The Mechanism of Degradation of Alizarin Red by a White-Rot Fungus (Lenzites Gibbosa)

Alizarin red (AR) is a typical anthraquinone dye, and the resulting wastewater is toxic and dicult to remove. A study showed that the white rot fungus Lenzites gibbosa (L. gibbosa) can degrade dye wastewater by decolorization and has evolved its own enzyme-producing traits. In this study, transcriptome sequencing was performed after alizarin red treatment for 0, 3, 7, 10, and 14 h. The key pathways and key enzymes involved in alizarin red degradation were found to be though the analysis of KEGG, GO and COG. The GST, MnP and Laccase enzyme activities of L. gibbosa treated with alizarin red for 0–14 h were detected. LC-MS and GC-MS analyses of alizarin red decomposition products after 7 h and 14 h were performed.

special properties of anthraquinone dyes, the wastewater produced is not only high in organic composition and not easily oxidized but also has high chromaticity, complex composition and toxicity, so it is di cult to oxidize and to degrade by chemical methods. It is easy to dissolve AR in water and di cult to remove it by conventional physical methods, which brings some di culty to wastewater treatment.
Anthraquinone dyes are di cult to remove as the main pollutants in wastewater, which affects the degradation of wastewater and the ecological environment [1][2][3]. The degradation methods of anthraquinone dye wastewater mainly include physical, chemical and biological methods. The chemical treatment of dyes easily causes secondary pollution, and physical bleaching has the disadvantages of high cost and low e ciency [4]; however, the biological treatment of dye wastewater is e cient and without secondary pollution [5]. According to previous research, white rot fungi have certain decolorization and degradation abilities in dye wastewater [6]. White rot fungi secrete a variety of intracellular and extracellular enzymes, such as extracellular manganese peroxidase (MnP), lignin peroxidase (LiP) and Laccase [7][8][9][10], and intracellular cytochrome P450 (CYP450) [11][12]. The extracellular oxidase produced in secondary metabolism can degrade all kinds of dyes in a broad spectrum, especially for many synthetic compounds such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyl compounds. However, the mechanism of dye degradation by white rot fungi is not well studied. Using transcriptome technology to study various biological phenomena at the molecular level is a popular research method [13].
On the basis of previous studies, HiSeq high-throughput sequencing technology was used to study the changes in the transcription level of Lenzites gibbosa treated at 0, 3, 7, 10, and 14 h in an anthraquinone dye alizarin red environment. To explore the key pathways, genes and their regulated enzymes of L. gibbosa involved in alizarin red dye degradation were studied. Enzyme activity related to alizarin red degradation was detected. AR degradation intermediate products were analysed by LC-MS and GC-MS to explore the degradation pathway of AR. For white rot bacteria, the mechanism of L. gibbosa degradation of anthraquinone dye AR provides molecular-level data support and a theoretical basis.

Fungal culture and the hyphal collection
Source of materials: Our limited collection of the Lenzites gibbosa (L. gibbosa) CB1 subentities for research is allowed. The strain was collected by Professor Yujie Chi in Changbai Mountain, Jilin Province, China, and then underwent tissue separation in the laboratory. Store in the Forest Pathology Laboratory of School of Forestry, Northeast Forestry University. Samsampling site is located in Changbai Mountain Nature Reserve, Northeast China ( 127 42 '55 "-128 16' 48" E,41 41 '49″-42 51' 18″N) with an average altitude of 802 m. Professor Yujie Chi sequenced the collected subentities and the isolated mycelium extract DNA for ITS, and the PCR product sequencing of the rDNA-ITS region of the strain CB1 obtained a 601bp nucleotide sequence. Submit this sequence to NCBI's GenBank (NCBI Accession No: JF279440).
Lenzites gibbosa(L. gibbosa)CB1( NCBI Accession No: JF279440) was obtained by self-separation subphysical processes from the Changbai Mountains. Samples were preserved in the Northeast Forestry University Laboratory. The preserved strains were inoculated on PDA plate medium (9 cm) and cultured at 25°C for 7 days and to obtain hyphae from the edge of the plate with a 5 mm 2 punch. Hyphae were inoculated into a triangle bottle (250 mL) containing 5 ml ltered and sterilized 15% glucose (V/V) and 70 mL LNAS medium. After static culture at 25°C for 10 days, alizarin red (AR, C 14 H 7 NaO 7 S·H 2 O) dye was added to each bottle, and the nal concentration of AR dye in the medium was 50 mg/L. Hyphae were extracted after 0 h, 3 h, 7 h, 10 h and 14 h of AR treatment. Among them, 0 h was set as the control group (no dye treatment) mark as CK, and the others were the experimental group, mark as QSH1, 2, 3 and 4. Every 5 bottles of hyphae of the same culture conditions were mixed as a repeat, with 3 replicates per group. After mixing the hyphae and then placed into the frozen storage tube, sealed, put into liquid nitrogen for freezing, and then put into the 80°C refrigerator and saved for future analysis.
2.2. RNA extraction, cDNA library sequencing of the transcriptome, and the assembly and splicing of sequences Frozen hyphal samples were sent to Beijing BMK Biotechnology Co., Ltd. for transcriptome sequencing by Illumina HiSeq X Ten high-throughput sequencing platform. Data were analysed after quality control of raw data obtained by sequencing (clean reads). Sequence alignment of clean reads with JGI Trametes gibbosa reference genomes using software. The genome was published in December 2016. (The ITS1 and ITS2 fragments of L. gibbosa strain CB-1 were 601 bp long (GenBank accession number: JF279440). A BLAST search of the 601 bp sequence indicated that the sequence showed 99% homology with 11 strains of Trametes gibbosa. Thus, Lenzites gibbosa===Trametes gibbosa.) The resulting mapped data and Cu inks were compared with mapped data reads. The transcripts were assembled according to the GFF documents of the reference genome, and the Cuffmerg transcripts were merged to obtain a complete set of transcript information and the expression value of each gene. Using BLAST [14] software, the genes obtained from the transcriptome were functionally annotated in the COG, eggNOG, GO, KEGG, NR, and SwissProt gene functional annotation databases.

Analysis of gene differential expression in AR
BMKCloud (www.biocloud.net) and edgeR software (https://bioconductor.org/packages/release/bioc/html/edgeR.html) were used, and FDR = 0.01 and FC = 2 were selected as the DEG screening criteria [15]. Analysis of differential expression was performed in the following groups: CK vs QSH1; CK vs QSH2;CK vs QSH3 CK vs QSH4 QSH1 vs QSH2; QSH1vs QSH3 QSH1 vs QSH4 QSH2 vs QSH3; QSH2vs QSH3 QSH3 vs QSH4. The differentially expressed gene (DEG) sets between the groups were obtained. Then, the total number of DEGs was obtained, and GO, COG and KEGG analyses of DEGs were performed.

Detection of AR decolorization rate
The maximum absorption peak and its wavelength were determined by scanning 50 mg/L AR between 300-800 nm with a spectrophotometer. The absorbance value of AR was measured at wavelength at the maximum absorption peak with the average of 3 sets of data, according to the absorption value of the test group minus the absorption value of the control group, as Ai and the absorptivity of the 50 mg/L AR minus the absorptivity of the control group as A 0 . The decolorization rate was calculated by the following formula.

Detection of extracellular enzyme MnP and Laccase activity
The extraction of 3 mL of culture medium was performed in 5 stages (three replicates per group), followed by 13000 r/min centrifugation for 10 minutes, and extraction of the supernatant. Extracellular enzyme Laccase and MnP activity related to AR degradation were detected. Laccase and MnP activity detection methods were refer to Y. P. Shi [16] and Y. L.Zhang [17].
2.6. Detection of intracellular GSH content and intracellular GST activity L. gibbosa hyphal treatment was extracted by AR at 5 stages and rinsed and dried in phosphate-buffered saline (PBS). Hyphae were ground into powder with liquid nitrogen, and then an equal proportion of PBS homogenate was used. Then, the samples were centrifuged to extract the supernatant and set aside. Glutathione S-transferase (GST), Glutathione (GSH) and the Total protein (TP) assay kit (Cargo Number: A004, A006-1-1 and A045-2, Nanjing Jiancheng Bioengineering Institute, China) methods and formulas were used to detect the GST and GSH activity of L. gibbosa mycelium at each stage.

LC-MS and GC-MS were used to detect decomposition products AR at different time points
Degradation of dye molecules during the Lenzites gibbosa decolourization process passes through some intermediate products that were analysed by LC-MS and GC-MS and identi ed by interpretation of their mass spectral data presenting their molecular ion peaks with respect to m/z (where m is the molecular weight of the intermediates in the mass spectra). Thus, the molecular cleavage pathway of the AR dye is deduced. The L. gibbosa culture medium (100 mL) was extracted after AR treatment for 7 and 14 h, and the culture medium was pre-treated.
The sample pre-treatment of culture medium was as follows: Dichloromethane were taken and activated in the C18 column stationary phase. Methanol was taken through the column to remove impurities.
Finally, the column was washed with deionized water. The culture medium over the C18 column and mixture solution were prepared using 2 mL methanol and 8 mL dichloromethane, and the intermediate product was eluted from the C18 column and coexisted in the test tube.
Descriptions of various test parameters and data processing procedure are found below. (2) GC-MS test parameters: Sampler: 7683B, The mass spectrometry used an EI ion source, electron energy of 70 eV, scanning range of 50 ~ 500, four-stage rod temperature of 150°C, ion source temperature of 230°C, and transmission line temperature of 280°C. For related parameters see Table 1.

Results of transcriptome data and quality control evaluation
A total of 109.29 Gb clean data were obtained from transcriptome analysis of 15 samples, the percentage of Q30 bases was 91.32% and above, and the quality of transcriptome data was up to standard. is the clean reads of each sample were compared with the designated reference genome, and the alignment e ciency was 87.22%-91.32%, and reference genome selection was correct and e cient (Table S1). A total of 12921 genes were obtained of which 10560 genes were annotated, and the annotation e ciency was 81.72%. There were 883 new genes, 162 of which were annotated. (Table S2). Three of the 15 samples (QSH1-0, QSH2-0, and QSH3-0) were signi cant outliers compared with the other 2 samples in the same group. Except for these 3 outlier samples, the other 12 samples showed good correlation ( Fig. 1a).
3.2. Gene Expression Pattern Analysis and Clustering of DEGs.
The transcriptome data were divided into 10 groups, among which 6 groups had more downregulated genes than upregulated genes (Table S3). Ten differential groups received a total of 1480 DEGs of which 1370 DEGs were annotated. A total of 4 expression trends were obtained by analysing 1480 DEG expression trends ( Fig. 1-b). Pro le 1 and Pro le 2 genes were downregulated, for a total of 761 genes.
Pro le 3 and Pro le 4 genes belonged to the upregulation type, for a total of 719 genes. There were more downregulated genes than upregulated genes. The expression of Pro le 4 genes increased at 0-3 h and decreased slightly at 3-14 h. Pro le 1 and Pro le 2 genes were inhibited after exposure to AR stimulation and stress. The expression of the Pro le 3 and Pro le 4 genes increased after AR induction.
According to the differential trend analysis of DEGs, it was found that for a total of 66

Differential grouping of DEGs in the GO database analysis
The DEGs of the different groups CK vs QSH1 and QSH1 vs QSH4 were GO enriched. The enrichment categories of log10(KS) ≥ 2 are intercepted (Table S4). KS: the signi cant statistics of the enrichment of the GO category, the smaller the KS value is the larger the log10(KS) is, indicating the more signi cant the enrichment. At 0-3 h, upregulated DEGs were signi cantly enriched to GO:0020037 (heme binding), GO:0000041 (transition metal ion transport), GO:0005741 (mitochondrial outer membrane), GO:0046274 (lignin catabolic process) and GO:0004601 (peroxidase activity) categories, and downregulated DEGs were signi cantly enriched to GO:0020037 (heme binding), GO:0004521 (endoribonuclease activity) and GO:0006536 (glutamate metabolic process) categories ( Fig. 2-a). At 3-14 h, upregulated DEGs were signi cantly enriched in the GO:0016620 (oxidoreductase activity, acting on the aldehyde or oxo group of donors, and NAD or NADP as the acceptor) and GO:0020037 (heme binding) categories, and downregulated DEGs were signi cantly enriched in the haem binding, lignin catabolic process and reactive oxygen species metabolic process categories ( Fig. 2-b). Four important categories were enriched in both phases: GO: 0020037 (heme binding), GO: 0046274 (lignin catabolical process), GO: 0016620 (oxidoreduction activity, acting on the aldehyde or oxo group of donors, and NAD or NADP as the acceptor) and GO: 0004601 (period activity). Haem is the cofactor of haemoglobin and myoglobin, cytochrome, peroxidase, and catalase, which controls the synthesis and expression of redox enzymes such as the MnP haem-binding category, which can be determined to be associated with redox reactions. Lignin is a polymer in which phenylpropanoid structural units are irregularly coupled by ether and carbon bonds [18]. However, most dyes are similar to lignin structures and are composed of heterocycles and aromatic rings, all of which are aromatic compounds. Therefore, the signi cant enrichment of the lignin catabolic process class can be determined to be related to AR degradation.
Based on two stages of GO enrichment analysis, at 0-3 h, L. gibbosa secondary metabolic activity and redox reactions become active, and basic life activities such as carbohydrate utilization and protein synthesis are inhibited. At approximately 14 h, the redox reaction activity decreased relative to that at 3 h and carbohydrate transport and metabolism as well as energy production and transformation functions at 14 h.
All 1480 DEGs were compared to the four important categories enriched above (  DEGs at 0-3 h were signi cantly enriched in alanine, aspartate and glutamate metabolism, regulation of mitophagy -yeast, arginine biosynthesis, starch and sucrose metabolism, glyoxylate and dicarboxylate metabolism, galactose metabolism and the nitrogen metabolism pathways (Fig. 2-c).
Upregulated DEGs at 3-14 h were signi cantly enriched in the pentose and glucuronate interconversion pathways. Downregulated DEGs at 3-14 h were enriched in biosynthesis of antibiotics, synthesis and degradation of ketone bodies, terpenoid backbone biosynthesis, tyrosine metabolism and valine, leucine and isoleucine degradation (Fig. 2-d).
According to DEG enrichment of KEGG pathways at 0-3 h, genes involved in glutathione metabolism expression were upregulated by AR at this stage. Downregulation of the gene enrichment pathway found that the addition of AR affected the metabolism of substances needed for basic life activities such as glucose metabolism and energy metabolism in L. gibbosa. At the 3-14 h stage, glucose metabolism genes began to be upregulated, and the metabolism of amino acids and lipids was downregulated. Terpenoid synthesis gene transcription began to be downregulated.
Glutathione metabolism and redox reactions are the main processes involved in AR degradation by COG and GO database analysis.

Redox reactions and glutathione metabolism are involved in AR degradation
Some important reactive redox reactions and glutathione metabolism pathway related to AR degradation were identi ed by transcriptome analysis (Fig. 3). Exogenous chemicals are generally eliminated in two ways: one is discharged directly from the body without metabolism, and the other is excreted in the form of metabolites after metabolism. The metabolism of exogenous chemicals in vivo mainly undergoes a two-step reaction. The rst step is the phase I reaction in which exogenous chemicals are oxidized, reduced or hydrolysed to more polar metabolites. The key enzyme to catalyse the phase I reaction is the CYP450 enzyme system. The second step is the phase II reaction in which exogenous chemicals and their metabolites are combined with endogenous substances and discharged in vitro. There are many enzymes that catalyse the phase II reaction. The important enzymes are glucuronic acid transferase, GST and N-phthalyl transferase. When AR enters L. gibbosa, it is rst oxidized by CYP450 and other phase I enzymes. Then, bound to reduced glutathione (GSH) under GST catalysis it is excreted (this process is an important detoxi cation process in organisms). AR metabolites were treated in vitro with CYP450 and GST and then oxidized and decomposed by the extracellular enzyme Laccase and MnP and other oxidases.

GST and GSH activity determination results
To verify whether the GST and GSH responds to AR, the GST activity and GSH content of hyphae in the 5 treatment stages were detected. The GSH content decreased slightly from 0 h 51.71 gGSH/L to 14 h 44.04 gGSH/L ( Fig. 4-a). The GST activity of 0-3 h decreased, 3-7 h increased sharply, 7-10 h decreased, and 10-14 h increased violently. GST activity showed an overall upward trend, from 0 h 117.55U/mg prot to 14 h 217.03U/mg prot, and enzyme activity increased 1.8 times (Fig. 4-b). GST activity and GSH content showed that the GSH content slightly decreased GST, and a large amount of secretion promoted the binding of GSH and AR. Thus, glutathione metabolism responds to AR and participates in intracellular AR degradation.

Detection of redox enzyme activity participates in AR degradation
Extracellular enzyme Laccase and MnP activity were determined at the 5 AR treatment stages. The results showed that Laccase and MnP enzyme activities decreased slightly at 0-3 h, increased sharply at 3-10 h and then the upward trend at 10-14 h slowed down (Fig. 4-c). Laccase activity increased 3.7 times from 0 h 7.22 U/L to 14 h 27.28 U/L. MnP activity from 0 h 6.45 U/L to 14 h 18.55 U/L, and enzyme activity increased 2.9 times. The results showed that with the increasing L. gibbosa time in the AR dye environment, the activities of Laccase and MnP increased. These two enzymes respond to AR and act extracellularly.

Analysis of AR degradation products by LC-MS and GC-MS techniques
To explore the possible degradation pathways of AR, LC-MS was carried out by sampling the intermediates as well as the nal and stable degraded products. The mass-to-load ratio (m/z) of particle fragments can be obtained by LC-MS. The results of LC-MS analysis showed that the types of intermediate products obtained at 7 h and 14 h were the same, but the contents of each substance at 14 h were obviously less than those at 7 h. Seven substances (Table 3 and Fig. 4-e, f, g and h) were obtained with mass-to-load ratios of 318.9, 117.1, 165.0, 179.1, 215.0, 225.0, and 304.9. On the basis of m/z, 318.9 is AR (alizarin red removal Na), and 304.9, 179.1, 225 and 165 are benzophenone-5, 1,1-diphenylethylene, bisphenol and phthalic acid, respectively. These four substances may be formed by AR from the benzene ring opening in the middle. The m/z 215.0 is 1,2-naphthalene dicarboxylic acid, and the carbonyl addition reaction may occur from AR and form ring openings from sul te roots. The m/z 117.1 is a 1,4-butene diacid, which is a small molecular substance formed by cracking of the benzene ring. Figure 4-e,f,g and h shows that 1,4-butene diacid is the most abundant AR decomposition intermediate and is the most important intermediate product of L. gibbosa degradation of AR. The m/z 179.1 can also be inferred to be 9,10-dihydroanthracene, which is derived from AR de-sodium ions, hydroxyl groups and sul te ions. The anthraquinone structure of AR is destroyed, and the anthraquinone structure is the hair colour group of AR. The anthraquinone structure was destroyed, and the dye colour disappeared.
Using GC-MS methods, the degradation products can be more comprehensively analysed to supplement LC-MS results. It can be observed in the above total ion chromatography ( Fig. 4-i,j) that the abundance of material absorption ranges from 10500 at 7 h to 9000 at 14 h, indicating that the pollutants that exist in the solution are decreasing. The detected substances in the GC-MS will be referenced to the NISETO mass spectrometry database for material matching. Intermediate products related to AR decomposition were detected as some small molecular substances (Table 4) such as 2-butene, acrylaldehyde and 1butylene.
The pathway of AR degradation was inferred from the analysis of L. gibbosa intermediate products of AR degradation by LC-MS and GC-MS ( Fig. 4-k). The AR hydroxyl groups and sul te roots were removed rst, then the anthraquinone structure was broken, and nally, the benzene ring cracked to form small molecular inorganic salts. At this point, AR is completely degraded.

Discussion
For the present study, we aimed to examine the gene transcriptional changes of L. gibbosa and AR metabolites at different processing times. To determine the degradation of key AR genes and enzymes and the AR degradation process, a transcriptomic analysis of L. gibbosa with different AR processing times was performed for the rst time of genes that encode enzymes from GST, MnP, Laccase, lignin peroxidases, and VP. Of the oxidoreductase types, AR treatment resulted in higher expression levels in L. gibbosa. White rot fungi are by far the most e cient ligninolytic organisms described to date. This capability to degrade lignin is due to their extracellular nonspeci c and nonstereoselective enzyme system is composed of Laccase, lignin peroxidases and manganese peroxidases, which function together with H 2 O 2 -producing oxidases and secondary metabolites [19]. The same unique nonspeci c mechanisms that give these fungi the ability to degrade lignin also allow them to degrade a wide range of pollutants. They are able to degrade polycyclic aromatic hydrocarbons (PAHs), chlorinated phenols, polychlorinated biphenyls, dioxins, pesticides, explosives and dyes [20][21]. Puri ed Laccases, lignin (LiP) and MnP are able to decolourize dyes of different chemical structures. It has been shown that these enzymes can degrade different dyes [22][23]. In our study, the activity of MnP and Laccase increased signi cantly after AR treatment. MnP, Laccase, LiP and other genes were differentially expressed, and most of them were upregulated.
Since one of the basic functions of GST is detoxi cation, GST in metabolic aromatic compounds may be expressed in conjunction with degradation enzyme genes, so they may be used as reporter genes to monitor the expression of degradation pathway genes [24]. L Loyd-Jones et al. believe that genes encoding GST may be widely present in PAH-degrading bacteria and can be used as molecular tools for the detection of such bacteria. The authors obtained ampli ed bands from PAH-degrading bacteria isolated from sources in the United States, New Zealand and Antarctica. Successful ampli cation in contaminated soils also makes GST coding genes a useful tool for evaluating PAH-contaminated soils [25]. This study used transcriptome differential analysis to enrich the glutathione metabolic pathway (ko00480) in which GST genes are the most differentially enriched genes in this pathway, and most of them are upregulated. GST activity is also rising, which is consistent with previous studies.

Conclusion
A transcriptome analysis of AR L. gibbosa showed the following: (1) The differentially expressed genes encoding oxidoreductase in the AR environment combined with the MnP and Laccase activity results indicate that oxidoreductase is the key enzyme for AR degradation. (2) The glutathione metabolic pathway (ko00480) was screened, and binding GST activity and GSH content indicated that the glutathione metabolic pathway is involved in AR degradation. The AR degradation process was inferred by LC-MS and GC-MS, and 1,4-Butene diacid was the most intermediate. This study explored the process of AR biodegradation at the molecular and biochemical levels and provided a theoretical basis for its application in practical production.
Abbreviations L. gibbosa: Lenzites gibbosa; AR:alizarin red; MnP:manganese peroxidase; LiP:lignin peroxidase; Cytochrome P450:CYP450; GSH:glutathione; GST:Glutathione S-transferase; TP:Total protein; VP:Versatile peroxidase Declarations Ethics approval and consent to participate This article does not contain any studies with human participants or animals that were performed by any of the authors.

Consent for publication
Not applicable.

Availability of data and material
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. This study data is being uploaded to the NCBI. NO. : SUB9461105.

Supplementary Files
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