- Metodología. ; [Background]: For ecological studies, it is crucial to count on adequate descriptions of the environments and samples being studied. Such a description must be done in terms of their physicochemical characteristics, allowing a direct comparison between different environments that would be difficult to do otherwise. Also the characterization must include the precise geographical location, to make possible the study of geographical distributions and biogeographical patterns. Currently, there is no schema for annotating these environmental features, and these data have to be extracted from textual sources (published articles). So far, this had to be performed by manual inspection of the corresponding documents. To facilitate this task, we have developed EnvMine, a set of text-mining tools devoted to retrieve contextual information (physicochemical variables and geographical locations) from textual sources of any kind. [Results]: EnvMine is capable of retrieving the physicochemical variables cited in the text, by means of the accurate identification of their associated units of measurement. In this task, the system achieves a recall (percentage of items retrieved) of 92% with less than 1% error. Also a Bayesian classifier was tested for distinguishing parts of the text describing environmental characteristics from others dealing with, for instance, experimental settings. Regarding the identification of geographical locations, the system takes advantage of existing databases such as GeoNames to achieve 86% recall with 92% precision. The identification of a location includes also the determination of its exact coordinates (latitude and longitude), thus allowing the calculation of distance between the individual locations. [Conclusion]: EnvMine is a very efficient method for extracting contextual information from different text sources, like published articles or web pages. This tool can help in determining the precise location and physicochemical variables of sampling sites, thus facilitating the performance of ecological analyses. EnvMine can also help in the development of standards for the annotation of environmental features. ; This work was supported by project generous grants from the Spanish Ministry of Science and Innovation (CONSOLIDER), the 7 th Framework programme of the European Union (Projects BACSINE and MICROME), and funds from Comunidad de Madrid, Spain. ; Peer reviewed

The expectations raised in the mid-1980s on the potential of genetic engineering for in situ remediation of environmental pollution have not been entirely fulfilled. Yet, we have learned a good deal about the expression of catabolic pathways by bacteria in their natural habitats, and how environmental conditions dictate the expression of desired catalytic activities. The many different choices between nutrients and responses to stresses form a network of transcriptional switches which, given the redundance and robustness of the regulatory circuits involved, can be neither unraveled through standard genetic analysis nor artificially programmed in a simple manner. Available data suggest that population dynamics and physiological control of catabolic gene expression prevail over any artificial attempt to engineer an optimal performance of the wanted catalytic activities. In this review, several valuable spin-offs of past research into genetically modified organisms with environmental applications are discussed, along with the impact of Systems Biology and Synthetic Biology in the future of environmental biotechnology. ; The work of our laboratories cited in this article was funded by grants of the Spanish Ministry of Education and Science, the European Union, and the Conservation Biology Program of the BBVA Foundation. ; Peer reviewed

The onset of techniques for both editing extant bacterial chromosomes and synthesizing long DNA sequences has enabled addressing the question on the smallest set of genes and biological functions that are required for running an operative cell. But this is not only a fundamental scientific endeavour: simpler genomes could be easier to understand and eventually reprogram for the sake of diverse applications. This has fostered efforts to eliminate apparently useless — if not annoying DNA segments from many biotechnologically relevant strains as well as attempts to (re)write complete genomes á la carte. Yet, the smallest number of genes that enable given functionalities do vary depending on the ultimate agenda, as both natural and engineered small-size genomes afford genetic simplicity by engaging in a suite of complex interactions with the surrounding medium. ; This work was supported by the CAMBIOS Project of the Spanish Ministry of Economy and Competitiveness (RTC-2014-1777-3), the EVOPROG (FP7-ICT-610730), ARISYS (ERC-2012-ADG-322797), and EmPowerPutida (EU-H2020-BIOTEC-2014-2015-6335536) Contracts of the European Union, and the PROMPT Project of the Autonomous Community of Madrid (CAM-S2010/BMD-2414). ; Peer reviewed

5 p.-3 fig. ; In the presence of m-xylene, the protein XylR encoded by the TOL plasmid of Pseudomonas putida, activates the GraphicGraphic-dependent promoter Pu. Early activation stages involve the release of the intramolecular repression caused by the signal reception N-terminal (A domain) of XylR on the central module of the protein. A genetic approach has been followed to locate the specific segment within A domain of XylR that is directly responsible for its down-regulation in the absence of inducer, as compared to that involved in effector (m-xylene) binding. For this, a reporter Escherichia coli strain carrying a monocopy transcriptional fusion of Pu to lacZ was transformed with a collection of plasmids encoding equivalent truncated varieties of XylR, consisting of nested and internal deletions throughout the entire A domain. Examination of the resulting phenotypes allowed the assignment of the A domain region near the central activation domain, as the portion of the protein responsible for the specific repression of XylR activity in the absence of m-xylene. ; This work was supported by Grant BIO95-0788 of the Spanish Comisión Interministerial de Ciencia y Tecnología (CICYT) and Contract BIOTECH BIO2-CT92-0084 of the European Union. ; Peer reviewed

© 2020 The Authors ; Co‐production of two or more desirable compounds from low‐cost substrates by a single microbial catalyst could greatly improve the economic competitiveness of many biotechnological processes. However, reports demonstrating the adoption of such co‐production strategy are still scarce. In this study, the ability of genome‐edited strain Pseudomonas putida EM42 to simultaneously valorize d‐xylose and d‐cellobiose – two important lignocellulosic carbohydrates – by converting them into the platform chemical d‐xylonate and medium‐chain‐length polyhydroxyalkanoates, respectively, was investigated. Biotransformation experiments performed with P. putida resting cells showed that promiscuous periplasmic glucose oxidation route can efficiently generate extracellular xylonate with a high yield. Xylose oxidation was subsequently coupled to the growth of P. putida with cytoplasmic β‐glucosidase BglC from Thermobifida fusca on d‐cellobiose. This disaccharide turned out to be a better co‐substrate for xylose‐to‐xylonate biotransformation than monomeric glucose. This was because unlike glucose, cellobiose did not block oxidation of the pentose by periplasmic glucose dehydrogenase Gcd, but, similarly to glucose, it was a suitable substrate for polyhydroxyalkanoate formation in P. putida. Co‐production of extracellular xylose‐born xylonate and intracellular cellobiose‐born medium‐chain‐length polyhydroxyalkanoates was established in proof‐of‐concept experiments with P. putida grown on the disaccharide. This study highlights the potential of P. putida EM42 as a microbial platform for the production of xylonate, identifies cellobiose as a new substrate for mcl‐PHA production, and proposes a fresh strategy for the simultaneous valorization of xylose and cellobiose. ; This work was funded by the SETH Project of the Spanish Ministry of Science (RTI 2018-095584-B-C42), MADONNA (H2020-FETOPEN-RIA-2017-1-766975), BioRoboost (H2020-NMBP-BIO-CSA2018) and SYNBIO4FLAV (H2020-NMBP/0500), contracts of the European Union and the S2017/BMD-3691 InGEMICS-CM funded by the Comunidad de Madrid (European Structural and Investment Funds) as well as by the Czech Science Foundation (19-06511Y).

Pseudomonas putida is a microorganism of biotechnological interest that—similar to many other environmental bacteria—adheres to surfaces and forms biofilms. Although various mechanisms contributing to the swim-attach decision have been studied in this species, the role of a 7-gene operon homologous to the wsp cluster of Pseudomonas aeruginosa—which regulates cyclic di-GMP (cdGMP) levels upon surface contact—remained to be investigated. In this work, the function of the wsp operon of P. putida KT2440 has been characterized through inspection of single and multiple wsp deletion variants, complementation with Pseudomonas aeruginosa's homologues, combined with mutations of regulatory genes fleQ and fleN and removal of the flagellar regulator fglZ. The ability of the resulting strains to form biofilms at 6 and 24 h under three different carbon regimes (citrate, glucose and fructose) revealed that the Wsp complex delivers a similar function to both Pseudomonas species. In P. putida, the key components include WspR, a protein that harbours the domain for producing cdGMP, and WspF, which controls its activity. These results not only contribute to a deeper understanding of the network that regulates the sessile-planktonic decision of P. putida but also suggest strategies to exogenously control such a lifestyle switch. ; This work was funded by the SETH Project of the Spanish Ministry of Science RTI 2018‐095584‐B‐C42, the MADONNA (H2020‐FET‐OPEN‐RIA‐2017‐1‐766975), BioRoboost (H2020‐NMBP‐BIO‐CSA‐2018), SYNBIO4FLAV (H2020‐NMBP/0500) and MIX‐UP (H2020‐Grant 870294) Contracts of the European Union and the S2017/BMD‐3691 InGEMICS‐CM Project of the Comunidad de Madrid (European Structural and Investment Funds).

© 2019 The Authors. ; Implementation of single‐stranded DNA (ssDNA) recombineering in Pseudomonas putida has widened the range of genetic manipulations applicable to this biotechnologically relevant bacterium. Yet, the relatively low efficiency of the technology hampers identification of mutated clones lacking conspicuous phenotypes. Fortunately, the use of CRISPR/Cas9 as a device for counterselection of wild‐type sequences helps to overcome this limitation. Merging ssDNA recombineering with CRISPR/Cas9 thus enables a suite of genomic edits with a straightforward approach: a CRISPR plasmid provides the spacer DNA sequence that directs the Cas9 nuclease ribonucleoprotein complex to cleave the genome at the wild‐type sequences that have not undergone the change entered by the mutagenic ssDNA oligonucleotide(s). This protocol describes a complete workflow of the method optimized for P. putida, although it could in principle be applicable to many other pseudomonads. As an example, we show the deletion of the edd gene that encodes one key enzyme that operates the EDEMP cycle for glucose metabolism in P. putida EM42. By combining two incompatible CRISPR plasmids with different antibiotic selection markers, we show that the procedure can be cycled to implement consecutive deletions in the same strain, e.g. deletion of the pyrF gene following that of the edd mutant. This approach adds to the wealth of genetic technologies available for P. putida and strengthens its status as a chassis of choice for a suite of biotechnological applications. ; This work was funded by the HELIOS Project of the Spanish Ministry of Science BIO 2015-66960-C3-2-R (MINECO/FEDER), the ARISYS (ERC-2012-ADG-322797), EmPowerPutida (EU-H2020- BIOTEC-2014-2015-6335536) and MADONNA (H2020-FET-OPENRIA-2017-1-766975) Contracts of the European Union, and InGEMICS-CM (B2017/BMD-3691) contract of the Comunidad de Madrid (FSE, FECER).

© 2019 The Authors. ; While prokaryotic promoters controlled by signal‐responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an OFF state of zero transcription—as ideally needed for synthetic circuits. To overcome this problem, we have modelled and implemented a simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation‐inhibitory sRNA with the translational coupling of the gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropyl ketone) and shown to perform effectively in both Escherichia coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of, e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo. ; This work was funded by the SETH Project of the Spanish Ministry of Science RTI2018‐095584‐B‐C42, MADONNA (H2020‐FET‐OPEN‐RIA‐2017‐1‐766975), BioRoboost (H2020‐NMBP‐BIO‐CSA‐2018) and SYNBIO4FLAV (H2020‐NMBP/0500) Contracts of the European Union, the S2017/BMD‐3691 InGEMICS‐CM funded by the Comunidad de Madrid (European Structural and Investment Funds) and the SynBio3D project of the UK Engineering and Physical Sciences Research Council (EP/R019002/1).

Biofilms are bacterial communities in the shape of exopolysaccharide matrix-encased aggregates attached onto interphases able to resist environmental aggressions. The development of bacteria in the shape of biofilms deeply affects the performance of many industrial processes which work with fluidic systems, where bacteria may settle and prosper. As a consequence industrial equipment experiments low performance issues and substantial maintenance costs. The study of how bacteria of industrial interest such as Pseudomonas putida spread in these fluidic systems is highly dependent on the chosen experimental system to retrieve such data, thus using scaled prototypes becomes an essential step towards the design of a more efficient system to handle biofilms, either to control them or to prevent them. This protocol describes how to assemble, operate and maintain a device to grow and monitor the biofilm spreading pattern of this bacterium (as a function of the fluid hydrodynamics) in a custom-made chamber larger than those typically used in laboratory environments, and how to analyze the information gathered from it in a straightforward fashion. Description of the protocol was thought to be used as a working template not only for the presented case study but for any other potential experiment in different contexts and diverse scales following similar design principles. ; This work was funded by the HELIOS Project of the Spanish Ministry of Economy and Competitiveness BIO 2015-66960-C3-2-R (MINECO/FEDER); the ARISYS (ERC-2012-ADG-322797), EmPowerPutida (EU-H2020-BIOTEC-2014-2015-6335536), MADONNA (H2020-FET-OPEN-RIA-2017-1-766975), BioRoboost (H2020-NMBP-BIO-CSA-2018), and SYNBIO4FLAV (H2020-NMBP/0500) Contracts of the European Union and the S2017/BMD-3691 InGEMICS-CM funded by the Comunidad de Madrid (Spain) and the European Structural and Investment Funds. ; Peer reviewed

Supporting information ; The metabolic versatility of the soil bacterium Pseudomonas putida is reflected by its ability to execute strong redox reactions (e.g., mono- and di-oxygenations) on aromatic substrates. Biodegradation of aromatics occurs via the pathway encoded in the archetypal TOL plasmid pWW0, yet the effect of running such oxidative route on redox balance against the background metabolism of P. putida remains unexplored. To answer this question, the activity of pyridine nucleotide transhydrogenases (that catalyze the reversible interconversion of NADH and NADPH) was inspected under various physiological and oxidative stress regimes. The genome of P. putida KT2440 encodes a soluble transhydrogenase (SthA) and a membrane-bound, proton-pumping counterpart (PntAB). Mutant strains, lacking sthA and/or pntAB, were subjected to a panoply of genetic, biochemical, phenomic and functional assays in cells grown on customary carbon sources (e.g., citrate) versus difficult-to-degrade aromatic substrates. The results consistently indicated that redox homeostasis is compromised in the transhydrogenases-defective variant, rendering the mutant sensitive to oxidants. This metabolic deficiency was, however, counteracted by an increase in the activity of NADP+ -dependent dehydrogenases in central carbon metabolism. Taken together, these observations demonstrate that transhydrogenases enable a redox-adjusting mechanism that comes into play when biodegradation reactions are executed to metabolize unusual carbon compounds. ; This work was supported by the EVOPROG (FP7-ICT-610730), ARISYS (ERC-2012-ADG-322797) and EmPowerPutida (EU-H2020-BIOTEC-2014-2015-6335536), Contracts of the European Union, and the CAMBIOS (RTC-2014-1777-3) and CONTIBUGS (PCIN-2013-040) projects of the Spanish Ministry of Economy and Competitiveness. ; Peer reviewed

© 2020 The Author(s) ; Application of single-stranded DNA recombineering for genome editing of species other than enterobacteria is limited by the efficiency of the recombinase and the action of endogenous mismatch repair (MMR) systems. In this work we have set up a genetic system for entering multiple changes in the chromosome of the biotechnologically relevant strain EM42 of Pseudomononas putida. To this end high-level heat-inducible co-transcription of the rec2 recombinase and P. putida's allele mutLE36KPP was designed under the control of the PL/cI857 system. Cycles of short thermal shifts followed by transformation with a suite of mutagenic oligos delivered different types of genomic changes at frequencies up to 10% per single modification. The same approach was instrumental to super-diversify short chromosomal portions for creating libraries of functional genomic segments—e.g., ribosomal-binding sites. These results enabled multiplexing of genome engineering of P. putida, as required for metabolic reprogramming of this important synthetic biology chassis. ; This work was funded by the MADONNA (H2020-FET-OPEN-RIA-2017-1-766975), BioRoboost (H2020-NMBP-BIO-CSA-2018), SYNBIO4FLAV (H2020-NMBP/0500), and MIX-UP (H2020-Grant 870294) contracts of the European Union and the S2017/BMD-3691 InGEMICS-CM Project of the Comunidad de Madrid (European Structural and Investment Funds).

© 2019 The Authors. ; The biological regime of Pseudomonas putida (and any other bacterium) under given environmental conditions results from the hierarchical expression of sets of genes that become turned on and off in response to one or more physicochemical signals. In some cases, such signals are clearly defined, but in many others, cells are exposed to a whole variety of ill‐defined inputs that occur simultaneously. Transcriptomic analyses of bacteria passed from a reference condition to a complex niche can thus expose both the type of signals that they experience during the transition and the functions involved in adaptation to the new scenario. In this article, we describe a complete protocol for generation of transcriptomes aimed at monitoring the physiological shift of P. putida between two divergent settings using as a simple case study the change between homogeneous, planktonic lifestyle in a liquid medium and growth on the surface of an agar plate. To this end, RNA was collected from P. putidaKT2440 cells at various times after growth in either condition, and the genome‐wide transcriptional outputs were analysed. While the role of individual genes needs to be verified on a case‐by‐case basis, a gross inspection of the resulting profiles suggested cells that are cultured on solid media consistently had a higher translational and metabolic activity, stopped production of flagella and were conspicuously exposed to a strong oxidative stress. The herein described methodology is generally applicable to other circumstances for diagnosing lifestyle determinants of interest. ; This work was funded by the HELIOS Project of the Spanish Ministry of Science BIO 2015-66960-C3-2-R (MINECO/FEDER), the ARISYS (ERC-2012-ADG-322797), EmPowerPutida (EU-H2020- BIOTEC-2014-2015-633 5536) and MADONNA (H2020-FET-OPENRIA-2017-1-766975) contracts of the European Union, the InGEMICS-CM (B2017/BMD-3691) contract of the Comunidad de Madrid (FSE, FECER) and NIH grant R01GM123609

Elimination or mitigation of the toxic effects of chemical waste released to the environment by industrial and urban activities relies largely on the catalytic activities of microorganisms—specifically bacteria. Given their capacity to evolve rapidly, they have the biochemical power to tackle a large number of molecules mobilized from their geological repositories through human action (e.g., hydrocarbons, heavy metals) or generated through chemical synthesis (e.g., xenobiotic compounds). Whereas naturally occurring microbes already have considerable ability to remove many environmental pollutants with no external intervention, the onset of genetic engineering in the 1980s allowed the possibility of rational design of bacteria to catabolize specific compounds, which could eventually be released into the environment as bioremediation agents. The complexity of this endeavour and the lack of fundamental knowledge nonetheless led to the virtual abandonment of such a recombinant DNA-based bioremediation only a decade later. In a twist of events, the last few years have witnessed the emergence of new systemic fields (including systems and synthetic biology, and metabolic engineering) that allow revisiting the same environmental pollution challenges through fresh and far more powerful approaches. The focus on contaminated sites and chemicals has been broadened by the phenomenal problems of anthropogenic emissions of greenhouse gases and the accumulation of plastic waste on a global scale. In this article, we analyze how contemporary systemic biology is helping to take the design of bioremediation agents back to the core of environmental biotechnology. We inspect a number of recent strategies for catabolic pathway construction and optimization and we bring them together by proposing an engineering workflow. ; PD is the holder of the Marie Sklodowska-Curie grant No. 704410 (FUTURE). The work in Authors' Laboratory was funded by the CAMBIOS Project of the Spanish Ministry of Economy and CompetitivenessRTC-2014-1777-3 (MINECO), HELIOS Project of the Spanish Ministry of Economy and Competitiveness BIO 2015-66960-C3-2-R (MINECO/FEDER) and the ARISYS (ERC-2012-ADG-322797), EmPowerPutida (EU-H2020-BIOTEC-2014-2015-6335536), and Raft4Biotech (720776) contracts of the European Union. JD is supported also by the Czech Ministry of Education (LQ1605, LO1214, LM2015055), and Czech Grant Agency (GA16-06096S). ; Peer Reviewed