Histone Methyltransferase LaeA And Related Proteins Involved In The Expression Of The Cellulase Genes In Penicillium Oxalicum

Cellulose is the rich and renewable resource,which could be digested by cellulase for production of biofuels and chemical products.It is helpful for environment protection and sustainable economic development.Among cellulase-producing microorganisms,intact cellulolytic enzyme components could be secreted with filamentous fungi.The reasonable target could be provided for the genetic transformation of strains with exploring the key factors in the transcriptional regulation and elucidating the regulation network of cellulase synthesis will,which is significant to increase the production and reduce the cost of enzymes.The putative methyltransferase LaeA/LAEl was reported to regulate the development of fungi,the expression of the cellulase genes and secondary metabolic gene clusters.In this paper,the function of LaeA was served as the starting point to screen proteins interacted with LaeA.And the mechanism how LaeA and its interacted proteins and transcription factors to activate expression of cellulase genes involved in Penicillium oxalicum.Major advances in this thesis are as follows:1.Screening of LaeA interaction proteins in Penicillium oxalicumYeast two-hybrid and tandem affinity purification-mass spectrometry methods were used to screen and identify proteins that interact directly with LaeA in Penicillium oxalicum.Yeast two-hybrid technique was used to screen the proteins interacted with LaeA in cDNA library.Four candidate proteins were identified,which may interact with LaeA in Penicillium oxalicum.With tandem affinity purification-mass spectrometry technology,15 LaeA candidate interaction proteins were identified,including members VeA and VelB in the VelB/VeA/LaeA complex,glucose repressor protein TUP 1/RcoA,and s-adenosylmethionine synthetase A,histone H2B,nitroreductase,transcription factor AmyR,elongation factor EF-2,subunit of F1F0-ATPase complex,chaperone DnaK,tyrosine 3-monooxygenase/tryptophan 5-monooxygenase,1-phosphatidylinositol-4.5-diphosphodiesterase,6-phosphofructo-2-kinase,beta subunit with putative calcium ion binding activity protein and ATP synthase.It is speculated that the heterochromatin protein HepA,glucose repressor protein TUP1/RcoA,s-adenosylmethionine synthetase A and histone H2B are related to the regulation of cellulase gene expression.2.Expression and chromatin structures of cellulolytic enzyme gene regulated by heterochromatin protein 1HepA is the protein interacted with LaeA,obtained by yeast two-hybrid technology,which is homologous protein of heterochromatic protein HP 1.Compared to the wild-type strain,the sporulation capacity was reduced in the deletion of hepA.The recomplement strain(RpohepA and RAnhepA)can recover the defects caused by hepA knockout,indicating that the HepA in Aspergillus nidulans and Penicillium oxalicum are highly conserved in their biological functions.HepA is actually an activated factor in cellulase gene expression.After four days of fermentation with ?hepA strains,the filter paper activity,endoglucanase activity,cellobiohydrolyase activity and?-glucosidase acctivity were decreased by 65.6%,77.3%,91.8%and 70.1%,respectively.The filter paper activity,endoglucanase activity,cellobiohydrolyase activity and ?-glucosidase acctivity of the strain with hepA overexpression were increased by 2.5-fold,2.9-fold,2.2-fold,and 11.9-fold,respectively.The Chromatin Accessibility by Real-Time PCR(CHART-PCR)technique was used to analyze the HepA-mediated changes in chromatin structure and the extent of chromatin opening in the upstream region of the cellulase gene in each mutant.In the ?hepA strain,the chromatins of the promoter regions(core promoter region and upstream region)of the two main cellulase genes cel7A/cbh1 and cel7B/eg1 were all open.Chromatin Accessibility Index(CAI)was significantly increased,indicating that HepA is required for chromatin condensation.3.LaeA-RcoA,RcoA-ClrB have intranuclear interactions and are required for the active expression of the cellulase gene of Penicillium oxalicumRcoA is the protein interacted with LaeA,which was identified with tandem affinity purification-mass spectrometry.A previous laboratory study found that RcoA interacts with the ClrB,the cellulase transcriptional activator.RcoA is highly conserved in eukaryotes and typically forms complexes with Cyc8.The interaction of RcoA with ClrB,LaeA,and Cyc8 was confirmed and subcellularly localized by bimolecular fluorescence complementation(BiFC)technology.The interaction between LaeA and ClrB and Cyc8 was studied.It shows that:(1)LaeA interacts with RcoA in the nucleus;(2)ClrB interacts with RcoA in the nucleus;(3)RcoA interacts with Cyc8 in the nucleus;(4)There is no intracellular interaction between LaeA and ClrB;(5)There is no intracellular interaction between LaeA and Cyc8;(6)There is no intracellular interaction between ClrB and Cyc8.Analysis of sporulation ability and cellulase biosynthetic ability of the clrB,rcoA,and laeA knockout and overexpression strains was performed.It was revealed that RcoA and LaeA can regulate the development of Penicillium oxalicum.However,the sporulation ability of Penicillium oxalicum was not related to the knockout and overexpression of ClrB.Knocking out rcoA significantly reduced the ability of cellulase synthesis.After 5 days cultivation at 30?,FPA activity,CMC activity,pNPC activity,and pNPG activity decreased by 86.5%,59.4%,72.0%,and 57.7%,respectively.The transcription level and cellulase activity of major cellulase genes were significantly reduced with knocking out of laeA and clrB,Double mutant strains,?laeAOEclrB,OEclrB?rcoA,?clrBOElaeA and?clrBOErcoA,were constructed.Compared with the wild type strain after 5 days cultivation at 30?,it showed that FPA activity,CMC activity,pNPCase activity,and pNPG activity in the ?laeAOEclrB strain decreased by 58.2%,67.9%,64.5%and 28.6%,respectively.The cellulase activity of OEclrB?rcoA strains decreased by 93.5%,88.1%,91.4%and 74.2%;The cellulase activity of ?clrBOElaeA strains decreased by 94.2%,98.2%,72.5%and 19.7%;The cellulase activity of?clrBOErcoA strains decreased by 95.1%,98.8%,77.7%and 13.9%.The results showed that ClrB,RcoA,LaeA in Penicillium oxalicum are all required for cellulase gene activation expression.4.Mechanism of activation of cellulase gene expression involved by LaeA,RcoA,ClrBThe crude proteins of LaeA were obtained by tandem affinity purification.S-adenosylmethionine was used as a methyl donor to catalyze human histone H2B.It was revealed the methylation of lysines at position 109 and position 117 of the human histone H2B with LC-MS/MS identification analysis.The identity between the P.oxalicum H2B and Human histone H2B is 61.7%,and the methylation sites corresponded to the position 123 and position 131 of Penicillium oxalicum H2B.Therefore,we speculated that,LaeA may change the structure of chromatin by methylating the lysine at position 123 and position 131 of histone H2B in Penicillium oxalicum.Then we constructed the H2B mutations strains H2BK123A,H2BK131A,and analyzed the phenotypes of the strains with ?laeA.Spore development,expression of secondary metabolic gene clusters,and regulation of cellulase gene expression could be affected by LaeA in Penicillium oxalicum.The development of the H2B point mutation strain was severely affected,the color of spores became lighter,and the amount of spores decreased.Besides,the H2B point mutant strain could silence three secondary metabolic gene clusters,while the laeA knockout strain could silence the expression of five secondary metabolic gene clusters.The ability of cellulase synthesis was reduced in H2B point mutation strains,which was similar to laeA knockout strains.At the same time,it was found that knockout of clrB,rcoA,and laeA resulted transcriptional reduction of the cellulase genes cel7A/cbhl and cel7B/eg1,which was accompanied by chromatin condensation in the promoter region.Based on results above,we proposed a mechanism model for the activation of cellulase gene expression by laeA,rcoA and clrB:the transcription factor clrB binds to the cellulase gene promoter region to recruit of RcoA-Cyc8 complex.RcoA was served as a mediator to recruit the histone methyltransferases,LaeA.The chromatin structure was altered and the transcription of cellulase genes was activated by methylating the histones H2B Lys123 and H2B Lys 131.