An Aerobic Hybrid Phthalate Degradation Pathway via Phthaloyl-Coenzyme A in Denitrifying Bacteria

Abstract

The degradation of the xenobiotic phthalic acid esters by microorganisms is initiated by the hydrolysis to the respective alcohols and ortho-phthalate (hereafter, phthalate). In aerobic bacteria and fungi, oxygenases are involved in the conversion of phthalate to protocatechuate, the substrate for ring-cleaving dioxygenases. In contrast, anaerobic bacteria activate phthalate to the extremely unstable phthaloyl-coenzyme A (CoA), which is decarboxylated by oxygen-sensitive UbiD-like phthaloyl-CoA decarboxylase (PCD) to the central benzoyl-CoA intermediate. Here, we demonstrate that the facultatively anaerobic, denitrifying Thauera chlorobenzoica 3CB-1 and Aromatoleum evansii KB740 strains use phthalate as a growth substrate under aerobic and denitrifying conditions. In vitro assays with extracts from cells grown aerobically with phthalate demonstrated the succinyl-CoA-dependent activation of phthalate followed by decarboxylation to benzoyl-CoA. In T. chlorobenzoica 3CB-1, we identified PCD as a highly abundant enzyme in both aerobically and anaerobically grown cells, whereas genes for phthalate dioxygenases are missing in the genome. PCD was highly enriched from aerobically grown T. chlorobenzoica cells and was identified as an identical enzyme produced under denitrifying conditions. These results indicate that the initial steps of aerobic phthalate degradation in denitrifying bacteria are accomplished by the anaerobic enzyme inventory, whereas the benzoyl-CoA oxygenase-dependent pathway is used for further conversion to central intermediates. Such a hybrid pathway requires intracellular oxygen homeostasis at concentrations low enough to prevent PCD inactivation but sufficiently high to supply benzoyl-CoA oxygenase with its cosubstrate. IMPORTANCE Phthalic acid esters (PAEs) are industrially produced on a million-ton scale per year and are predominantly used as plasticizers. They are classified as environmentally relevant xenobiotics with a number of adverse health effects, including endocrine-disrupting activity. Biodegradation by microorganisms is considered the most effective process to eliminate PAEs from the environment. It is usually initiated by the hydrolysis of PAEs to alcohols and o-phthalic acid. Degradation of o-phthalic acid fundamentally differs in aerobic and anaerobic microorganisms; aerobic phthalate degradation heavily depends on dioxygenase-dependent reactions, whereas anaerobic degradation employs the oxygen-sensitive key enzyme phthaloyl-CoA decarboxylase. We demonstrate that aerobic phthalate degradation in facultatively anaerobic bacteria proceeds via a previously unknown hybrid degradation pathway involving oxygen-sensitive and oxygen-dependent key enzymes. Such a strategy is essential for facultatively anaerobic bacteria that frequently switch between oxic and anoxic environments.

Expanding the current knowledge and biotechnological applications of the oxygen‐independent ortho‐phthalate degradation pathway

“…It is worth mentioning that during the evaluation of this work a similar conclusion has been achieved by Ebenau‐Jehle et al . (2020) using a biochemical approach, thus reinforcing our results. Hence, the pht pathway may constitute an evolutionary acquisition for PA degradation by bacteria that thrive either in anoxic environments, or in environments that face oxygen limitations and that rely on benzoyl‐CoA, rather than on catecholic central intermediates, for the aerobic catabolism of aromatic compounds.…”

The occurrence, fate, toxicity, and biodegradation of phthalate esters: An overview

“…Other pathway involves the formation of 3,4‐dihydroxybenzoate (Protocatechuate) by dioxygenase and dehydrogenase enzyme. The anoxic conditions decrease the rate of biodegradation of PAEs (Ebenau‐Jehle et al, 2017, 2020).…”

“…The facultative denitrifying anaerobe Thauera chlorobenzoica 3CB‐1 was used. In PAE degradation, the unstable phthaloyl‐CoA is converted to benzoyl CoA intermediate by decarboxylation through PCD (Ebenau‐Jehle et al, 2020). Bacillus mojavensis B1811 strain was isolated from the oil‐polluted soil.…”

Isophthalate:coenzyme A ligase initiates anaerobic degradation of xenobiotic isophthalate

“…Biodegradation of phthalate esters is possible under aerobic or anaerobic conditions. In both cases, microbes initiate degradation by hydrolysis of PAE, yielding the corresponding alcohols and the free phthalate moiety [15,16]. Under anaerobic conditions, the key step in phthalate degradation is the removal of one of the carboxylic acid moiety [11].…”