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16 p.-8 fig.-1 tab., We have identified and characterized the AccS multidomain sensor kinase that mediates the activation of the AccR master regulator involved in carbon catabolite repression (CCR) of the anaerobic catabolism of aromatic compounds in Azoarcus sp. CIB. A truncated AccS protein that contains only the soluble C-terminal autokinase module (AccS′) accounts for the succinate-dependent CCR control. In vitro assays with purified AccS′ revealed its autophosphorylation, phosphotransfer from AccS′∼P to the Asp60 residue of AccR, and the phosphatase activity toward its phosphorylated response regulator, indicating that the equilibrium between the kinase and phosphatase activities of AccS′ may control the phosphorylation state of the AccR transcriptional regulator. Oxidized quinones, e.g., ubiquinone 0 and menadione, switched the AccS′ autokinase activity off, and three conserved Cys residues, which are not essential for catalysis, are involved in such inhibition. Thiol oxidation by quinones caused a change in the oligomeric state of the AccS′ dimer resulting in the formation of an inactive monomer. This thiol-based redox switch is tuned by the cellular energy state, which can change depending on the carbon source that the cells are using. This work expands the functional diversity of redox-sensitive sensor kinases, showing that they can control new bacterial processes such as CCR of the anaerobic catabolism of aromatic compounds. The AccSR two-component system is conserved in the genomes of some betaproteobacteria, where it might play a more general role in controlling the global metabolic state according to carbon availability., This work was supported by grants BIO2016-79736-R and PCIN-2014-113 from the Ministry of Economy and Competitiveness of Spain; by a grant of Fundación Ramón-Areces XVII CN; by grant CSIC 2016 2 0E 093 from the CSIC; and by European Union H2020 grant 760994.

43 p.-7 fig., Most of the current methods for controlling the formation rate of a key protein or enzyme in cell factories rely on the manipulation of target genes within the pathway. In this article, we present a novel synthetic system for post-translational regulation of protein levels, FENIX, which provides both independent control of the steady-state protein level and inducible accumulation of target proteins. The FENIX device is based on the constitutive, proteasome-dependent degradation of the target polypeptide by tagging with a short synthetic, hybrid NIa/SsrA amino acid sequence in the C-terminal domain. Protein production is triggered via addition of an orthogonal inducer ( i.e., 3-methylbenzoate) to the culture medium. The system was benchmarked in Escherichia coli by tagging two fluorescent proteins (GFP and mCherry), and further exploited to completely uncouple poly(3-hydroxybutyrate) (PHB) accumulation from bacterial growth. By tagging PhaA (3-ketoacyl-CoA thiolase, first step of the route), a dynamic metabolic switch at the acetyl-coenzyme A node was established in such a way that this metabolic precursor could be effectively redirected into PHB formation upon activation of the system. The engineered E. coli strain reached a very high specific rate of PHB accumulation (0.4 h-1) with a polymer content of ca. 72% (w/w) in glucose cultures in a growth-independent mode. Thus, FENIX enables dynamic control of metabolic fluxes in bacterial cell factories by establishing post-translational synthetic switches in the pathway of interest., This study was supported by The Novo Nordisk Foundation (Grant NNF10CC1016517) and the Danish Council for Independent Research (SWEET, DFF-Research Project 8021-00039B) to P.I.N. This study was also supported by the HELIOS Project of the Spanish Ministry of Economy and Competitiveness BIO2015-66960-C3-2-R (MINECO/FEDER), and the ARISYS (ERC-2012-ADG-322797), EmPowerPutida (EUH2020-BIOTEC-2014-2015-6335536), and MADONNA (H2020-FET-OPEN-RIA-2017-1-766975) contracts of the European Union to V.D.L.