Light Regulation of Two New Manganese Peroxidase-Encoding Genes in Trametes polyzona KU-RNW027

To better understand the light regulation of ligninolytic systems in Trametes polyzona KU-RNW027, ligninolytic enzymes-encoding genes were identified and analyzed to determine their transcriptional regulatory elements. Elements of light regulation were investigated in submerged culture. Three ligninolytic enzyme-encoding genes, mnp1, mnp2, and lac1, were found. Cloning of the genes encoding MnP1 and MnP2 revealed distinct deduced amino acid sequences with 90% and 86% similarity to MnPs in Lenzites gibbosa, respectively. These were classified as new members of short-type hybrid MnPs in subfamily A.2 class II fungal secretion heme peroxidase. A light responsive element (LRE), composed of a 5′-CCRCCC-3′ motif in both mnp promoters, is reported. Light enhanced MnP activity 1.5 times but not laccase activity. The mnp gene expressions under light condition increased 6.5- and 3.8-fold, respectively. Regulation of laccase gene expression by light was inconsistent with the absence of LREs in their promoter. Blue light did not affect gene expressions but impacted their stability. Reductions of MnP and laccase production under blue light were observed. The details of the molecular mechanisms underlying enzyme production in this white-rot fungus provide useful knowledge for wood degradation relative to illumination condition. These novel observations demonstrate the potential of enhancing ligninolytic enzyme production by this fungus for applications with an eco-friendly approach to bioremediation.

The effect of light on the production of ligninolytic enzymes such as lignin peroxidase (LiP) and laccase has been investigated previously [14,15]. Green light enhanced lignin peroxidase production in Phanerochaete chrysosporium under submerged culture, whereas red, blue, and UV light reduced

Microorganism and Inoculum Preparation
The white rot fungus, T. polyzona KU-RNW027, was maintained on potato dextrose agar (PDA) and stored in 20% glycerol at -20 • C for long-term preservation. For inoculum preparation, this fungus was cultured in 50 mL Kirk's liquid medium for 3 days to prepare fungal pellet inoculum following Lueangjaroenkit et al. [21]. Fungal pellets (0.05 g/L) were used as inoculum in all experiments.

Plate Assay of Light-Driven Ligninolytic Enzyme Activity
A plug of mycelial tip with a 0.5 cm diameter was inoculated on PDA plates supplemented with 1 mM 2,6-dimethoxy phenol (2,6-DMP). The fungus was cultured under continuous lighting with a tungsten incandescent bulb. Light intensity was set to 500 lux. For the dark control, plates were fully covered with aluminum foil. PDA without 2,6-DMP was used as the control. All cultures were incubated at 30 • C for 3 days. Ligninolytic enzymes oxidized 2,6-DMP to an orange product, allowing the visualization of enzyme activity.

Effect of Light on Transcription Levels and Enzyme Production
T. polyzona KU-RNW027 was grown in 50 mL of Kirk's liquid medium and exposed to light sources for 7 days. Tungsten incandescent bulb and blue light emitting diode (LED) were used. Tungsten incandescent bulbs had a broad spectrum of 320-1100 nm. The wavelength of the blue LED was 465 nm and the light intensity was set to 500 lux. The cultures were incubated under shaking at 130 rpm, 30 • C. The control was incubated in the dark and flasks were fully covered with aluminum foil to prevent light penetration. Ligninolytic enzyme activities, gene expression at the transcription level, and fungal growth were determined. All experiments were performed in triplicate.

Enzyme Stability under Different Wavelengths of Light
Enzyme stability toward different light wavelengths was evaluated at 30 • C by pre-incubating the ligninolytic enzyme under continuous lighting with a tungsten incandescent bulb for visible light and blue LED light for 72 h. Light intensity was set to 500 lux. Residual activities were determined at 1, 3, 6, 24, 48, and 72 h. All experiments were performed in triplicate.

Enzyme Assay
MnP and laccase activity assays were performed according to the method of Lueangjaroenkit et al. [21] and Kondo et al. [23]. MnP and laccase activities were determined by monitoring the oxidation of 2,6-DMP at 469 nm, and its absorbency change followed within 3 min. For laccase assay, the reaction mixture consisted of 1 mM 2,6-DMP in 50 mM malonate buffer (pH 4.5). For the MnP assay, the reaction mixture consisted of 1 mM 2,6-DMP, 1 mM MnSO 4 , and 0.1 mM H 2 O 2 in malonate buffer (pH 4.5). The result for the latter procedure is expressed as total activity (laccase + MnP activity). Differences between activities of laccase and total activity were used to calculate MnP activity. Enzyme denaturation at 100 • C for 15 min was performed as a control reaction in parallel. One unit (U) of either MnP or laccase was defined as activity oxidizing 1 µmol of substrate per min.

Cloning of Ligninolytic Enzyme-Encoding Genes and Promoter Region
Genomic DNA was extracted from the mycelia cultivated in Kirk's liquid medium as previously described [21]. In brief, mycelia were ground in liquid nitrogen and lysis extraction buffer was then added. After incubation on ice for 30 min, 50 µL of 5 M potassium acetate (pH 4.8) was added and the mixture was further incubated on ice for 15 min before centrifuging. The supernatant was transferred into a new tube. DNA precipitation was conducted using isopropanol. Genomic DNA was washed with 70% ethanol. Finally, DNA pellets were resuspended in ultrapure water.
Degenerate primers for mnp were designed according to LC-MS/MS based protein fingerprints of MnPs [22], whereas degenerate primers for lac were designed based on the conserved nucleotide sequence of lac from related species in genus Trametes and are listed in Table S1. The fragments were amplified by PCR with LA Taq DNA polymerase (Takara Bio, Kusatsu, Japan). To obtain the 5 and 3 flanking of the ligninolytic enzyme-encoding genes mnp1, mnp2, and lac1, inverse PCR was performed with the primers listed in Table S1. Genomic DNA of T. polyzona KU-RNW027 was digested with restriction enzymes BglII, StyI, and BamHI, and self-ligated using DNA ligation kit version 2.1 (Takara Bio, Kusatsu, Japan). Inverse PCR was performed with LA Taq DNA polymerase (Takara Bio, Kusatsu, Japan) using self-ligated DNA as the template and inverse PCR primer pairs (Table S1). The inverse primers were designed based on known sequences of mnp1, mnp2, and lac1. All PCR products were purified and cloned into pUC19 vector. Escherichia coli DH5α was transformed with pUC19 recombinants and cultured on Luria-Bertani medium supplemented with 50 µg/mL ampicillin at 37 • C for 16-18 h Total RNA was extracted using TRIzol Reagent ® (Molecular Research Center Inc., Cincinnati, OH, USA). Total cDNA was synthesized from the total RNA using ReverTra Ace ® qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). Using the total cDNA as a template with degenerate primer pairs (MnPf and MnPr for mnp and Lacf and Lacr for lac), 0.8 kb mnp and 1 kb lac fragments were amplified by PCR with LA Taq DNA polymerase (Takara Bio, Kusatsu, Japan). Based on the nucleotide sequences of these fragments, specific primers for 5 and 3 rapid amplification of cDNA ends (5 /3 -RACE Kit, Roche Diagnostics, Mannheim, Germany) were designed to perform cloning of the full-length of cDNA, as shown in Table S1. All PCR products were purified and cloned into pT7blue vector (Novagen, Madison, WI, USA). Sequences were analyzed. The cDNA sequences of ligninolytic enzyme-encoding genes were aligned with those of other fungi by Blast analysis in GenBank database (http://www.ncbi.nlm.nih.gov/). A phylogenetic tree of the deduced amino acid sequences was constructed using the Mega6 program using the maximum likelihood method with 1000 bootstrap resampling replicates [24]. A putative signal peptide was analyzed using SignalP 4.0 [25].

Analysis of Gene Expression by Quantitative Real-Time PCR (qRT-PCR)
Total cDNA was synthesized as mentioned in Section 2.7. The template cDNA was diluted five times with 10 mM Tris-HCl (pH 8.0). RNA was quantified by measuring its absorbency at A260 using NanoDrop One (Thermo Fisher Inc., Waltham, MA, USA). RNA purity and quality were checked by A260/280 and A260/A230 ratios and gel electrophoresis, respectively. mnp1, mnp2, and lac1 expressions were quantified using StepOnePlus Real-Time PCR system (Thermo Fisher Scientific Inc., Waltham, MA, USA). qRT-PCR was performed using the primer sets listed in Table S2. Reactions were conducted using Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan) following the manufacturer's instructions. Briefly, qRT-PCR was performed with pre-denaturation at 95 • C for 60 sec followed by 40 cycles of denaturation at 95 • C for 15 s and annealing and extension at 60 • C for 30 s. In the final step, the melting curve was analyzed.
Standard curves of mnp1, mnp2, and lac1 amplification were generated using PCR products amplified from total cDNA of T. polyzona KU-RNW027. Transcription levels are expressed as induction fold. This is a copy number of target genes, mnp1, mnp2, or lac1, referred to as the copy number of the housekeeping gene. In this study, the rRNA gene was used as the housekeeping gene. All experiments were performed in triplicate.

Statistical Analysis
All experiments were conducted in triplicate. A pairwise multiple comparison procedure following Student-Newman-Keuls post-hoc tests was used to identify sample means that were significantly different from each other. SigmaPlot 11 (Systat Software Inc., San Jose, CA, USA) was used to calculate statistical probabilities. A p-value < 0.05 indicated statistically significant differences between means of maximum enzyme activities, transcription level, and fungal growth at a 95% confidence interval.

Ligninolytic Enzyme-Encoding Genes of T. polyzona KU-RNW027
Using MnPf and MnPr as the degenerate primers, 1014 and 1016 bp fragments of mnp1 and mnp2, respectively, were obtained by PCR. Inverse PCR was then conducted to clone the 5 and 3 flanking sequences of these fragments. For mnp1, an amplified product of 1661 bp was obtained from inverse PCR. After dividing the 1661 bp into two parts at the BglII restriction site and reintegrating with the 1014 bp fragment, a 2379 bp fragment was obtained that encompassed an intact 5 and 3 flanking sequence of mnp1. In parallel, RT-PCR and 5 /3 -RACE were performed to amplify the cDNA sequence of mnp1. Full-length cDNA sequences of the mnp1 gene consisted of 1331 nucleotides. Four short introns were found in mnp1 sized between 60 and 80 bp. All intron splice junction regions belonged to the GT-AG rule [26]. The mnp1 gene showed 86% nucleotide sequence identity with mnp1 from Lenzites gibbosa CB-1 (FJ848739.2). The mnp1 corresponded to a protein of 364 amino acids. Sequence analysis of the deduced amino acids of MnP1 by SignalP 4.0 [25] revealed the presence of a putative signal peptide of 21 amino acids, with a cleavage site between Ser-21 and Ala-22 residues. The mature protein of MnP1 contained 343 amino acids.
For mnp2, an amplified product of 1711 bp was obtained from inverse PCR. After dividing the 1711 bp into two parts at the StyI restriction site and reintegrating with the 1016 bp fragment, a 2312 bp fragment containing intact 5 and 3 flanking sequence of mnp2 was obtained. In parallel, RT-PCR and 5 /3 -RACE were performed to amplify the cDNA sequence of mnp2. Full-length cDNA sequences of the mnp2 gene consisted of 1306 nucleotides. The alignment of cDNA and intact sequence mnp2 revealed five introns. The nucleotide sequence of mnp2 showed 84% identity with mnp2 from L. gibbosa CB-1 (JQ388597.1). The mnp2 gene predicted 366 amino-acid-encoded protein, and a putative 21 amino acid signal peptide was identified with a putative cleavage site between Gln-21 and Ala-22 residues. The mature protein of MnP2 contained 345 amino acids.
A 1625 bp fragment of lac1 was obtained by PCR using Lacf and Lacr as degenerate primers. To clone the 5 and 3 flanking of Lac1 sequence, inverse PCR was performed, yielding a 1623 bp segment. After dividing the 1523 bp into two parts at the BamHI restriction site and reintegrating with the 1625 bp fragment, a 3016 bp fragment of lac1 was obtained. RT-PCR and 5 /3 -RACE were performed in parallel to amplify the cDNA sequence of lac1. Full-length cDNA sequences of the lac1 gene consisted of 1809 nucleotides. The alignment of cDNA and intact sequence lac1 revealed 10 introns. The nucleotide sequence of lac1 has 97% identity with lac from T. polyzona MUCL:38443 (KT802746.1). The lac1 corresponded to a protein of 520 amino acids. The deduced amino acid sequence of Lac1 showed 99% identity with laccase from T. polyzona MUCL:38443 (AOZ19964.1). SignalP 4.0 analysis revealed that pre-mature protein of Lac1 of T. polyzona KU-RNW027 consisted of a putative 21 amino acid signal peptide with a cleavage site between Ala-21 and Ala-22 residues. The mature Lac1 protein had 499 amino acids.
Phylogenetic analysis of the deduced amino acid sequence of Lac1 was constructed with the other 35 complete amino acid sequences of multiple copper oxidases from different sources, including Basidiomycetes, Ascomycetes, plants, insects, and bacteria. The results revealed that Lac1 is clustered in the Basidiomycetes laccase clade in the same branch as laccases from the other T. polyzona strains ( Figure 1B).
The ligninolytic system of T. polyzona KU-RNW027 consisted of two MnP-encoding genes and one laccase-encoding gene. According to their deduced amino acid sequences, MnP1 and MnP2 are new manganese peroxidases in class II fungal secretion heme peroxidase, whereas Lac1 is similar to other T. polyzona laccases. The intact nucleotide sequences of mnp1, mnp2, and lac1 were deposited in GenBank under accession numbers LC340363, LC464698, and LC464699, respectively, whereas full-length cDNA sequences of mnp1, mnp2, and lac1 are LC340360, LC340361, and LC340362, respectively.
The promoter regions of mnp1, mnp2, and lac1 showed low sequence similarity with those of the other fungi. We found unique sequences in the respective genes. However, all promoters shared common regulatory elements, such as MRE, XRE, and HSE [19,28,30,31]. Conserved sequences, TATA boxes, and CAAT boxes were commonly found in the core promoter region as essential for gene expression. The TATA box is known as a transcription initiator site [34], whereas the CAAT box is an RNA polymerase recognition site [35]. The promoter regions of ligninolytic enzyme-encoding genes usually contain one TATA box and at least one CAAT box at positions between -30 and -1145 from the start codon to initiate the transcription [18]. In contrast, no direct CAAT box was found in the promoter regions of mnp1 and mnp2. Two inverted CAAT boxes were identified in both promoters, in accordance with results reported by Chen et al. [28].
In previous reports, no LRE was identified on the promoters of ligninolytic enzyme-encoding genes. Interestingly, we found one and two LREs in the promoters of T. polyzona KU-RNW027 mnp1 and mnp2, respectively. These may drive regulation by light.

Preliminary Plate Assay Study
Under continuous lighting, the size of the orange zone that indicates ligninolytic enzyme activities was larger than in the dark control (Figure 4). This implied that light enhances the ligninolytic enzyme production of T. polyzona KU-RNW027. Therefore, we determined the ligninolytic enzyme activities and their gene expressions under different wavelengths of light. However, light did not affect fungal growth, since the diameters of fungal colonies on agar plates were no different under light and dark conditions. common regulatory elements, such as MRE, XRE, and HSE [19,28,30,31]. Conserved sequences, TATA boxes, and CAAT boxes were commonly found in the core promoter region as essential for gene expression. The TATA box is known as a transcription initiator site [34], whereas the CAAT box is an RNA polymerase recognition site [35]. The promoter regions of ligninolytic enzyme-encoding genes usually contain one TATA box and at least one CAAT box at positions between -30 and -1145 from the start codon to initiate the transcription [18]. In contrast, no direct CAAT box was found in the promoter regions of mnp1 and mnp2. Two inverted CAAT boxes were identified in both promoters, in accordance with results reported by Chen et al. [28].
In previous reports, no LRE was identified on the promoters of ligninolytic enzyme-encoding genes. Interestingly, we found one and two LREs in the promoters of T. polyzona KU-RNW027 mnp1 and mnp2, respectively. These may drive regulation by light.

Preliminary Plate Assay Study
Under continuous lighting, the size of the orange zone that indicates ligninolytic enzyme activities was larger than in the dark control ( Figure 4). This implied that light enhances the ligninolytic enzyme production of T. polyzona KU-RNW027. Therefore, we determined the ligninolytic enzyme activities and their gene expressions under different wavelengths of light. However, light did not affect fungal growth, since the diameters of fungal colonies on agar plates

Effect of Light on Transcription Levels and Enzyme Production
Light of tungsten (320-1100 nm) significantly stimulated MnP activity but not laccase activity. MnP activities were observed as being 1.5 times higher under light compared with the dark control ( Figure 5A). In the dark, T. polyzona KU-RNW027 produced both MnP and laccase at 2.2 and 1.9 U/mL, respectively. Reduction of both enzyme activities was caused by blue light. Only 0.81 and 0.59 U/mL of MnP and laccase activities were detected under blue light, respectively. However, tungsten light and blue light did not affect fungal growth. The biomass of the fungus averaged 1.2 ± 0.07 g/L in all the conditions tested.

Effect of Light on Transcription Levels and Enzyme Production
Light of tungsten (320-1100 nm) significantly stimulated MnP activity but not laccase activity. MnP activities were observed as being 1.5 times higher under light compared with the dark control ( Figure 5A). In the dark, T. polyzona KU-RNW027 produced both MnP and laccase at 2.2 and 1.9 U/mL, respectively. Reduction of both enzyme activities was caused by blue light. Only 0.81 and 0.59 U/mL of MnP and laccase activities were detected under blue light, respectively. However, tungsten light and blue light did not affect fungal growth. The biomass of the fungus averaged 1.2 ± 0.07 g/L in all the conditions tested.
The highest levels of mnp1 and mnp2 transcripts were observed under tungsten light irradiation at 6.5 and 3.8-fold induction of mnp1 and mnp2, respectively, compared to dark condition ( Figure 5B). This result showed the correlation of the induction of both mnp gene transcriptions by tungsten light illumination with increasing MnP activity. However, tungsten light only up-regulated the mnp genes but not the lac gene. Under blue light illumination, levels of mnp1, mnp2, and lac1 transcripts were not significantly different from those observed under the dark control. Blue light had no effect on either mnp or lac gene expressions. However, blue light affected both enzyme activities ( Figure 5A,B).
Tungsten light up-regulated MnP activity and its gene expression compared with the dark condition. Laccase activity and its gene transcription levels were not affected by light, in good agreement with the observation that no LRE was identified on the lac1 promoter region. Previous studies reported the effect of light on MnP, laccase, and lignin peroxidase production in submerged cultures of C. unicolor, P. sanguineus, P. lindtneri, and P. chrysosporium [14,15]. Light also stimulated the production of MnP from solid-state ash sawdust C. unicolor culture [36]. However, analysis of ligninolytic enzymes gene expression in C. unicolor revealed that wood degradation properties may not only be dependent on lighting conditions but also result from overall stimulation of fungal metabolism by daylight [37]. The highest levels of mnp1 and mnp2 transcripts were observed under tungsten light irradiation at 6.5 and 3.8-fold induction of mnp1 and mnp2, respectively, compared to dark condition ( Figure 5B). This result showed the correlation of the induction of both mnp gene transcriptions by tungsten light illumination with increasing MnP activity. However, tungsten light only up-regulated the mnp genes but not the lac gene. Under blue light illumination, levels of mnp1, mnp2, and lac1 transcripts were not significantly different from those observed under the dark control. Blue light had no effect on either mnp or lac gene expressions. However, blue light affected both enzyme activities ( Figures  5A,B).
Tungsten light up-regulated MnP activity and its gene expression compared with the dark condition. Laccase activity and its gene transcription levels were not affected by light, in good agreement with the observation that no LRE was identified on the lac1 promoter region. Previous studies reported the effect of light on MnP, laccase, and lignin peroxidase production in submerged cultures of C. unicolor, P. sanguineus, P. lindtneri, and P. chrysosporium [14,15]. Light also stimulated the production of MnP from solid-state ash sawdust C. unicolor culture [36]. However, analysis of ligninolytic enzymes gene expression in C. unicolor revealed that wood degradation properties may not only be dependent on lighting conditions but also result from overall stimulation of fungal metabolism by daylight [37]. The highest levels of mnp1 and mnp2 transcripts were observed under tungsten light irradiation at 6.5 and 3.8-fold induction of mnp1 and mnp2, respectively, compared to dark condition ( Figure 5B). This result showed the correlation of the induction of both mnp gene transcriptions by tungsten light illumination with increasing MnP activity. However, tungsten light only up-regulated the mnp genes but not the lac gene. Under blue light illumination, levels of mnp1, mnp2, and lac1 transcripts were not significantly different from those observed under the dark control. Blue light had no effect on either mnp or lac gene expressions. However, blue light affected both enzyme activities ( Figures  5A,B).
Tungsten light up-regulated MnP activity and its gene expression compared with the dark condition. Laccase activity and its gene transcription levels were not affected by light, in good agreement with the observation that no LRE was identified on the lac1 promoter region. Previous studies reported the effect of light on MnP, laccase, and lignin peroxidase production in submerged cultures of C. unicolor, P. sanguineus, P. lindtneri, and P. chrysosporium [14,15]. Light also stimulated the production of MnP from solid-state ash sawdust C. unicolor culture [36]. However, analysis of ligninolytic enzymes gene expression in C. unicolor revealed that wood degradation properties may not only be dependent on lighting conditions but also result from overall stimulation of fungal metabolism by daylight [37]. Transcriptional levels of the mnp1 ( ), mnp2 ( ), and lac1 ( ) genes were determined using mycelia harvested from the cultures after 7 days of cultivation under different wavelengths of light. Transcription levels of mnp1, mnp2, and lac1 are shown as induction fold values relative to those observed under the dark condition. Error bars represent standard deviation (n = 3). a, b, and c represent significant enzyme production and gene expression compared to the control (p < 0.05).
The highest levels of mnp1 and mnp2 transcripts were observed under tungsten light irradiation at 6.5 and 3.8-fold induction of mnp1 and mnp2, respectively, compared to dark condition ( Figure 5B). This result showed the correlation of the induction of both mnp gene transcriptions by tungsten light illumination with increasing MnP activity. However, tungsten light only up-regulated the mnp genes but not the lac gene. Under blue light illumination, levels of mnp1, mnp2, and lac1 transcripts were not significantly different from those observed under the dark control. Blue light had no effect on either mnp or lac gene expressions. However, blue light affected both enzyme activities ( Figures  5A,B).
Tungsten light up-regulated MnP activity and its gene expression compared with the dark condition. Laccase activity and its gene transcription levels were not affected by light, in good agreement with the observation that no LRE was identified on the lac1 promoter region. Previous studies reported the effect of light on MnP, laccase, and lignin peroxidase production in submerged cultures of C. unicolor, P. sanguineus, P. lindtneri, and P. chrysosporium [14,15]. Light also stimulated the production of MnP from solid-state ash sawdust C. unicolor culture [36]. However, analysis of ligninolytic enzymes gene expression in C. unicolor revealed that wood degradation properties may not only be dependent on lighting conditions but also result from overall stimulation of fungal metabolism by daylight [37]. Transcriptional levels of the mnp1 ( ), mnp2 ( ), and lac1 ( ) genes were determined using mycelia harvested from the cultures after 7 days of cultivation under different wavelengths of light. Transcription levels of mnp1, mnp2, and lac1 are shown as induction fold values relative to those observed under the dark condition. Error bars represent standard deviation (n = 3). a, b, and c represent significant enzyme production and gene expression compared to the control (p < 0.05).
The highest levels of mnp1 and mnp2 transcripts were observed under tungsten light irradiation at 6.5 and 3.8-fold induction of mnp1 and mnp2, respectively, compared to dark condition ( Figure 5B). This result showed the correlation of the induction of both mnp gene transcriptions by tungsten light illumination with increasing MnP activity. However, tungsten light only up-regulated the mnp genes but not the lac gene. Under blue light illumination, levels of mnp1, mnp2, and lac1 transcripts were not significantly different from those observed under the dark control. Blue light had no effect on either mnp or lac gene expressions. However, blue light affected both enzyme activities ( Figures  5A,B).
Tungsten light up-regulated MnP activity and its gene expression compared with the dark condition. Laccase activity and its gene transcription levels were not affected by light, in good agreement with the observation that no LRE was identified on the lac1 promoter region. Previous studies reported the effect of light on MnP, laccase, and lignin peroxidase production in submerged cultures of C. unicolor, P. sanguineus, P. lindtneri, and P. chrysosporium [14,15]. Light also stimulated the production of MnP from solid-state ash sawdust C. unicolor culture [36]. However, analysis of ligninolytic enzymes gene expression in C. unicolor revealed that wood degradation properties may not only be dependent on lighting conditions but also result from overall stimulation of fungal metabolism by daylight [37]. The highest levels of mnp1 and mnp2 transcripts were observed under tungsten light irradiation at 6.5 and 3.8-fold induction of mnp1 and mnp2, respectively, compared to dark condition ( Figure 5B). This result showed the correlation of the induction of both mnp gene transcriptions by tungsten light illumination with increasing MnP activity. However, tungsten light only up-regulated the mnp genes but not the lac gene. Under blue light illumination, levels of mnp1, mnp2, and lac1 transcripts were not significantly different from those observed under the dark control. Blue light had no effect on either mnp or lac gene expressions. However, blue light affected both enzyme activities ( Figures  5A,B).
Tungsten light up-regulated MnP activity and its gene expression compared with the dark condition. Laccase activity and its gene transcription levels were not affected by light, in good agreement with the observation that no LRE was identified on the lac1 promoter region. Previous studies reported the effect of light on MnP, laccase, and lignin peroxidase production in submerged cultures of C. unicolor, P. sanguineus, P. lindtneri, and P. chrysosporium [14,15]. Light also stimulated the production of MnP from solid-state ash sawdust C. unicolor culture [36]. However, analysis of ligninolytic enzymes gene expression in C. unicolor revealed that wood degradation properties may not only be dependent on lighting conditions but also result from overall stimulation of fungal metabolism by daylight [37]. The highest levels of mnp1 and mnp2 transcripts were observed under tungsten light irradiation at 6.5 and 3.8-fold induction of mnp1 and mnp2, respectively, compared to dark condition ( Figure 5B). This result showed the correlation of the induction of both mnp gene transcriptions by tungsten light illumination with increasing MnP activity. However, tungsten light only up-regulated the mnp genes but not the lac gene. Under blue light illumination, levels of mnp1, mnp2, and lac1 transcripts were not significantly different from those observed under the dark control. Blue light had no effect on either mnp or lac gene expressions. However, blue light affected both enzyme activities ( Figures  5A,B).
Tungsten light up-regulated MnP activity and its gene expression compared with the dark condition. Laccase activity and its gene transcription levels were not affected by light, in good agreement with the observation that no LRE was identified on the lac1 promoter region. Previous studies reported the effect of light on MnP, laccase, and lignin peroxidase production in submerged cultures of C. unicolor, P. sanguineus, P. lindtneri, and P. chrysosporium [14,15]. Light also stimulated the production of MnP from solid-state ash sawdust C. unicolor culture [36]. However, analysis of ligninolytic enzymes gene expression in C. unicolor revealed that wood degradation properties may not only be dependent on lighting conditions but also result from overall stimulation of fungal metabolism by daylight [37].
) genes were determined using mycelia harvested from the cultures after 7 days of cultivation under different wavelengths of light. Transcription levels of mnp1, mnp2, and lac1 are shown as induction fold values relative to those observed under the dark condition. Error bars represent standard deviation (n = 3). a, b, and c represent significant enzyme production and gene expression compared to the control (p < 0.05).

Effect of Light on Ligninolytic Enzyme Stability
Compared to the equivalent dark controls, both MnP and laccase activities were relatively stable under tungsten light for the first six hours of exposure, retaining 90%-95% initial activity respectively. Thereafter, both enzymes were progressively effected to a greater extent, but still retained 60%-75% initial activity even after 72 h of exposure. Conversely, over the same time course, blue light had a much stronger negative effect, with the retained activity of both enzymes falling to 70% after only 6 h of exposure. For MnP, retained activity progressive declined to 39% after 72 h of exposure, whereas for laccase the detrimental effect became much more noticeable with increased exposure such that the retained activity of this enzyme had declined to 6% of its initial level at 72 h. (Figure 6) The biochemical basis of the detrimental effects of exposure to blue light on both the tested MnP and laccase from KU-RNW027 remains currently uncharacterized. However, blue light has been previously shown to effect the biological functions of many proteins [1]; specifically, blue light can change the activity of the endonuclease PvuII by changing the sulfur-sulfur bond configuration of the enzyme in a way that reduces enzyme-substrate interaction [38]. Multiple cysteine residues, and therefore, potentially disulfide bonds, exist in both the tested MnP and laccase enzymes (Figure 2), and so disruption of these particular sulfur-sulfur bonds could be a possible explanation for the observed effects of blue light on these enzymes. Confirmation of the actual mode of action of blue light on KU-RNW027 requires much more extensive investigation. exposure such that the retained activity of this enzyme had declined to 6% of its initial level at 72 h. (Figure 6) The biochemical basis of the detrimental effects of exposure to blue light on both the tested MnP and laccase from KU-RNW027 remains currently uncharacterized. However, blue light has been previously shown to effect the biological functions of many proteins [1]; specifically, blue light can change the activity of the endonuclease PvuII by changing the sulfur-sulfur bond configuration of the enzyme in a way that reduces enzyme-substrate interaction [38]. Multiple cysteine residues, and therefore, potentially disulfide bonds, exist in both the tested MnP and laccase enzymes (Figure 2), and so disruption of these particular sulfur-sulfur bonds could be a possible explanation for the observed effects of blue light on these enzymes. Confirmation of the actual mode of action of blue light on KU-RNW027 requires much more extensive investigation.  and laccase from KU-RNW027 remains currently uncharacterized. However, blue light has been previously shown to effect the biological functions of many proteins [1]; specifically, blue light can change the activity of the endonuclease PvuII by changing the sulfur-sulfur bond configuration of the enzyme in a way that reduces enzyme-substrate interaction [38]. Multiple cysteine residues, and therefore, potentially disulfide bonds, exist in both the tested MnP and laccase enzymes (Figure 2), and so disruption of these particular sulfur-sulfur bonds could be a possible explanation for the observed effects of blue light on these enzymes. Confirmation of the actual mode of action of blue light on KU-RNW027 requires much more extensive investigation. ), tungsten light ( and laccase from KU-RNW027 remains currently uncharacterized. However, blue light has been previously shown to effect the biological functions of many proteins [1]; specifically, blue light can change the activity of the endonuclease PvuII by changing the sulfur-sulfur bond configuration of the enzyme in a way that reduces enzyme-substrate interaction [38]. Multiple cysteine residues, and therefore, potentially disulfide bonds, exist in both the tested MnP and laccase enzymes (Figure 2), and so disruption of these particular sulfur-sulfur bonds could be a possible explanation for the observed effects of blue light on these enzymes. Confirmation of the actual mode of action of blue light on KU-RNW027 requires much more extensive investigation. and laccase from KU-RNW027 remains currently uncharacterized. However, blue light has been previously shown to effect the biological functions of many proteins [1]; specifically, blue light can change the activity of the endonuclease PvuII by changing the sulfur-sulfur bond configuration of the enzyme in a way that reduces enzyme-substrate interaction [38]. Multiple cysteine residues, and therefore, potentially disulfide bonds, exist in both the tested MnP and laccase enzymes (Figure 2), and so disruption of these particular sulfur-sulfur bonds could be a possible explanation for the observed effects of blue light on these enzymes. Confirmation of the actual mode of action of blue light on KU-RNW027 requires much more extensive investigation.

Conclusions
Our findings suggested two new manganese peroxidases and one laccase in the ligninolytic system of T. polyzona KU-RNW027. Ligninolytic enzyme production and gene expression in T. polyzona KU-RNW027 can be regulated by light. This is the first report on LREs identified in the mnp promoter region in fungi. Tungsten light enhanced MnP production but not for laccase, whereas blue light impacted enzyme stability. Our findings effectively supported improvement of ligninolytic enzyme production in the white rot fungus T. polyzona KU-RNW027. This result benefits the application of an eco-friendly approach, especially in toxic aromatic compound bioremediation. Author Contributions: Conceptualization, T.K., K.S. and L.C.; methodology, P.L., E.K., T.K. and L.C.; software, P.L. and E.K.; validation, P.L., E.K. and M.S.; formal analysis, P.L., E.K., M.S. and C.T.; investigation, T.K., K.S. and L.C.; resources, K.S. and L.C.; data curation, P.L. and E.K.; writing-original draft preparation, P.L.; writing-review and editing, P.L., E.K., M.S., T.K., K.S., C.T. and L.C.; visualization, T.K., K.S. and L.C.; supervision, T.K., K.S. and L.C.; project administration, K.S. and L.C.; funding acquisition, K.S. and L.C. All authors have read and agreed to the published version of the manuscript.