Chassis organism from Corynebacterium glutamicum: the way towards biotechnological domestication of Corynebacteria.

For a long time, Corynebacteria have been the organisms of choice for industrial bioproduction of amino acids. Later on, Corynebacteria have also been used for making biofuels and a suite of added-value chemicals. In this issue of Biotechnology Journal, Unthan et al. provide a splendid example of how systems and synthetic biology approaches are instrumental for significantly increasing the value of Corynebacterium glutamicum as a platform strain for industrial applications. To this end, genomic segments that appeared to be non-essential for maintaining every desirable trait in C. glutamicum were delineated. A massive, recursive deletion of each (or most) of such apparently useless DNA was then carried out to verify the relevance of the excised sequences, and the resulting C. glutamicum variants were tested under various growth conditions. This approach produced not only interesting new strains but also raised new questions on how to design reliable microbial chassis that fulfil the biotechnological promise of synthetic biology.Peer reviewe

When biology became engineering : adopting standards for living systems

For decades, molecular biologists have been removing or inserting genes into all kinds of organisms with biotechnological intent or simply to generate fundamental knowledge. Synthetic biology (SynBio) goes one step further by incorporating conceptual frameworks from computing, electronics, and industrial design. This change makes it possible to conceive the creation of complex biological objects that were previously considered too difficult to assemble. To do this, the stages of any industrial production process must be adopted: design, construction of the components, assembly, and final manufacture. This objective requires standardisation of the physical and functional formats of the components involved, DNA assembly methods, activity measurements, and descriptive languages

Expanding the Boolean logic of the prokaryotic transcription factor XylR by functionalization of permissive sites with a protease-target sequence

The σ54-dependent prokaryotic regulator XylR implements a one-input/one-output actuator that transduces the presence of the aromatic effector m-xylene into transcriptional activation of the cognate promoter Pu. Such a signal conversion involves the effector-mediated release of the intramolecular repression of the N-terminal A domain on the central C module of XylR. On this background, we set out to endow this regulator with additional signal-sensing capabilities by inserting a target site of the viral protease NIa in permissive protein locations that once cleaved in vivo could either terminate XylR activity or generate an effector-independent, constitutive transcription factor. To find optimal protein positions to this end, we saturated the xylR gene DNA with a synthetic transposable element designed for randomly delivering in-frame polypeptides throughout the sequence of any given protein. This Tn5-based system supplies the target gene with insertions of a selectable marker that can later be excised, leaving behind the desired (poly) peptides grafted into the protein structure. Implementation of such knock-in-leave-behind (KILB) method to XylR was instrumental to produce a number of variants of this transcription factor (TF) that could compute in vivo two inputs (m-xylene and protease) into a single output following a logic that was dependent on the site of the insertion of the NIa target sequence in the TF. Such NIa-sensitive XylR specimens afforded the design of novel regulatory nodes that entered protease expression as one of the signals recognized in vivo for controlling Pu. This approach is bound to facilitate the functionalization of TFs and other proteins with new traits, especially when their forward engineering is made difficult by, for example, the absence of structural data.This study was supported by the BIO and FEDER CONSOLIDER-INGENIO Program of the Spanish Ministry of Science and Innovation, the MICROME, ST-FLOW and ARYSIS Contracts of the EU, and the PROMT Project of the CAM.Peer reviewe

Genetically modified organisms for the environment: stories of success and failure and what we have learned from them

The expectations raised in the mid-1980s on the potential of genetic engineering for in situ remediation of environmental pollution have not been entirely fulfilled. Yet, we have learned a good deal about the expression of catabolic pathways by bacteria in their natural habitats, and how environmental conditions dictate the expression of desired catalytic activities. The many different choices between nutrients and responses to stresses form a network of transcriptional switches which, given the redundance and robustness of the regulatory circuits involved, can be neither unraveled through standard genetic analysis nor artificially programmed in a simple manner. Available data suggest that population dynamics and physiological control of catabolic gene expression prevail over any artificial attempt to engineer an optimal performance of the wanted catalytic activities. In this review, several valuable spin-offs of past research into genetically modified organisms with environmental applications are discussed, along with the impact of Systems Biology and Synthetic Biology in the future of environmental biotechnology. [Int Microbiol 2005; 8(3):213-222

From the phosphoenolpyruvate phosphotransferase system (PTS) to selfish metabolism: a story retraced in Pseudomonas putida

Although DNA is the ultimate repository of biological information, deployment of its instructions is constrained by the metabolic and physiological status of the cell. To this end, bacteria have evolved intricate devices that connect exogenous signals (e.g. nutrients, physicochemical conditions) with endogenous conditions (metabolic fluxes, biochemical networks) that coordinately influence expression or performance of a large number of cellular functions. The phosphoenolpyruvate:carbohydrate-phosphotransferase system (PTS) is a bacterial multi-protein phosphorylation chain which computes extracellular (e.g. sugars) and intracellular (e.g. phosphoenolpyruvate, nitrogen) signals and translates them into post-translational regulation of target activities through protein-protein interactions. The PTS of Pseudomonas putida KT2440 encompasses one complete sugar (fructose)-related system and the 3 enzymes that form the so-called nitrogen-related PTS (PTSNtr), which lacks connection to transport of substrates. These two PTS branches cross-talk to each other, as the product of the fruB gene (a polyprotein EI-HPr-EIIA) can phosphorylate PtsN (EIIANtr) in vivo. This gives rise to a complex actuator device where diverse physiological inputs are ultimately translated into phosphorylation or not of PtsN (EIIANtr) which, in turn, checks the activity of key metabolic and regulatory proteins. Such a control of bacterial physiology highlights the prominence of biochemical homeostasis over genetic ruling –and not vice versa.This study was supported by the BIO Program of the Spanish Ministry of Science and Innovation, the ST-FLOW and ARYSIS Contracts of the EU, the ERANET-IB Program and the PROMT Project of the CAMPeer reviewe

Engineering multicellular logic in bacteria with metabolic wires

Aromatic biodegradation pathways of environmental bacteria are vast sources of matching trios of enzymes, substrates and regulators that can be refactored to run logic operations through cell-to-cell communication. As a proof of concept, the connection between two Pseudomonas putida strains using benzoic acid as the wiring molecule is presented. In this system, a sender strain harboring the TOL pathway for biodegradation of aromatics processed toluene as input and generated benzoate as the output signal. Diffusion of such metabolic intermediate to the medium was then sensed by a second strain (the receiver) that used benzoate as input for a new logic gate producing a visual output (i.e., light emission). The setup was functional irrespective of whether sender and receiver cells were in direct contact or in liquid culture. These results highlight the potential of environmental metabolic pathways as sources of building blocks for the engineering of multicellular logic in prokaryotic systems. © 2013 American Chemical Society.This study was supported by the BIO and FEDER CONSOLIDER-INGENIO program of the Spanish Ministry of Science and Innovation, the MICROME, ST-FLOW and ARYSIS Contracts of the EU, and the PROMT Project of the Autonomous Community of Madrid.Peer Reviewe

Robustness of Pseudomonas 1 putida KT2440 as a host for ethanol 2 biosynthesis

Expansion of the burgeoning biofuels agenda involves not only the design of suitable genetic and metabolic devices but also their deployment into suitable hosts that can endure the stress brought about by the products themselves. The microorganisms easiest to genetically manipulate for these endeavours (e.g., Escherichia coli) are often afflicted by an undesirable sensitivity to the very product that they are engineered to synthesize. In this context, we have examined the resistance to the stress arising from ethanol synthesis and/or its addition to cultures of recombinant Pseudomonas putida, using as a benchmark the same trait in an E. coli strain. To this end, ethanologenic strains of these two species were constructed by functionally expressing pdc (pyruvate decarboxylase) and adhB (alcohol dehydrogenase) from Zymomonas mobilis. Recombinants were compared under anoxic conditions as ethanol producers, and cell survival, stress resistance, and phenotypic stability were quantified in each case. P. putida consistently outperformed E. coli in every ethanol tolerance test conducted – whether the alcohol was produced endogenously or added exogenously. These results highlight the value of such bacterium as a microbial cell factory for the production of biofuels owing to its naturally preevolved ability to withstand different kinds of chemical stresses.This study was supported by the BIO Program of the Spanish Ministry of Economy and Competitiveness, the ST-FLOW and ARIAYS Contracts of the EU, and the PROMT Project of the CAM.Peer reviewe

Biología sintética: la ingeniería al asalto de la complejidad biológica

Synthetic Biology is not just a contemporary update of the recombinant DNA technologies of the past 30 years along with a descriptive language imported from electrical and industrial engineering. It is also a new key to interpreting living systems, as well as a declaration of intent about the use and reprogramming of biological objects for human benefit. In the same way that scientific chemistry initiated by Lavoisier evolved into the Chemical Engineering that is the basis of our industrial society, Biology has acquired a transforming potential that will possibly lead to a type of industry and economy very different from the current paradigm. To this end, it is essential to identify bottlenecks that limit the design of biological objects from first principles and not to miss the train of Synthetic Biology at its current foundational stage, when talent -and not muscle- is what really matters.La Biología sintética no es solo una reformulación contemporánea de las tecnologías del ADN recombinante de los últimos 30 años junto con un lenguaje descriptivo importado de la ingeniería eléctrica e industrial. Es también una nueva clave interpretativa de los sistemas vivos y una declaración de intenciones sobre la utilización y reprogramación de los objetos biológicos en beneficio humano. De la misma forma que la Química científica iniciada por Lavoisier devino en la Ingeniería química que está en la base de nuestra sociedad desarrollada, la Biología ha adquirido un potencial transformador que posiblemente nos lleve a un tipo de industria y de economía muy distinta de la actual. Para ello es esencial identificar los cuellos de botella que limitan el diseño de objetos biológicos desde sus primeros principios y no perder el tren de la Biología sintética en su etapa fundacional, cuando el talento -y no el músculo- es lo determinante

Effector specificity mutants of the transcriptional activator NahR of naphthalene degrading Pseudomonas define protein sites involved in binding of aromatic inducers

This work reports a genetic analysis of the interactions between NahR, the LysR-type regulator of the NAH operons for biodegradation of naphthalene in Pseudomonas, and its aromatic effectors. Six mutants encoding NahR variants responsive to salicylate analogs such as benzoate, which is not an inducer for the wild type regulator, were isolated with a polymerase chain reactionbased saturation mutagenesis protocol. Most mutants displaying a specific change of effector profile bore single amino acid substitutions within a short protein segment of 60 residues located at the central portion of the NahR sequence. Some of the protein variants exhibited an increased affinity for salicylate and also for otherwise suboptimal effectors, with apparent Ks * values 5–100-fold lower than those of the wild type NahR protein. In addition, all mutants were activated by inducers bearing novel substituents at positions 1 or 2 of the aromatic ring and displayed also an enhanced tolerance to changes at positions 3 and 4. Correlation between mutations in NahR and the structures of the new effectors suggested that protein sites Met116, Arg132, Asn169, and Arg248 are involved in effector recognition and binding during the earlier steps of the process leading to transcriptional activation of cognate NAH promoter

Pyridine nucleotide transhydrogenases enable redox balance of Pseudomonas putida during biodegra dation of aromatic compounds

Supporting informationThe metabolic versatility of the soil bacterium Pseudomonas putida is reflected by its ability to execute strong redox reactions (e.g., mono- and di-oxygenations) on aromatic substrates. Biodegradation of aromatics occurs via the pathway encoded in the archetypal TOL plasmid pWW0, yet the effect of running such oxidative route on redox balance against the background metabolism of P. putida remains unexplored. To answer this question, the activity of pyridine nucleotide transhydrogenases (that catalyze the reversible interconversion of NADH and NADPH) was inspected under various physiological and oxidative stress regimes. The genome of P. putida KT2440 encodes a soluble transhydrogenase (SthA) and a membrane-bound, proton-pumping counterpart (PntAB). Mutant strains, lacking sthA and/or pntAB, were subjected to a panoply of genetic, biochemical, phenomic and functional assays in cells grown on customary carbon sources (e.g., citrate) versus difficult-to-degrade aromatic substrates. The results consistently indicated that redox homeostasis is compromised in the transhydrogenases-defective variant, rendering the mutant sensitive to oxidants. This metabolic deficiency was, however, counteracted by an increase in the activity of NADP+ -dependent dehydrogenases in central carbon metabolism. Taken together, these observations demonstrate that transhydrogenases enable a redox-adjusting mechanism that comes into play when biodegradation reactions are executed to metabolize unusual carbon compounds.This work was supported by the EVOPROG (FP7-ICT-610730), ARISYS (ERC-2012-ADG-322797) and EmPowerPutida (EU-H2020-BIOTEC-2014-2015-6335536), Contracts of the European Union, and the CAMBIOS (RTC-2014-1777-3) and CONTIBUGS (PCIN-2013-040) projects of the Spanish Ministry of Economy and Competitiveness.Peer reviewe