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.

The soil bacterium Pseudomonas putida KT2440 lacks a functional Embden-Meyerhof-Parnas (EMP) pathway, and glycolysis is known to proceed almost exclusively through the Entner-Doudoroff (ED) route. To investigate the raison d'être of this metabolic arrangement, the distribution of periplasmic and cytoplasmic carbon fluxes was studied in glucose cultures of this bacterium by using ¹³C-labeled substrates, combined with quantitative physiology experiments, metabolite quantification, and in vitro enzymatic assays under both saturating and non-saturating, quasi in vivo conditions. Metabolic flux analysis demonstrated that 90% of the consumed sugar was converted into gluconate, entering central carbon metabolism as 6-phosphogluconate and further channeled into the ED pathway. Remarkably, about 10% of the triose phosphates were found to be recycled back to form hexose phosphates. This set of reactions merges activities belonging to the ED, the EMP (operating in a gluconeogenic fashion), and the pentose phosphate pathways to form an unforeseen metabolic architecture (EDEMP cycle). Determination of the NADPH balance revealed that the default metabolic state of P. putida KT2440 is characterized by a slight catabolic overproduction of reducing power. Cells growing on glucose thus run a biochemical cycle that favors NADPH formation. Because NADPH is required not only for anabolic functions but also for counteracting different types of environmental stress, such a cyclic operation may contribute to the physiological heftiness of this bacterium in its natural habitats.

Summary: Glucose catabolism of P seudomonas putida is carried out exclusively through the E ntner– D oudoroff ( ED ) pathway due to the absence of 6‐phosphofructokinase. In order to activate the E mbden– M eyerhof– P arnas ( EMP ) route we transferred the pfkA gene from E scherichia 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. P seudomonas 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.

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