Faculty of Biology Microbiology, Albert Ludwigs University Freiburg, Freiburg, Germany.
Faculty of Biology Microbiology, Albert Ludwigs University Freiburg, Freiburg, Germany
Appl Environ Microbiol. 2020 May 19;86(11). doi: 10.1128/AEM.00498-20.
The degradation of the xenobiotic phthalic acid esters by microorganisms is initiated by the hydrolysis to the respective alcohols and -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 3CB-1 and KB740 strains use phthalate as a growth substrate under aerobic and denitrifying conditions. assays with extracts from cells grown aerobically with phthalate demonstrated the succinyl-CoA-dependent activation of phthalate followed by decarboxylation to benzoyl-CoA. In 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 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. 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 -phthalic acid. Degradation of -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.
邻苯二甲酸酯类化合物（Xenobiotic phthalic acid esters）由微生物降解时，首先会被水解为相应的醇和邻苯二甲酸（phthalate）。在好氧细菌和真菌中，氧合酶参与邻苯二甲酸转化为原儿茶酸（protocatechuate）的过程，原儿茶酸是裂解双加氧酶的底物。相比之下，厌氧细菌将邻苯二甲酸激活为极不稳定的邻苯二甲酰辅酶 A（phthaloyl-coenzyme A，CoA），然后由氧敏感的 UbiD 样邻苯二甲酰辅酶 A 脱羧酶（phthaloyl-CoA decarboxylase，PCD）将其脱羧，生成中央苯甲酰辅酶 A 中间体。在这里，我们证明兼性厌氧脱氮菌 3CB-1 和 KB740 菌株可以在有氧和缺氧条件下以邻苯二甲酸作为生长基质。用邻苯二甲酸培养好氧细胞提取的实验表明，邻苯二甲酸在琥珀酰辅酶 A 依赖性作用下被激活，然后脱羧生成苯甲酰辅酶 A。在 3CB-1 中，我们发现 PCD 是好氧和厌氧生长细胞中高度丰富的酶，而基因组中缺失邻苯二甲酸双加氧酶基因。PCD 从好氧生长细胞中高度富集，并被鉴定为在缺氧条件下产生的相同酶。这些结果表明，在脱氮细菌中，好氧邻苯二甲酸降解的初始步骤是由厌氧酶库完成的，而苯甲酰辅酶 A 加氧酶依赖的途径则用于进一步转化为中央中间体。这种混合途径需要细胞内氧平衡，氧浓度既要足够低以防止 PCD 失活，又要足够高以向苯甲酰辅酶 A 加氧酶提供其辅酶。邻苯二甲酸酯（phthalic acid esters，PAEs）每年的工业产量超过 100 万吨，主要用作增塑剂。它们被归类为具有多种不良健康影响的环境相关外源化合物，包括内分泌干扰活性。微生物的生物降解被认为是从环境中消除 PAEs 最有效的方法。它通常由邻苯二甲酸酯的水解引发，生成醇和邻苯二甲酸。好氧和厌氧微生物中邻苯二甲酸的降解有根本的不同；好氧邻苯二甲酸的降解主要依赖于加氧酶依赖性反应，而厌氧降解则采用氧敏感的关键酶邻苯二甲酰辅酶 A 脱羧酶。我们证明，兼性厌氧细菌中的好氧邻苯二甲酸降解是通过一种以前未知的混合降解途径进行的，该途径涉及氧敏感和氧依赖的关键酶。对于经常在好氧和缺氧环境之间切换的兼性厌氧细菌来说，这种策略是必不可少的。
