Why Pseudomonas putida?

The workhorse and experimental system par excellence in our Laboratory is the Gram-negative soil bacterium and plant-colonizer Pseudomonas putida, in particular the strain called KT2440. But in fact, we are not as interested in the lifestyle of this bacterium in its natural niches as on its awesome physiological strength and stress-tolerance properties. We try to domesticate and repurpose these features with a biotechnological aim with the tools of contemporary Synthetic Biology. While Escherichia coli constitutes an optimal model in Biology, many reactions and processes of environmental and industrial interest proceed with the accumulation of toxic intermediates that E. coli can hardly cope with. In contrast, P. putida seems to fulfill nearly all conditions required in an ideal cell factory, as this bacterium adapt easily to very different physicochemical and nutritional niches and it is well equipped to endure both endogenous and exogenous stresses. The origin of such heftiness is to be found in its central biochemical network, which is well geared to produce the metabolic currency required to overcome all types of physical and chemical insults. Our starting point is thus a natural bacterium that we have tried over the years to tame for our own sake and to redesign in many different ways.


Development of Pseudomonas putida as a Synthetic Biology chassis

We attempt to use this soil bacterium as the starting point for building an all-purpose, reliable chassis that is instrumental for engineering a large number of new-to-nature biochemical traits and physicochemical properties. This is because P. putida KT2440 is already endowed with a default metabolic core that ensures an adequate supply of of reducing power and tolerance to a whole range on environmental insults. But many other traits are not optimal or are altogether missing. 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—what we call cyborgyzation. Also, we pursue programming alternative lifestyles; e.g. aerobic vs. anaerobic, growth at high vs. low temperatures, and tolerance to DNA damage and understanding where things are inside cells.

People involved: Esteban Martínez-García, Tomás Aparicio, Elena Algar, Sofía Fraile

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 R. Espeso, Sofía Fraile, Angeles Hueso

Operative (genetic) systems for programming 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 endeavour has various sides. One is the generation of a minimal collection of connectable ORN gates based on repressor/promoter pairs on the basis of which one can design complex decision-making circuits and shaping á la carte control layers between specific signals and given actuators. This effort faces the challenge of inter-species portability and context-sensitivity. A second aspect is the need to digitalize transcriptional responses for making typical ON/OFF promoter outputs as close as possible to binary 1/0 states.

People involved: Belen Calles, Huseyin Tas, Angeles Hueso

SynBio standards: 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, Tomás Aparicio, Belén Calles, Sofía Fraile

Accelerated diversification of whole genomes and genomic segments

Understanding and ultimately taming Evolution is not only a major scientific endeavour, but also a very practical biotechnological challenge. Full rational design of new traits in biological systems is typically limited by the dearth of known parameters for an effective and predictable engineering. In order to optimize a synthetic pathway (or modified biological object thereof) without controlling all components at stake we apply the principle of heterotic computing i.e. embodying the problem in a physical object, apply physical forces and identify the best performers. We need molecular strategies—and matching bioreactors— for diversifying the DNA sequences either whole genomes or specific parts of them. Natural mechanisms for such an endeavour (e.g. generation of metabolic reactive oxygen species, mutator variants, suppression of mismatch repair) can be nurtured to this end. But also we can develop a la carte diversity-generation tools based on single-stranded DNA recombineering or cytosine-deamination. Their applicability ranges from diversification of genome-encoded and surface-displayed antibodies to optimization of complex pathways for degradation of nitroaromatic compounds.

People involved: Yamal Alramahi, Tomas Aparicio, Esteban Martinez, Elena Velazquez, David R. Espeso

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] Genome assembly and genome shuffling/transplantation platforms. 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, Elena Algar, Angeles Hueso, Huseyin Tas