Transcriptome and proteome profiling reveals complex adaptations of Candida parapsilosis cells assimilating hydroxyaromatic carbon sources

Many fungal species utilize hydroxyderivatives of benzene and benzoic acid as carbon sources. The yeast Candida parapsilosis metabolizes these compounds via the 3-oxoadipate and gentisate pathways, whose components are encoded by two metabolic gene clusters. In this study, we determine the chromosome level assembly of the C. parapsilosis strain CLIB214 and use it for transcriptomic and proteomic investigation of cells cultivated on hydroxyaromatic substrates. We demonstrate that the genes coding for enzymes and plasma membrane transporters involved in the 3-oxoadipate and gentisate pathways are highly upregulated and their expression is controlled in a substrate-specific manner. However, regulatory proteins involved in this process are not known. Using the knockout mutants, we show that putative transcriptional factors encoded by the genes OTF1 and GTF1 located within these gene clusters function as transcriptional activators of the 3-oxoadipate and gentisate pathway, respectively. We also show that the activation of both pathways is accompanied by upregulation of genes for the enzymes involved in β-oxidation of fatty acids, glyoxylate cycle, amino acid metabolism, and peroxisome biogenesis. Transcriptome and proteome profiles of the cells grown on 4-hydroxybenzoate and 3-hydroxybenzoate, which are metabolized via the 3-oxoadipate and gentisate pathway, respectively, reflect their different connection to central metabolism. Yet we find that the expression profiles differ also in the cells assimilating 4-hydroxybenzoate and hydroquinone, which are both metabolized in the same pathway. This finding is consistent with the phenotype of the Otf1p-lacking mutant, which exhibits impaired growth on hydroxybenzoates, but still utilizes hydroxybenzenes, thus indicating that additional, yet unidentified transcription factor could be involved in the 3-oxoadipate pathway regulation. Moreover, we propose that bicarbonate ions resulting from decarboxylation of hydroxybenzoates also contribute to differences in the cell responses to hydroxybenzoates and hydroxybenzenes. Finally, our phylogenetic analysis highlights evolutionary paths leading to metabolic adaptations of yeast cells assimilating hydroxyaromatic substrates.


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yeast Candida parapsilosis metabolizes these compounds via the 3-oxoadipate and gentisate 34 pathways, whose components are encoded by two metabolic gene clusters. In this study, we determine Benzene and its derivatives are simple aromatic compounds representing key substances for the 58 chemical industry. While benzene itself is toxic and carcinogenic, benzoic acid is commonly used in the 59 food industry and some of its derivatives are used in pharmacology (aspirin) or cosmetics (parabens).

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The benzene ring of aromatic molecules is relatively stable, but many microorganisms including yeasts     (Fig 1). Alignments with the reference genome sequence of the strain 133 CDC317 (21) cover 99.5% of the assembly and have a 99.9% identity. Compared to the CDC317 134 assembly, there is a single large-scale translocation between chromosomes 4 and 5 (CDC317 contigs 135 HE605208.1 and HE605204.1). Annotation of the nuclear chromosomes contains 5,856 predicted 136 protein-coding genes; 5,797 of them overlap with protein coding genes mapped from the CDC317 137 strain. The six genes coding for the GP components (i.e. MNX2, HBT1, GDX1, FPH1, GFA1, GTF1) 138 are localized in a single MGC which is present in the subtelomeric region of chromosome 6. The 3-OAP 139 components are encoded by a cluster comprising four genes (i.e. FRD1, HDX1, OSC1, OTF1) located 140 on chromosome 5, as well as by several additional loci on chromosomes 1 and 2 (Fig 1).

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The Venn diagrams show numbers of upregulated (log 2 fold change ≥ 2; p ≤ 0.05; (A)) or downregulated 166 (log 2 fold change ≤ -2; p ≤ 0.05; (B)) genes in CLIB214 cells assimilating 3-hydroxybenzoate, 4-167 hydroxybenzoate or hydroquinone compared to galactose. The results are based on the lists of 168 differentially expressed genes (S1 Table). The diagrams were drawn with a web tool 169 (http://bioinformatics.psb.ugent.be/webtools/Venn/). In line with our previous reports (7,9,10), the RNA-Seq analysis showed that the genes encoding the 173 enzymes catalyzing reactions in each pathway as well as the plasma membrane carriers facilitating the 174 transport of hydroxybenzoates (Fig 3A) are co-regulated in a substrate-specific manner. Specifically, 175 the GP cluster genes are highly upregulated (i.e. between 267-(GFA1) and 3,061-fold (HBT1)) in the 176 cells assimilating 3-hydroxybenzoate, which is metabolized via the GP. These genes exhibit only minor 8 177 changes in media containing 4-hydroxybenzoate, except for GTF1 and HBT1 showing about 12.6-and 178 4.7-fold induction on this substrate, respectively (Fig 3B, S1 Table). The genes for the 3-OAP enzymes 179 and two plasma membrane transporters (HBT2 and its paralog HBT3) are highly upregulated on both 180 4-hydroxybenzoate (i.e. between 46.5-(OSC1) to 1,090-fold (HBT2)) and hydroquinone (i.e. between 181 8.1-(HBT3) and 208-fold (HDX1)). Expression of these genes changes only slightly on the GP 182 substrate, except MNX1 which exhibits about 19-fold increase (Fig 3B, S1 Table).   Table). For proteins that were not identified on all carbon sources the LFQ values imputed 202 from a normal distribution were used in the calculation (indicated in parentheses).  Table). However, we did not identify several proteins (i.e. Hbt3p, Hbt4p, Gtf1p, Otf1p). We presume 212 that this is caused by overall low abundance of these polypeptides in the cells or their depletion from 213 the prepared extracts due to insolubility or subcellular localization. assimilated hydroxyaromatic substrates (Fig 6A). The RNA-Seq analysis of the cells utilizing 3-332 hydroxybenzoate, 4-hydroxybenzoate or hydroquinone show that although there is a group of ninety 333 nine genes upregulated on any of the three carbon sources, many genes are selectively induced only 334 on a single substrate (Fig 2A). downregulated genes on these substrates is even greater (Fig 2)

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While its growth is impaired in media containing hydroxybenzoates (i.e. 4-hydroxybenzoate, 2,4-401 dihydroxybenzoate, 3,4-dihydroxybenzoate), we did not observe a growth defect in media containing 402 hydroxybenzenes (resorcinol, hydroquinone). On the other hand, the ∆gtf1/∆gtf1 mutant is unable to 403 grow in media containing 3-hydroxybenzoate or gentisate, which are degraded via the GP (Fig 8). The in the ∆gtf1/∆gtf1 mutant compared to the parental strain (Fig 3, S2 Table, S6 Fig). In addition, we 421 found that the transcript(s) derived from CANPARB_p44920-A and CANPARB_p44910-A ORFs coding 422 for a predicted amidohydrolase superfamily protein is almost absent in this mutant.

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A comparison of the ∆otf1/∆otf1 mutant and CPL2H1 cells revealed more subtle differences in 424 the expression of genes for the 3-OAP enzymes. We found that MNX1 and HBT2 are downregulated 425 by 6.37-and 1.97-fold, respectively (Fig 3, S2  the ∆otf1/∆otf1 mutant has impaired growth on hydroxybenzoates (Fig 7).  Fig, S8 Fig) which are co-induced in the cells grown in 448 media with 4-hydroxybenzoate or hydroquinone (Fig 3). which along with the GP cluster genes is also highly induced in media containing 3-hydroxybenzoate 461 (Fig 3).

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As our previous studies (7,9,10) indicated that the 3-OAP and GP genes are repressed in media 463 containing glucose, we also searched the promoter sequences for sequence motifs potentially the cells grown on 4-hydroxybenzoate compared to those assimilating hydroquinone (Fig 3) and the 490 growth phenotypes (Fig 8) and