However, the possible physiological mechanism underlying H2S-alleviated salt stress in cucumber remains unclear. Here, a pot experiment was conducted with an aim to examine the possible mechanism of H2S in enhancement of cucumber salt stress tolerance. The results showed that H2S ameliorated salt-induced growth inhibition and alleviated the reduction in photosynthetic attributes, chlorophyll fluorescence and stomatal parameters. Meanwhile H2S increased the endogenous H2S level concomitant with increased activities of D/L-cysteine desulfhydrase and β-cyanoalanine synthase and decreased activities of O-acetyl-L-serine(thiol)lyase under excess NaCl. Notably, H2S maintained Na+ and K+ homeostasis via regulation of the expression of +-ATPasePM H, SOS1 and SKOR at the transcriptional level under excess NaCl. Moreover, H2S alleviated salt-induced oxidative stress as indicated by lowered lipid peroxidation and reactive oxygen species accumulation through an enhanced antioxidant system. Altogether, these results demonstrated that application of H2S could protect cucumber seedlings against salinity stress, likely by keeping the Na+/K+ balance, controlling the endogenous H2S level by regulating the H2S synthetic and decomposition enzymes, and preventing oxidative stress by enhancing the antioxidant system under salinity stress.Salinity stress from soil or irrigation water can significantly limit the growth and development of plants. Emerging evidence suggests that hydrogen sulfide (H Salt stn and Tr . Chloropn and Tr . Under a of PSII . In genearameter . The facarameter . Under s+ in plants cells, which competitively inhibit the uptake of K+, thus leading to a deficiency of K+  is able to remove excess Na+ from cells  is an economically important crop as well as a model plant for systematic investigations on many aspects.Evidence in some plant species has proven that Hrawberry . Deng ett stress . But the2S enhances tolerance of salinity stress in the important vegetable crop cucumber, with particular emphasis on maintenance of the Na+/K+ balance, and regulating of H2S metabolism and oxidative stress response.In this study, a pot experiment was conducted with an aim to evaluate the possible mechanism by which H2S, the ameliorative effects of H2S will be easy to judge. Therefore, the sensitive cucumber (C. sativus L.) cultivar “Chunxiaqiuwang” was used in our experiments. Uniform seeds were sterilized in 10 % (v/v) sodium hypochlorite solution for 5 min followed by washing three times with distilled water. Sterilized seeds were germinated on moist filter paper in petri dishes at 25°C in the dark. After germination, seedlings were transplanted into perlite-filled plastic pots  and watered with Hoagland’s solution. 10 mL Hoagland’s solution was applied for every other day after germination, which contained 5 mM KNO3, 5 mM Ca(NO3)2, 1 mM NH4H2PO4, 2 mM MgSO4, 10 μM MnSO4, 50 μM H3BO3, 0.7 μM ZnSO4, 0.2 μM CuSO4, 0.01 μM (NH4)6Mo7O24 and 70 μM Fe-EDTA-Na2. The seedlings of cucumber were maintained in a controlled growth chamber with a light/dark regime of 14/10 h, relative humidity of 70%, temperature of 25°C and a PAR of 800 μmol.m-2.s-1. When the first true leaf emerged, uniform and healthy seedlings were selected and divided into three groups for the following treatments. (i) Hoagland’s nutrient solution (as Control); (ii) Hoagland’s nutrient solution + 200 mM NaCl (as NaCl); (iii) Hoagland’s nutrient solution + 200 mM NaCl + 5 or 10, or 15 or 20 μM NaHS (as NaCl + NaHS). In our preliminary experiment, the phenotypic characteristics of plants with treated by H2S alone were no significant difference compared with the control group (data not shown). Thus there is no only H2S treatment group in our study. NaHS is commonly used as an H2S donor since it dissociates to water produce HS- and Na+, and then combination of HS- with H+ produces H2S  and leaf area index were determined after 7 days of treatment. Leaf area index was measured using graph paper according to the method of n and Tr were measured using an LI-6400 portable photosynthesis system . The leaf chlorophyll fluorescence parameters were measured using a portable chlorophyll fluorometer . The Fo emission was determined on dark-adapted leaves. The Fm was obtained during a subsequent saturating light pulse. A second saturating pulse of white light was imposed to determine the Fm′. The Fo′ was determined by illuminating the leaf with far-red light for 3 s. The fluorescence parameters were calculated according to v/Fm = (Fm-Fo)/Fm; effective quantum-use efficiency of PSII in light-adapted state, Fv′/Fm′ = (Fm′-Fo′)/Fm′; quantum yield of PSII photochemistry, ΦPSII = (Fm′-Ft)/Fm′; qP = (Fm′-Ft)/(Fm′-Fo′); and NPQ = (Fm-Fm′)/Fm′. The contents of Chlorophyll a (Ca), chlorophyll b (Cb), and Cx+c were measured on fresh leaves as described by The first true leaf was chosen to determine the parameters of photosynthesis, chlorophyll fluorescence and chlorophyll Content. And the PThe first true leaves were selected to measure the stomatal opening by scanning electron microscopy . The leaf epidermis and stomata were observed and photographed under 15 kv. The diameter of the stomata was measured by Motic Images Advanced 3.2  software at 300× magnification.g for 10 min at 4°C. To measure MDA, 1.5 mL of the supernatant was added into 1.5 mL of 0.5% (w/v) TBA made in 5 % TCA. The mixture was heated at 100°C for 20 min and then quickly cooled in an ice bath. After centrifuging at 7, 888 × g for 10 min, the absorbance of the supernatant was measured at 450 nm, 532 nm, and 600 nm, respectively. The MDA content of leaves or roots  was calculated using the formula: C (nmol. g-1 FW) = 20 × [6.45 (OD532 – OD600) – 0.56 OD450].Membrane integrity was evaluated in the leaves and roots by measuring electrolyte leakage. Fresh leaf or root samples (50 mg) were cut in small pieces followed by repeated washings with double distilled water. And then the samples were incubated at 28°C for 2 h in test tubes containing 10 mL of double distilled water for determining EC1 using a conductivity meter . The same samples were again kept in water bath at 100°C for 20 min and the EC2 was recorded at room temperature. The electrolyte leakage was calculated as electrolyte leakage (%) = (EC1/EC2) × 100%. MDA content in the leaves or roots was measured by TBA reaction according to 2•- was determined using the protocol described by g for 10 min at 4°C. The supernatant was reacted with 1 mL of hydroxylamine hydrochlorides for 1 h, then 1 mL of p-aminobenzene sulfonic acid and 1 ml of α-naphthylamine were added. The solution was kept at 25°C for 20 min. The optical density value of the solution was measured with a spectrophotometer at 530 nm using NaNO2 as the standard curve and the corresponding calibration curves was Y = 552.16 X + 4.365 (R2 = 0.9903) H2O2 was measured spectrophotometrically according to g for 20 min at 4°C. The supernatant was used for measuring H2O2 content. The reaction mixture consisted of 0.5 mL of the extracted supernatant, 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M potassium iodide. The reaction was developed for 1 h in darkness before the absorbance was measured at 390 nm. The blank was 0.1% TCA in the absence of root extract. The amount of H2O2 was calculated using a standard curve prepared with known concentrations of H2O2. The corresponding calibration curves was Y = 82.69 X – 1.11 (R2 = 0.9959).The production of O2S content was determined by the formation of methylene blue from dimethyl-p-phenylenediamine in H2SO4 according to the method described previously  zinc acetate was used to absorb H2S. After 30 min reaction, 0.3 mL 5 mM N,N-dimethyl-p-phenylenediamine dihydrochloride dissolved in 3.5 mM H2SO4 was injected into the trap. Then 0.3 mL of 50 mM ferric ammonium sulfate in 100 mM H2SO4 was injected into the trap. The amount of H2S in zinc acetate traps was determined spectrophotometrically at 670 nm, after leaving the mixture for 15 min at room temperature. Solutions with different concentrations of Na2S were prepared, treated in the same way as the assay samples and were used for the quantification of H2S.Endogenous Heviously . Samplesg at 4°C for 15 min. The resulting supernatant was used for the determination of the activities of L/D-CD, CAS, and OAS-TL. Total L-CD activity was determined by the release of H2S from L-cysteine as described in 3 dissolved in 1.2 N HCl and 0.1 mL of 20 mM N, N-dimethyl-p-phenylenedi-amine dihydrochloride dissolved in 7.2 N HCl. The formation of methylene blue was determined at 670 nm, and known concentrations of Na2S were used in the calibration curve. D-CD activity was determined in the same way as L-CD activity except with D-cysteine instead of L-cysteine and the pH of the Tris–HCl buffer was 8.0. One unit of enzyme activity is defined as the amount of enzyme that produces 1.0 mmol/min H2S under the stated assay conditions and expressed as U g-1 FW. The total OAS-TL activity was determined following the method described 2S from L-cysteine in the same way as L-CD activity with the following modifications: the 1.0 mL volume of reaction mixture contained 0.1 mL of 10 mM L-cysteine, 0.7 mL 100 mM Tris–HCl (pH 9.0), 0.1 mL of 7.5 mM KCN and 0.1 mL of protein solution, as described in Samples (0.3 g) were ground with a mortar and pestle in liquid nitrogen and soluble proteins were extracted with 1.5 mL of cold extraction buffer containing 20 mM Tris–HCl (pH 8.0), 0.1% (w/v) DTT and 0.2% (w/v) sodium ascorbate. The homogenate was centrifuged at 13, 000 × 4: HNO3: H2SO4 ] on a hot plate (120°C) until the volume was reduced to 1 mL. After diluting with double distilled water, the Na+ and K+ contents were estimated using the flame photometer  was subjected to acid digestion in 10 mL digestion mixture [HClOotometer .SOS1, SKOR, and +-ATPasePM H genes in cucumber leaves and roots in response to salt stress, 18-day-old seedlings treated with either Hoagland’s nutrient solutions (control group), 200 mM NaCl, or 200 mM NaCl + 15.0 μM NaHS for a week were harvested. Total RNA of cucumber leaves or roots (100 mg) was isolated by grinding with mortar and pestle in liquid nitrogen to a fine powder and using TriZol Reagent (Invitrogen) in accordance with the manufacturer’s protocol. The total RNA samples were treated with RNAase-free DNase  to eliminate traces of DNA, followed by quantification using the NanoDrop 2000 . Total RNA (2 μg) was reverse-transcribed using an oligo d (T) primer  and M-MLV reverse transcriptase  . Real-time quantitative RT-PCR reactions were performed using an ABI 7000 (Applied Biosystems) with SYBR®Premix ExTaqTM  and the cycling conditions of denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 15 s. Using specific primers . Three independent replicates were performed for each sample. The comparative threshold cycle (Ct) method was used to determine the relative amount of gene expression. Relative gene expression levels were calculated via the 2-ΔΔCT method. The relative gene expression was determined as previously described by To study the effect of NaHS treatment on the expression of the 4 and 0.50 mL of 15% K4Fe(CN)6⋅3H2O. The volume of the extract was adjusted to 10.0 mL with 1% oxalic acid, followed by centrifugation at 10, 000 × g for 10 min. Then 3 mL of the supernatant was mixed with 2.0 mL of dye solution (0.25% DCIP and 0.20% NaHCO3) and 3 mL of dimethylbenzene. The absorbance was read at 500 nm, and the content of ASA was calculated using a standard curve. Total GSH was estimated using a kit . The absorbance of GSH was measured at 420 nm according to manufacturer’s instructions. CAT activity was measured according to the methods described by 2O2. H2O2 was assayed from the decrease in absorbance in optical density at 240 nm, and the activity was calculated using the extinction coefficient of 40 mM cm-1 of H2O2. GR activity was determined by measuring the absorbance change at 340 nm due to oxidation of NADPH (The ASA content was determined using the DCIP method. Samples (0.50 g) were homogenized in 3.0 mL of 2 % oxalic acid supplemented with 0.50 mL of 30 % ZnSOof NADPH . SOD andP < 0.05 level.Data were analyzed by one-way ANOVA using SPSS . Different letters on the graphs or in the tables indicated that the mean values were statistically different at the P < 0.05) reduced the survival rate by 89 %, the rate of expansion of the second leaf by 62 % (P < 0.05) affected general growth (P < 0.05) reduced by 66%, 58%, 65%, 43%, 50%, 53%, 90%, and 48% under salt stress, respectively (P < 0.05) reduced shoot fresh weight, root fresh weight, seedling fresh weight, seedling total length, shoot dry weight, root dry weight, leaf area and root length by 28%, 40%, 31%, 14%, 17%, 42%, 41%, and 15%, respectively, as compared to the control  decline in the Pn (by 14.3%) and a sharp decline in the Tr (by 94%) were recorded in the leaves of salt-stressed cucumber plants (n (by 2.4%) and Tr (by 90.4%) compared with the control, respectively  reduced Fv/Fm (by 6%), Fv/Fo (by 25%),ΦPSII (by 7%) and qP (by 12%) compared with the control, while 15 μM NaHS treatment reduced these by 0.5%, 4.5%, 2% and 3.1%, respectively, compared with the control. In contrast, comparing with the control, the parameters of Fo and NPQ significantly (P < 0.05) increased by 39% and 78% under salt stress. As expected, 15 μM NaHS significantly reversed the increase in Fo (by 10.4%) and NPQ (by 34.2%) under salt stress  increased electrolyte leakage by 1.1 fold in leaves  (2S by 9.9% in leaves (P < 0.05) decreased the activity of L-CD by 19% in leaves (P < 0.05) improved the activities of L-CD (38.7% in roots) and D-CD (by 52.7% in leaves and 4.6 fold in roots) compared with the control   and root(by 49%)  of salt-n leaves  and by 9n leaves  comparedn leaves  and 12% n leaves . The samn leaves  and 33 %n leaves , As expe control –F. TheseP < 0.05) improved the activity of CAS in leaves by 19% (P < 0.05) increased in both leaves (by 65%)   and root(by 65%)  under sa(by 65%) .+/K+ ratio is critical to its ability to tolerate salt stress. Compared with the control, a sharp increase in the Na+ content was observed in leaves (by 2.13 fold) and roots (1.35 fold) under salt stress (+ content was recorded in leaves (by 30%) and roots (by 41%) under salt stress as compared to the control (+/K+ ratio significantly increased in leaves (by 3.4 fold) and roots (by 3 fold) of salt-treated plants than the control  and the activities of antioxidant enzymes  were analyzed in leaves and roots of cucumber plants. As show in To evaluate the role of H2S donor NaHS, numerous results demonstrated that H2S participates in plant adaptive responses against multiple abiotic stresses  H2S increases endogenous H2S levels via increasing the activities of L-CD and D-CD and decreasing the activities of CAS and OAS-TL in leaves or roots; (2) H2S maintains the Na+ and K+ balance through regulating the expression of +-ATPase, SOS1PM H and SKOR; and (3) H2S enhances the accumulation of antioxidants and the activities of antioxidant enzymes, leading to decline ROS accumulation and membrane lipid peroxidation. As summarized in 2S alleviates salt stress in cucumber through cross talk among the mechanisms that maintain Na+/K+ homeostasis and regulating H2S metabolism and oxidative stress response. In addition, the roots and leaves of cucumber showed different responses to exogenous H2S treatment. Taken together, suitable concentration H2S could be used to alleviate NaCl-stress induced toxicity in cucumber seedlings.In conclusion, exogenous application of HJ-LJ designed the experiment and wrote the manuscript. YT and X-MR conducted the experiment. MY and R-PH helped in data analysis and presentation. LL revised the manuscript. All authors listed have made a substantial, direct and intellectual contribution to the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cerrena unicolor BBP6 fused with α-factor prepro-leader that was engineered through random mutagenesis followed by in vivo assembly in Saccharomyces cerevisiae. Three evolved fusion variants selected from ~3500 clones presented 31- to 37-fold increases in total laccase activity, with better thermostability and broader pH profiles. The evolved α-factor prepro-leader enhanced laccase expression levels by up to 2.4-fold. Protein model analysis of these variants reveals that the beneficial mutations have influences on protein pKa shift, subunit interaction, substrate entrance, and C-terminal function.Laccases have great potential for industrial applications due to their green catalytic properties and broad substrate specificities, and various studies have attempted to improve the catalytic performance of these enzymes. Here, to the best of our knowledge, we firstly report the directed evolution of a homodimeric laccase from  Laccase is a multicopper oxidase that is capable of oxidizing a wide range of substrates, including phenol and aromatic compounds, using only oxygen from air. Laccases are considered as green catalysts of great biotechnological potential with applications in different industrial processes . In the catk of the engineered variant was improved two-fold after evolution. In another case, the sinapic acid binding pocket of a high redox potential chimeric laccase was delimited by performing iterative saturation mutagenesis (ISM) on six residues. The new variants showed altered pH activity profiles and increased turnover rates for sinapic acid and its methyl ester . L. LCerrenS. cerevisiae to generate the best combinations. This strategy reduced random mutagenesis to one round and significantly minimized the library size for site-directed mutagenesis. If specific activity towards ABTS can be further improved, then the present strategy will be more reasonable and convincing.In the present study, random mutagenesis with a low mutation rate followed by in vivo assembly was demonstrated. A mutagenic library with a mutation rate of 0–4.5 mutations per 1000 bp was generated by error-prone PCR (epPCR). Thereafter, a few positive variants were selected, and limited mutations were identified as positive on each variant. Finally, all of the positive mutations were re-assembled in http://www.kazusa.or.jp/codon/), four of the six synonymous mutations favored codon usage in S. cerevisiae, which improved the translation and expression of the laccase gene in yeast [A random mutagenic library was generated by epPCR while using the GeneMorph II Random Mutagenesis kit  B. Approxin yeast ,26.S. cerevisiae was used as a cell factory rather than an expression host for the final evolved laccase, all of the synonymous mutations would not be integrated into the laccase gene for the next round of evolution despite the fact that these mutations might contribute to activity improvements. As the objective of evolution is performance improvement instead of function alteration, mutations are predicted to occur in non-conserved regions [Since  regions ,28. A coS. cerevisiae machinery to ligate fragments and assemble mutations [According to the results that are shown in utations . These tS. cerevisiae was almost constant, 15.1 ± 1.5 µg/mL, for the parent-type laccase and its variants. However, the laccase activity varied from ~0.3 to ~12 U L−1. Because of such low protein content, crude enzymes were used for laccase characterization and kinetic studies. The pH profiles, thermostability, and kinetics parameters of 13 selected variants were investigated.Throughout the experiments, the protein content in the crude enzyme that was generated by Cerrena unicolor BBP6, and 41.8% relative activity was detected at pH 4.0  in S. cerevisiae. To this end, the supernatant of the positive variants obtained from two-round evolution was kinetically characterized. All of the kinetics parameters and specific activities of parent-type and mutated laccases to ABTS are listed in mK), turnover rate (catk), and catalytic efficiency (catk/mK) were 205.7 µM, 2208.2 s−1 and 10.9 µM−1 s−1, respectively for the recombinant αLacA expressed in S. cerevisiae. Regardless of the mutant libraries, the mK values of the most evolved laccases were enhanced to 60–90 µM, except for the mutants RM811, RM1905, and RM2067, which displayed mK values over 140 µM. Therefore, R157P and I486M were screened out after assembly. The mutants RM243 and RM1524 displayed 3.9- and 3.5-fold increases in catk and 11.9- and 12.3-fold increases in catk/mK, respectively, when compared to those of the parent type. As shown in mK) was 69.8, 85.3 and 64.0 µM, respectively. The turnover rates (catk) of such variants were 9.3-, 9.4-, and 7.3-fold greater, respectively, than that of the parent-type laccase. Accordingly, the catk/mK values were increased up to 27.0-fold. The above results suggest that the performance of the mutants is not proportional to the number of substitutions. Interestingly, the kinetics performance of the laccase variants towards ABTS was considerably enhanced, while their affinity constants were similar to those of other fungal laccases [The kinetics parameters of the evolved laccases were investigated at pH 4.0 using ABTS as the substrate. Unfortunately, we were not able to obtain purified laccases due to the low expression level of laccase  from the α-factor library was tested to investigate its effect on the secretion of the evolved laccase mutants. The best three evolved variants  were fused to the evolved α-factor. The fusion genes were generated by in vivo ligation and named αF7I-189 (αF7I/V160D/T292N/A491S), αF7I-205 (αF7I/V160D/T290N/T292N/P303-S/A491S), and αF7I-264 (αF7I/V160D/T290N/T292N/P303S/T412S). As shown in −1 after 27 h of cultivation, which was 2.4-fold that of native αLacA. Similar results were obtained with αF7I-189 and αF7I-205, showing approximate 1.5-fold total activities while using the evolved α-factor. Compared with that of the parent-type αLacA, the TAI were improved 33-fold and 37-fold, respectively  and the energy of intra-residue van der Waals torsional clashes . The energy term of van der Waals clashes may represent physically relevant unfavorable interactions, as well as errors that are inherent to the geometry of the homology model [After repairing the protein modeling structures, the free energy of folding for the parent laccase (ΔG) and the relative Gibbs free energies of folding between the variants and the parent laccase (ΔΔG) were calculated by using FoldX, and the results are listed in gy model . The lowIn addition to ΔG, the pKa value is also critical to the pH dependence of enzyme . In the mK (RM1524).The homodimeric structure of LacA indicates any mutations in the gene will result in substitutions on both subunits. As shown in T785N and T787N (T290N and T292N) were located at the interface of the two subunits and at the sides of substrate entrance (mK of mutant RM2067 (αF7I/P303S) (mK and an increase in the catk as compared to those of the parent type.Pro303 (798) is located in a turn next to the entrance of the substrate channel, and Thr412 (907) is located in a neighboring coil in the same subunit (I/P303S) . FurtherMelanocarpus albomyces revealed that the extension of the C-terminus opens the solvent channel for the entrance of oxygen molecules and the subsequent exit of water molecules [Ala491 (986) is located in the C-terminal coil at the protein surface  was amplified by using Marathon cDNA Amplification Kit . Gene lacA fused with prepro-leader of α-factor  and an ampicillin resistance shuttle vector pYES2-αlacA under the control of GAL1 promoter was obtained from a previous experiment in the lab. Protease-deficient S. cerevisiae BJ5465, ATCC 208289  was used as the host for laccase heterologous expression. Escherichia coli DH5α  cells were used for plasmid amplification.A novel w/v) sterile yeast nitrogen base, 1.92 g yeast synthetic dropout medium supplement without uracil, 20 g Bacto agar, 100 mL 20% (w/v) sterile raffinose, 100 mL 20% (w/v) sterile galactose, 1 M sorbitol, 0.2 mM CuSO4, 0.4 mM ABTS and double-distilled H2O (ddH2O). The growth medium (1 L) contained 50 mL 13.4% (w/v) yeast nitrogen base, 1.92 g of yeast synthetic dropout medium supplement without uracil, 100 mL sterile 20% (w/v) raffinose, and 850 mL ddH2O. The expression medium had the same composition as the screening medium without Bacto agar, sorbitol and ABTS. All the chemicals were of the analytical grade and they were obtained from Sigma-Aldrich (Singapore) unless otherwise stated.The screening medium that was used for selective plates (1 L) contained 50 mL 13.4% (lacA as the template. The library was constructed at a mutation rate of between 0 to 4.5 mutations per 1000 bp. Error-prone PCR (epPCR) was performed in a T100 thermocycler  at a final volume of 50 µL containing 500 nM each primer, 814 ng template, dNTPs (0.2 mM each), and 2.5 units of Mutazyme II DNA polymerase. PCR was performed under the following conditions: 95 °C for 2 min (1 cycle); 95 °C for 0.5 min, 50 °C for 0.5 min, and 72 °C for 2 min (30 cycles); and, 72 °C for 10 min (1 cycle). The primers OL_pYEα-F and OL_lac-R (EcoRI and XbaI) were purified and concentrated after electrophoresis by using a gel extraction kit . Subsequently, 300 ng of PCR fragments were mixed with the linearized vector (100 ng) and transformed into S. cerevisiae BJ5465 competent cells by using the Gene Pulser Xcell System . All of the transformed cells were spread onto selection plates and incubated for three days at 30 °C.A mutagenic library  was generated with a GeneMorph II Random Mutagenesis kit  using αOL_lac-R  used forS. cerevisiae taking advantage of its efficient DNA in vivo homologous recombination capacity [Libraries were constructed by in vivo assembly in capacity . PrimerslacA (without α-factor) for in vivo ligation was created by digesting vector pYES2-αlacA with EcoRI and XhoI  at 37 °C for 3 h. The fragments αF2 and pYES2-lacA were further analyzed on an agarose gel by electrophoresis and purified by using a gel extraction kit . The mixture of αF1 (17 ng), αF2 (30 ng), and pYES2-lacA (100 ng) was transformed into S. cerevisiae BJ5465 competent cells by using the Gene Pulser Xcell System. All of the transformed cells were spread onto selective plates and incubated for three days at 30 °C.The short fragments αF1 was generated by modified PCR using degenerate primers αF_1F and αF_1R. PCR was conducted in a final volume of 20 µL containing 5 µM each primer, dNTPs (0.2 mM each), and five units of Bestaq DNA polymerase  Inc., Vancouver, BC, Canada). The PCR conditions for αF1 were 94 °C for 3 min (1 cycle); 60 °C for 0.5 min; and, 72 °C for 0.5 min (1 cycle); 72 °C for 5 min (1 cycle). The 20 µL of reaction mixture was directly used for transformation. The fragment αF2 was amplified using degenerate primers αF_2F and αF_2R . The 50 lacA fragments  was achieved using the same procedure of αF2 amplification, as previously described. The primers used are listed in lacA with XhoI and XbaI  at 37 °C for 3 h. The mixture of lac1 (50 ng), lac2 (40 ng), lac3 (30 ng), lac4 (25 ng), and pYES2-αfactor (100 ng) was transformed into S. cerevisiae BJ5465 competent cells using the Gene Pulser Xcell System. All of the transformed cells were spread onto the selective plates and incubated at 30 °C for three days.Amplification of short HTP screening was performed according to previous reports, with some modifications .S. cerevisiae transformants were selected on selection plates containing ABTS. Colonies showing green halos after three-day incubation at 30 °C were sub-cultured on 96-well microplates  for high-throughput screening.Positive g and room temperature for 3 min, and 20 μL aliquots of the supernatant were transferred to new plates. A mixture of one millimolar ABTS in 100 mM acetate buffer (pH 4.0) was brought to a final volume of 200 μL, and the absorbance was measured at 420 nm  in kinetics mode in a microplate reader . To confirm the results, positive variants having higher laccase activity than the parent type were selected for the second batch of screening. Each positive variant was grown in three wells, wells H7–H9 were inoculated with the parent laccase, and wells H10–H12 were not inoculated. The plates were assessed by using the same cultivation and assay protocols described above.Colonies selected from pre-screening were transferred with sterile crystal tips to a 96-well microplate containing 50 μL of growth medium in each well. After 1–2 days of incubation at 28 °C and 700 rpm in a thermo-shaker , 150 μL of the expression medium was added, and the plates were incubated for another 2–3 days. Each variant was grown in two wells; wells H9–H10 were inoculated with the parent type, and wells H11-H12 were not inoculated. The plates were sealed with Parafilm to prevent evaporation. To determine laccase activity in each well, the plates were centrifuged at 4500× 600 of 0.4. The flasks were incubated at 28 °C and 200 rpm for 1–2 days. Crude broth was harvested, and the cells were precipitated by centrifugation at 4500× g for 5 min. The supernatant was used for enzyme activity assays, characterization, and plasmid extraction.The best variants selected from the second batch of microplate screening were inoculated to a 50 mL centrifuge tube containing 5 mL of the growth medium and were incubated at 30 °C and 200 rpm for two days. An aliquot of the cells was transferred to 20 mL expression medium in a 100 mL flask to an OD−1 cm−1) at room temperature. One unit of enzyme activity (U) was defined as the amount of enzyme required to produce 1 µmol oxidized substrate per min.The activity of laccase was determined according to the previously published methods, with slight modifications . A mixtuPolyacrylamide gel electrophoresis under non-denaturing conditions (native-PAGE) was conducted with a 10% Mini-PROTEAN TGX precast gel  by using the Precision Plus Protein Dual Color Standards  in a Mini Protein Tetra System  according to the manufacturer’s instructions. The laccase band was visualized by incubating the gel in 1 mM ABTS and 100 mM sodium acetate buffer (pH 4.0) at room temperature for 20 min.The optimal pH for laccase activity was evaluated by the varying pH values in 100 mM citrate buffer (pH 2.0–3.0) and 100 mM acetate buffer (pH 3.5–5.5) with ABTS as the substrates. The supernatants (30 µL) of the best variants mixed with 180 µL of 100 mM acetate buffer (pH 4.0) containing 1 mM ABTS were used to determine the laccase activity. Oxidation of ABTS was spectrophotometrically monitored at 420 nm at room temperature in the microplate reader.The thermostability of the best variants were investigated by incubating supernatants for 0, 5, 10, 15, 30, 45, and 60 min at 50 °C, followed by chilling samples at 4 °C for 10 min and incubating them at room temperature for an additional 5 min. Subsequently, the residual laccase activity was measured, as described above.The substrate specificities of the variants were estimated by measuring the absorption change at 420 nm in a final volume of 210 µL containing 30 µL supernatant, 100 mM acetate buffer, and 10–1000 µM ABTS at pH 4.0 and 30 °C. The kinetics parameters of the Michaelis-Menten equation were calculated according to Lineweaver-Burk plots. All the assays were conducted in duplicate wells on one 96-well plate.lacA were extracted  and transferred into E. coli DH5α cells. The E. coli transformants were spread onto Luria-Bertani (LB) agar plates with 200 µg L−1 ampicillin as the selective marker. Subsequently, single colonies were selected and used to inoculate 5 mL LB ampicillin medium overnight at 37 °C and 200 rpm. Subsequently, the plasmids were extracted . Pure plasmids were sequenced by using a BigDye Terminator (version 3.1) cycle sequencing kit  and were performed at a DNA Sequencing Service . The primers used for sequencing were designed by using Primer 5 software (http://www.premierbiosoft.com/primerdesign/) and are listed in Plasmids with the evolved αEcoRI and XbaI. Fragment α-factor (20 ng), evolved laccases (100 ng), and pYES2 backbone (100 ng) were mixed and transformed into S. cerevisiae BJ5465. The PCR conditions, electroporation methodology, transformants selection, and inoculation were previously described. Flasks were incubated at 28 °C and 200 rpm for 30 h. Aliquots of the culture supernatant were sampled at different time intervals to measure the enzyme activity.The evolved prepro-leader of α-factor and the evolved laccases were amplified using the primers αF54_F/R and Lac_uni_F/R, respectively. The linearized plasmid backbone was generated by double digestion using https://www.ncbi.nlm.nih.gov/) with the parent laccase was conducted using ClustalW . The 3D structure of the evolved and parent-type laccase LacA were generated by homology modeling using DeepView Project Mode on SWISS-MODEL web server (http://swissmodel.expasy.org/). The template laccase with 68.7% identity to LacA was obtained from Lentinus sp. at 1.8-Å resolution (PDB ID: 3X1B) according to similarity alignment of the proteins.Alignment of 100 laccase proteins that were obtained from NCBI (http://www.yasara.org/) by using FoldX (version 4.0) [M − ΔGPT. Here, ΔGM and ΔGPT are the free energies of folding for the mutant and the parent-type proteins, respectively [The 3D models of the parent-type and evolved laccase were analyzed with YASARA viewer (ion 4.0)  as a pluectively .M − pKaPT. Here, pKaM and pKaPT are, respectively, the pKa values for the mutant and parent-type proteins.Calculation of pKa values was performed with the DelPhiPKa web server, in which a Poisson-Boltzmann based approach was used to calculate the pKa values ,45. The The solvent accessible surface of the protein structure is shown by PyMOL viewer using repaired protein models.S. cerevisiae. This approach reduced the workload and thereby the screening time. Positive variants with up to 37-fold TAI at pH 4.0 with better thermostability and broader pH profiles were obtained. Furthermore, the fusion of α-factor prepro-leader and mature laccase made laccase expression levels detectable [Laccases are being increasingly evaluated for use in a variety of biotechnological applications due to their green catalytic properties and broad substrate specificities ,41. In ttectable . The pro


Deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and cluster of differentiation 31 (CD31) staining were used to assess cell death and vascular content, respectively. Further, tumours receiving a single combined USMB and HT treatment exhibited decreased tumour volumes (p<0.05) compared to those receiving either treatment alone when monitored over the duration of 30 days. Additionally, tumour response monitored weekly up to 4 weeks demonstrated a reduced vascular index and tumour volume, increased fibrosis and lesser number of proliferating cells with combined treatment of USMB and HT. Thus in this study, we characterize a novel therapeutic approach that combines USMB with HT to enhance treatment responses in a prostate cancer xenograft model in vivo.It is now well established that for tumour growth and survival, tumour vasculature is an important element. Studies have demonstrated that ultrasound-stimulated microbubble (USMB) treatment causes extensive endothelial cell death leading to tumour vascular disruption. The subsequent rapid vascular collapse translates to overall increases in tumour response to various therapies. In this study, we explored USMB involvement in the enhancement of hyperthermia (HT) treatment effects. Human prostate tumour (PC3) xenografts were grown in mice and were treated with USMB, HT, or with a combination of the two treatments. Treatment parameters consisted of ultrasound pressures of 0 to 740 kPa, the use of perfluorocarbon-filled microbubbles administered intravenously, and an HT temperature of 43°C delivered for various times (0–50 minutes). Single and multiple repeated treatments were evaluated. Tumour response was monitored 24 hours after treatments and tumour growth was monitored for up to over 30 days for a single treatment and 4 weeks for multiple treatments. Tumours exposed to USMB combined with HT exhibited enhanced cell death ( This resulted in a protective effect, diminishing cell death, due to the activation of several heat shock proteins (HSPs), which were overexpressed in the tumours after HT treatment  [16 [1615] . More spin vivo and in vitro  [21 [2120] . Previouin vitro . Thus onin vivo prostate cancer xenograft model was investigated. Tumour responses were assessed at 24 hours and longitudinally with single treatments and multiple treatments for up to over 30 days and 4 weeks respectively. Histopathological techniques including the terminal dUTP nick-end labeling (TUNEL) for cell death, cluster of differentiation 31 (CD31) for vascular index, Masson's trichrome staining for fibrosis, and Ki-67 staining for cell viability were used to characterize tumour response. The combination of USMB with HT resulted in increased cell death, decreased vascularity and superior tumour growth inhibition when compared to USMB or HT alone for 24 hour cohort animals. Additionally, long-term data from combined USMB and HT treatment demonstrated a reduced vascular index and decreased tumour volume. Further the results indicated areas of fibrosis in addition to a reduction of proliferating cells with combined treatment.In the work here, the combined effect of USMB with HT in an 6 cells.Prostate cancer cells (PC3) from the American Type Culture Collections  were maintained in RPMI1640 medium from Multicell (cat# 350–000), containing 10% FBS  and 1% Penicillin-Streptomycin (Gibco 15140). Cells were allowed to reach confluency while incubated at 37°C and 5% CO2. In preparation for injection, cells were washed with PBS, detached and collected using 0.05% Trypsin-EDTA (v/v)  at room temperature. Cells were centrifuged at 200g for 10 min at 4°C and cell pellets were isolated and re-suspended in 100 μl phosphate buffered saline (PBS) per 5×106 PC3 cells subcutaneously in the hind leg of male severe combined immuno-deficient (SCID) CB-17 mice . The tumours were allowed to grow for 3–4 weeks, at which point they reached approximately 8–10 mm in size.Tumours were induced by injecting 5 × 10All mice were anesthetized prior to treatment by an intraperitoneal injection of a mixture consisting of ketamine (100 mg/kg), xylazine (5 mg/kg) and acepromazine (1 mg/kg) . Anesthetized mice were monitored visually and kept near heat lamps to maintain mouse body temperature.in vivo animal experiments were conducted in accordance with policies of the animal care committee at Sunnybrook Health Science Centre (Comparative Research), under animal use protocol # 18–395 and in accordance with the Canadian Council on Animal Care Guidelines. This study was approved by the animal care committee at Sunnybrook Health Science Centre (Comparative Research), at Sunnybrook Health Science Centre, Toronto, Canada. Animals were monitored and handled properly throughout the experimental procedure to minimize suffering or pain. After experiments animals were sacrificed using standard operating procedures stated by comparative research that included anesthetic overdose and cervical dislocation.All In this study, peak negative ultrasound pressures of 0 kPa, 126 kPa, 246 kPa, 570 and 740 kPa were used with MB and various HT treatment durations including 0, 10, 20, 30, 40 and 50 min were tested. Experiments consisted of a short-term study lasting 24 hours, where cell death and vascular effects on tumour were assessed, and two long-term studies (over 4 weeks each), where effects on tumour growth delay were observed with repeated measurements of tumour size. For the 24 hour study, all permutations of the above parameters were tested and conditions were selected for multiple treatment experimentation. In those experiments 5 animals per group were used with 30 conditions for 150 animal’s total. In the long term studies one study used 25 animals (n = 5 per group) for evaluation of single treatments over 4 weeks.For the long term studies, only ultrasound pressures of 0 kPa and 570 kPa and HT treatment durations of 0 min and 50 min were used. The other study here used 4 experimental treatment groups with 20 animals in each group with 5 animals sacrificed each week. This then used 20 animals treated for 1 week, 15 animals treated for 2 weeks, 10 animals treated for 3 weeks, and 5 animals treated for 1 week) for an additional 80 animals in the study. In total this works overall used 255 animals.Definity® bubbles  were activated by shaking using a Vialmix®  device at 3000 rpm for 45 seconds. MB was administered to a final concentration of 1% (v/v). The MB solution was injected intravenously in mice using a 26-gauge tail vein catheter and was followed immediately by a 150 μl 0.2% heparin/saline flush. During the time of injection, mice were mounted using a custom-built mounting plate and partially submerged in a degassed water bath at 37.5°C, ensuring the tumour bearing limb was completely immersed in water.For the ultrasound-stimulated MB procedure, an ultrasound transducer with 500 kHz central frequency  and an aperture diameter of 28.6 mm was used. The transducer was focused at 85 mm with a focal zone -6 dB beam-width of 31 mm. Animals received a 5 min exposure to 16 cycles tone burst at 500 kHz with a 3 kHz pulse repetition frequency and with a total ultrasound exposure time of 750 milliseconds.Hyperthermia treatment was administered using a 43°C water bath. Mice were mounted upright in a tube with an opening at the closed end to provide an air vent and an opening in the lid for the leg and tail to pass through. The tail of the mouse was taped to the side of the tube so only the tumour-baring limb was submerged in the water. Hyperthermia exposure time ranged from 0 to 50 min.Tumours were excised 24 hours after treatments, dissected in half and fixed for 2 hours in room temperature in 1% paraformaldehyde (PFA). Subsequently, they were immersed in 70% ethanol (v/v) for 48 hours at 4°C. Fixed specimens were embedded in paraffin blocks and sectioned (5 μm thick) and placed on glass slides for staining.For the detection of apoptosis, TUNEL staining, a widely accepted in situ technique that labels nuclear DNA fragmentation was used. The stained sections were then digitized using a low-magnification light microscope . The cell death percentage was quantified from digitized TUNEL image files using an in-house program developed in MATLAB  . For theColor-based segmentation of histologically-stained digital images was performed in order to quantify objectively the amount of fibrosis and tumour cell proliferation, associated with USMB and HT therapy. Automated analysis was performed using the roicolor method in MATLAB to select ROI based on the specified color threshold. Masson’s trichrome-stained histological images depict areas with collagen fiber deposits, resulting from fibrosis, as regions with blue/green color. The percentage of collagen was obtained by dividing the number of pixels with blue/green color by the total number of pixels in the image. Similarly, cell proliferation marker Ki-67-stained images depict the nuclear area in actively proliferating cells as a brown region. The Ki-67 labeling index was obtained by dividing the number of brown pixels with a total number of pixels in the image.Longitudinal studies were performed in order to verify the efficacy of multiple combined treatments compared to either USMB or HT alone and were composed of two experimental cohorts. The first was conducted over 4 weeks at the start of which each animal received a single treatment consisting of either USMB only, HT only, or a combined treatment consisting of USMB + HT (30 min) or USMB + HT (50 min) . A seconFor the multiple treatment studies , only on2/2). The change in volume for each tumour measurement was calculated relative to initial tumour volume for over 30 day-study and the means and standard errors were plotted for each treatment for 4 weeks study.Tumour growth was assessed for long term cohort studies, which lasted for over 4 weeks for the single treatment group and a similar time frame for the multiple treatment groups. Tumour size was measured using digital calipers every 2–3 days and weekly in order to determine the length (L) and width (W). Tumour volume was determined by using the modified ellipsoidal formula  were regarded as statistically significant and are indicated by asterisk*.Statistical analysis was conducted using Graph Pad/InStat 3.0). Statistical significance was determined by analysis of variance (ANOVA) followed by a Bonferroni’s selected comparisons test. This work aimed to study the acute (after 24 hours) and long-term effects (at 4 weeks) of USMB and HT in a mouse model of prostate cancer (PC3). For the 24 hour cohort, animals were submitted to treatment with USMB consisting of 1% (v/v) Definity MB stimulated at various ultrasound pressures of 0, 126, 246, 570 and 740 kPa followed with heat treatment, 43°C for 0, 10, 20, 30, 40, and 50 min. A total of 30 different treatment conditions were investigated and a total of 150 animals  were included for the 24 hour cohort. For the 4 week cohort, animals were divided into five groups consisting of USMB (n = 4), 30 min HT (n = 5), USMB+ 30 min HT (n = 5), 50 min HT (n = 5), and USMB+ 50 min HT (n = 5). For this cohort, a total of 4–5 animals per week were included for each treatment group including control, USMB, 50 min HT, and USMB+50 min. Untreated mice were labeled as control (0 kPa+ 0 min).p≤0.05) when compared to the untreated control (1.80 ± 0.37%), 10 min heat only (4.93 ± 1.62%) and 40 min heat only (9.64 ± 2.43%) treatments (p≤0.05) and 50 min (52.08 ± 4.82%) (p≤0.05) heat caused a significant increase in cell death compared with the same USMB treatment combined with 0 min (2.55 ± 0.92%), or 10 min (4.32 ± 1.99%) of heat. Furthermore, USMB treatments at 570 kPa combined with 40 min of heat exhibited a significantly higher amount of cell death (27.50 ± 3.66%) (p≤0.05) compared to 0 min (5.84 ± 3.63%) and 10 min (6.30 ± 2.60%) of heat. At the same pressure, 50 min heat did not cause any further drastic increases in cell death (30.07 ± 10.07%) compared to 40 min (27.50 ± 3.66%), however the cell death index was significantly higher than what was observed at 0 min (5.84 ± 3.63%) and 10 min (6.30 ± 2.60%) of heat. Comparisons were also made to verify the differences in cell death between the different treatment conditions. Fifty minutes of heat produced a significant level of cell death regardless of ultrasound pressure, indicating that this duration of heat application is not enhanced by the USMB treatment. A 40 min heat treatment, however, is significantly enhanced (p<0.05) by USMB at a pressure of 570 kPa.Results indicated an increase in cell death with increasing HT treatment duration. Cell death, confirmed by the presence of dark brown regions in TUNEL stained tumour sections  increaseeatments . SimilarInterestingly, 30 min of heat combined with 126 kPa indicated significant cell death  however p≤0.05) of heat compared to the untreated control (VI = 19.63 ± 2.42). In the USMB treated tumours, at 246 kPa, a significant reduction in vascular labeling was observed with 40 min (VI = 12.93± 0.6960) (p≤0.05) and 50 min (VI = 11.10 ± 1.156) (p≤0.05) compared with 0 min (VI = 21 ± 3). At 570 kPa, 40 min (VI = 7.380± 2.460) (p≤0.05) and 50 min (VI = 7.933± 1.184) (p≤0.05) of heat reduced the vascular labeling significantly when compared to 0 min (VI = 17.43 ± 2.185). When comparing between the different ultrasound pressures, the combined treatment at 570 kPa with 40 min of heat indicated decreased vascularity (VI = 7.380± 2.460) (p≤0.05), which was significant compared to 40 min of heat alone (VI = 17.20 ± 3.811). Further increases in the heating time did not change the effect on the vasculature while heating times of 20 and 30 min had effects intermediate to 10 and 40 min. The lower and higher pressures used, 126 kPa, and 740 kPa showed a similar trend of reduction in vascular labeling as 570 kPa (The disruption of blood vessels resulting from USMB and HT treatments was investigated using CD31 immunohistochemistry . In the  570 kPa .p≤0.05) compared to the group receiving 30 min of HT only with a fold change of (3.98 ± 0.19). Similarly, this persisted to day 24, 28 and 32 with a fold change of (3.21 ± 0.29) (p≤0.05), (4.12 ± 0.30) (p≤0.05) and (4.81 ± 0.35) (p≤0.05) respectively compared to 30 min of HT only with a fold change of (4.52 ± 0.40) at day 24, (6.18 ± 1.09) at day 28 and (7.00 ± 1.24) at day 32. Within the group receiving the combined treatment of USMB + HT (50 min), tumour growth was delayed longer, for example at day 16, tumour volume fold change was found to be (1.26 ± 0.14) (p≤0.05), when compared to the group receiving 50 min of HT only with a fold change of (3.02 ± 0.23). Tumours grew more slowly after the combined treatment USMB + HT (50 min) with a fold change at day 20 (1.73 ± 0.26) (p≤0.05), day 24 (2.53 ± 0.50) (p≤0.05), day 28 (2.92 ± 1.19) (p≤0.05) and day 32 (4.00 ± 1.12) (p≤0.05) compared to HT (50 min) alone with a fold change of (3.96 ± 0.24), (4.50 ± 0.19), (4.93 ± 0.24) and (5.41 ± 0.83) at day 20, 24, 28 and 32 respectively.In the single treatment over 30 day-study , tumour p≤0.05) compared to those receiving USMB only (1.06 ± 0.40%) or HT (50 min) only (4.50 ± 1.88%).Quantification of TUNEL staining indicated tumours receiving USMB only treatment demonstrated no appreciable increase in areas of cell death at weeks 1, 2, 3 and 4. Further in the heat (50 min) only treated tumours, negligible area of cell death was visible at weeks 1, 2, 3 and 4. Tumours receiving the combined treatment of USMB + heat (50 min) exhibited large areas of cell death at week 1, however, this was less with subsequent treatments. Specifically, after one week of treatment, a significant increase in cell death was observed in tumours receiving the combined treatment of USMB + HT (50 min) (17.87 ± 7.36%) (p≤0.05), week 2 (VI = 7.47 ± 0.19) (p≤0.05), week 3 (VI = 6.90 ± 1.88) (p≤0.05) and week 4 (VI = 7.70 ± 0.70) (p≤0.05) compared to USMB only indicating vascular labeling at week 1 (VI = 11.31 ± 1.12), week 2 (VI = 11 ± 0.59), week 3 (VI = 10.65 ± 0.50) and week 4 (VI = 15.38 ± 2.19). Differences compared to HT alone were less  at week 1 (VI = 6.92 ± 0.87)  and (462.3 ± 47.52 mm3) at week 4 compared to pre-treatment (217.6 ± 42.07 mm3) and (208.1 ± 9.74 mm3) respectively. However, in the case of HT alone, no significant changes in tumour volume were noticed at week 1, 2 and 3. At week 4 reduction in tumour volume was observed but it was not statistically significant compared to pre-treatment. Combination treatment USMB+ HT (50 min) resulted in substantial tumour volume reduction with (103.0 ± 27.16 mm3) (p≤0.05) at week 2, (135.5 ± 46.99 mm3) (p≤0.05) at week 3 and (161.2 ± 6.123 mm3) at week 4 compared to pre-treatment group (212.8 ± 49.05 mm3).Tumour size after multiple treatments experiments was assessed up to 4 weeks after treatment . Mean tuSpecifically, since tumours receiving the combined treatment did not indicate any significant cell death, Masson's trichrome staining of tumour sections was performed to test whether the cells converted viable tissue to fibrosis resulting in collagen deposition. Fibrosis was characterized by staining for the accumulation of abnormal extracellular matrix components resulting in enormous tissue repair. Trichrome staining is mostly used to detect the presence of collagen . Trichrop≤0.05) at week 1, (0.13 ± 0.04%) (p≤0.05) at week 2, (0.20 ± 0.02%) (p≤0.05) at week 3 and (0.16 ± 0.05%) (p≤0.05) at week 4 compared to USMB. A significant reduction in Ki-67 index was observed at week 4 when compared between HT alone and USMB + HT (50 min).In order to better characterize treatment effects on cell activity, Ki-67 immunostaining was conducted to analyze the fraction of tumour cell proliferation and growth for further investigation of multiple treatment effects on tumour regression . TreatmeSeveral studies have demonstrated that ultrasound-stimulated microbubble (USMB) treatment can cause the mechanical disruption of tumour vasculature resulting in enhanced cell death  14] [15 [1514]  17]. A . A 17]. Despite the wide use and effectiveness of radiotherapy and chemotherapy to sensitize tumours, surviving tumour cells often acquire resistance towards these therapies  31]. Mo. Mo31]. Thus in this study, the rationale for combining USMB with HT is to enhance the efficacy of both modalities by targeting tumour vascular endothelium contributing overall to tumour response. Tumours were subjected to USMB treatment preceding HT by 5 hours in order to maximize responses based on previous work done with USMB. The results indicated that when combined with USMB, HT treatment resulted in cell death, which increased with HT treatment duration  production . Th. Thin vioduction  37]  [27 [2728] . On the nd Bcl-2  along wind Bcl-2 . Here, iIn a second longitudinal study as part of the work here, PC3 tumours received multiple treatments, two days a week over a four-week period. Results demonstrated a significant increase in cell death with the combined USMB + HT (50 min) treatment at week 1, but no significant difference in cell death was noticed followed week 2 . NeverthPreviously, Goetz and group studied the longitudinal effects (over a duration of 5 weeks) of USMB treatment on the size of PC3 tumours implanted in athymic mice and demonstrated that USMB treatment alone did not impact the size of tumours significantly. However, combining USMB with the chemotherapy drug docetaxel resulted in significant tumour size reduction. The change in tumour size was interpreted being a result of vascular shutdown . SimilarIn order to further study these biological responses to treatment were further assessed using Masson's trichrome staining and Ki-67 staining. The results here from Masson's trichrome staining demonstrated that in tumours treated with USMB only there was a little or no collagen deposition at weeks 1, 2, 3 and 4. In the HT (50 min) only treatment group, the presence of collagen was visible as early as week 2 and continued into weeks 3 and 4 . Similarly, the combined treatment USMB + HT (50 min) exhibited a clear increase in collagen staining in weeks 2, 3 and 4. These results indicate that treating tumours multiple times with HT either alone or in combination with USMB causes tumour cells to undergo fibrosis resulting in collagen accumulation . This woThe Ki-67 immunohistological assessment of tumours from combined USMB + HT (50 min) treatment group demonstrated the lowest level of actively dividing cells compared to USMB only treated group or HT (50 min) only treated animals . Hence, in vivo and in vitro studies have suggested that simultaneous application of heat with other cancer treatments is known to have superior tumour response because of maximum thermal enhancement achieved by reducing the interval between two treatments [The study here investigated the new combination of USMB and HT in PC3 tumours in a mouse xenograft model. While using HT adjuvant with other cancer treatment the sequence and time interval between the two modalities is known to have a profound effect on tumour response  55]. Th. Th55]. eatments  and henceatments .The primary finding of this study is that the combination of USMB and HT caused acute and long-term effects on prostate xenograft model. Significant effects in cellular response measured by cell death and vascular damage were noted within 24 hours followed by tumour growth inhibition. Additionally, 4 weeks of USMB and HT exposure showed no measurable apoptotic cell death which may have been a result of fibrosis due to multiple treatments indicating vascular disruption. Furthermore, the combined treated group demonstrated a significant reduction in tumour volume which was associated with a relatively low percentage of proliferating cells. It is likely that the long-term tumour response observed in this study might be dependent on the vascular damage noted in the acute study.in vivo respond to the combination of USMB and HT. A limitation of the work here involves addressing any tissue heating heterogeneity which can be carried out using more sophisticated hyperthermia methods. Hence the absorbed thermal dose and any non-thermal effects remain undetermined. Modern HT techniques for example modulated electro-hyperthermia technology, and MRI-guided hyperthermia using focused ultrasound enables several technical adaptation to tissue heterogeneity and can records the total absorbed dose with the ability to control the selective deposition of energy by the tumour tissue [There is abundant evidence demonstrating the potential of using ultrasound and microbubbles to enhance tumour response. In this study, we investigated ultrasound-stimulated microbubbles (USMB) to enhance the effects of hyperthermia (HT). Tumours treated with the combination of USMB and HT revealed increased cell death, reduced vascularity, and superior inhibition in tumour growth and tumour cell proliferative activity when compared with either treatment alone or untreated group. The enhanced tumour response demonstrated in this study provides a basic understanding of how tumours r tissue . In addir tissue .Furthermore for this technique to be successfully implemented with clinical utility further preclinical research should be conducted that includes testing this technique in large animals and or an orthotropic tumour model. In addition, the optimal time interval between HT and USMB should be investigated and the research should provide a more definitive answer to whether HT treatment should be given sequentially or simultaneously with USMB.S1 ARRIVE checklist(DOCX)Click here for additional data file.S1 FigP-values (p≤0.05) are indicated by an asterisk*.Quantified cell death staining at 24 hours following treatment of PC3 xenografts with varying ultrasound pressure and hyperthermia duration: (A) 0 kPa 20 and 30 min; (B) 246 kPa 20 and 30 min; (C) 570 kPa 20 and 30 min (upper row); (D) 126 kPa 0, 10, 20, 30, 40 and 50 min; (E) 740 kPa 0, 10, 20, 30, 40 and 50 min (lower row). (TIF)Click here for additional data file.S2 FigP-values (p≤0.05) are indicated by an asterisk*.Quantified CD31 labeling at 24 hours in mice bearing human PC3 xenografts treated with different ultrasound pressure and hyperthermia duration: (A) 0 kPa 20 and 30 min; (B) 246 kPa 20 and 30 min; (C) 570 kPa 20 and 30 min (upper row); (D) 126 kPa 0, 10, 20, 30, 40 and 50 min; (E) 740 kPa 0, 10, 20, 30, 40 and 50 min (lower row). (TIF)Click here for additional data file.S3 FigQuantification of untreated tumor sections stained with (A) TUNEL (B) anti-CD31 antibody (C) Masson's trichrome and (D) Ki-67 staining at week 4.(TIF)Click here for additional data file.


Trichoderma reesei is widely used for cellulase production and accepted as an example for cellulase research. Cre1-mediated carbon catabolite repression (CCR) can significantly inhibit the transcription of cellulase genes during cellulase fermentation in T. reesei. Early efforts have been undertaken to modify Cre1 for the release of CCR; however, this approach leads to arrested hyphal growth and decreased biomass accumulation, which negatively affects cellulase production.T. reesei genome, which generated several transformants, named Kuace3, Kuclr2, Kuace2, and Kuxyr1. No obvious differences in growth were observed between the parent and transformant strains. However, the transcription levels of cel7a, a major cellulase gene, were significantly elevated in all the transformants, particularly in Kuace2 and Kuxyr1, when grown on lactose as a carbon source. This suggested that CCR inhibition was released or attenuated in the transformant strains. The growth of Kuace2 and Kuxyr1 was approximately equivalent to that of the parent strain in fed-batch fermentation process. However, we observed a 3.2- and 2.1-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of the parent strain. Moreover, we observed a 6.1- and 3.9-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of Δcre1 strain.In this study, novel fusion transcription factors (fTFs) were designed to release or attenuate CCR inhibition in cellulase transcription, while Cre1 was left intact to maintain normal hyphal growth. Four designed fTFs were introduced into the T. reesei to improve cellulase titers without impairing fungal growth. This study will be valuable for lignocellulosic biorefining and for guiding the development of engineering strategies for producing other important biochemical compounds in fungal species.A new strategy based on fTFs was successfully established in  Despite being the most abundant natural material, lignocellulose is a considerably under-utilized biomass , 2. Lignd-glucose or d-xylose during cellulase fermentation triggers carbon catabolite repression (CCR), which significantly decreases cellulase transcription in filamentous fungi [Δcre1 strain was 1.45-fold higher than that of its parent strain of Trichoderma orientalis EU7-22. Furthermore, Sun and Glass [cre1 in Neurospora crassa improved the expression of most cellulases and increased endocellulase activity by approximately 50%. Therefore, the deletion of cre1 is widely accepted as an effective strategy for releasing CCR. However, several studies have revealed that cre1 deletion impairs filamentous fungal growth, which leads to decreased final product titers. For example, the deletion of cre1 in T. reesei or its replacement with a cre1 mutant inhibited hyphal growth and affected final biomass accumulation, which led to decreased cellulase production [Δcre1 strain exhibited slower growth than its parent strain [T. reesei Rut-C30 and contributed to a partial release from CCR [cre1-96 could improve cellulase activity without impairing strain growth [One promising strategy for decreasing enzyme costs is to increase cellulase production by filamentous fungi . Howeverus fungi . Therefous fungi –14. In fus fungi  reportednd Glass  demonstroduction , 17, 18.t strain . Howeverfrom CCR . Furthern growth . Howevern growth . These ocre1 led to serious growth defects. Furthermore, Cre1 is required, albeit indirectly, for the positive regulation of key genes involved in fundamental life processes, such as energy biosynthesis and general metabolism [The construction of synthetic transcription factors involves the fusion of transcription factor zinc finger domains with the activation domains of well-studied activators or repressors. They have demonstrated significant potential as a tool for creating phenotypic alterations in yeast and mammalian cells –24. Repltabolism . TherefoT. reesei, N. crassa, and Penicillium oxalicum [T. reesei has the highest potential for the overproduction and secretion of cellulases [T. reesei CL847 can reach levels of 243 FPU/L/h in a 3000-L fermenter using lactose as a carbon source [T. reesei [The industrial production of cellulase involves the fermentation of filamentous fungi, such as oxalicum –28. Of tllulases , 30. Forn source . Further. reesei .T. reesei has been extensively explored in the last few years, and the functions of cellulase transcriptional repressors and activators in T. reesei have been comprehensively characterized [T. reesei is a suitable system for screening novel synthetic transcription factors owing to their ability to release or attenuate CCR while retaining intact Cre1 to allow effective fungal growth. Such efforts will also accelerate the industrialization of biorefining processes.The cellulase transcriptional regulatory network in cterized . Cre1 ancterized , and Acecterized , 33. TheT. reesei that was based on the construction of fusion transcription factors (fTFs). Four fTFs were constructed and integrated into the T. reesei genome and the phenotypic characteristics of the transformants were investigated in detail. Furthermore, we examined the transcription levels of the cellulase transcription factors and the major cellulase gene cel7a in the fTF transformants to confirm the effectiveness of our strategy for activating cellulase transcription. In addition, the cellulase titers of the transformants were compared with those of the parent and Δcre1 strains using fed-batch culture in 3-L fermenters. This approach could also be employed in other fungal strains to release CCR while maintaining normal strain growth.Here, we proposed a novel strategy to alleviate CCR in Sace3, Sclr2, Sace2, and Sxyr1. Each fTF was under the control of the T. reesei cre1 promoter and the Aspergillus nidulans trpC terminator , the zinc finger regions of Cre1 and Ace1, and a peptide used to link each domain . Phenotypic analysis of each strain was performed after incubating the plates for 6 days at 30 °C. Figure T. reesei [The growth of Kuace3, Kuclr2, Kuace2, or Kuxyr1 was not obviously impaired on plates containing lactose. Lactose is considered to be an economically feasible carbon source for inducing cellulase expression in . reesei –37. Therxyr1, which encodes the main cellulase activator in T. reesei [Next, we used qRT-PCR to investigate the expression levels of genes encoding cellulase transcription factors in the Kuace3, Kuclr2, Kuace2, Kuxyr1, and parent strains following their incubation in media containing 2% lactose for 1, 6, 8, and 12 h. The transcriptional levels of most cellulase transcription factors were unaltered (data not shown). However, . reesei , was sigT. reesei Rut-C30 was cultured in a two-stage system with lactose as a carbon source and a controlled pH of 4.8, the major secreted cellulase was cellobiohydrolase 1, which can account for approximately 50% of the total extracellular protein [cel7a gene. Therefore, we examined the transcriptional level of cel7a in all our strains. As shown in Fig. cel7a in Kuace3, Kuclr2, Kuace2, and Kuxyr1 were all significantly upregulated at 6  among the transformants  activities of Kuace2, Kuxyr1, and the parent strain. For each of these strains, pNPCase activities were determined, and the results were expressed as enzyme unit per dry mycelium weight. Although the pNPCase activities in Kuace2 and Kuxyr1 were significantly increased compared with that of the parent strain  2SO4 as a nitrogen source was added after 66 h of cultivation. The intracellular protein concentration was measured to determine the biomass concentration in each culture [As cellobiohydrolase 1 expression in the Kuace2 or Kuxyr1 strains was significantly elevated in shake-flask cultures Figs.  and 5, w culture , 40. In pNPCase activities were significantly increased in the Kuace2 and Kuxyr1 strains compared with those in the parent and Δcre1 strains after feeding was complete . The biomass accumulation of the Kuace2, Kuxyr1, and parent strains significantly increased after 108 h of cultivation and was noted to be maximum at 180 h. In contrast, the biomass concentration of the Δcre1 strain slowly increased throughout its cultivation  produced by T. reesei hydrolyzes lactose into d-galactose and d-glucose, and bga1 overexpression abolishes the expression of the two prominent cellulase-encoding genes cbh1 and cbh2 in T. reesei [cel7a, we also observed that the expression of the cellulase genes such as cel6a, cel5a, and cel7b in T. reesei was significantly elevated in all fTF-containing transformants cultured on plates containing lactose [cre1, cre1-96, into T. reesei [cre1-96 or fTF has no significant effect on T. reesei growth and exerts a positive effect on cellulase production by attenuating the effect of CCR.Designing new strategies is important for the production of protein or other biochemicals in fungal species. Recent studies to increase cellulase production have primarily focused on the manipulation of Cre1/Mig1 (a yeast Cre1 homolog), for example, the use of gene knockouts or replacement of the gene with a non-functional allele , 41, 42.. reesei –37, whic. reesei –45. Howe. reesei . Therefo lactose   a heterodimer composed of Xyr1 and Ace2 has been demonstrated to function as an activator of cellulase expression; (ii) a Xyr1 homodimer has been implicated in the formation of a cellulase induction-specific complex [T. reesei; and (iv) it is possible that Sace2 and Sxyr1 may form more hetero- or homodimers with Xyr1 at the cel7a promoter region than with Sace3 or Sclr2. Future studies will involve the introduction of the four fTFs  into T. reesei Δxyr1  or T. reesei Δace2 . The transcriptional levels of cellulase genes will be compared among all the transformants to investigate the relationship between fTFs and homodimer/heterodimer formation. These will be helpful for further understanding the role of the Xyr1 and Ace2 activation domains on cellulase transcription.Although the four fTFs possess the same zinc finger motifs, the  complex , 51;  were found to be lower than those of the wild type strain; however, a 1.35-fold lipid increase in lipid content was observed [Synthetic or fusion transcription factors have been prepared for exploring the transcriptional regulation network and phenotypic engineering in yeast and mammalian cells , 22. Howobserved .T. reesei cellulase titers. Four fTFs  were designed to release or attenuate the CCR inhibition of cellulase genes, and Cre1 was left intact to maintain strain growth. The Kuace3, Kuclr2, Kuace2, and Kuxyr1 strains were produced by transforming Sace3, Sclr2, Sace2, and Sxyr1, respectively, into the T. reesei genome. The growth of these transformants was roughly equivalent to that of the parent strain. However, the transcription levels of cel7a, which encodes the major cellulase protein cellobiohydrolase 1, were significantly elevated in all transformants, particularly those in Kuace2 and Kuxyr1, when lactose was used as the carbon source. This suggests that a complete or partial release from CCR occurred. Furthermore, the use of fed-batch fermentation resulted in a significant increase in the pNPCase titers of the Kuace2 or Kuxyr1 strains compared with those of the parent and Δcre1 strains.Here, we developed a new strategy for increasing T. reesei. In addition, this study provides valuable information for designing new fTFs for biochemical compounds production in other fungal species.To summarize, a new strategy based on fTFs was successfully established to improve cellulase titers in Trichoderma reesei Δtku70 (ATCC MYA-256), a nonhomologous end joining pathway-deficient strain, was used as the parent strain in this study [E. coli strain GB05-dir was used for constructing all the plasmids [is study . All theplasmids .p-nitrophenyl-β-d-cellobioside (pNPC) was purchased from Sigma-Aldrich . Uridine, PEG6000, sorbitol, and lactose were purchased from Sangon Biotech Co., Ltd. . KOD FX DNA polymerase  was used for all polymerase chain reaction amplifications. RNAiso™ reagent, PrimeScript® RT reagent Kit With gDNA Eraser  and SYBR® Premix Ex Tag™ (Tli RNase H Plus) were purchased from Takara Bio Inc. . The DIG High Prime DNA Labeling and Detection Starter Kit I  was used for Southern blotting analysis. The restriction enzymes Ase I and Xho I used for genome digestion were purchased from Thermo Fisher Scientific Inc. . All other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. .Wheat bran was kindly provided by Longlive Bio-Technology Co., Ltd. . The E. coli GB05-dir [Sxyr1 replacement cassette are as follows: 2 kb of sequence up- and downstream of the ku70 gene, the promoter and zinc finger regions of cre1 (comprising nucleotides − 1364 to 417 bp), the activation domain of xyr1 (445 to 2947 bp), and the zinc finger region of ace1 (1276 to 1716 bp) were each amplified from the T. reesei genome. A GSGGSGTS peptide was then used to link each domain. The pyr4 selective marker was obtained as previously described [trpC terminator was cloned from pSilent-1. The pUG6 plasmid was used as the accepter plasmid. A similar strategy was used to construct the pSace3, pSclr2, and pSace2 plasmids. The activation domains of ace3 (comprising nucleotides 220–2035 bp), clr2 (100–2003 bp), and ace2 (118–1023 bp) were inserted into pSace3, pSclr2, and pSace2, respectively. A schematic diagram of the Sace3, Sclr2, Sace2, and Sxyr1 replacement cassettes is shown in Additional file cre1 deletion cassette included the 2 kb up- and downstream of the ku70 gene, the pyr4 selective marker, and the pUG6 plasmid. A schematic diagram of the cre1 deletion cassette is shown in Additional file All plasmids were constructed by linear–linear homologous recombination, which was mediated by full-length RecET in GB05-dir , 54. Theescribed , and theSace3, Sclr2, Sace2, and Sxyr1 replacement cassettes and the cre1 deletion cassette into T. reesei Δtku70 was performed using previously described methods [The transformation of  methods . The tra methods . Followi3 of agar containing parent or mutant mycelia was inoculated on a solid plate containing PDA, wheat bran, MM with 2% glucose, or MM with 2% lactose. All media used for T. reesei Δtku70 culture were supplemented with 0.001% uridine. Plates were incubated at 30 °C for 6 days, and colony diameters were measured from the 2nd day. All experiments were performed in triplicates. Microscopic observation of hyphal morphology was based on a previously described method [3 spores were inoculated on coverslips with solidified medium containing PDA, wheat bran, MM with 2% glucose, or MM with 2% lactose at 30 °C for 48 h. Microscopic images of hyphae  were then captured using a Nikon D5000 camera.Approximately 1 cmd method . Approxi6 spores of parent strain and transformants were inoculated on PDA and wheat bran plates and incubated at 30 °C for 6 days. Spores were then harvested from the plates using a solution containing 0.9% NaCl and 0.5% Tween-80 and enumerated with a hemocytometer. Colony diameters were measured on the 6th day of incubation. The number of spores produced was normalized to the colony area.Approximately, 1 × 107 spores of the parent strain and transformants containing fTFs were inoculated into liquid medium containing MM + 2% glucose + 0.001% tryptone and cultured at 30 °C and 200 rpm for 48 h. The mycelia were filtered and washed with MM solution. Equal amounts of mycelia were transferred into submerged medium containing MM + 105 mM glycerol and the samples were incubated at 30 °C and 200 rpm for 24 h. The mycelia were harvested, and approximately 0.1 g of harvested mycelia was transferred to 100 mL liquid medium containing MM + 2 g lactose. The mycelia were cultured at 30 °C and 200 rpm. Samples were taken at 1, 6, 8, and 12 h after inoculation and immediately frozen at − 80 °C. These samples were used for determining the transcriptional levels of cellulase transcription factors and cellulase genes.Approximately, 1 × 10−ΔΔCT method [The extraction of total RNA and subsequent synthesis of cDNA were performed as previously described . Real-tiT method . The pripNPCase activities, mycelia were precultured as described above. Approximately 0.2 g of mycelia was inoculated in 200 mL submerged medium containing MM solution + 4 g lactose and incubated at 30 °C and 200 rpm. Culture samples (30 mL) were collected on days 4, 5, and 6 after inoculation and centrifuged at 12,000 rpm for 10 min. The solid sediment was extracted and dried at 100 °C for 24 h [pNPCase activity was measured as previously described [To determine for 24 h . The supescribed  and normTrichoderma reesei was cultured using a protocol based on a previously published method [7 spores) were inoculated in 100 mL liquid medium containing MM solution + 2% glucose + 0.001% tryptone and incubated at 30 °C and 200 rpm. After 48 h of cultivation, the samples were used as seed cultures for fermentation. The seed culture (200 mL) was used to inoculate a liquid medium containing 1.8 L of MM solution, 6 g glucose, 10 g Avicel, and 2 g tryptone in a 3-L fermenter . The feeding solutions contained 400 mL of carbon source (150 g lactose/L) and 400 mL of nitrogen source (160 g (NH4)2SO4/L). The initial pH value was regulated at pH 4.5 and maintained at pH 4.0–5.0 by the automatic addition of 3 M H3PO4 and 3 M KOH during the entire fermentation process. The airflow was 1.5 L/min and the agitation rate was adjusted to maintain the dissolved oxygen level above 30%. The media were fed following 66 h of cultivation. The injection rates of the carbon and nitrogen solutions were 0.72 g/h and 0.23 g/h, respectively. Samples were periodically collected to measure the pNPCase titer and intracellular protein concentration. The measurements of intracellular protein concentration to determine the biomass concentration were performed according to a previously described method [d method . Brieflyd method .t tests, and p values of < 0.05 were considered statistically significant.All experiments were performed in triplicates. Statistical analyses were performed using two-tailed Student’s Additional file 1: Figure S1. Southern blotting analysis of parent and fTF-containing transformants. A) Schematic diagram of Southern blotting. B) Southern blot of the parent, Kuace3, and Kuclr2 strains. C) Southern blot of the parent, Kuace2, and Kuxyr1 strains. 1: 2 kb upstream of ku70; 2: cre1 promoter; 3: Sace3; 4: trpC terminator; 5: pyr4; 6: 2 kb downstream of ku70; 7: Sclr2; 8: Sace2; 9: Sxyr1. Lane M: 1 kb molecular weight marker, Lane 1: parent strain, Lane 2: Kuace3 strain, Lane 3: Kuclr2 strain, Lane 4: Kuace2 strain, Lane 5: Kuxyr1 strain. The arrows indicate the predicted size of each band.Additional file 2: Figure S2. Southern blotting analysis of the Δcre1 strain. A) Schematic diagram of Southern blotting. B) Southern blot of parent and Δcre1 strains. 1: 2 kb upstream of cre1 gene; 2: pyr4; 3: 2 kb downstream of cre1. Lane M: 1 kb molecular weight marker, Lane 1: parent strain, Lane 2: Δcre1 strain. The arrows indicate the predicted size of each strain.Additional file 3: Figure S3. Colony diameters of parent strain and transformants on plates containing different carbon sources. Black represents cultivation for 2 days, white represents cultivation for 3 days, dark gray represents cultivation for 4 days, and light gray represents cultivation for 5 days.Additional file 4: Figure S4. Microscopic observation of the hyphae of the parent strain and transformants. Scale: 100 μm.Additional file 5: Figure S5. Sporulation of the parent strain and transformants. Black represents wheat bran as the culture medium; white represents PDA as the culture medium.Additional file 6: Figure S6. Transcriptional levels of fTFs among the transformants. The transcriptional level of fTFs was normalized to that of actin. Dark gray represents Kuace3, white represents Kuclr2, black represents Kuace2, and light gray represents Kuxyr1.Additional file 7: Figure S7. Transcriptional levels of cel6a, cel5a, and cel7b among the transformants. The transcriptional levels of cel6a, cel5a, and cel7b were normalized to that of actin . A left slash represents the parent strain, dark gray represents Kuace3, white represents Kuclr2, black represents Kuace2, and light gray represents Kuxyr1.Additional file 8: Table S1.T. reesei strains used in this study.Additional file 9: Table S2. Primers used for real-time PCR.


Irpex lacteus exhibits a great potential in biopretreatment of lignocellulose as well as in biodegradation of xenobiotic compounds by extracellular ligninolytic enzymes. Among these enzymes, the possible involvement of dye-decolorizing peroxidase (DyP) in lignin degradation is not clear yet.The white rot fungus I. lacteus CD2 produced DyPs as the main ligninolytic enzymes when grown in Kirk’s medium supplemented with lignin. Further transcriptome analysis revealed that induced transcription of genes encoding DyPs was accompanied by the increased expression of transcripts for H2O2-generating enzymes such as alcohol oxidase, pyranose 2-oxidase, and glyoxal oxidases. Meanwhile, accumulation of transcripts for glycoside hydrolase and protease was observed, in agreement with abundant proteins. Moreover, the biochemical analysis of IlDyP2 and IlDyP1 confirmed that DyPs were able to catalyze the oxidation of typical peroxidases substrates ABTS, phenolic lignin compounds DMP, and guaiacol as well as non-phenolic lignin compound, veratryl alcohol. More importantly, IlDyP1 enhanced catalytic activity for veratryl alcohol oxidation in the presence of mediator 1-hydroxybenzotriazole, which was similar to the laccase/1-hydroxybenzotriazole system.Based on the extracellular enzyme activities and secretome analysis, The results proved for the first time that DyPs depolymerized lignin individually, combining catalytic features of different peroxidases on the functional level. Therefore, DyPs may be considered an important part of ligninolytic system in wood-decaying fungi.The online version of this article (10.1186/s13068-018-1303-9) contains supplementary material, which is available to authorized users. Lignin is the second most abundant constituent of lignocellulosic biomass, amounting to 15–30% by weight or up to 40% by energy . However2+, which is attributed to high redox potential peroxidase LiP/VP and MnP, respectively [Dye-decolorizing peroxidase (DyP) is a member of the novel heme peroxidase family (DyP-type peroxidase superfamily), showing no homology to classic fungal heme peroxidases including MnP, VP, and LiP , 9. So fectively , 10, 11.Irpex lacteus is a white rot fungus with a significant potential for various biotechnological applications such as bioremediation of organopollutants in water and soil environments and biopretreatment of lignocellulose [I. lacteus CD2 could degrade all kinds of lignocellulose and dyes [I. lacteus CD2 has seven mnp genes, two lip genes, and four dyp genes, without lac gene [I. lacteus, DyP is scarcely known for catalytic properties and substrate specificities, especially in lignin degradation. Herein, the main ligninolytic enzymes of I. lacteus CD2 grown in lignin medium were determined, combining extracellular enzyme activities and secretome analysis. Furthermore, the mechanisms of lignin degradation by the main ligninolytic enzymes DyPs were elucidated using transcriptomics and biochemical analysis.ellulose , 13. Itsellulose –18. Our and dyes . Genome lac gene . CompareI. lacteus in lignin medium , copper radical oxidase, and cellobiose dehydrogenase (CDH)  Table , which coxidases .Table 1MIn addition to ligninolytic enzymes, some proteins involving in fungal growth, such as glycosidase hydrolases  and proteases were identified in the extracellular proteins. Chitinases, glucanases, and mannosidases are involved in hyphal cell wall biosynthesis . ProteasI. lacteus CD2.A total of 10,167 genes were determined from the transcriptomes of four samples: LIG3d (3 days in lignin), GLU3d (3 days in glucose), LIG6d (6 days in lignin) and GLU6d (6 days in glucose). To identify key genes and pathways associated with lignin degradation, four pairwise comparisons were performed, including LIG3d versus GLU3d, LIG6d versus GLU6d, LIG6d versus LIG3d , GLU6d versus GLU3d. The results indicated that 4603 and 3816 genes showed at least twofold differences in comparisons of groups LIG3d versus GLU3d, and LIG6d versus GLU6d, respectively, whereas only 300 genes exhibited not less than twofold differences in the comparison of group LIG6d versus LIG3d. Moreover, GO-enrichment analysis between LIG6d versus GLU6d and LIG6d versus LIG3d revealed that these differential expression genes were significantly enriched in lignocellulose-degrading process, including lignin, carbohydrate, polysaccharide, hemicellulose, hydrogen peroxide, and phenylpropanoid metabolic/catabolic process Fig. a. Beside2O2-generation enzymes in I. lacteus in lignin medium  as well as other proteins were upregulated in lignin medium. 47 CAZYs genes of glycoside hydrolase, carbohydrate esterase (CE) or polysaccharide lyase (PL) families had significantly different transcript abundance in LIG3d relative to GLU3d , DyPs also contain heme group judging from the absorbance at 410 nm [410/A280) ratios of purified DyPs were 2.5 and 1.0, respectively. Meanwhile, in order to identify corresponding DyP isoenzymes expressed in I. lacteus CD2, purified DyPs were separated from SDS-PAGE to digested for peptide mass fingerprinting analysis. Based on the number of unique peptides matched in DyPs from I. lacteus, IlDyP2 and IlDyP1 corresponded to genes 0808.621 and 0821.105, respectively , lignin model compounds  and ABTS , while IlDyP1 was found to show less acidic optima . However, only IlDyP2 could oxidize RB5 in the narrow pH range from 3.0 to 4.0, which was slightly different from DyP in I. lacteus CCBAS 238 with optimal pH 3 and retaining 60% of residual activity at pH 2 for RB5. The optimal pH of 3.0 and 4.0 for IlDyP2 and IlDyP1 against VA and ABTS, respectively, and pH 4 for IlDyP1 could retain 80% of residual activity against VA and ABTS, while DyP in I. lacteus CCBAS 238 had optimal pH 2 for VA and pH 3 for ABTS, and retained 10% and 60% of residual activities at pH 4 for VA and ABTS, respectively. IlDyP2 and IlDyP1 showed the same optimal pH 3.0 for DMP, in accordance with DyP in I. lacteus CCBAS 238 [IlDyP2 showed acidic optima of DMP , while IlDyP1 was found to show less acidic optima . The optimal values of DyPs in this study were very near to those of DyP from Auricularia auricula-judae  [The optimal pH for CBAS 238 . IlDyP2 CBAS 238 . IlDyP2 and RB5) . Liers eIlDyP2 and IlDyP1 isoenzymes might result in wider pH range for efficient lignin degradation.These differences in biochemical properties of DyP isoenzymes might be attributed to their divergent evolutionary origin. Furthermore, DyP isoenzymes had complementary effects on different pH values, the combination of IlDyP2 and IlDyP1 for classic fungal heme peroxidases substrates ABTS, DMP, and guaiacol were presented in Table IlDyP2 and IlDyP1 exhibited significant differences in catalytic efficiencies for different substrates. Km of IlDyP1 toward three substrates was higher than that of IlDyP2, suggesting that IlDyP2 had higher affinity for three substrates. IlDyP2 and IlDyP1 had similar activity for ABTS and guaiacol, while IlDyP2 had higher activity for DMP than for IlDyP1. IlDyP2 had sixfold and threefold higher catalytic efficiencies than IlDyP1 for ABTS and guaiacol. This suggested that IlDyP2 had better substrate fitting at the oxidation site, which was in accordance with the lack of activity of IlDyP1 on the recalcitrant dye RB5 [IlDyP2 and IlDyP1 displayed higher catalytic efficiencies for the oxidation of ABTS and DMP [The catalytic properties of  dye RB5 . In comp and DMP , 35.TablIlDyP2 and IlDyP1 were similar to that of classic fungal heme peroxidases. IlDyP2 and IlDyP1 were both able to decolorize the high redox potential anthraquinone dye RB19, which is the common feature of all DyP-type peroxidases [IlDyP2 had the ability to decolorize the recalcitrant dye RB5, while IlDyP1 could not decolorize RB5. Although IlDyP2 and IlDyP1 could not oxidize Mn2+ to Mn3+, they could catalyze the oxidation of typical peroxidases substrates ABTS as well as phenolic lignin compounds DMP and guaiacol. Moreover, they exhibited low activity to oxidize non-phenolic lignin compound VA through peroxidase activity, belonging to the classic high redox potential peroxidases LiP and VP [I. lacteus CCBAS 238 showed low activities for VA and RB5 [IlDyP1. This phenomenon is similar to the oxidation of a non-phenolic lignin model compound by the laccase/1-HBT redox system [IlDyP2 and IlDyP1 might be a part of the alternative biocatalytic system for lignin degradation by I. lacteus, combining the catalytic properties of heme peroxidases and laccase.In addition, the data shown in Fig. oxidases . NotablyP and VP . It was P and VP , 35. DyP and RB5 . In thisx system , 37. Witkcat/Km) with a few orders of magnitude [Thermomonospora curvata showed high catalytic efficiency with ABTS, close to that of fungal DyPs [Raoultella ornithinolytica OKOH-1 can directly decolorize melanin, and immobilization can improve its activity and stability [Some proteins in DyP family have low amino acid sequence identity (lower than 15%), and significant differences in catalytic efficiency  (ABTS), 2,6-dimethylphenol (DMP), guaiacol, reactive black 5 (RB5), veratryl alcohol (VA), and 1-hydroxybenzotriazole (1-HBT) were purchased from Sigma-Aldrich . Reactive blue 19 (RB19) was purchased from Sinopharm Chemical Reagent Company .Irpex lacteus CD2 [2PO4, 2 g/L; MgSO4·7H2O, 0.71 g/L; CaCl2, 0.1 g/L; and 70 mL trace element solution. The trace element solution contains NaCl, 1 g/L; CoCl2·6H2O, 0.184 g/L; FeSO4·7H2O, 0.1 g/L; ZnSO4·7H2O, 0.1 g/L; CuSO4, 0.1 g/L; H3BO3, 0.01 g/L; Na2MoO4·2H2O, 0.01 g/L; KAl(SO4)2·12H2O, 0.01 g/L; and nitrilotriacetic acid, 1.5 g/L.teus CD2  was mainε420 = 36,000 M−1 cm−1) at 420 nm, in 50 mM sodium tartrate buffer (pH 4.0) containing 1 mM ABTS, 1 mM Mn2+, and 0.1 mM H2O2. Manganese-independent peroxidase activity was also determined by ABTS oxidation in the absence of Mn2+. DyP activity was assayed by the decolorization of an anthraquinone dye RB19  at 595 nm. The reaction was performed in the same buffer containing 125 μM RB19 and 0.1 mM H2O2. One unit of enzyme activity was defined as the amount of enzyme that oxidized 1 μmol of ABTS or RB19 per minute at 25 °C.Total ligninolytic enzyme activities were measured by monitoring the oxidation of ABTS  according to the manufacturer’s instructions. The total RNA was sent to Annoroad Genomics  for sample preparation and sequencing. All samples were in duplicate. The cDNA was synthesized and prepared for sequencing using the Illumina mRNA-Seq Sample Prep Kit . The samples were run on independent lanes, and paired-end sequences of 150 bp were obtained at 4 Gb clean data for each sample using the Illumina Hiseq 2500. The raw reads were trimmed and filtered using Trimmomatic software to remove adapters and low-quality bases [I. lacteus CD2 genome as a Ref. [q value < 0.05 [ty bases . Then cls a Ref. , 44. Alls a Ref. , and reae < 0.05 .I. lacteus CD2 grown in alkali lignin for 3 days were collected and concentrated by 80% ammonium sulfate at 4 °C. 20 mM sodium acetate buffer (pH 5.0) was used to dissolve the pellets and dialyzed using 30-kDa cutoff membrane. Then IlDyPs were purified using a HiTrap Q HP anion exchange column  pre-equilibrated with the same acetate buffer. The IlDyPs were eluted with a linear gradient of 0–1.0 M NaCl, and fractions containing active enzymes were pooled after SDS-PAGE. Meanwhile, the bands were excised and identified by peptide mass fingerprinting.The liquid cultures of IlDyP2 and IlDyP1 were considered to be 100%. For catalysis properties, the reactions were performed in optimal pH at 25 °C using 50–4000 μM substrates by monitoring corresponding oxidation products. The nonlinear least square fitting method was used to calculate the Km, kcat, and kcat/Km parameters of IlDyP2 and IlDyP1 using the GraphPad Prism 5 software.To determine the optimal pH, 50 mM sodium tartrate buffers with pH ranging from 2.0 to 7.0 were used for all substrates including ABTS, DMP, guaiacol, VA, RB19, and RB5 at 25 °C. The maximum activities of IlDyP1 and IlDyP2 to oxidize the non-phenolic lignin compound VA. The oxidation of VA was performed in 50 mM sodium tartrate buffer (pH 4.0 or pH 3.0) containing 1 mM VA, 1 mM 1-HBT, 0.1 mM H2O2, and 0.5 U/mL IlDyP1 or IlDyP2, without 1-HBT or DyP as corresponding control. The reaction proceeded at 30 °C. After 24 h, the reaction products were analyzed by HPLC using a reverse-phase C18 column, 4.6 mm × 250 mm, 5-m particle size (Waters XTerra). The isocratic elution condition was performed with 55% methanol containing 0.1% formic acid at a flow rate of 1 mL/min. The elution peaks were monitored at 310 nm.1-HBT was used as the mediator in evaluating the abilities of Additional file 1. Proteins identification from I. lacteus secretome in kirk’s medium supplemented with lignin on the third day.Additional file 2. Transcript levels of I. lacteus genes encoding carbohydrate active enzymes in GLU3d, GLU6d, LIG3d and LIG6d.Additional file 3. Transcript levels of I. lacteus genes involved in mobilizing and recycling nitrogen in GLU3d, GLU6d, LIG3d and LIG6d.