Final Report Summary - ALLEGRO (Biotechnological exploitation of Pseudomonas putida: Lego-lizing and refactoring central metabolic blocks through rational genome engineering) ALLEGRO (Biotechnological exploitation of Pseudomonas putida: Lego-lizing and re-factoring central metabolic blocks through rational genome engineering) aimed at the large-scale, rational modification of basic biological functions in the model environmental bacterium Pseudomonas putida KT2440. The project proposed a flawless road-map from the wild-type bacterium towards the implementation of a chassis specifically tailored for biotechnological purposes through the use of synthetic biology and metabolic engineering tools. P. putida is one of the best studied species of the metabolically versatile and ubiquitous genus of the Pseudomonads. As a species, it exhibits interesting biotechnological potential, with numerous strains, some of them solvent-tolerant, able to efficiently produce a range of bulk and fine chemicals. These features, along with their entire lack of pathogenesis determinants, amenability to genetic manipulation, and suitability as a host for heterologous gene expression, make P. putida particularly attractive as a potential bacterial host for the design of robust biocatalysis. P. putida KT2440 is a spontaneous restriction-deficient derivative of the P. putida isolate originally called mt-2, which has been cured of the catabolic TOL plasmid. This bacterial strain serves as a workhorse of basic and applied research worldwide given the fact that it is a Generally Recognized as Safe microorganism (i.e. GRAS certified), and it constituted the starting point of ALLEGRO. Yet, many of the potential P. putida-based applications were in an early stage of progress largely due to a lack of knowledge of the genotype/phenotype relationships in these bacteria under conditions relevant for both industrial and environmental endeavors. On the other hand, the design of biocatalysts using this bacterium has been seriously afflicted by the lack of genetic engineering tools specifically designed for P. putida. As a first step towards the rational construction of a suitable P. putida genomic and metabolic chassis, some basic functions of P. putida KT2440 were edited, streamlined, and re-factored. The issue at stake in this case has been the removal of elements encoded in the extant chromosome that are not necessary for catalytic functions while maintaining the robustness and tolerance to stress of the natural strain. Most of the targeted DNA segments were those causing genomic instability, in particular active or cryptic mobile elements. Non-necessary energy-consuming structures on the cell envelope (e.g. flagella) were deleted. Such operations rendered the genome chassis genetically and metabolically more stable, enabling a considerable simplification for subsequent manipulations and, hence, a better understanding and control of the metabolic and regulatory hierarchies in the microbial host. Another trait explored in ALLEGRO has been the targeted manipulation of the central metabolism in P. putida KT2440. This bacterium is remarkably adapted to use a large number of carbon sources and to colonize a wide variety of habitats, thus reflecting its metabolic diversity and its adaptability to many different physicochemical conditions. Pseudomonads mostly operates the Entner-Doudoroff pathway for hexoses breakdown, but the metabolic network involved in the use of other carbon sources has not been elucidated. Along the line, gaining a deeper insight into the regulation of central catabolism in this bacterium is of paramount interest for the design of a flawless catabolic pathways. Even when hexoses are the archetypal carbon substrates used in bioprocesses, some interesting alternatives have emerged in the last few years. Glycerol, for instance, became a very cheap and abundant carbon source for Biotechnology, as it is a by-product of the biodiesel industry. The biochemical network involved in the processing of hexoses is also intertwined with the metabolic functions needed for glycerol catabolism. The compound simultaneously elicited a gluconeogenic response that indicated an efficient channeling of carbon skeletons back to biomass build-up rather than energization of the cells through downwards catabolic pathways. The simultaneous glycolytic and gluconeogenic metabolic regimes on glycerol, paradoxical as they seem, make sense from an ecological point of view by favoring prevalence versus exploration. This metabolic situation was accompanied by a considerably low expression of stress markers as compared with other carbon sources, the manipulation of which has been a key aspect of ALLEGRO. To cope with changing (and often harsh) conditions, P. putida has developed a suite of molecular and physiological assets for counteracting environmental stress. As proposed in ALLEGRO, the re-factoring of these stress-resistance properties calls for an in-depth knowledge on how stress-resistance networks operate, as the mechanisms involved in robustness have been only partially elucidated. Cataloging them is thus important not just for the specific purposes of manipulating stress endurance, but also to understand the abundance of Pseudomonas strains in sites afflicted by adverse environmental conditions. Ultimately, this knowledge translates in the ability of manipulating these features for biotechnological applications (e.g. biodegradation of xenobiotic compounds and/or biocatalysis through the expression of strong oxidative enzymes). In this regard, ALLEGRO explored the functions of one of the most intriguing players in stress resistance, i.e. inorganic polyphosphate. Polyphosphate accumulation is a persistent trait throughout the whole Tree of Life, and is claimed to play a fundamental role in enduring environmental insults in a large variety of microorganisms, however, the extent of this role in environmental bacteria has not been assessed before. It was found that the accumulation of polyphosphate is essential to maintain metabolic robustness, and hence this biochemical process became the target for further manipulations. Finally, ALLEGRO envisioned the design and application of new genetic tools for P. putida, specifically tailored for the needs herein proposed.ALLEGRO launched P. putida as a flawless microbial cell factory useful in a number of relevant applications. In particular, the heavily re-factored strains became biological chassis of reference for genetic and metabolic (re-)programming of bacterial catalysts à la carte. These traits have been documented and summarized in a recent review article [Nikel et al. (2015) Nature Reviews Microbiology 12: 368-379]. Apart of the explicit biotechnological agenda of ALLEGRO, a central aspect emerged as a consequence of the implementation of the project, namely, the deconstruction/reconstruction of the component parts of a system as the ultimate proof of its understanding. Such situation echoes the celebrated remark by the 1965 Nobel Prize winner Richard Feynman "...what I cannot create, I do not understand". Very scarce examples concerning the experimental design and implementation of re-factored bacterial chasses existed before ALLEGRO. This is not unexpected, because the fine-tuning of core biological functions towards a desired phenotype (i.e. efficient managing of energy resources) often involves complexities which are not encountered in the optimization of a single gene or metabolic function. Moreover, the challenge afforded by ALLEGRO has been the emergence of a completely novel and re-factored version of P. putida and not a mere combination of individual gene deletions or replacements, thereby moving the field of Systems Biotechnology significantly forward, since no previous example of such a deep metabolic manipulation has been reported.Globally, the results from this project have a clear impact on a society demand which has been formulated in various European documents, particularly, the increasingly urgent need to undertake new and innovative White Biotechnology approaches in order to better preserve natural resources and biodiversity of natural ecosystems - a most relevant aspect in our World nowadays, in which volatile oil-pricing policies call for alternative catalysis processes. On the other hand, owing to their intrinsic general applicability (as they are based on first principles), the concepts developed and insights gained in ALLEGRO, particularly in regards of cellular reprogramming, will open up new avenues and possibilities in other significant areas of application such as the production of high-added value compounds in the field of Industrial Biotechnology. It is expected that the framework developed in ALLEGRO will significantly contribute to merge Synthetic Biology principles with Systems Biology setups for the sake of applications both in White Biotechnology and Industrial Biocatalysis. The widely recognized European Union scientific excellence in the area of Environmental Microbiology has been potentiated by undertaking this project, that joined a highly qualified and motivated young researcher, well versed in the Metabolic Engineering field, with an innovative and world's leading group that exploits Systems and Synthetic Biology approaches to face biotechnological applications of environmental bacteria.