Bioconversion of lignin-derived aromatics into the building block pyridine 2,4-dicarboxylic acid by engineering recombinant Pseudomonas putida strains.

2,4 pyridine dicarboxylic acid (2,4 PDCA) is an analogue of terephthalate, and hence a target chemical in the field of bio-based plastics. Here, Pseudomonas putida KT2440 strains were engineered to efficiently drive the metabolism of lignin-derived monoaromatics towards 2,4 PDCA in a resting cells-based bioprocess that alleviates growth-coupled limitations and allows biocatalysts recycling. Native ß-ketoadipate pathway was blocked by replacing protocatechuate 3,4-dioxygenase by the exogenous LigAB extradiol dioxygenase. Overexpression of pcaK encoding a transporter increased 8-fold 2,4 PDCA productivity from protocatechuate, reaching the highest value reported so far (0.58 g L-1h-1). Overexpression of the 4-hydroxybenzoate monooxygenase (pobA) speed up drastically the production of 2,4 PDCA from 4-hydroxybenzoate (0.056 g L-1h-1) or p-coumarate (0.012 g L-1h-1) achieving values 15-fold higher than those reported with Rhodococcus jostii biocatalysts. 2,4 PDCA was also bioproduced by using soda lignin as feedstock, paving the way for future polymeric lignin valorization approaches.

A Novel Redox-Sensing Histidine Kinase That Controls Carbon Catabolite Repression in Azoarcus sp. CIB.

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.IMPORTANCE Two-component signal transduction systems comprise a sensor histidine kinase and its cognate response regulator, and some have evolved to sense and convert redox signals into regulatory outputs that allow bacteria to adapt to the altered redox environment. The work presented here expands knowledge of the functional diversity of redox-sensing kinases to control carbon catabolite repression (CCR), a phenomenon that allows the selective assimilation of a preferred compound among a mixture of several carbon sources. The newly characterized AccS sensor kinase is responsible for the phosphorylation and activation of the AccR master regulator involved in CCR of the anaerobic degradation of aromatic compounds in the betaproteobacterium Azoarcus sp. CIB. AccS seems to have a thiol-based redox switch that is modulated by the redox state of the quinone pool. The AccSR system is conserved in several betaproteobacteria, where it might play a more general role controlling their global metabolic state.

Combination of degradation pathways for naphthalene utilization in Rhodococcus sp. strain TFB.

Rhodococcus sp. strain TFB is a metabolic versatile bacterium able to grow on naphthalene as the only carbon and energy source. Applying proteomic, genetic and biochemical approaches, we propose in this paper that, at least, three coordinated but independently regulated set of genes are combined to degrade naphthalene in TFB. First, proteins involved in tetralin degradation are also induced by naphthalene and may carry out its conversion to salicylaldehyde. This is the only part of the naphthalene degradation pathway showing glucose catabolite repression. Second, a salicylaldehyde dehydrogenase activity that converts salicylaldehyde to salicylate is detected in naphthalene-grown cells but not in tetralin- or salicylate-grown cells. Finally, we describe the chromosomally located nag genes, encoding the gentisate pathway for salicylate conversion into fumarate and pyruvate, which are only induced by salicylate and not by naphthalene. This work shows how biodegradation pathways in Rhodococcus sp. strain TFB could be assembled using elements from different pathways mainly because of the laxity of the regulatory systems and the broad specificity of the catabolic enzymes.