Genomic, Transcriptomic and Suspect/Non-Target Screening Analyses Reveal the Role of CYP450s in the Degradation of Imazalil and Delineate Its Transformation Pathway by Cladosporium herbarum

Abstract

Imazalil (IMZ), a major surface water contaminant characterised by high environmental recalcitrance and toxicity, is used in fruit-packaging plants to control fungal infestations during storage. This leads to the production of wastewaters which should be treated on site before their environmental release. We previously isolated a Cladosporium herbarum strain, the first microorganism that could degrade IMZ. Here we describe the genetic network utilised by the fungus to degrade IMZ and its detailed transformation. Genomic and transcriptomic analysis of C. herbarum pointed to the involvement of strongly upregulated CYP450s in IMZ degradation, as further verified by cessation of its biodegradation by CYP450 inhibitors. LC-QTOF-HRMS analysis and suspect/non-target screening identified nine transformation products (TPs) of IMZ. IMZ biotransformation mainly proceeded through O-dealkylation, while other less important paths, most probably controlled by upregulated oxidases, were operative involving successive hydroxylation reactions. These lead to the formation of TPs like IMZ_313 and IMZ_331, with the former being further transformed through imidazole ring scission to IMZ_288, a TP reported for the first time. We provide first evidence for the transformation mechanism of IMZ by C. herbarum and the potential genes/enzymes involved, paving the way for the use of C. herbarum in the biodepuration of agro-industrial effluents.

Keywords: Cladosporium herbarum; CYP450 monoxygenases; Imazalil; O‐dealkylation; transformation products.

FIGURE 1

Heatmap of the log2‐fold change (LogFC) values (blue‐green‐red) of the read counts of the 5% most upregulated genes in each treatment. LogFC values are clustered according to complete linkage hierarchical clustering method. Gene names reflect their annotation ID and the gene product. The annotation column (left: dark purple to yellow) illustrates the average Log2 counts per million of each gene. The annotation row (top) corresponds to the experimental treatments (IMZ⁻: cyan, IMZ⁺: yellow).

FIGURE 2

Heatmap of the log2‐fold change (LogFC) values (blue‐green‐red) of the read counts of the genes annotated as CYP450 according to InterPro. LogFC values are clustered according to complete linkage hierarchical clustering method. Gene names reflect their annotation ID and the gene product. The annotation column (left: dark purple to green) illustrates the average Log2 counts per million of each gene. The annotation row (top) corresponds to the experimental treatments (IMZ⁻: cyan, IMZ⁺: yellow).

FIGURE 3

The degradation of ΙΜΖ by Cladosporium herbarum in the presence or absence of the CYP450 inhibitors piperonyl butoxide and ABS at three different concentrations. Each value is the mean of three replicates ± the standard deviation of the mean.

FIGURE 4

The degradation pattern of IMZ (Green line, Left panel) in cultures of C. herbarum and the formation patterns of its detected transformation products through RPLC‐ESI(+)‐QToF analysis.

FIGURE 5

Extracted Ion Chromatograms of the identified TPs of IMZ acquired by RPLC in positive ionisation mode. TPs are indicated by different colours with the main TPs, IMZ_M257 (red) and IMZ_331A (purple) exhibiting higher intensity.

FIGURE 6

The proposed transformation pathway of IMZ by Cladosporium herbarum , showing the proposed structures of the transformation products (TPs) detected and the type of reaction involved in each step of the pathway. The major TP, IMZ_257, was depicted in a green frame. The functional groups are indicated with different colours.