New branches in the degradation pathway of monochlorocatechols by Aspergillus nidulans: a metabolomics analysis

A collective view of the degradation of monochlorocatechols in fungi is yet to be attained, though these compounds are recognised as key degradation intermediates of numerous chlorinated aromatic hydrocarbons, including monochlorophenols. In the present contribution we have analysed the degradation pathways of monochlorophenols in Aspergillus nidulans using essentially metabolomics. Degradation intermediates herein identified included those commonly reported (e.g. 3-chloro-cis,cis-muconate) but also compounds never reported before in fungi revealing for 4-chlorocatechol and for 3chlorocatechol unknown degradation paths yielding 3-chlorodienelactone and catechol, respectively. A different 3-chlorocatechol degradation path led to accumulation of 2chloromuconates (a potential dead-end), notwithstanding preliminary evidence of chloromuconolactones and protoanemonin simultaneous formation. In addition, some transformation intermediates, of which sulfate conjugates of monochlorophenols/chlorocatechols were the most common, were also identified. This study provides critical information for understanding the role of fungi in the degradation of chlorinated aromatic hydrocarbons; furthering their utility in the development of innovative bioremediation strategies. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 New branches in the degradation pathway of monochlorocatechols by Aspergillus nidulans: a metabolomics analysis Tiago M. Martins, a Oscar Núñez, b Hector Gallart-Ayala, b Maria Cristina Leitão, a Maria Teresa Galceran, b and Cristina Silva Pereira, a* a Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal b Department of Analytical Chemistry, University of Barcelona, Diagonal 645, E-08028

Introduction Chlorinated aromatic hydrocarbons are widely distributed in the environment and many are highly toxic and persistent [1,2]. Globally, their environmental dissemination is a consequence of their extensive use in man´s activities (e.g. pesticides, solvents and plastics [1,2]), notwithstanding that some can be also produced in natural processes [3,4]. Their toxicity may increase as a result of degradation, either abiotic or biotic [5][6][7]. Photolysis has been shown to increase the toxicity of monochlorophenols (mCPs) due to transformation into the corresponding chlorobiphenyls [6]. Biodegradation of substituted derivatives of biphenyls has been shown to produce chlorinated phenyl lactones as dead-end metabolites [8,9]. Even bacterial degradation of the least chlorinated and less toxic phenols may end in the accumulation of several dead-end metabolites, including the antibiotic protoanemonin or its chlorinated derivative [7,10].

Decay of monochlorophenols during A. nidulans growth
2CP, 3CP and 4CP MICs on A. nidulans germination and growth were respectively >1.00, 1.00 and 0.85 mM. Accordingly at an initial concentration of 0.80 mM, all the mCPs were completely transformed by the fungus within the fourteen days of cultivation (Fig. 1a). Their degradation followed a sigmoid decay response for 2CP and 3CP and an exponential decay response for 4CP (Table S1). Preliminary data indicated that mycelial bio-accumulation was negligible (data not shown). mCPs abiotic degradation in the controls was below 10% at the end of fourteen days. 8

Quantification of monochlorophenols degradation intermediates
The mCPs degradation intermediates produced by A. nidulans along the incubation time (UPLC analyses) are depicted in Table 1 (Fig. S1). Data revealed the transient accumulation of monochlorocatechols ( Fig. 2c and d), as previously reported in other fungi [17,[21][22][23]. 2CP and 4CP degradation pathways yielded essentially 3CC and 4CC ( Fig. 1c and d) respectively and that of 3CP yielded the two former mCCs ( Fig. 1c and d). 3CC and 4CC were completely transformed at the same time point as their corresponding parent compounds (Fig. 1a, c and d). The formed 4CC was further metabolised to 3-chloro-cis,cis-muconate (Fig. 1b), which accumulated at a similar rate to a compound herein named as X211 ( max 211 nm) (Fig. 1h). As the concentration of both decreased, trans-acetylacrylate accumulated (Fig. 1f). In the 4CP cultures, cisdienelactone was also detected in small quantities (not quantified).
Near the end of the incubation period the concentration of all the identified degradation intermediates of 3CP and 4CP decreased, except that of transacetylacrylate. Its MIC was at least 500 fold higher than the maximum concentration detected (10 mM < MIC  15 mM). trans-Acetylacrylate was fully co-metabolised in glucose MM within two weeks (5 µM to 2 mM) and used as a sole carbon and energy source (5 and 10 mM). Albeit in these cultures no degradation intermediates of transacetylacrylate could be detected by UPLC.
3CC presumably yielded X280a and X280b ( max ca. 280 nm), which showed a late accumulation profile (Fig. 1g) fourteen days of incubation (Fig. 1e). This compound has never been identified as a degradation intermediate of monochlorophenols in fungi. In the abiotic controls only mCCs (<2 µM) were detected ( Table 1).

Discussion
The selection of A. nidulans, a reference strain, should reflect a pollutant unbiased environmental background. This opposes to the recurrent selection of environmental strains showing high degradation ability [17,21,22]. Analysis of 2CP and 3CP degradation profiles (Fig. 1a, Table S1) showed that after an initial lag phase (probably for induction of degradation enzymes) [41], accumulation of inhibitory degradation intermediates, if any, was inconsequential. On the contrary, during 4CP degradation, no lag phase could be observed, yet some inhibitory degradation intermediates likely accumulated, one of which was probably 3-chloro-cis,cis-muconate (Fig. 1b) . Levels were very low, probably due to reduced phenol 2-monooxygenase activity against catechols [44].
3-Chloromuconolactone was not detected in any of these cultures, probably because it was either rapidly hydrolysed abiotically at neutral pHs to 3-hydroxymuconolactone [39] or isomerised enzymatically to 3-chlorodienelactone through a pathway branch that might differ from that yielding cis-dienelactone (Fig. 2). 3-Chlorodienelactone concentration profile along time (Fig. 1h)  14 nidulans [49]. Likely the spontaneous decarboxylation of maleylacetate originated the cis-acetylacrylate [12] which in turn yielded trans-acetylacrylate (Fig. 1f) . Apart from the present study, only one used an alkaline growth media, pH 7.8 [17], yet the degradation products were not comprehensively characterised.

Pathway branch of 3-chlorocatechol yielding catechol
The downstream degradation intermediates of 3CC (Fig. 1c) included catechol and its respective ring fission product cis,cis-muconate (Table 1 and 2). Catechol is a preferred substrate for the catechol 1,2-dioxygenase activity (EC 1.13.11.1) detected in crude enzyme extracts of A. nidulans (Fig. S3) and previously reported e.g. in P. frequentans [17]. Most likely the degradation of cis,cis-muconate involved the catechol branch of the -ketoadipate pathway [46]. This has never been reported in fungi before, probably because catechol and cis,cis-muconate concentrations in media were very low. Some

Alternative paths to the degradation of monochlorophenols: conjugation reactions
Apart from the degradation intermediates of mCPs herein detected (at the heart of this study), some transformation intermediates (compounds involving oxidation and conjugation reactions) were also observed. Conjugation reactions are well established as common intracellular mechanisms of detoxification [32]. Not surprisingly, several conjugates of mCPs and mCCs were detected (

Conclusions
Chlorinated aromatic hydrocarbons degradation by fungi have been largely ignored but well studied in bacteria [1].
Challenged by this, we resolved here major monochlorocatechols degradation paths through a comprehensive analysis of the degradation intermediates being formed in A. nidulans cultures. These, which included those commonly reported, but also some never observed before, revealed for 4CC and 3CC new degradation paths yielding 3-chlorodienelactone and catechol, respectively.