The ‘Standard European Vector Architecture’ database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.

Background: Glucose metabolism in many bacteria is based on the standard linear Embden-Meyerhof-Parnas pathway. Results: Pseudomonas putida operates a cycle merging components of the Entner-Doudoroff and pentose phosphate pathways along with gluconeogenic reactions from the upper glycolysis to process glucose (EDEMP cycle). Conclusion: This unusual glycolytic cycle nucleates the central metabolism of P. putida. Significance: The environmental lifestyle of P. putida is reflected in its central metabolic map.

The itinerary followed by Pseudomonas putida from being a soil-dweller and plant colonizer bacterium to become a flexible and engineer-able platform for metabolic engineering stems from its natural lifestyle, which is adapted to harsh environmental conditions and all sorts of physicochemical stresses. Over the years, these properties have been capitalized biotechnologically owing to the expanding wealth of genetic tools designed for deep-editing the P. putida genome. A suite of dedicated vectors inspired in the core tenets of synthetic biology have enabled to suppress many of the naturally-occurring undesirable traits native to this species while enhancing its many appealing properties, and also to import catalytic activities and attributes from other biological systems. Much of the biotechnological interest on P. putida stems from the distinct architecture of its central carbon metabolism. The native biochemistry is naturally geared to generate reductive currency [i.e., NAD(P)H] that makes this bacterium a phenomenal host for redox-intensive reactions. In some cases, genetic editing of the indigenous biochemical network of P. putida (cis-metabolism) has sufficed to obtain target compounds of industrial interest. Yet, the main value and promise of this species (in particular, strain KT2440) resides not only in its capacity to host heterologous pathways from other microorganisms, but also altogether artificial routes (trans-metabolism) for making complex, new-to-Nature molecules. A number of examples are presented for substantiating the worth of P. putida as one of the favorite workhorses for sustainable manufacturing of fine and bulk chemicals in the current times of the 4th Industrial Revolution. The potential of P. putida to extend its rich native biochemistry beyond existing boundaries is discussed and research bottlenecks to this end are also identified. These aspects include not just the innovative genetic design of new strains but also the incorporation of novel chemical elements into the extant biochemistry, as well as genomic stability and scaling-up issues.

Much of contemporary synthetic biology research relies on the use of bacterial chassis for plugging-in and plugging-out genetic circuits and new-to-nature functionalities. However, the microorganisms that are the easiest to manipulate in the laboratory are often suboptimal for downstream industrial applications, which can involve physicochemical stress and harsh operating conditions. In this Review, we advocate the use of environmental Pseudomonas strains as model organisms that are pre-endowed with the metabolic, physiological and stress-endurance traits that are demanded by current and future synthetic biology and biotechnological needs.

SummaryBy the time the complete genome sequence of the soil bacterium Pseudomonas putida KT2440 was published in (Nelson et al., 2002 this bacterium was considered a potential agent for environmental bioremediation of industrial waste and a good colonizer of the rhizosphere. However, neither the annotation tools available at that time nor the scarcely available omics data-let alone metabolic modeling and other nowadays common systems biology approaches-allowed them to anticipate the astonishing capacities that are encoded in the genetic complement of this unique microorganism. In this work we have adopted a suite of state-of-the-art genomic analysis tools to revisit the functional and metabolic information encoded in the chromosomal sequence of strain KT2440. We identified 242 new protein-coding genes and re-annotated the functions of 1548 genes, which are linked to almost 4900 PubMed references. Catabolic pathways for 92 compounds (carbon, nitrogen and phosphorus sources) that could not be accommodated by the previously constructed metabolic models were also predicted. The resulting examination not only accounts for some of the known stress tolerance traits known in P. putida but also recognizes the capacity of this bacterium to perform difficult redox reactions, thereby multiplying its value as a platform microorganism for industrial biotechnology.

Glucose catabolism of Pseudomonas putida is carried out exclusively through the Entner-Doudoroff (ED) pathway due to the absence of 6-phosphofructokinase. In order to activate the Embden-Meyerhof-Parnas (EMP) route we transferred the pfkA gene from Escherichia coli to a P. putida wild-type strain as well as to an eda mutant, i.e. lacking 2-keto-3-deoxy-6-phosphogluconate aldolase. PfkA(E. coli) failed to redirect the carbon flow from the ED route towards the EMP pathway, suggesting that ED was essential for sugar catabolism. The presence of PfkA(E. coli) was detrimental for growth, which could be traced to the reduction of ATP and NAD(P)H pools along with alteration of the NAD(P)H/NADP(+) ratio. Pseudomonas putida cells carrying PfkA(E. coli) became highly sensitive to diamide and hydrogen peroxide, the response to which is very demanding of NADPH. The inhibitory effect of PfkA(E. coli) could in part be relieved by methionine, the synthesis of which relies much on NADPH. These results expose the role of the ED pathway for generating the redox currency (NADPH) that is required for counteracting oxidative stress. It is thus likely that environmental bacteria that favour the ED pathway over the EMP pathway do so in order to gear their aerobic metabolism to endure oxidative-related insults.

The last few years have witnessed an unprecedented increase in the number of novel bacterial species that hold potential to be used for metabolic engineering. Historically, however, only a handful of bacteria have attained the acceptance and widespread use that are needed to fulfil the needs of industrial bioproduction - and only for the synthesis of very few, structurally simple compounds. One of the reasons for this unfortunate circumstance has been the dearth of tools for targeted genome engineering of bacterial chassis, and, nowadays, synthetic biology is significantly helping to bridge such knowledge gap. Against this background, in this review, we discuss the state of the art in the rational design and construction of robust bacterial chassis for metabolic engineering, presenting key examples of bacterial species that have secured a place in industrial bioproduction. The emergence of novel bacterial chassis is also considered at the light of the unique properties of their physiology and metabolism, and the practical applications in which they are expected to outperform other microbial platforms. Emerging opportunities, essential strategies to enable successful development of industrial phenotypes, and major challenges in the field of bacterial chassis development are also discussed, outlining the solutions that contemporary synthetic biology-guided metabolic engineering offers to tackle these issues.

Although the flagellar machinery of environmental bacteria endows cells with a phenomenal survival device, it also consumes much of the metabolic currency necessary for fuelling such a vigorous nano-motor. The physiological cost of flagella-related functions of the soil bacterium Pseudomonas putida KT2440 was examined and quantified through the deletion of a ≈ 70 kb DNA segment of the genome (≈ 1.1%), which includes relevant structural and regulatory genes in this micro-organism. The resulting strain lacked the protruding polar cords that define flagella in the wild-type P. putida strain and was unable of any swimming motility while showing a significant change in surface hydrophobicity. However, these deficiencies were otherwise concomitant with clear physiological advantages: rapid adaptation of the deleted strain to both glycolytic and gluconeogenic carbon sources, increased energy charge and, most remarkably, improved tolerance to oxidative stress, reflecting an increased NADPH/NADP(+) ratio. These qualities improve the endurance of non-flagellated cells to the metabolic fatigue associated with rapid growth in rich medium. Thus, flagellar motility represents the archetypal tradeoff involved in acquiring environmental advantages at the cost of a considerable metabolic burden.