Deep refactoring of the central metabolism of Pseudomonas putida

We attempt to develop this bacterium as a reliable chassis for engineering a large number of new-to-Nature biochemical traits. The starting point is a very good one, as P. putida KT2440 is already endowed with a default metabolic core that ensures an adequate supply of of reducing power. But many other traits are not optimal or are altogether missing. Also, the architecture of the central carbon metabolism is this bacterium needs to be disentangled in order to be able to manipulate the pathways involved. We are exploiting the wealth of tools made available by contemporary Synthetic Biology to edit the genome of P. putida in order to remove non-desired properties (e.g. instability determinants), replace others by better counterparts and knock-in modules recruited or assembled from other bacteria. In particular, a collection of GlucoBricks is being developed to ease transport and metabolism of sugars –and the exchange of the reactions involved depending on specific needs. Also, we pursue programming alternative lifestyles; e.g. aerobic vs. anaerobic, growth at high vs. low temperatures, and tolerance to DNA damage.

People involved: Pablo Nikel, Alberto Sanchez-Pascuala, Yamal Al-Ramahi, Esteban Martínez-García

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Pseudomonas putida as a chemical micro-factory

The metaphor of microorganisms as Cell Factories has inspired us the view of individual bacteria as authentic chemical plants in which reactions occur in time and space and are organized following a relational logic of pipes, valves and pumps. To this end, we first need to understand the 3D arrangement of the key components of the gene expression flow (DNA, RNA polymerase, mRNA, ribosomes) and visualize them with advanced microscopy. But we also need fresh approaches to examine intracellular micro-heterogeneity. For this, we have created in vivo genetic probes that deliver different fluorescent outputs depending on the trajectories and obstacles followed by two molecular partners in their way to meet each other (e.g. transcription factors and promoters). Furthermore, we are developing ways to detect protein complex location, substrate channeling, and redox activity in given sites. Eventually, we will identify intracellular addresses that can optimally accommodate synthetic constructs.

People involved: Angel Goñi, Juhyun Kim, Alberto Sanchez-Pascuala, Ilaria Benedetti, Belén Calles.

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Engineering cellulolytic Pseudomonas putida

Lignocellulose is the most abundant organic matter on Earth and an important constituent of agricultural, urban and industrial wastes. Lignocellulose-derived monomeric sugars and aromatic compounds can serve as cheap substrates for biotechnological production of numerous value-added chemicals (VAC). We are pursuing the expansion of the biocatalytic abilities of P. putida by genetically programming cells to express functional cellulosomes. These are efficient enzymatic nanomachines displayed on the surface of certain cellulolytic microorganisms. We are adopting state-of-the-art approaches and tools of Synthetic Biology, Systems Biology and Metabolic Engineering in order to construct P. putida variants displaying designer cellulosomes on their cell surface and forming valuable biopolymers directly from cellulosic waste. This task requires a rational orchestration of many distinct physiological features of the host in order to achieve the desired qualities without compromising cell viability. This project is a veritable testbed for many of the genetic tools developed in house.

People involved: Pavel Dvorak, Pablo Nikel, Esteban Martínez-García

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3D self-assembly of bacterial origami for distributed catalysis

Synthetic Biology allows rational reprogramming of every biological trait, including the shape and the physical characteristics of a live system, in what has been called Synthetic Morphologies. We argue that by combining the optimization of a given pathway (whether in one or more strains) with an ideal and malleable 3D structure of the catalytic biomass we can multiply the efficiency of any desired biotransformation. Our efforts to this end take 3 directions. First, overtaking the endogenous network that causes biofilm formation with tightly controlled expression systems for genes encoding di-c-GMP-specific diaguanylate cyclase and phosphodiesterase. Second, deleting all innate determinants of biofilm formation, including cell surface structures and displaying artificial adhesins (e.g. nanobodies) on the envelope of the resulting naked strain. Finally, developing assembly models and rules for generating predetermined 3D structures on the basis of the distribution of such adhesins on the bacterial surface.

People involved: Esteban Martínez-García, David Rodriguez-Espeso, Angel Goñi, Ilaria Benedetti, Pablo I. Nikel

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Operative (genetic) systems to program Pseudomonas putida

Bacteria are naturally able to compute a large number of endogenous cues and exogenous signals and produce distinct responses following a pre-determined logic. We aim at erasing many (if not all) of such built-in decision-making circuits and replace them by synthetic alternatives based on robust and connect-able Boolean gates made with transcriptional factors. In this way, we expect to submit P. putida to our own operative system so that the biochemical and physical properties of the cells can deployed according to a given program at user’s will. This endeavor has two sides. One is the complete elimination of Two-Component Systems and cdGMP-producing protein domains as they are the main sensing/responding devices allowing communication with the environment (i.e. resulting in blind and deaf bacteria). The other is the setup of a complete collection of rewire-able transcriptional logic gates for shaping á la carte control layers between given signals and given actuators.

People involved: Angel Goñi, Huseyin Tas

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Towards a bacterial-based artificial immune system

This is one of the most complex artificial biological systems ventured thus far. This all-bacterial immune-like platform will permit the application of single-chain camel antibodies (nanobodies) by themselves or displayed on bacterial cells, to many biotechnological challenges (e.g. in vivo adhesins). The work involves the assembly and validation of autonomous functional modules for [i] displaying antibody/affibody (AB) scaffolds attached to the surface of bacterial cells, [ii] conditional diversification of target-binding sequences of the ABs, [iii] contact-dependent activation of gene expression, [iv] reversible bi-stable switches, and [v] clonal selection and amplification of improved binders. These modules are then assembled in the genomic chassis of streamlined Escherichia coli and Pseudomonas putida strains. The resulting molecular network makes the antibodies expressed and displayed on the cell surface to proceed spontaneously (or at the user’s decision) through cycles of affinity and specificity maturation towards desired antigens.

People involved: Yamal Al-Ramahi, Angeles Hueso, Sofía Fraile, Belén Calles

Figureimm

The Standard European Vector Architecture (SEVA)

The SEVA platform is a coherent resource of molecular tools subjected to a concise, minimalist, and standardized format and nomenclature, fully compatible with old and new cloning protocols and DNA assembly methods. Simple assembly rules allow the generation of a huge collection of vectors that cover virtually all genetic and metabolic engineering necessities of P. putida KT2440 and other Gram-negative bacteria: genome editing (i.e. deletions and insertions), heterologous gene expression, protein secretion, metabolic pathways, logic circuits, biosensors with optical readouts, among many others. With these tools in hand, we are developing and adapting to P. putida a large number of Synthetic Biology resources, both computational (e.g. the Synthetic Biology Open Language, CELLO in vivo programming) and experimental (MAGE, CRISPR/Cas9, proteomic and transcriptional switches). These will allow setting the catalytic properties of P. putida to be deployed following given specifications of time, space and environmental conditions.

People involved: Esteban Martínez-García, Angel Goñi, Tomás Aparicio, Belén Calles, Sofía Fraile

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Oxidative stress as a driver of metabolic evolution

During the study of 2,4-dinitrotoluene biodegradation by Burkholderia sp. DNT we noticed that reactive oxygen species (ROS) brought about by the faulty performance of the first enzyme of the pathway (DntA dioxygenase) on the xenobiotic substrate, translated into DNA mutagenesis. Physiological stress caused by such ROS triggers also the genetic diversification necessary for exploring the solution space to the same problem. In order to examine this question in more detail, we have transplanted the key components of the phenomenon to both E. coli and Pseudomonas putida, two hosts with very different basal metabolism, poles apart in tolerance to environmental stress and quite unlike systems to deal with insults to DNA. Our data is fully consistent with the notion that stress caused by faulty metabolism of new substrates accelerates the rate of bacterial evolution in a fashion reminiscent of anti-fragile systems –yet, the phenomenon is species-specific.

People involved: Özlem Akkaya, Pablo Nikel, Danilo Pérez-Pantoja

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The TOL plasmid

The precursor of P. putida KT2440 is a natural isolate (called P. putida mt-2) which carries a large plasmid (named pWW0) that encodes all the regulatory and biochemical complement for complete biodegradation of toluene and m-xylene. This so-called TOL plasmid has been an extraordinary source of biological questions and genetic parts both for addressing fundamental features of the bacterial world (e.g. regulatory networks, transcriptional regulation, stochasticity, division of labor, catabolite repression) and very practical biotechnological endeavors (e.g. inducible expression systems, biotransformations). More recently, the interplay between the metabolic and regulatory circuit borne by the plasmid and those of the host have become an extraordinary experimental system for studying the retroactivity between a pre-existing chassis and an incoming genetic implant. These studies are revealing natural mechanisms through which such implants avoid biochemical and regulatory conflicts with the host e.g. by separating problematic reactions in time and space.

People involved: Danilo Pérez-Pantoja, Juyhun Kim, Pablo I.Nikel

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Tools for analysis & construction of Gram-negative bacterial phenotypes

We do not only use others’ great SynBio tools, but also produce our own for specific projects. Yet, many of these have later a general and unexpected utility beyond the original purpose of their creation. These include [i] recruitment of transcriptional factors of environmental bacteria for biosensing and heterologous expression devices [ii] engineering of ON/OFF transcriptional switches for transient expression of genes of interest (e.g. light-driven and nutrient-driven switches), [iii] Proteomic switches for conditional production / destruction of target enzymes at a post-translational level, [iv] scaffolds for heterologous protein display on the surface of P. putida cells, [v] protein folding reporters [vi] constitutive promoter collections [vii] Synthetic transposon vectors based on Tn5 and Tn7. These tools, many of them assembled following the SEVA format, allow a degree of genetic manipulation in P. putida that is impossible to achieve in bacteria other than E. coli.

People involved: Tomás Aparicio, Esteban Martínez-García, Belén Calles, Ilaria Benedetti

Figuretools

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