1. Introduction
Laccase is a multi-copper oxidase that catalyzes the oxidation of various phenolic and non-phenolic substrates associated with the lignin structure, while reducing oxygen to water [
1,
2,
3]. Due to the wide range of natural substrates, laccase is used in several fields of industrial and biotechnological applications, such as improvement of fiber properties, degradation of antibiotics and other pharmaceutical products, fuels, detoxification of environmental pollutants, stabilization of lignin by providing chemical precursors, and pulp bleaching paper industry [
4,
5,
6,
7,
8]. Most of the known laccase enzymes originated from fungi, especially white rot fungi that can perform lignin degradation [
9,
10,
11].
Fungal laccase is encoded by a multigene family and there are multiple isozymes that are mainly involved in physiological processes such as lignin degradation, fruiting body formation, pigment formation in asexual development and pathogenesis [
12,
13,
14,
15,
16]. Jiao et al. [
17] analyzed the expression patterns of 12 laccase encoding genes distributed in the genome of
P. ostreatus PC15 strain and hypothesized that
PoLac2 might be closely related to lignin degradation by
P. ostreatus. While
PoLac3 and
PoLac5 may be related to the formation of fruiting primordium and fruiting body, respectively,
PoLac12 may be related to the development of
P. ostreatus fruiting body, and overexpression of
PoLac2 gene was found to enhance the laccase activity and lignin degradation of
P. ostreatus. Lu et al. [
18] performed qRT-PCR on 11 Laccase genes of
Volvariella volvacea, and hypothesized that
Vvlcc3 was involved in stipe elongation by combining with heterologous expression in
Pichia pastoris. Analysis of the transcriptome of different developmental stages and different tissues of
Flammulina velutipes revealed that the expression of
lac4 gene was higher than other laccase genes at all developmental stages, suggesting it is involved in the biotransformation of lignin [
19]. Successful overexpression of the
Pblac1 gene in white-rot fungus
Polyporus brumalis revealed that the transformed strain exhibited higher lignin degradation activity and effective decolorization of RBBR dye compared to the wild type [
20]. Homologous overexpression of the
Gtlcc3 gene in brown-rot fungus
Gloeophyllum trabeum resulted in transformed strains with significantly higher lignin degradation and ethanol production than the wild type [
21]. At present, the functional roles of laccase genes are mostly at the speculative stage, and the specific functional roles of each isoenzyme are not clearly defined, and there are fewer studies related to functional verification and molecular mechanisms.
Crop straw is mainly composed of cellulose, hemicellulose and lignin, in which cellulose molecules are embedded in a barrier formed by covalent bonding of lignin and hemicellulose, making it difficult for enzymes to contact with cellulose molecules, reducing the utilization and nutritional value of straw, which is the main obstacle to the effective conversion of lignocellulosic [
22,
23]. Cotton as one of the important economic crops in China, its harvest will also produce a large number of byproducts-cotton straw [
24,
25]. With the development of science and technology, urbanization and the improvement of living standards, the application of cotton straw as a living fuel decreases, and is burned in the fields by cotton farmers, both wasting resources and polluting the environment. On the contrary, the application of cotton straw as a base material has been further strengthened. However, cotton straw is similar to branch of tree, and compared with corn, rice and wheat straws, they have higher lignin content, which greatly limits the degradation by microorganisms [
26].
In a previous study, it was found that
Pleurotus ostreatus Suping 1 could be produced on cotton straw substrates [
17], but the optimal production conditions and lignin degradation mechanism of cotton straw were not clear. Therefore, in this study, the optimal conditions for lignin degradation by
P. ostreatus Suping 1 on cotton straw substrate were firstly screened by medium optimization experiments. Then, the
Lacc1 gene of
P. ostreatus Suping 1 was obtained by homologous cloning, and the molecular mechanism of
Lacc1 in lignin degradation of cotton straw was analyzed by overexpression technique. The results of this study provide new insights into the mechanism of lignin degradation by white-rot fungi and provide useful guidance for the promotion and application of the laccase in
P. ostreatus.
3. Discussion
Studies have shown that the substrates cellulose: lignin ratios were found to be positively correlated to mycelial growth rates and to mushroom yield of
P. ostreatus [
30,
31], and cotton straw is a substrate with high lignin content. In reports on cotton straw culture of edible mushrooms, it was found that higher yields could be obtained by applying to cotton straw culture of edible mushrooms relative to other crop substrates [
32,
33,
34,
35], in which lignin degrading enzymes played a crucial role [
36,
37,
38]. In this study, we first screened the optimum culture conditions for laccase enzyme activity of
P. ostreatus Suping 1. The final culture conditions for the highest lignin degradation efficiency of the
P. ostreatus Suping 1 were determined by single-factor and orthogonal experiments: the size of cotton straw particle size in the medium was 0.75 mm, the medium had a solid–liquid ratio of 1:3, and contained 0.25 g/L of Tween, as well as the incubation temperature was 26 °C.
Two overexpression strains (OE L1-1 and OE L1-4) of
Lacc1 gene were obtained by
Agrobacterium tumefaciens-mediated method, and the gene expression increased 12.08 and 33.04-fold at day 30, respectively, and the enzyme activity of OE L1-4 strain increased 71.05% at day 30 compared to WT strain, whereas the enzyme activity of OE L1-1 strain was not significantly different from WT strain, showing the difference between gene expression level and gene product yields, probably due to the time required for gene translation into protein [
39,
40]. The lignin degradation rate of the medium was increased by 6.86% and 5.23% for OE L1-1 and OE L1-4 strains, respectively, and there was inequivalence between the lignin degradation ability and the increase in the fold to gene expression, probably due to the different mRNA translation efficiency of the two overexpression strains [
41,
42].
The lignin in cotton straw were characterized by wet chemistry (carbohydrate analysis) and spectroscopy methods (FT-IR, 13C and 1H-13C HSQC NMR spectroscopy) as well as gel permeation chromatography (GPC), which showed that the lignin in cotton straw belonged to typical G-S lignin, mainly composed of G-type units (59%) and distinct S-type units (40%), and the inter-unit linkages were mainly composed of β-O-4’ (75.6%) and β-β’ (12.2%) [
43]. To investigate the details of lignin biodegradation, Dong et al. used FTIR with CP/MAS13C -NMR to study the characteristics and process of sugarcane bagasse degradation by three lignin degrading fungi,
Phanerochaete chrysosporium PC2,
Lentinula edode LE16 and
P. ostreatus PO45, and found that all three strains preferentially degraded the S-unit of sugarcane bagasse lignin [
44]. In contrast, Zhang et al. used FTIR and 2D HSQC NMR to determine the lignin composition of degraded sacrau poplar and found that the G-unit lignin was more susceptible to degradation by
Trametes pubescens C7571 and
T. versicolor C6915 than the S-unit lignin, and found that the cleavage of β-O-4 linkages and the degradation of β-5 and β-β linkages clearly occurred [
27]. It is observed that the preferential degradation of S- or G-units in lignin, as well as the cleavage of linkages of different bond types, is related to the strain class. In this study, the G/S of acetylated lignin in cotton straw after 30 days of incubation with the wild-type strain was 1.94, and the G/S of acetylated lignin in cotton straw with OE L1-4 was 1.42. Compared with the wild-type control, the G/S of acetylated lignin in the overexpressed strain was reduced, indicating that the
Lacc1 gene accelerates the degradation of lignin G-units. In addition, the G/S ratio of acetylated lignin in cotton straw medium inoculated with OE L1-4 strain was 1.42, which was lower than that of acetylated lignin in cotton straw medium inoculated with wild-type strain (1.94), but still higher than that of original acetylated lignin (1.20). It indicates that the
Lacc1 gene only slowed down the degradation of S-unit and accelerated the degradation of G-unit, but the degradation of S-unit was still faster than that of G-unit, that is, overexpression of
Lacc1 gene did not change the preferential degradation of S-unit in cotton straw lignin by
P. ostreatus. Meanwhile, the
Lacc1 gene was found to be involved in the cleavage of β-O-4-type linkages.
From the ratio of H proton of aromatic acetates content to H proton of aliphatic acetates content, the ratio of phenolic to alcoholic hydroxyl groups in lignin can be obtained [
29]. The ratio of phenolic to alcoholic hydroxyl groups in the acetylated lignin of cotton straw after 30 days of incubation with wild-type strains was 0.23, and the ratio of phenolic to alcoholic hydroxyl groups in the acetylated lignin of cotton straw of OE L1-4 increased to 0.36. It was demonstrated that the degradation of lignin by the
Lacc1 gene was mainly reflected in the demethylation of lignin units and thus promoting the depolymerization of the units, which is consistent with the inference that the degradation of lignin by
Lacc1 may be mainly reflected in the demethylation of lignin units rather than the oxidative breaking of the chemical bonds between the lignin benzene ring structures [
45].
4. Materials and Methods
4.1. Strains
The Pleurotus ostreatus Suping 1 strain was provided by Vegetable Research Institute, Jiangsu Academy of Agricultural Sciences and preserved in the Institute of Horticulture, Anhui Academy of Agricultural Sciences. The Escherichia coli DH5α and Agrobacterium tumefaciens EHA105 strain were preserved in the laboratory. The pCAMBIA1304-Pogpd-PoLac1 plasmids were constructed with the pCAMBIA1304 plasmids also kept in the same laboratory.
4.2. Single-Factor Optimization of Lignin Degradation in Cotton Straw by Pleurotus ostreatus
Straw particle size. Five grams of cotton straw with particle sizes of 5 mm, 0.75 mm, 0.425 mm and 0.25 mm were weighed into culture flasks, 22 mL of nutrient solution was added and autoclaved at 121 °C for 30 min. The nutrient solution was prepared by ammonium tartrate (22.0 g/L), macronutrients (20 g/L KH2PO4, 13.8 g/L MgSO4·7H2O, 1.0 g/L CaCl2, 0.6 g/L NaCl), trace elements (0.35 g/L MnSO4·H2O, 60 g/L FeSO4·7H2O, 110 mg/L CoCl2·6H2O, 60 mg/L ZnSO4·7H2O, 95 mg/L CuSO4·5H2O, 6 mg/L AlK(SO4)2·12H2O, 6 mg/L H3BO3, 6 mg/L Na2MoO4·2H2O), VB1 (100 mg/L) and water in the ratio of 1:15:15:3:16. Three P. ostreatus Suping 1 pieces of about 1 cm in diameter from PDA medium (boiled juice of 200 g/L potato, 20.0 g/L glucose, and 15.0 g/L agar added to solid medium) were inoculated into each culture flask and incubated at 25 °C for 20 days in the dark to determine the remaining medium lignin content.
Solid–liquid ratio. Five grams of cotton straw with particle sizes of 0.425 mm were weighed into culture flasks and 10 mL, 15 mL, 20 mL and 25 mL of nutrient solution were added to each treatment to make the solid–liquid ratio (w/v) 1:2, 1:3, 1:4 and 1:5, respectively, and autoclaved at 121 °C for 30 min. Different treatments were inoculated with P. ostreatus Suping 1 and the remaining medium lignin content was measured after 20 days of dark incubation at 25 °C.
Temperature. Five grams of cotton straw with a particle size of 0.425 mm were weighed into the culture flask, 22 mL of nutrient solution was added, sterilized and inoculated with P. ostreatus Suping 1. Then, the remaining lignin content of the medium was measured after 20 days of incubation under dark conditions at 20 °C, 25 °C and 27 °C, respectively.
Tween. Five grams of cotton straw with a particle size of 0.425 mm were weighed into the culture flask and 22 mL of nutrient solution containing different concentrations of Tween 80 was added, with final concentrations of Tween 80 are 0.1 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L and 0.5 g/L, respectively, and inoculated with P. ostreatus Suping 1 after sterilization. Then, after 20 days of culture at 25 °C in the dark, the remaining lignin content in the medium was measured.
4.3. Orthogonal Array Optimization of Lignin Degradation in Cotton Straw by P. ostreatus
The orthogonal experiment was performed with four factors of straw particle size (A), solid–liquid ratio (B), incubation temperature (C) and Tween content (D), and three levels of each factor were selected, as in
Table 5, using orthogonal test L9 (3
4) with nine treatments, each treatment was inoculated with five bottles and replicated three times. After 20 days of incubation, five bottles of medium were mixed and tested for lignin content, and the lignin degradation rate of
P. ostreatus Suping 1 was statistically calculated, and the effect of each treatment factor on the lignin degradation rate of
P. ostreatus Suping 1 was analyzed by ANOVA and Duncan’s multiple comparisons using SPSS version 17.0 statistical analysis software.
4.4. Determination of Lignin Content
After culture of
P. ostreatus Suping 1 strains, the solid residue substrates were dried in an oven at 60 °C to a constant weight. 100 mL of acid washing buffer (4 mol/L HCl) was used to digest 1 g of the dried substrate residue for 60 min. The samples were then washed with acetone and petroleum ether using a cold leaching device. Subsequently, the sample was washed with 12 mol/L sulfuric acid solution for 3 h. Then, the sample was washed to neutral and dried in a vacuum oven at 130 °C to a constant weight. The dried samples were ashed in a muffle furnace at 550 °C for 2 h. After cooling, the samples were weighed and the lignin content in the substrate residue was calculated [
46].
4.5. Optimization of Laccase Production by Orthogonal Array Method
Orthogonal array design was adopted for four culture conditions using Minitab 16 software to evaluate the factors influencing the yield of laccase. Each independent variable was tested at three levels such as high, middle, and low level. The symbol code and actual level of the variables and the experimental design are shown in
Table 2. Four factors such as straw particle size, solid–liquid ratio, temperature, and Tween contents were studied in 9 experiments to calculate the standard error. The triplicate verification test was performed to check the optimum condition and the average value was taken as the response.
4.6. Laccase Activity Assay
The samples were removed every 5 days and 15 mL of purified water was added in batches and extracted overnight at 4 °C. Thereafter, shaking extraction was performed at 200 r/min for 1 h, followed by freezing centrifugation at 12,000 r/min for 10 min. Finally, the supernatant was obtained as the crude enzyme solution. The laccase activity was calculated by examining the oxidation of 2,2-azobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at 420 nm. One unit of activity was defined as the amount of enzyme converting 1 μmol of substrate per minute [
47].
4.7. Cloning and Expression Pattern Analysis of Lacc1 in P. ostreatus Suping 1
The genomic DNA and RNA of
P. ostreatus Suping 1 were extracted using the EasyPure
®Plant Genomic DNA Kit (Trans, Beijing) and RNAprep Pure Plant Kit (Tiangen, China), respectively. Subsequently, the CDS sequence of LACC1 (KDQ27217.1) from strain
P. ostreatus PC15 (GCA_000697685.1) submitted to NCBI was used to design primers (Lac1-F and Lac1-R,
Table 6) to amplify the
Lacc1 gene sequence of
P. ostreatus Suping 1 using the DNA and cDNA of
P. ostreatus Suping 1 as templates, respectively. The amplified PCR products were purified, ligated to the pMD18-T vector (Takara, Dalian) and sequenced. The CDS sequence of
Lacc1 gene of
P. ostreatus Suping 1 was used to design qRT-PCR primers (Lac1-qF and Lac1-qR) to detect the expression pattern of
Lacc1 gene in different growth stages of
P. ostreatus Suping 1. RNA reverse transcription was performed using TransScript
® One-Step RT-PCR SuperMix (Trans, China) and qRT-PCR was performed using the Bio-rad Cfx96 Touch™ Deep Well Real-Time PCR detection system. The PCR volume was 20 μL, and
cyph (transcript ID: 1058252) was set as the reference gene [
48]. Three parallel replicates were set for each sample. Finally, relative gene expression was calculated by the 2
−ΔΔCt method. The reactions were contained in the following: 10 μL of TransStart Tip Green qRT-PCR SuperMix (2×) (Trans, China), 2 μL of template cDNA, 0.8 μL of forward and reverse primers and ddH
2O to 20 μL. The PCR amplification conditions were performed as follows: 98 °C for 2 min, followed by 40 cycles of 98 °C for 10 s, 60 °C for 10 s and 68 °C for 30 s.
4.8. Agrobacterium Tumefaciens-Mediated Transformation of P. ostreatus
The glyceraldehyde-3-phosphate dehydrogenase (
gpd) promoter was cloned from
P. ostreatus [
49]. Then, it was inserted into the expression plasmids pCAMBIA1304 to replace its CAMV35S promoter by using the restriction sites of
Hind III and
Nco I. Subsequently,
Lacc1 were cloned from
P. ostreatus Suping 1 using specific primers (Lac1-eukF and Lac1-eukR,
Table 5), respectively. The PCR products were digested with
Bgl II and
Spe I (Takara), then inserted into the pCAMBIA1304-
Pogpd vector. Final vector plasmids were designated as pCAMBIA1304-
Pogpd-
PoLac1.
The A. tumefaciens strains EHA105, harboring pCAMBIA1304-Pogpd-PoLac1, were cultivated at 28 °C in LB medium (containing antibiotics kanamycin and rifampicin) to an OD600 of 0.6–0.8. Bacterial cells were then collected, centrifugated, and suspended in an induction medium (IM, boiled juice of 200 g L−1 potato, 20.0 g L−1 glucose, and including 200 μmol/L acetosyringone) to an OD600 of 0.5–0.6, pH 5.5, and the virulence of A. tumefaciens was induced by shaking at 150 rpm for 6 h at 28 °C.
The mycelia masses (0.9 × 0.9 cm) of
P. ostreatus were grown on PDA for 7 days was immersed in pre-inducing bacterial culture for 30 min and then placed on solid induction medium for 4 days. Subsequently, the co-cultured mycelia were transferred to PDA with 100 μg/mL hygromycin and 300 μg/mL cefotaxime. The stability of the transformation was confirmed by subculturing colonies onto selective medium three times, then cultured on PDA for three generates to rejuvenate mycelia [
50].
4.9. PCR Analysis and Visual Detection of β-Glucuronidase (GUS)
The genomic DNA of the putative transformants was extracted using the EasyPure
®Plant Genomic DNA Kit (Trans, China). To confirm that the gene had been integrated into the genome, primers GUS-F and GUS-R (
Table 1) for the
GUS gene were used to verify whether the T-DNA was inserted by PCR (conditions: 94 °C for 3 min, followed by 35 cycles of amplification, 94 °C for 30 s, 59 °C for 30 s, 72 °C for 100 s, and ending after 10 min at 72 °C). To verify the expression of the introduced
GUS reporter gene, the presumptive transformants were detected with the GUS Histochemical Assay Kit (MKbio, Shanghai).
4.10. Fourier Transform Infrared Spectroscopy (FTIR) Analysis
After cultivating for 30 days with the wild type and transformants, cotton stalks were removed and dried at 40 °C. According to the previous methods [
51], milled wood lignin was extracted and purified. FTIR analysis was performed to determine the functional groups converted during the degradation of lignin by
Lacc1-encoded laccase. Local microscopic FTIR with a KBr wafer was used, for which the milled wood lignin samples are homogeneous and representative. The micro-FTIR spectra of local areas of the wafer specimen were measured using a NICOLET iN10 MX spectrometer (Thermos Nicolet Corporation, Madison, WI, USA); connected to a Nicolet NicPlan IR microscope and an MCT detector. The spectral range was taken from 4000 to 650 cm
−1 at a resolution of 4 cm
−1.
4.11. 1H-NMR
Lignin was extracted from the uncultured substrates and the substrates that were cultured with
P. ostreatus after 30 days. A total of 20 mg of milled wood lignin samples was weighed and dissolved in 2 mL of pyridine: acetic anhydride (1:1) mixture. Nitrogen was charged into the reaction bulb, which was placed in the dark at room temperature for 72 h. After the reaction was completed, the reactant was dripped into diethyl ether until precipitate was formed, after which the solution was centrifuged to separate the precipitate. The precipitate was then washed with diethyl ether six to eight times to remove the pyridine odor from the precipitate. Finally, the completely acetylated lignin was obtained. The acetyl-treated lignin samples were dissolved in 0.5 mL of DMSO, with tetramethylsilane as the internal standard. Finally, the
1H-NMR assay was conducted with a Bruker-400 superconducting NMR spectrometer (Bruker, Fällanden, Switzerland) at a frequency of 400 MHz [
44].