Toward Characterizing the Genetic Basis of Trace Organic Contaminant Biotransformation in Activated Sludge: The Role of Multicopper Oxidases as a Case Study

Activated sludge treatment leverages the ability of microbes to uptake and (co)-metabolize chemicals and has shown promise in eliminating trace organic contaminants (TrOCs) during wastewater treatment. However, targeted interventions to optimize the process are limited as the fundamental drivers of the observed reactions remain elusive. In this work, we present a comprehensive workflow for the identification and characterization of key enzymes involved in TrOCs biotransformation pathways in complex microbial communities. To demonstrate the applicability of the workflow, we investigated the role of the enzymatic group of multicopper oxidases (MCOs) as one putatively relevant driver of TrOCs biotransformation. To this end, we analyzed activated sludge metatranscriptomic data and selected, synthesized, and heterologously expressed three phylogenetically distinct MCO-encoding genes expressed in communities with different TrOCs oxidation potentials. Following the purification of the encoded enzymes, we screened their activities against different substrates. We saw that MCOs exhibit significant activities against selected TrOCs in the presence of the mediator compound 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid and, in some cases, also in the presence of the wastewater contaminant 4′-hydroxy-benzotriazole. In the first case, we identified oxidation products previously reported from activated sludge communities and concluded that in the presence of appropriate mediators, bacterial MCOs could contribute to the biological removal of TrOCs. Similar investigations of other key enzyme systems may significantly advance our understanding of TrOCs biodegradation and assist the rational design of biology-based water treatment strategies in the future

Isoprenoid biosynthesis in the diatom <i>Haslea ostrearia</i>

International audienceDiatoms are eukaryotic, unicellular algae that are responsible for c. 20% of the Earth's primary production. Their dominance and success in contemporary oceans have prompted investigations on their distinctive metabolism and physiology. One metabolic pathway that remains largely unexplored in diatoms is isoprenoid biosynthesis, which is responsible for the production of numerous molecules with unique features.We selected the diatom species Haslea ostrearia because of its characteristic isoprenoid content and carried out a comprehensive transcriptomic analysis and functional characterization of the genes identified.We functionally characterized one farnesyl diphosphate synthase, two geranylgeranyl diphosphate synthases, one short‐chain polyprenyl synthase, one bifunctional isopentenyl diphosphate isomerase – squalene synthase, and one phytoene synthase. We inferred the phylogenetic origin of these genes and used a combination of functional analysis and subcellular localization predictions to propose their physiological roles.Our results provide insight into isoprenoid biosynthesis in H. ostrearia and propose a model of the central steps of the pathway. This model will facilitate the study of metabolic pathways of important isoprenoids in diatoms, including carotenoids, sterols and highly branched isoprenoids

MOESM1 of Production of the forskolin precursor 11β-hydroxy-manoyl oxide in yeast using surrogate enzymatic activities

Additional file 1: Table S1. List of primers. Table S2. Experimental and bibliographic 1H and 13C NMR data (in CDCl3) of 11β-hydroxy-manoyl oxide (4). Figure S1. 1H NMR spectrum of 11β-hydroxy-manoyl oxide (4). Figure S2. 13C NMR spectrum of 11β-hydroxy-manoyl oxide (4)