Functional Identification Of Translation Elongation Factor EEF1A In Regulation Of Plant-Biomass-Degrading Enzyme Biosynthesis In Penicillium Oxalicum

Plant biomass such as cellulose,hemicellulose,starch,etc.,is the richest renewable biomass resource on the earth.It can be used as feedstock to produce clean and renewable bioenergy through biorefinergy.Enzymatic hydrolysis of plant biomass is a key step for biorefinergy.Filamentous fungus Penicillium oxalicum can produce integative plant-biomass-degrading enzyme system with high enzymatic activity,but having problems low enzyme yield and high cost.The knowledge on regulation of biosynthesis of plant-biomass-degrading enzymes in P.oxalicum is rather limited.Previous studies found a novel transcription factor PoxCxrC negatively regulating the biosynthesis of plant-biomass-degrading enzymes in P.oxalicum,but its regulatory mechanism is still unclear.The aim of this study is to find the interacting protein of PoxCxrC and further identify its function in biosynthesis of plant-biomass-degrading enzymes.In this study,the proteins interacting with PoxCxrC were screened by tandem affinity purification-mass spectrometry(TAP-MS)and immunoprecipitationmass spectrometry(IP-MS).As a result 26 candidate proteins were found.Notably,the proteins POX06702 and POX00101 were verified to interact physically with PoxCxrC via GST pull-down assay.Sequence alignment analysis revealed that POX00101 was 94.17% identical to GTP-binding protein Ran in Neurospora crassa strain bd-A;POX06702 was 94.98% identical to EF-1α in Aspergillus niger strain CBS 101883,indicating that POX00101 and POX06702 are small GTPase Ran and translation elongation factor eEF1 A,respectively.The overexpression strain ΔPox Ku70::eEF1 A was constructed through homologous recombination,in the P.oxalicum parental strain ΔPox Ku70.When cultivated in medium containing Avicel or soluble corn starch for 2-4 d after transfer from glucose,the production of cellulse(i.e.,filter paper cellulase [FPase],carboxymethylcellulase [CMCase] and p-nitrophenyl-D-glucopyranosidase[p NPGase]),xylanase and amylase in the ΔPox Ku70::eEF1 A considerably ehanced by 31.03%-58.64%,30.49%-40.61% and 68.84%-100.48%,respectively,in comparious with those in the parental strain ΔPox Ku70,which suggested that eEF1 A positively regulated the production of plant-biomass-degrading enzymes in P.oxalicum.In addition,the engineered strain ΔPoxCxrC::eEF1 A and double gene overexpression strain ΔPox Ku70::PoxCxrC::eEF1 A were constructed.When cultivated in medium containing Avicel,wheat bran plus rice straw,or soluble corn starch,the production of cellulase,xylanase and amylase in the engineered strain ΔPoxCxrC::eEF1 A were 50.40%-98.43%,45.6%-120.76% and 55.4%-314.6% more than those in the ΔPoxCxrC,respectively.The results abtained above clearly confirmed that Simultaneous manipulation of transcriptional regulator PoxCxrC and translational elongation factor eEF1 A ehancing the production of plant-biomass-degrading enzyme is feasible in P.oxalicum.By contrast,the production of cellulase,xylanase and amylase by theΔPox Ku70::PoxCxrC::eEF1 A exhibited no significant alteration,compared to those in the mutant ΔPox Ku70::PoxCxrC,suggesting that large amount of m RNA is prerequisite for the enhancement of plant-biomass-degrading enzyme production.Protein subcellular localization analysis displayed that the fusion protein eEF1A-GFP was in cytoplasm and around nucleus in the overexpression strainΔPox Ku70::eEF1 A.The fusion protein PoxCxrC-GFP was located in nucleus in the double gene overexpression strain ΔPox Ku70::PoxCxrC::eEF1 A.Analysis of phenotype,sporulation and biomass accumulation revealed that colony diameter of the engineered strain ΔPoxCxrC::eEF1 A was larger than that of the mutant ΔPoxCxrC on PDA plates,while similar to that of the parentalΔPox Ku70.The overexpression strain ΔPox Ku70::eEF1 A exhibited increased spore production by 19.3%-270.0% on solid plates containing PDA,Avicel,wheat bran plus rice straw,or soluble corn starch.The biomass of the engineered strainΔPoxCxrC::eEF1 A and mutant ΔPoxCxrC increased by 20.6%-30.4% compared with the ΔPox Ku70 in the late period of cultivation(60 h-72 h).Therefore,the eEF1 A is involved in the growth and sporulation of P.oxalicum.Real-time quantitative PCR assay showed that the expression of major plantbiomass-degrading enzyme genes and sporulation-related genes exhibited remarkably alteration in the overexpression strain ΔPox Ku70::eEF1 A and engineered strain ΔPoxCxrC::eEF1 A,relative to those in the parental strainΔPox Ku70.For instance,the transcriptional levels of cellulase and xylanase genes cbh1,cbh2,eg1,Cel12 A and bgl1 were up-regulated by 1.0-33.0-fold;amylase genes Pox GA15 A,POX02412 and amy13 A and their regulatory gene amy R were up-regulated by 3.9-62.2 times;the genes brl A,aba1 and flb D related to spore production improved by 3.5-20.0-fold.When cultived in medum containing wheat bran plus rice straw or soluble corn starch for 4 d after transfer,the secred curde enzymes by the engineered strainΔPoxCxrC::eEF1 A were collected and used to hydrolyze the pretreated sugarcane bagasse and raw cassava flour,respectively.The results displayed that the hydrolysis effiency of crude cellulase from the ΔPoxCxrC::eEF1 A against the pretreated sugarcane bagasse was similar to that by the ΔPox Ku70.Remarkably,the relased glucose during hydrolysis of raw cassava flour by crude amylase from the ΔPoxCxrC::eEF1 A for 96 h was 9.3%-15.5% more than that by the ΔPox Ku70,and the glucose concentration at 96 h reached 101.0 g/L with a hydrolysis effiency of 89.1%.Studies on the proteins interacted by PoxCxrC and their roles in the regulation of plant-biomass-degrading enzyme biosynthesis in P.oxalicum has the important reference significance on deeply understanding of regulatory mechanism of fungal plant-biomass-degrading enzyme biosynthesis,and also provide feasible targets for improving the production of plant biomass-degrading enzymes through synthetic biology.