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.

SUMMARY Aromatic compounds belong to one of the most widely distributed classes of organic compounds in nature, and a significant number of xenobiotics belong to this family of compounds. Since many habitats containing large amounts of aromatic compounds are often anoxic, the anaerobic catabolism of aromatic compounds by microorganisms becomes crucial in biogeochemical cycles and in the sustainable development of the biosphere. The mineralization of aromatic compounds by facultative or obligate anaerobic bacteria can be coupled to anaerobic respiration with a variety of electron acceptors as well as to fermentation and anoxygenic photosynthesis. Since the redox potential of the electron-accepting system dictates the degradative strategy, there is wide biochemical diversity among anaerobic aromatic degraders. However, the genetic determinants of all these processes and the mechanisms involved in their regulation are much less studied. This review focuses on the recent findings that standard molecular biology approaches together with new high-throughput technologies (e.g., genome sequencing, transcriptomics, proteomics, and metagenomics) have provided regarding the genetics, regulation, ecophysiology, and evolution of anaerobic aromatic degradation pathways. These studies revealed that the anaerobic catabolism of aromatic compounds is more diverse and widespread than previously thought, and the complex metabolic and stress programs associated with the use of aromatic compounds under anaerobic conditions are starting to be unraveled. Anaerobic biotransformation processes based on unprecedented enzymes and pathways with novel metabolic capabilities, as well as the design of novel regulatory circuits and catabolic networks of great biotechnological potential in synthetic biology, are now feasible to approach.

The genome of the soil bacterium Pseudomonas putida KT2440 bears two virtually identical arsRBCH operons putatively encoding resistance to inorganic arsenic species. Single and double chromosomal deletions in each of these ars clusters of this bacterium were tested for arsenic sensitivity and found that the contribution of each operon to the resistance to the metalloid was not additive, as either cluster sufficed to endow cells with high-level resistance. However, otherwise identical traits linked to each of the ars sites diverged when temperature was decreased. Growth of the various mutants at 15°C (instead of the standard 30°C for P. putida) uncovered that ars2 affords a much higher resistance to As (III) than the ars1 counterpart. Reverse transcription polymerase chain reaction of arsB1 and arsB2 genes as well as lacZ fusions to the Pars1 and Pars2 promoters traced the difference to variations in transcription of the corresponding gene sets at each temperature. Functional redundancy may thus be selected as a stable condition - rather than just as transient state - if it affords one key activity to be expressed under a wider range of physicochemical settings. This seems to provide a straightforward solution to regulatory problems in environmental bacteria that thrive under changing scenarios.

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The BzdR transcriptional regulator that controls the P N promoter responsible for the anaerobic catabolism of benzoate in Azoarcus sp. CIB constitutes the prototype of a new subfamily of transcriptional regulators. Here, we provide some insights about the functional-structural relationships of the BzdR protein. Analytical ultracentrifugation studies revealed that BzdR is homodimeric in solution. An electron microscopy three-dimensional reconstruction of the BzdR dimer has been obtained, and the predicted structures of the respective N-and C-terminal domains of each BzdR monomer could be fitted into such a reconstruction. Gel retardation and ultracentrifugation experiments have shown that the binding of BzdR to its cognate promoter is cooperative. Different biochemical approaches revealed that the effector molecule benzoyl-CoA induces conformational changes in BzdR without affecting its oligomeric state. The BzdRdependent inhibition of the P N promoter and its activation in the presence of benzoyl-CoA have been established by in vitro transcription assays. The monomeric BzdR4 and BzdR5 mutant regulators revealed that dimerization of BzdR is essential for DNA binding. Remarkably, a BzdR⌬L protein lacking the linker region connecting the N-and C-terminal domains of BzdR is also dimeric and behaves as a super-repressor of the P N promoter. These data suggest that the linker region of BzdR is not essential for protein dimerization, but rather it is required to transfer the conformational changes induced by the benzoyl-CoA to the DNA binding domain leading to the release of the repressor. A model of action of the BzdR regulator has been proposed.

3Most of the current methods for controlling the formation rate of a key protein or enzyme in cell 4 factories rely on the manipulation of target genes within the pathway. In this article, we present a 5 novel synthetic system for post-translational regulation of protein levels, FENIX, which provides both 6 independent control of the steady-state protein level and inducible accumulation of target proteins. 7 The FENIX device is based on the constitutive, proteasome-dependent degradation of the target 8 polypeptide by tagging with a short synthetic, hybrid NIa/SsrA amino acid sequence in the C-terminal 9 domain. Protein production is triggered via addition of an orthogonal inducer (i.e. 3-methylbenzoate) 10 to the culture medium. The system was benchmarked in Escherichia coli by tagging two fluorescent 11 proteins (GFP and mCherry), and further exploited to completely uncouple poly(3-hydroxybutyrate) 12 (PHB) accumulation from bacterial growth. By tagging PhaA (3-ketoacyl-CoA thiolase, first step of the 13 route), a dynamic metabolic switch at the acetyl-coenzyme A node was established in such a way 14 that this metabolic precursor could be effectively re-directed into PHB formation upon activation of the 15 system. The engineered E. coli strain reached a very high specific rate of PHB accumulation (0.4 h -1 ) 16 with a polymer content of ca. 72% (w/w) in glucose cultures in a growth-independent mode. Thus, 17FENIX enables dynamic control of metabolic fluxes in bacterial cell factories by establishing post-18 translational synthetic switches in the pathway of interest.One of the main challenges in contemporary metabolic engineering is to develop systems for 4 controlling protein production in a spatial-temporal fashion, leading to the highest possible catalytic 5 output 1-2 . The problem can be tackled by manipulating genes and proteins in cell factories at different 6 levels of regulation. Transcriptional and translational regulation mechanisms, for instance, have been 7 studied in great detail in many biotechnologically-relevant microorganisms, and several studies 8 describe synthetic circuits exploiting these cellular processes for bioproduction purposes 3-6 . More 9 recently, the adoption of CRISPR/Cas9-mediated technologies has opened up countless possibilities 10 for targeted regulation at the gene/genome level 7-8 . The conditional and dynamic control of protein 11 levels in vivo, in contrast, has received less attention thus far, and the majority of the currently 12 available tools designed to modulate protein activity target mRNAs and protein synthesis rates (e.g. 13 by using specific transcriptional repressors, RNA interference strategies, and riboregulators). Some 14 synthetic devices for the tunable control of protein synthesis and degradation have been developed 15 over the last few years 9 , e.g. systems triggered by small molecules 10-12 or indirect degradation 16 processes 13-15 . From a practical perspective, these strategies allow for a tight and accurate control of 17 metabolic pathways...

Organic wastes are a suitable feedstock for the production of value‐added products that have been insufficiently exploited due to their complexity, which challenges their transformation by conventional procedures. Gasification and pyrolysis of organic wastes can reduce this complexity by producing syngas (CO plus H2 and other C1 gases), which can be used as a valuable commodity by catalytic conversion into chemicals. However, the high cost and susceptibility to poisoning of chemical catalysts have encouraged research on biocatalysts that convert C1 components of syngas into different multi‐carbon compounds. Nowadays, research on syngas fermentation is receiving much attention in order to enhance the productivity of microorganisms by remodeling their metabolism and by optimizing the bioreactor operational conditions. This review highlights the new technical achievements of pyrolysis as well as the new biotechnological uses of syngas for the production of bulk chemicals and biopolymers, discussing the major bottlenecks that challenge syngas fermentation. © 2015 Society of Chemical Industry

The role of oxygen in the transcriptional regulation of the P N promoter that controls the bzd operon involved in the anaerobic catabolism of benzoate in the denitrifying Azoarcus sp. strain CIB has been investigated. In vivo experiments using P N ::lacZ translational fusions, in both Azoarcus sp. strain CIB and Escherichia coli cells, have shown an oxygen-dependent repression effect on the transcription of the bzd catabolic genes. E. coli Fnr was required for the anaerobic induction of the P N promoter, and the oxygen-dependent repression of the bzd genes could be bypassed by the expression of a constitutively active Fnr* protein. In vitro experiments revealed that Fnr binds to the P N promoter at a consensus sequence centered at position ؊41.5 from the transcription start site overlapping the ؊35 box, suggesting that P N belongs to the class II Fnr-dependent promoters. Fnr interacts with RNA polymerase (RNAP) and is strictly required for transcription initiation after formation of the RNAP-P N complex. An fnr ortholog, the acpR gene, was identified in the genome of Azoarcus sp. strain CIB. The Azoarcus sp. strain CIB acpR mutant was unable to grow anaerobically on aromatic compounds and it did not drive the expression of the P N ::lacZ fusion, suggesting that AcpR is the cognate transcriptional activator of the P N promoter. Since the lack of AcpR in Azoarcus sp. strain CIB did not affect growth on nonaromatic carbon sources, AcpR can be considered a transcriptional regulator of the Fnr/Crp superfamily that has evolved to specifically control the central pathway for the anaerobic catabolism of aromatic compounds in Azoarcus.