Functional Identification Of A New Transcription Factor PoxCxrC In Penicillium Oxalicum And Its Homologous Protrin TP05746 In Talaromyces Pinophilus

Plant biomass is the most abundant renewable resource on earth.The biorefinery of plant biomass can produce high value-added products.Plant-biomass-degrading enzymes（PBDE）secreted by filamentous fungi are green catalysts for biorefinery,but their low yields severly hinder industrial development of biorefinery.Penicillium oxalicum and Talaromyces pinophilus can secrete complete PBDE under induction of carbon sources.The expression of PBDE-encoding genes is strictly regulated by transcription factor（TF）,but understanding of molecular regulatory mechanism of TF is rather limited.Previous work constructed the mutantΔPox Ku70,in which the gene Pox Ku70 involved in non-homologous recombination was knocked-out in P.oxalicum HP7-1.Futhermore,Pox Cxr A,a new key regulatory gene regulating the biosynthesis of major PBDE,was deleted in theΔPox Ku70.Comparative transcriptomic analysis found that the transcriptional level of POX01387（named as PoxCxrC,P.oxalicum cellulase and xylanase regulator C）in the mutant?Pox Cxr A significantly increased by 1.6-fold compared with that in the parental strain?Pox Ku70.In this study,it was confirmed that Pox Cxr A directly and dynamically regulated the expression of PoxCxrC.Sequence anlignment analysis showed that all the homologous proteins of PoxCxrC in Gen Bank were hypothetical.PoxCxrC was localized in the nucleus and showed transcription activation function.In this study,the mutant?PoxCxrC was obtained by knocking-out PoxCxrC gene in?Pox Ku70,and its complementation strain?PoxCxrC::PoxCxrC was constructed.When these three strains were transfered into liquid medium containing Avicel or wheat bran plus rice straw（WR）and cultivated for 2-4 days,the production of filter paper cellulase（FPase）and xylanase of the mutant?PoxCxrC significantly increased by 42.2%-119.6%.Similarly,the amylase production significantly increased by 48.9%-81.6%under soluble corn starch（SCS）induction.Compared withΔPox Ku70,the biomass of the mutant?PoxCxrC significantly increased by 59.2%-93.8%at 36 h-84 h in the liquid medium containing carboxyl methyl cellulose（CMC）;the conidiospore production of the mutant?PoxCxrC increased by 6.2-and 1.6-fold（p<0.01）in solid medium containing CMC and Avicel for 11 and 5 days,respectively.The phenotypes of the complementation strain were similar to those of the parental strain.These results indicated that PoxCxrC negatively regulated the biosynthesis of major PBDE,fungal mycelial growth and conidiospore production of P.oxalicum.Comparative analysis of transcriptomes showed that the transcription levels of 1545 genes in the mutant?PoxCxrC changed significantly compared with those of the parental strainΔPox Ku70 cultivated on Avicel for 24 h.For example,transcription levels of the key cellulase and xylanase genes such as cbh1,cbh2,eg1,eg2,Cel12A,bgl1,xyn11A increased by 1.3-2.9 folds,and the key regulatory genes and genes related to fungal growth and development such as POX02484,Pox Cxr B,Pox Nsd D,brl A and flb D increased by 41.3%-258.6%in the mutant?PoxCxrC.EMSA results showed that PoxCxrC directly bound to the promoter region of the above-described genes.These results indicated that PoxCxrC directly regulated the expression of these genes.The DNA sequences encoding PoxCxrC and seven regions with different lengths of PoxCxrC(PoxCxrC_1-853,PoxCxrC_1-417,PoxCxrC_1-288,PoxCxrC_1-248,PoxCxrC_12-924,PoxCxrC_200-924and PoxCxrC_{12-200/248-288})under the control of Avicel-inducing promoter p Pox Eg Cel5B were used to respectively replace one protease gene inΔPox Ku70 to construct the corresponding strains.Deletion of the protease gene inΔPox Ku70 didn’t affect the PBDE production.Under Avicel induction for 4 days,compared withΔPox Ku70,the production of FPase by the strainΔPox Ku70::PoxCxrC_1-288 andΔPox Ku70::PoxCxrC_12-924 significantly reduced by 53.9%and 87.7%,respectively,but the strainsΔPox Ku70::PoxCxrC_1-248,ΔPox Ku70::PoxCxrC_200-924 andΔPox Ku70::PoxCxrC_{12-200/248-288} showed no significant changes,indicating that PoxCxrC_12-288 is required for normal regulatory function of PoxCxrC.The strainΔPoxCxrC::PoxCxrC_1-288 was constructed by introducing the DNA sequence coding PoxCxrC_1-288 into the mutantΔPoxCxrC.Compared with the mutantΔPoxCxrC,the production of FPase byΔPoxCxrC::PoxCxrC_1-288 significantly decreased by 14.0%,further confirming that PoxCxrC_12-288 is necessary for PoxCxrC.Sequence alignment analysis revealed that PoxCxrC contained two conserved amino acid sequences PYQLPPPR（16-23）and LPSVRSLLTP（65-74）.The DNA sequences encoding PoxCxrC_1-288 lacking the conserved amino acids PoxCxrC_1-288?16-23 or PoxCxrC_1-288?65-74 under the control of Avicel-inducing promoter p Pox Eg Cel5B were used to respectively replace one protease gene inΔPox Ku70 to obtain the corresponding strains.After 4 days of Avicel induction,the production of FPase by the strainΔPox Ku70::PoxCxrC_1-288?65-74 was similar to that ofΔPox Ku70,indicating that PoxCxrC_1-288?65-74 lost regulatory function.The production of FPase by the strainΔPox Ku70::PoxCxrC_1-288?16-23significantly reduced by 53.9%compared with that ofΔPox Ku70,indicating that PoxCxrC_1-288?16-23 still possessed partial regulatory function.The coding sequence of PoxCxrC with deletion of amino acids LPSVRSLLTP（65-74）was introduced into the mutantΔPoxCxrC.The FPase production of the obtained strainΔPoxCxrC::PoxCxrCΔ65-74 was similar to that ofΔPoxCxrC,confirming the necessity of LPSVRSLLTP（65-74）for PoxCxrC.GST-pull down analysis showed that PoxCxrC could form homopolymer,but did not when lacking LPSVRSLLTP（65-74）.These results indicated that LPSVRSLLTP（65-74）was essential for the proper function of PoxCxrC by mediating the formation of PoxCxrC polymer.A hypothetical protein TP05746 in T.pinophilus 1-95 was found to share the highest homology with PoxCxrC,and its similarity to PoxCxrC and PoxCxrC_12-288 was 70.65%and 66.55%,respectively.The gene Tp Ku70,which is involved in non-homologous recombination,was knocked out in T.pinophilus 1-95 to obtain the mutantΔTp Ku70.Futhermore,the gene TP05746 was deleted in theΔTp Ku70.Compared with that of theΔTp Ku70,the production of amylase,FPase and xylanase byΔTP05746 significantly increased by 112.9%-323.9%under SCS or wheat bran plus Avicel（WA）induction for 2-4 days.The biomass ofΔTP05746significantly increased by 58.2%-323.6%（24 h-60 h）and 15.8%-31.3%（24 h-36h）induced by SCS and WA.The conidiospore production ofΔTP05746respectively reduced by 63.4%and 52.6%in solid medium containing SCS or WA for 6 days.These results indicated that TP05746 negatively regulated the biosynthesis of major PBDE and mycelial growth,positively regulated conidiospore production of T.pinophilus.Comparative analysis of transcriptomes showed that the transcription levels of 4429 genes in the mutantΔTP05746 changed significantly compared with those in the parental strainΔTp Ku70 induced by SCS for 12 h.Among them,the transcription levels of amylase genes such as TP03368,amy13A and TP12319 as well as regulatory genes such as Tp Rfx1 and Tp Amy R increased by 1.1-58.7 folds,while regulatory genes Ve A,Wet A and Flb A significantly reduced by 56.2%-70.0%.EMSA results showed that TP05746 directly bound to the promoter region of the above-described genes.These results indicated that TP05746 directly regulated the expression of these genes in T.pinophilus.The strainΔPox Ku70::TP05746 was obtained by introducing TP05746 into P.oxalicum strainΔPox Ku70.Compared with that in theΔPox Ku70 under Avicel induction for 1-3 days,amylase,cellulase and xylanase production ofΔPox Ku70::TP05746 significantly reduced by 15.6%-84.6%except forβ-glucosidase,indicating that TP05746 regulated major PBDE production in P.oxalicum.In conclusion,the novel TF PoxCxrC of P.oxalicum inhibited the major PBDE biosynthesis,fungal mycelial growth and conidiospore production by negatively regulating the expression of key PBDE genes and genes related to fungal growth and development.PoxCxrC_12-288 and LPSVRSLLTP（65-74）were required for the normal function of PoxCxrC,and LPSVRSLLTP（65-74）mediated the polymerization of PoxCxrC.TP05746,a PoxCxrC homolog found in T.pinophilus 1-95,negatively regulated the production of major PBDE and fungal mycelial growth as well as positively regulated the conidiospore production in T.pinophilus by regulating the expression of key PBDE genes and genes related to fungal growth and development.The gene TP05746 expressed regulatory function in P.oxalicum.These results provide new insights into molecular mechanisms of regulation of PBDE gene expression in filamentous fungi,and also provide theoretical guidance for rational design and directed genetic modification of fungi to improve PBDE yield.