Regulation Of Extracellular Laccase Activity By Heat Shock Transcription Factor HSF2 In Trametes Trogii

Fungal laccase?EC 1.10.3.2?is a copper-containing enzyme,as known as polyphenol oxidase,which is an important lignocellulose degrading enzyme that can oxidize various phenolic compounds such as phenolic compounds and aromatic amines using molecular oxygen as a terminal electron acceptor.It is widely used in the fields of biopulping,food industry,biosynthesis,bioenergy,bioassay and bioremediation.In the previous study,we screened a strain of T.trogii S0301,which has the characteristics of fast growth,high laccase production and outstanding laccase properties.It is ideal for commercial laccase production strains.The characteristics of high laccase production and short fermentation cycle have good theoretical research and industrial application prospects.However,at present,the genomic information of this strain has not been reported,and the genetic transformation method has not yet been established.The understanding of the regulation of laccase isoenzyme gene expression in white rot fungi represented by this strain is also quite limited.In view of this,our research based on this strain,the main results are as follows:1.Genomic sequencing of Thermotolerant T.trogii and laccase isozyme family gene analysisBased on the isolation of thermotolerant T.trogii homo-19,we obtain about 220×clean reads date of the strain's geome by third generation sequencing technology.The results of genomic data analysis showed that about 39.88 Mb clean reads,2,4 Mb overlapping group N50 were obtained from each library.The number of overlapping genome groups was 29 Mb,the longest overlapping group length was 4.82 Mb,and the shortest overlapping group length was 20,367 bp.The percentage of GC content was 55.47%;14,508 protein-coding genes were predicted,which 542CAZymes-related genes and 9 possible laccase genes were identified.The gene structure of 9 laccase isozyme genes and the cis-acting elements in the upstream promoter region were predicted and analyzed,which laid a foundation for the study of the expression pattern of laccase isozyme gene family and the utilization of gene resources in this strain.2.Construction of genetic transformation system in thermotolerant TrametesIn order to improve the conversion rate,we referred to the reports of the genetic transformation methods in filamentous fungi,and optimized various conditions such as the starting strain for transformation,formulation of enzymatic hydrolysate,osmotic buffer,enzymatic hydrolysis time and carrier for transformation and finally,1.5%lysozyme,4 h enzymatic hydrolysis,0.75 M sucrose concentration,PEG/CaCl₂-conversion method were selected as the suitable conditions for the strain.The level of gene overexpression depends on the strength of the promoter,and we optimized the promoter in the starting plasmid.On the basis of the establishment of the transformation method,we cloned different sections of the glyceraldehyde triphosphate dehydrogenase?Gpd?promoter in the strain,and constructed them to drive the green fluorescent protein?GFP?and transform the protoplast of Trametes.By detecting the fluorescence intensity of GFP in the transformed strain,Gpd-T2could better drive the expression of GFP gene.The laccase isoenzymes Lac13 and Lac53 were successfully overexpressed using the Gpd-T2 promoter,and the Laccase secretion occurs earlier in Lac13 and Lac53 overexpressed positive strain which they had higher laccase activity than the wild-type strain.3.Regulation of extracellular laccase activity by heat shock transcription factor HSF2 in T.trogii.The study found that there is an alternative splicing mutant of HSF2 in T.trogii homo-19.According to the gene domain analysis of the transcription factor HSF2,we selected four fragments.Through the genetic transformation system,we overexpressed these four fragments in the strain T.trogii homo-19,respectively.25fragments of HSF2-1 overexpressed transformants;10 fragments of HSF2-2overexpressed transformants;40 fragments of HSF2-3 overexpressed transformants;and 27 fragments of HSF2-4 overexpressed transformants.were obtained by hygromycin resistance and hygromycin gene screening.Then,the transcriptional level of HSF2-2 overexpressed transformant was detected.We found that the overexpression of HSF2-2 transformants could regulate the extracellular total laccase activity.On the 8th day,the relative laccase activity of transformer OE-2-20 was more than three multiple that of wild type.Moreover,Lac13?2.12 and 1.61 multiple?,Lac2-2?3.14 and 1.95 multiple?,Lac2-8?1.94 and 3.74 multiple?,Lac1?1.43 and 3.87multiple?,Lac7?1.18 and 1.14 multiple?and Lac53?1.42 and 2.56 multiple?can be regulated by transcriptional level.The thermal stability of laccase from HSF2-2overexpressing transformants at 50,60 and 70 can prove this point.In summary,this study obtained the third generation genomic information of Thermotolerant T.trogii and completed preliminary annotations.At the same time,we established a method for overexpression of functional genes in this strain,and completed the analysis of the laccase isozyme gene family in this strain.The molecular mechanism of heat shock transcription factor HSF2 of this strain involved in the regulation of extracellular laccase activity was also preliminarily explored,which proved that the overexpression of HSF2 can affect the expression level of different laccase isozymes,thus regulating the total activity of extracellular laccase,the properties of laccase and the process of laccase production.These results provide the possibility to study the expression regulation of laccase and other genes in this strain,and provide the research basis for exploring the overexpression and early expression of induced enzymes at secondary metabolic stages such as laccase in white rot fungi by genetic manipulation of transcription factors.This study laid the foundation for improving the production of white rot laccase,accelerating the process of producing laccase,and promoting the industrial application of laccase.