Degradación de compuestos aromáticos y alicíclicos en la bacteria Azoarcus sp. CIB, y sus aplicaciones biotecnológicas

Abstract

The β-proteobacteria Azoarcus sp. CIB is a model organism for the study of the anaerobic/aerobic degradation of aromatic compounds. In this doctoral thesis, the knowledge on these metabolic pathways has been expanded, broadening the degrading capacity of the CIB strain over new contaminant compounds (PA) and studying the metabolism of alicyclic compounds, such as cyclohexane carboxylate (CHC), whose degradation converge with that of aromatic compounds at the level of the lower pathway of aliphatic dicarboxylic acids β-oxidation (the (hydroxy)pimeloyl-CoA catabolon). The development of a pht cassette (from the Aromatoleum aromaticum EbN1 genes) was the first objective of this work. This cassette includes both the catabolic genes needed for the peripheral anaerobic metabolism of PA to benzoyl-CoA, and the genes phtTaTb that encode for the first TAXI-TRAP transporter described for the transport of PA and that are essential for the anaerobic growth of the recombinant CIB strain in PA. Unexpectedly, the pht pathway has been demonstrated to be functional in the presence of oxygen, and it has been confirmed the subsequent mineralization of the benzoyl-CoA generated through the box hybrid aerobic pathway. The expression of the pht cassette in the bacteria Cupriavidus necator H16 has allowed us to develop a biocatalyst able to valorize PA for polyhydroxybutyrate (PHB) production. This represents a pioneer approach for the recycling of plasticizing substances that are also emerging contaminants. The characterization of the bad genes involved in the aerobic/anaerobic metabolism of CHC in Azoarcus sp. CIB was the second objective of this thesis. Unlike what was described for phototroph bacteria or strict anaerobic degraders of aromatic compounds, the bad pathway of denitrificant bacteria, such as the CIB strain, does not converge with the central anaerobic pathway of benzoyl-CoA (bzd pathway). The specific regulation of the Azoarcus sp. CIB bad cluster, mediated by the BadR transcriptional repressor and the CHC-CoA inductor molecule, has been characterized. The presence of bad clusters with a similar genetic organization in diverse proteobacteria suggests a conserved transcriptional regulation and reveals that the capacity to metabolize CHC is more widespread than what it was initially thought. The development of a bad cassette and its expression in E. coli to produce pimeloyl-CoA (a biotin precursor) from CHC is an example of the biotechnological value of these discoveries. The third objective of this work was to identify the aab genes, involved in the lower pathway for the β-oxidation of (hydroxy)pimeloyl-CoA, which is derived from the anaerobic central pathway of benzoyl-CoA (bzd pathway) and the bad pathway in Azoarcus sp. CIB. A second β-oxidation cluster (pim genes), that is similar to the previous one, is responsible for the degradation of pimelate in the CIB strain. Although the aab and pim clusters are specifically induced in the presence of their diverse substrates, the inactivation of one of them is compensated with the induction of the other. The characterization of the catabolon of (hydroxy)pimeloyl-CoA for the metabolism of aromatic (anaerobic conditions), alicyclic (CHC) and aliphatic (pimelate) compounds has allowed us to construct diverse metabolic cassettes that, in combination with the bad genes, allow to widespread CHC and pimelate degradation to heterologous hosts, such as Pseudomonas putida, and that can be of great biotechnological interest for the synthesis of new biopolymers