Microbial Degradation of Phthalates: Biochemistry and Environmental Implications

Environmental Microbiology Reports (2020) 12(1), 3–15 doi:10.1111/1758-2229.12787

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Microbial degradation of phthalates: biochemistry and environmental implications

Matthias Boll, 1* Robin Geiger,1 Madan Junghare2 Introduction and Bernhard Schink2 Phthalic acids (benzene dicarboxylic acids; phthalates) 1Faculty of Biology, Microbiology, University of Freiburg, consist of a benzene ring to which two carboxylic groups Freiburg, Germany. are attached. Depending on the position of the carboxylic 2Department of Biology and Microbial Ecology, groups to each other, three isomers are possible, namely University of Konstanz, Constance, Germany. phthalic acid (PA; ortho-positioned), isophthalic acid (IPA; meta-positioned) and terephthalic acid (TPA; para-posi- Summary tioned). In colloquial usage, the term ‘phthalate’ refers mostoftentotheortho isomer and is also used for its The environmentally relevant xenobiotic esters of esters. Phthalic acids are essential constituents of synthetic phthalic acid (PA), isophthalic acid (IPA) and ter- organic polymers that are commonly called plastics. In ephthalic acid (TPA) are produced on a million ton scale these materials, phthalates are either part of the polymeric annually and are predominantly used as plastic poly- structure as in poly(ethylene terephthalate) (PET), or mers or plasticizers. Degradation by microorganisms is they are non-covalently dissolved as low-molecular-weight considered as the most effective means of their elimina- di-esters within the polymeric structure to act as softeners tion from the environment and proceeds via hydrolysis or plasticizers. to the corresponding PA isomers and alcohols under Plastic materials are used in almost every aspect of mod- oxic and anoxic conditions. Further degradation of PA, ern life and offer solutions and features that other materials IPA and TPA differs fundamentally between anaerobic were not able to provide before (Andrady and Neal, 2009). and aerobic microorganisms. The latter introduce Due to their low production costs and lack of cheap alterna- hydroxyl functionalities by dioxygenases to facilitate tives, there is an increasing demand of synthetic organic subsequent decarboxylation by either aromatizing polymers worldwide (Thompson et al., 2009). For instance, dehydrogenases or cofactor-free decarboxylases. In contrast, anaerobic bacteria activate the PA isomers to the packaging industry alone accounts for approximately fi the respective thioestersusingCoAligasesorCoA 42% of the global use of all non- bre plastics, followed by transferases followed by decarboxylationtothecentral the building and construction sector accountable for about fi intermediate benzoyl-CoA. Decarboxylases acting on 19% of non- bre plastics (Geyer et al., 2017). It is estimated the three PA CoA thioesters belong to the UbiD enzyme that the worldwide annual production of plastics will double family that harbour a prenylated flavin mononucleotide to 600 million tons within 20 years (Kumar, 2018). Despite (FMN) cofactor to achieve the mechanistically challeng- their numerous advantages, plastics cause increasing envi- ing decarboxylation. Capture of the extremely instable ronmental problems and health concerns worldwide PA-CoA intermediate is accomplished by a massive (Thompson et al., 2009). Some of these problems start only overproduction of phthaloyl-CoA decarboxylase and a at the end of their designated purpose when plastics balanced production of PA-CoA forming/decarboxylating become waste. Europe disposes approximately 50% of its fi enzymes. The strategy of anaerobic phthalate degrada- plastic waste in engineered land lls, while in other parts of tion probably represents a snapshot of an ongoing the world it is still incinerated openly, emitting large amount evolution of a xenobiotic degradation pathway via a of toxic or greenhouse gases. Huge freights of plastic mate- short-lived reaction intermediate. rials end up in the oceans where they persist for long times and endanger birds and other marine animals. Although recycling might be an option to limit production of new plastics, this material primarily ends up in secondary products *For correspondence. E-mail [email protected]; with less demanding material properties and unclear fate, Tel. (+49) 761 2032649; Fax (+49) 761 2032626. and is hence not a permanent solution. Furthermore,

© 2019 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 4 M. Boll, R. Geiger, M. Junghare and B. Schink recycling will not prevent the rise of microplastics in marine environments which is a growing problem that is difﬁcult to solve (Hahladakis et al., 2018). Therefore, it is important to develop new strategies of waste management and to better understand plastic biodegradation as a sustainable per- spective for removal of plastics in nature.

Phthalic acid ester use and biodegradation Phthalic acid polyesters. Phthalates can form essential building blocks inside plastic polymers (1). In these cases, phthalates (most often TPA) are bound via ester linkages to divalent alcohols such as ethylene glycol, 1,4-butanediol, 1,6-hexanediol, and so on. Needless to say, these covalently linked TPA residues are rather difficult to release from their polymeric mother backbones, as this is true for synthetic polymers in general. Although ester bonds are comparably easy to cleave by hydrolysis and require only water as a co-substrate, degradation of such polymers is difficult because a hydrolytic enzyme first has to get access to such polymer fibres, and this would require swelling of the plastic material by excessive water uptake. Plastics are typically produced as water-resis- tant, rather hydrophobic structures that are hardly accessible Fig. 1. Examples of industrially produced esters of the three phthalic to hydrolytic enzymes. Moreover, hydrolytic enzymes pro- acid isomers. A. Polyethylenglycol terephthalate ester polymer (PET). duced by microbes are typically synthesized rather specifically B. Polyethylenglycol IPA esters used as co-polymer of PET. C. PA esters used as plasticizers, for typical alcohols residues (=R) see Table 1. for attack on specific ester structures and do not interact freely with novel ester linkages. Last but not least, synthesis of extracellular enzymes for polymer degradation is an expensive covers in agriculture, or for plastic shopping bags. The BASF fi ® effort for a microbial cell and has to guarantee a suf cient pay- company produces Ecoflex , a copolymer of 1,4-butanediol back by easily degradable breakdown products that can cover with alternating terephthalate and adipate (hexane dioic acid) the energy expenditure for enzyme synthesis on the side of residues (poly butylene-adipate-terephthalate; PBAT). The ‘ the producer. Thus, the breakdown products should be palat- material decomposes in compost and agricultural soil within ’ able and should easily provide metabolic energy for a broad several months and thus fulfils its task to survive one vegeta- community of microbes of which then only few would dare to tion period and to disappear before the next one starts. In invest into the development of suited enzymes for polymer our lab, we could confirm that this material decomposes hydrolysis. As we will see later, the biochemistry of phthalate under oxic conditions and also anaerobically in the presence degradation is a rather demanding task, and the aromatic of nitrate, although nitrate-dependent degradation turned out derivatives formed in its breakdown are energetically not too to be far slower with terephthalate-containing polymers than rewarding to justify every effort into such an enterprise. Poly- with adipate-containing ones. There was no significant deg- saccharides, proteins, nucleic acids and so on are much more radation of either type of polymer in incubations under strictly appealing in this respect. This is true also for the natural poly- anoxic conditions with either sulfate or CO2 as the only elec- esters, e.g., poly-3-hydroxybutyric acid, a bacterial storage tron acceptor (Schink, unpublished). This is surprising as polymer which is easily degradable by a broad community of hydrolytic depolymerization of such synthetic material should aerobic and anaerobic bacteria (Jendrossek and Handrick, be possible under any condition, independent of the avail- 2002; Hazer and Steinbüchel, 2007). able electron acceptor. We assume that the probability of PET (Fig. 1A) is produced at high rate (56 million tons in development of efficient polymer-degrading hydrolases may 2013; Kawai et al., 2019), mainly as containers for soft drinks be higher in the aerobic and nitrate-dependent microbial and so on. Its microbial degradation is very slow and incom- world than among the strict anaerobes. Moreover, production plete. It appears that only surface-exposed residues are of extracellular hydrolases for unusual polymeric substrates degraded by microbes to a limited extent through the action of may be too ‘expensive’ for strict anaerobes with their limited cutinases, lipases and other carboxylesterases (Kawai et al., energy budgets. We have to conclude that phthalate resi- 2019). As this plastic material is applied mainly as drink con- dues that are covalently bound inside polymeric plastic tainers it has little tendency to take up water, and this limits materials are released only slowly, and their release fi severely a suf cient interaction of microbial esterases with the depends largely on the physicochemical properties of the fi micro bers of this plastic material (Tokiwa et al., 2009; Wei and plastic material and the incubation conditions. Zimmermann, 2017). Slow degradation of low-crystallinity PET to TPA and ethylene glycol was reported for a new aerobic bacterial isolate, Ideonella sakaiensis (Yoshida et al., 2016). Occurrence and use of isophthalic acid. IPA is an important Also ‘biodegradable’ synthetic phthalate polyesters are isomer produced on the million ton per year scale by oxida- commercially available, especially for use as protecting tion of m-xylene (Sheehan, 2005). It is mainly used as a

© 2019 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology Reports, 12, 3–15 Anaerobic degradation of phthalates 5 feedstock for the production of unsaturated polyesters, anal- and endocrine disruptors (Fushiwaki et al., 2003). Thus, from ogous to terephthalate, and is also a co-monomer in some the beginning there has been great concern about the release common plastics such PET (Fig. 1B). Furthermore, iso- of PAEs into the environment (Mayer et al., 1972; Giam et al., phthalate is an important constituent of the extremely heat- 1978). Furthermore, many PAEs have been blacklisted by the stable polymer polybenzimidazole (Sheehan, 2005). U.S. Environmental Protection Agency and the Chinese Envi- ronment Monitoring Center and banned, especially for their use in children’s toys (Net et al., 2015). Phthalic acid esters as synthetic plasticizers. Low-molecu- – lar-weight PA isomer esters nearly all of them PA esters Abiotic and biotic degradation of PA esters. PAEs can be – (PAE) (Fig. 1C) are essential constituents of synthetic plas- degraded in the environment by abiotic or biotic reactions such tics where they act as plasticizers. These are additives that as hydrolysis (Jonsson et al., 2006), UV light photolysis (Lau are freely dissolved within the polymeric plastic resins and et al., 2005), TiO2-enhanced photocatalysis (Sin et al., 2012) increase the plasticity or elasticity of a material (Giam et al., and microbial degradation (Chao et al., 2006; Li et al., 2006). 1984; Nilsson, 1994). PAEs were introduced in the 1920s to However, abiotic (non-biological) degradation of PAEs is very replace camphor-based plasticizers. In order to provide the slow, e.g., butyl-benzyl phthalate has an aqueous photolysis required elasticity to plastic products, they are not bound half-life of >100 days, for dimethyl phthalate it is about 3 years, covalently to the plastic material and can easily leach out and for ethyl hexylphthalate 2000 years (Gledhill et al., 1980; (Nilsson, 1994). Today, more than 60 kinds of PAEs are pro- Staples et al., 1997). Compared to non-biological destruction, duced for different commercial applications such as plastic microbial degradation is rather fast and a promising, cost- manufacturing, additives in paints, lubricants, adhesives, effective and environmentally friendly method for removal of insecticides, material packaging, and cosmetics (Vats et al., phthalates from contaminated sites. For instance, removal of 2013; Benjamin et al., 2015). Their estimated global produc- PAEs has been reported from various environments, including tion is growing rapidly with 1.8 million tons in 1975, reaching natural water, soils (Carrara et al., 2011), sediments (Chang 6.2 million tons in 2009 and more than 8 million tons in 2011 et al., 2005), wastewater (Camacho-Munoz et al., 2012), and (Peijenburg and Struijs, 2006; Meng et al., 2014; Net et al., landfills (Boonyaroj et al., 2012). 2015). The most frequently utilized PAEs for different appli- PAE-degrading microbes are either aerobic, facultatively cations are listed in Table 1. anaerobic or strictly anaerobic microorganisms. In most cases, Due to the high global utilization of plasticizers, they are the primary step is the initial hydrolysis of PAEs by esterases released during production and distribution of plastics in nearly that release the free phthalate moiety and side chain alcohols. every environment (Giam et al., 1984) such as air, freshwater, In addition, transesterification or the conversion of longer alkyl oceans, sediments, soil, wastewater, household dust and food residues to shorter ones by unknown enzymes have been products (Gao and Wen, 2016), independent of the degrada- reported occasionally (Liang et al., 2008). Esterases acting on tion of the mother polymer itself. Their continuous release from PAEs have been characterized from several bacteria: plastics leads to environmental contamination. PAEs are e.g., esterases purified from Micrococcus sp. strain YGJ1, and described as man-made priority pollutants due to their high from Bacillus sp. hydrolyze medium-chain (C3-C5) mono/di- exposure to humans and adverse effects on their health. Cer- alkyl phthalate esters (Niazi et al., 2001; Maruyama et al., tain PAEs have adverse effects on the development of the 2005). Most often the aliphatic or aromatic side chain alcohols male reproductive system due to their anti-androgenic proper- are easily utilized by bacteria, and phthalate accumulates due ties (Latini et al., 2006; Lambrot et al., 2009). They are also to a lack of essential enzymes for its degradation. At least considered as potential carcinogens, teratogens, mutagens, under anoxic conditions, degradation of phthalate is considered the rate-limiting step in degradation of PAEs (Gao and Wen, Table 1. Applications of selected phthalate esters that are commonly 2016). The complete mineralization of PAEs requires a combi- used in industry. nation of the metabolic capacities of diverse microbes that could accomplish the degradation of side chain alcohols as well as of the phthalate residues. For instance, degradation of Phthalate ester Application di-n-octyl phthalate is carried out by a metabolic cooperation Dimethyl phthalate Insect repellent/ectoparasiticide, e.g., between two bacteria: e.g., Gordonia sp. strain JDC-2 and against mosquitoes and flies Arthrobacter sp. strain JDC-32 (Wu et al., 2010). In this co-cul- Diethyl phthalate Often used in cosmetics, fragrances and other industrial uses including ture, Gordonia sp. converts di-n-octyl phthalate (DOP) into PA, plasticizers, aerosol sprays etc. which is degraded by Arthrobacter sp. strain JDC-32 that can- Butyl-benzyl phthalate Mostly used as a plasticizer for PVC. not degrade DOP alone. Other uses include traffic cones, food conveyor belts, artificial leather etc. Diisononyl phthalate Mostly used as a plasticizer Aerobic biodegradation of PA isomers Di(2-ethylhexyl) Used as plasticizer in medical devices, phthalate e.g., intravenous tubing, IV catheters, The aerobic biodegradation of PA, IPA and TPA has nasogastric tubes, dialysis bags and tubing, blood bags and transfusion been studied since almost 60 years (Ribbons and tubing, air tubes Evans, 1960; Keyser et al., 1976), and has meanwhile Diisodecyl phthalate Mostly used as a plasticizer been demonstrated in a large number of gram-positive Dibutyl phthalate Used as a plasticizer or as an additive in adhesives or printing inks and -negative bacteria, as well as in some fungi. For detailed lists of species that use the three PA isomers as

© 2019 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology Reports, 12, 3–15 6 M. Boll, R. Geiger, M. Junghare and B. Schink sole source of carbon and energy we refer to previous 4,5-dihydroxy-PA (mostly in gram-negative bacteria) or reviews (Vamsee-Krishna and Phale, 2008; Gao and 3,4-dihydro-cis-3,4-dihydroxy-PA (mostly in gram-positive bac- Wen, 2016). Here, we summarize the common enzymatic teria) (Fig. 2). In both products a hydroxyl group is positioned in ortho or para to one of the two carboxyl functionalities, which is strategies of aerobic PA, IPA and TPA degradation in essential for the down-stream decarboxylation reaction. order to compare them with the alternative ones employed TPA is initially attacked by a 1,2-dioxygenase yielding by anaerobic bacteria. 2-hydro-cis-1,2-dihydroxy-TPA (Schläfli et al., 1994; Choi Uptake of PA has been studied only in a few cases on et al., 2005; Fukuhara et al., 2008), which can be directly the biochemical level. In Burkholderia multivorans ATCC decarboxylated by a cis-1,2-dihydroxy dehydrogenase. Less 17616, there are two transport systems for PA uptake: the is known about the ring-hydroxylating oxygenase involved in OphD permease and an ABC transporter-type system IPA degradation. It has been shown that it specifically forms (Chang et al., 2009). Here, the specific porin OphP medi- the 4-hydro-cis-3,4-dihydroxy-IPA product (Vamsee-Krishna et al., 2006). ates PA transport across the outer membrane. An ABC transporter-type system has also been reported to be Aromatizing cis-diol-dehydrogenases. Decarboxylating, involved in PA uptake during PAE degradation in NAD+-dependent 2-hydro-cis-1,2-dihydroxy-TPA dehydroge- Rhodococcus jostii RHA1 (Hara et al., 2010). In contrast, a nases have been purified and structurally characterized from tripartite tricarboxylate transporter-type is involved in TPA different bacteria (Saller et al., 1995; Bains et al., 2012). Here, uptake in Comamonas sp. Strain E6 (Hosaka et al., 2013). dehydrogenation proceeds via elimination of a hydride from C2 In almost all aerobic microorganisms the three phthalate accompanied by decarboxylation at C1 (Fig. 2B). A number of decarboxylating 2-hydro-cis-dihydro-dihydroxy-PA dehydroge- isomers are converted to protocatechuate (3,4-dihydroxy- nases have been identified based on the corresponding benzoate), a central intermediate of aerobic degradation of phthalate-induced genes (Chang and Zylstra, 1998; Stingley aromatic compounds (Fig. 2). The conversion of PA to pro- et al., 2004; Choi et al., 2005; Carbone et al., 2008). tocatechuate in aerobic microorganisms involves three steps: (i) dioxygenation to cis-dihydroxy-dihydro-PA, (ii) dehydroge- Dihydroxyphthalate decarboxylases. So far, two 4,5- nationtodihydroxy-PAand(iii)decarboxylationtopro- dihydroxy-PA decarboxylases have been purified and initially tocatechuate. In contrast, formation of protocatechuate from characterized that both form the common protocatechuate intermediate. Here, decarboxylation of the carboxyl functionality in TPA and most possibly also IPA affords only two steps as para-position to a phenolic group is largely facilitated, and conse- decarboxylation is achieved directly in step (ii). Thus, the quently these decarboxylases do not require a special cofactor mandatory decarboxylation of the three phthalic acid isomers for catalysis (Fig. 2C), (Nakazawa and Hayashi, 1978; Pujar and is accomplished either by special decarboxylases (PA), or Ribbons, 1985). by cis-dihydrodiol dehydrogenases that abstract CO2 instead of a proton during hydride transfer (IPA, TPA). The Environmental importance of anaerobic biodegradation protocatechuate formed is finally cleaved by dioxygenases and converted to central intermediates either via the ortho- Being surrounded by air containing 21% of molecular oxygen, or meta-cleavage pathways (Dagley, 1971; Stanier and we consider aerobic processes to be most important for deg- Ornston, 1973). A variant of these canonical PA isomer degra- radation of organic compounds. For this reason microbial dation pathways has been described recently in Pseudomo- degradation of novel synthetic compounds has always been nas sp. strain PTH10 during growth with PA involving a studied under oxic conditions in the first place. However, oxy- phthalate-2,3-dioxygenase and a decarboxylating 2,3- gen can quickly become limiting wherever its supply depends dihydro-cis-2,3-dihydroxy-PA dehydrogenase (Kasai et al., on diffusive rather than convective transport. In the late 2019). The ring system of the cis-2,3-dihydroxybenzoate inter- 1970s, Julian Wimpenny and coworkers showed that even mediate formed is then directly cleaved by a 3,4-dioxygenase. colonies of aerobic bacteria on a Petri dish may be entirely The enzymes involved in the degradation of the three PA iso- anoxic inside, i.e., that a film of about 50 μm thickness (about mers are briefly described in the following. 50 layers of bacterial cells) is sufficient to consume the atmo- spheric oxygen entirely (Wimpenny and Coombs, 1983). In Two-component dioxygenases. The initial dihydroxylation of sediments of freshwater lakes or marine water bodies, oxy- PA isomers has been studied most intensively with PA as sub- gen contents decrease from air saturation to zero within few strate. Its dihydroxylation to cis-dihydrodiol-PA is catalysed by millimetres, depending on their trophic status and on their two-component Rieske non-heme iron oxygenase systems supply with easily degradable organic matter. In soil, oxygen comprising (i) a NAD(P)H-dependent dioxygenase reductase may reach down into the ground by few centimetres, strictly component that harbours FMN and a plant-type [2Fe-2S] ferre- depending on the water content. Water-logged soils, e.g., rice doxin, and (ii) a non-heme dioxygenase component binding a paddies, are largely oxygen-free, similar to lake sediments. Rieske-type [2Fe-2S] cluster and an active site Fe atom [some fi selected references: (Batie et al., 1987; Correll et al., 1992; Waste dump sites such as land lls contain a broad variety of Tarasev and Ballou, 2005; Karandikar et al., 2015)]. There are organic materials including various types of plastics. Their ini- two types of PA dioxygenases yielding either 4,5-dihydro-cis- tial aerobic biodegradation will consume the available oxygen

Fig. 2. Aerobic degradation of phthalic acids. A. Left three panels: degradation pathways of TPA, PA and IPA (from left to right) via the central protocatechuate intermediate; right panel: degradation of PA via the 2,3-hydroxybenzoate meta-cleavage pathway. Enzymes involved are (I) two- component Rieske non-heme iron dioxygenases, (II) decarboxylating or non-decarboxylating cis-(di)hydrodiol dehydrogenases and (III) dihydroxy-PA decarboxylases. B. Mechanism of decarboxylation by (II). C. Mechanism of decarboxylation by (III).

© 2019 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology Reports, 12, 3–15 8 M. Boll, R. Geiger, M. Junghare and B. Schink quickly, resulting in an entirely anoxic zone below a thin cover Thauera chlorobenzoica 3CB-1, Aromatoleum aromaticum layer. It is obvious from these few examples that microbial EbN1 and Azoarcus evansii KB740 [now reclassified as degradation of plastic wastes often has to face oxygen limita- Aromatoleum evansii KB740, (Rabus et al., 2019)], (Ebenau- Jehle et al., 2017). All denitrifying phthalate degraders grow with tion or even entirely anoxic conditions, be it in soils (protective phthalate and nitrate at doubling times in the range of 10 to 15 h foils in agriculture), sediments (accidental spills and losses) which opened the door for obtaining sufficient biomass to study fi or land lls. Thus, considerations on the (bio)degradability of the genes, enzymes and metabolites involved in anaerobic PA plastic constituents under entirely anoxic (reducing) condi- degradation. tions are of global relevance. Even if the polymeric backbone In vitro assays with extracts of these phthalate-degrading, structures may be hardly degraded under anoxic conditions, denitrifying pure cultures provided evidence for the succinyl- the slow but steady release of low-molecular-mass plasti- CoA-dependent conversion of phthalate to the central intermedi- cizers such as phthalate di-esters continues in any case. ate benzoyl-CoA indicating the following reaction sequence: (i) activation of phthalate to phthaloyl-CoA by a succinyl-CoA dependent CoA transferase and (ii) decarboxylation of the Anaerobic biodegradation of PA isomers formed phthaloyl-CoA to benzoyl-CoA by a decarboxylase (Junghare et al., 2016; Ebenau-Jehle et al., 2017). Differential The use of dioxygen as substrate for ring-hydroxylating and proteome analyses of Aromatoleum EbN1 (Ebenau-Jehle et al., ring-cleaving dioxygenases is not an option for anaerobic 2017) and Azoarcus sp. strain PA01 (Junghare et al., 2016) of phthalate degradation. In anaerobic bacteria most monocy- cells grown with phthalate versus benzoate revealed phthalate- clic aromatic compounds, includingthethreePAisomers, induced gene clusters comprising genes encoding transporters and enzymes involved in uptake and degradation of PA. In are converted to the central intermediate benzoyl-CoA A. aromaticum EbN1 the genes involved in anaerobic phthalate (Carmona et al., 2009; Fuchs et al., 2011; Rabus et al., degradation are located on the conjugative p2A plasmid 2016). Exceptions are only aromatics that contain at least (0.22 Mb), whereas in other denitrifying bacteria they are inte- two meta-positioned hydroxyl functionalities which substan- grated into the chromosome. The genes, enzymes and reac- tially weaken the aromatic character (Philipp and Schink, tions involved in phthalate degradation in selected anaerobic 2012). Benzoyl-CoA is dearomatized to cyclohexa-1,- bacteria are depicted in Fig. 3, and will be discussed in the 5-diene-1-carboxyl-CoA either by ATP-dependent class following. I benzoyl-CoA reductases (BCRs) present in facultative Uptake of PA All phthalate-induced gene clusters of denitrifying phthalate degraders contain genes putatively encoding anaerobes (Boll et al., 2014; Tiedt et al., 2018), or by ATP- a tripartite ATP-independent periplasmic (TRAP) transporter independent class II BCRs that occur in strict anaerobes (Junghare et al., 2016; Ebenau-Jehle et al., 2017), a kind of (Weinert et al., 2015; Huwiler et al., 2019). The further degra- transport system typically involved in the uptake of dicarboxylic dation to acetyl-CoA units and CO2 proceeds in the so- acids (Forward et al., 1997; Mulligan et al., 2011). These sec- called benzoyl-CoA degradation pathway by a series of ondary transporters involved in phthalate uptake comprise a modified β-oxidation like reactions including a hydrolytic ring periplasmic binding protein and a permease subunit with two cleavage step (Carmona et al., 2009; Fuchs et al., 2011; Boll fused domains. In A. aromaticum EbN1 and A. toluclasticus, genes for an additional three-component ABC transporter sys- et al., 2014). Thus, the basic question of anaerobic PA, IPA tem were identified including a periplasmic binding protein, a and TPA degradation is how they are converted to the cen- permease and an ATPase domain. It is unknown whether the tral intermediate benzoyl-CoA. TRAP- and ABC-type components both transport phthalate under different conditions or whether they transport different substrates. Anaerobic degradation of phthalate Succinyl-CoA:phthalate CoA transferase (SPT) and insta- Degradation of phthalate in denitrifying bacteria. bility of phthaloyl-CoA. In denitrifying bacteria PA degrada- Biodegradability of PA in denitrifying bacteria was reported tion is initiated by a class III CoA transferase that specifically already in the early eighties (Aftring et al., 1981). It was soon uses succinyl-CoA as CoA donor, and PA as CoA acceptor postulated that in anaerobic bacteria PA is decarboxylated to (Junghare et al., 2016; Ebenau-Jehle et al., 2017). The benzoate followed by thioesterification, e.g. by an ATP- heterodimeric enzyme from A. aromaticum EbN1 was heter- dependent benzoate CoA ligase (Eaton and Ribbons, 1982; ologously produced in Escherichia coli by expression of the Taylor and Ribbons, 1983). However, decarboxylation of any encoding sptAB genes. The isolated enzyme predominantly of the three phthalate isomers to benzoate would afford an catalysed the phthalate-dependent hydrolysis of succinyl- extremely unfavourable dianionic intermediate. As an alter- CoA, whereas phthaloyl-CoA was formed only at sub- native, initial activation to phthaloyl-CoA was suggested, micromolar concentrations (Mergelsberg et al., 2018). This followed by decarboxylation to benzoyl-CoA (Nozawa and finding is rationalized by the extreme instability of phthaloyl- Maruyama, 1988). Experimental evidence for such a sce- CoA (half-life <7 min) that is prone to intracellular substitu- nario was lacking for almost another 20 years. In two recent stud- tion yielding CoA and phthalic anhydride, the latter is then ies, complete PA degradation to CO2 coupled to denitrification immediately hydrolyzed to phthalate (Fig. 4). The extreme was reported for the newly isolated Azoarcus sp. strain PA01 instability is also reflected by the low cellular phthaloyl-CoA (Junghare et al., 2015; Junghare et al., 2016) and for a number of concentration which is more than three orders of magnitude known aromatic compound degrading denitrifyers such as below that of benzoyl-CoA (Mergelsberg et al., 2018).

Fig. 3. Genes, enzymes and metabolites involved in anaerobic PA degradation. Encoding structural genes are depicted in identical colour as gene products. The multimerization state of individual components is indicated based on puriﬁed or closely related proteins. Upper panel: phthalate-induced gene clusters in two nitrate- and one sulfate-reducing bacterium; lower panel: uptake of phthalate and its conversion to the central intermediate benzoyl-CoA. 1, periplasmic binding protein; 2, TRAP transporter; 3a, Heterodimeric SptAB subunits of succinyl-CoA:phthalate CoA transferase; 3b phthalate-CoA ligase of unknown multimerization state; 4, hexameric UbiD-like PCD; 4a, inactive PCD apo-form; 4b, active PCD with prFMN cofactor; 5, dodecameric UbiX-like subunit of prFMN forming prenyltransferase (for better presentation the FMN cofactor has only been shown for four subunits); 6, Nudix-like hydrolase putatively involved in synthesis of DMAP from DMAPP.

UbiD-like phthaloyl-CoA decarboxylase and UbiX-like cofactor is formed by a second component, UbiX, that uses prenyltransferase The second step in anaerobic PA degra- dimethylallyl-monophosphate (DMAP) as prenylating co- dation is catalysed by phthaloyl-CoA decarboxylase (PCD) substrate for the conversion of a FMN to prFMN (White et al., belonging to the UbiD enzyme family of (de)carboxylases with 2015) (Fig. 3). The gene annotated as a Nudix hydrolase 3-octaprenyl-4-hydroxybenzoate carboxy-lyase involved in might be involved in hydrolysis of DMAPP to DMAP + Pi.The ubiquinone biosynthesis serving as name-giving enzyme (Cox prFMN formed is then transferred to the apo-form of UbiD et al., 1969). UbiD family members contain a modified, involving an oxidative maturation step to its active imine form. prenylated flavin adenosine mononucleotide (prFMN) cofactor All PA-induced gene clusters in denitrifying bacteria contain at that is involved in covalent catalysis via a 1,3-dipolar cycload- least one gene encoding an UbiD-like PCD and an UbiX-like dition (Payne et al., 2015; Leys, 2018). The prenylated prenyltransferase. There are three copies of ubiD-like genes on the p2A plasmid of A. aromaticum EbN1 (Fig. 3): two of the deduced gene products code for almost identical PCDs (99% amino acid sequence identity), whereas a third one of unknown function shows only low amino acid sequence identity (25%) (Ebenau-Jehle et al., 2017). PCD specifically catalyses the decarboxylation of phthaloyl-CoA to benzoyl-CoA and can be regarded as key Fig. 4. Spontaneous decay of phthaloyl-CoA to phthalate and CoA enzyme of anaerobic phthalate degradation; it has so far via intramolecular substitution. been isolated only from T. chlorobenzoica cells grown with

© 2019 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology Reports, 12, 3–15 10 M. Boll, R. Geiger, M. Junghare and B. Schink phthalate (Mergelsberg et al., 2017). Due to the instability of Degradation of phthalate in sulfate-reducing bacteria. Until the phthaloyl-CoA substrate, PCD is routinely measured in a recently, knowledge of anaerobic degradation of PA in obli- coupled assay together with SPT and the formation of gately anaerobic bacteria was restricted to a few studies with benzoyl-CoA from phthalate and succinyl-CoA is followed. sediment slurries and enrichment cultures (Kleerebezem et al., Hexameric PCD contains prFMN, K+ and Fe2+ as cofactors. 1999a; Kleerebezem et al., 1999b; Liu and Chi, 2003; Liu In the presence of oxygen, the Fe2+ is oxidized to Fe3+ et al., 2005a; Liu et al., 2005b). Further, a Pelotomaculum iso- resulting in a loss of the prFMN cofactor and activity. It is pthalicum species has been described that grows with all three proposed that K+ and Fe2+ (evidenced by Fe-determination phthalate isomers in syntrophic association with a methanogen and EPR spectroscopy) are both involved in prFMN binding (Qiu et al., 2006). In a recent study, the previously overseen (Mergelsberg et al., 2017), with the Fe2+ found in PCD capacity of the sulfate-reducing, benzoate-degrading Des- substituting for the Mn2+ found in UbiD-like enzymes from ulfosarcina cetonica to use phthalate as carbon and together aerobic organisms (Payne et al., 2015). It is unknown with sulfate as energy source was reported (Geiger et al., 2019). whether all UbiD-like enzymes from anaerobes contain Fe2+ A differential proteome analysis identified a gene cluster that was instead of Mn2+, and whether the metal is involved in a redox induced in D. cetonica cells grown with phthalate versus benzo- process during maturation of the prFMN cofactor. ate. In this cluster a number of genes putatively encoding a PCD is the only member of the UbiD-like family of (de)car- TRAP transporter, a periplasmic binding protein, an UbiD-like boxylases that use a CoA-ester substrate. The CoA-ester func- PCD, and the corresponding UbiX-like prenyltransferase were tionality may contribute to the stabilization of a putative anionic found, indicating significant similarities to the gene products intermediate in the course of catalysis. However, it is unknown involved in phthalate degradation in denitrifying bacteria (Fig. 3). how the prFMN cofactor promotes phthaloyl-CoA decarboxyl- However, a gene encoding a CoA transferase was missing, ation. The 1,3-dipolar cycloaddition mechanism proposed for instead a gene encoding a putative ATP-dependent CoA ligase the decarboxylation of cinnamic acid and related derivatives was identified. In vitro enzyme activity assays confirmed that cannot be applied for PCD as the direct cleavage of a carboxyl indeed phthaloyl-CoA is formed by an ATP-dependent phthalate functionality from the aromatic ring would necessitate an CoA ligase. Similar as in denitrifying bacteria, phthaloyl-CoA unfavourable dearomatization step (Mergelsberg et al., 2017). accumulated only to submicromolar concentrations as a result of Capture of labile phthaloyl-CoA The formation of the its rapid decay by intramolecular hydrolysis. In the coupled extremely labile phthaloyl-CoA intermediate is a major chal- assays with enriched PCD, the ready formation of benzoyl-CoA lenge of anaerobic PA degradation. The relatively high appar- from ATP, CoA and PA was demonstrated (Geiger et al., 2019). ent Km of PCD for phthaloyl-CoA of around 120 μM, 200-fold The rationale for using a phthalate CoA ligase instead of a higher than the cellular phthaloyl-CoA concentration, together less energy consuming SPT in sulfate-reducing bacteria −1 −1 with the low specific activity (Vmax ≈ 35 nmol min mg )are could be that they oxidize fatty acids via the Wood-Ljungdahl not indicative for an efficient phthaloyl-CoA turnover by PCD pathway rather than via the Krebs cycle (Schauder et al., (Mergelsberg et al., 2017). For this reason, it had been 1986; Janssen and Schink, 1995). As a consequence, suggested initially that SPT and PCD may form a complex succinyl-CoA is not available to significant amounts to serve without releasing the phthaloyl-CoA intermediate. However, a as CoA donor for phthalate activation. Indeed, the cellular series of assays with SPT and PCD, separated by dialysis succinyl-CoA concentrations in D. cetonica grown with membranes, ruled out a necessary direct interaction of both phthalate are several orders of magnitude lower than in den- enzymes to efficiently form benzoyl-CoA, CO2 and CoA from itrifying bacteria (Geiger et al., 2019). phthalate and succinyl-CoA (Mergelsberg et al., 2018). Instead, A phthalate degradation gene cluster highly similar to that PCD was found as the most abundant enzyme in phthalate- of D. cetonica was identified in Desulfobacula toluolica,a grown cells of denitrifiers with an estimated cellular concentra- sulfate-reducing δ-proteobacterium known to degrade sev- tion of around 140 μM per active site-containing PCD monomer eral monocyclic aromatic compounds (Wöhlbrand et al., (10%–15% of soluble protein). Thus, the concentration of PCD 2013), leaving little doubt that this organism has the capacity is around 250-fold higher than that of its substrate phthaloyl- to growth with phthalate and sulfate. However, no complete CoA which guarantees an efficient capture and decarboxylation phthalate catabolic gene cluster was found in other known of the substrate (Mergelsberg et al., 2018). aromatic compound degrading strictly anaerobic bacteria To avoid phthaloyl-CoA accumulation and its subsequent (Geiger et al., 2019). A number of putative δ-proteobacterial hydrolysis balanced SPT and PCD activities are mandatory. PCD encoding genes (amino acid sequence identity ≥66%) In vitro titration experiments with purified SPT and PCD indi- are present in the metagenomes of a shallow sediment- cated that the optimal PCD:SPT ratio for the conversion of hosted perennially suboxic/oxic aquifer (Anantharaman phthalate + succinyl-CoA to benzoyl-CoA + CO2 + succinate et al., 2016), suggesting that phthalate degradation capacity was 4:1 (normalized to active site subunits). Remarkably, is present in many so far unknown δ-proteobacteria. the relative cellular abundance of both enzymes was almost in the same ratio (Mergelsberg et al., 2018). Thus, highly balanced SPT and PCD production levels are essential to mini- Anaerobic degradation of isophthalate mize phthaloyl-CoA hydrolysis. Even under these optimized conditions found in T. chlorobenzoica cells, still around 20% Anaerobic degradation of isophthalate has been studied of the succinyl-CoA added to the coupled SPT/PCD assays only with very few bacterial cultures. Syntrophorhabdus was hydrolyzed rather than converted to benzoyl-CoA, indi- aromaticivorans degrades isophthalate and other aro- cating that phthalate degradation via phthaloyl-CoA repre- matic compounds in syntrophic association with a H2- sents a poorly optimized xenobiotic degradation pathway. consuming partner organism (Qiu et al., 2004; Qiu et al.,

© 2019 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology Reports, 12, 3–15 Anaerobic degradation of phthalates 11 2008). Further, a defined co-culture of strictly anaerobic, Concluding remarks syntrophically isophthalate fermenting bacteria belonging The predominantly xenobiotic esters of the three PA iso- to the genus Pelotomaculum (family Peptococcaceae), mers play a globally important role as plasticizers and i.e. P. isophthalicum has been described (Qiu et al., 2006). polymer constituents. Though massively produced only A recent study of anaerobic isophthalate degradation in since around 60 years, a remarkable variety of degrada- S. aromaticivorans provided experimental evidence of the tion strategies have evolved in a relatively short time span. genes, key enzymes and intermediates involved in anaero- Degradation pathways are usually initiated by hydrolysis to bic degradation of IPA (Junghare et al., 2019). Similar to PA the individual PA isomers plus the corresponding alcohols, degradation by sulfate-reducing bacteria (Junghare et al., followed by decarboxylation of one of the two carboxylic 2016; Ebenau-Jehle et al., 2017), S. aromaticivorans acti- acid functionalities. The decarboxylation of non-activated vates isophthalate by an ATP-dependent isophthalate:CoA phthalic acids via negatively charged intermediates is ligase enzyme to isophthaloyl-CoA which is subsequently mechanistically difficult. For this reason, two aerobic and decarboxylated to benzoyl-CoA (Junghare et al., 2019). one anaerobic strategy have evolved to accomplish decar- Decarboxylation of isophthaloyl-CoA to benzoyl-CoA is boxylation: (i) during aerobic degradation of IPA and TPA, a catalysed by the UbiD enzyme family member isophthaloyl- dioxygenase introduces hydroxyl groups directly at a car- CoA decarboxylase that requires an UbiX-like flavin boxyl functionality yielding cyclic cis-diol-dienes that can prenyltransferase to generate the active site prFMN cofactor directly be decarboxylated during the rearomatizing dehy- (Junghare et al., 2019). Thus, anaerobic isophthalate degra- drogenation reaction; (ii) aerobic PA degradation proceeds dation appears to employ a highly similar enzyme inventory via dihydroxyphthalates with phenolic functionalities in as anaerobic phthalate degradation (Junghare et al., 2016; ortho-orpara-position to a carboxylic group which greatly Ebenau-Jehle et al., 2017). A major difference is that facilitates their decarboxylation; (iii) anaerobic degradation isophthaloyl-CoA is not prone to intramolecular hydrolysis of most likely all three phthalic acid isomers proceeds via such as phthaloyl-CoA, and has a half-life time of several CoA thioesterification followed by decarboxylation to hours (Junghare et al., 2019). benzoyl-CoA by UbiD-like decarboxylases. The mechanism of how this activation promotes the subsequent decarboxylation is unknown, and there is a high research demand to elucidate the structure and function of UbiD-like decar- Anaerobic degradation of terephthalate boxylases involved in the degradation of all three PA iso- In comparison to the anaerobic degradation of PA and mers. The mechanism of phthaloyl-CoA and terephthaloyl- IPA, much less is known about the genes and enzymes CoA decarboxylase should be similar but markedly differ involved in anaerobic TPA degradation. It has been from that of isophthaloyl-CoA decarboxylase due to the dif- reported to occur in syntrophic methanogenic consortia fering reactivities of the carboxyl functionalities. in bioreactors (Qiu et al., 2004; Lykidis et al., 2011), and The phthaloyl-CoA intermediate during anaerobic PA Pelotomaculum species have been described that were degradation is possibly the most unstable CoA thioester capable of degrading TPA in syntrophic co-culture with a in biology, which demands additional adaptations to cir- methanogen (Qiu et al., 2006). Further, ubiD-like genes cumvent its immediate futile hydrolysis. The solution to have been identified as potentially encoding a decarbox- overproduce PCD up to 140 μM is accompanied by a ylase involved in TPA degradation, either directly or after high energetic burden for its synthesis. Taken into activation to terephthaloyl-CoA (Lykidis et al., 2011, account that even under optimized conditions a signifi- Nobu et al., 2015). However, any experimental evidence cant phthaloyl-CoA hydrolysis of up to 20% occurred, for the involvement of the proposed gene products in anaerobic phthalate degradation via phthaloyl-CoA is a anaerobic TPA is missing. It is generally difficult,ifnot non-optimized, rather wasteful strategy. PA represents impossible to make any prediction to deduce a specific an intermediate during the aerobic degradation of natu- PA, IPA or TPA degradation potential purely based on rally occurring polycyclic aromatic compounds. Thus, its the presence of ubiD-like genes. As the reactivity of the complete aerobic degradation pathways cannot be ortho-orpara-positioned carboxyl-functionalities in PA and assigned to recent evolutionary inventions. However, PA TPA should be equivalent, there remains little doubt that is not known as an intermediate of an anaerobic degra- anaerobic TPA degradation proceeds via activation to dation pathway. In conclusion, the current anaerobic terephthaloyl-CoA followed by decarboxylation. As the solution may indeed represent an early stage of the predicted terephthaloyl-CoA intermediate is much more sta- ongoing evolution of a degradation pathway for an essen- ble than phthaloyl-CoA, the necessity of highly balanced tially xenobiotic compound. Future evolution of the anaer- terephthaloyl-CoA formation/decarboxylation activities obic pathway may result in complex formation between appears to be less mandatory. SPT and PCD that would allow a direct transfer of

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