Construction And Application Of A FLP/FRT Mediated Conditionally Gene Knockout System In Penicillium Oxalicum

Biorefining is the process of converting biomass including lignocellulose as raw materials to high value-added bio-products through biomass pretreatment,enzymatic hydrolysis,and further fermentation,which is a green and environment-friendly biological process.Penicillium oxalicum carries a complete secretion system of plant-biomass-degrading enzymes and high?-glucosidase activity,which has attracted much attention,but with low yield.Molecular genetic engineering is an effective method to increase the production of plant-biomass-degrading enzymes from P.oxalicum.However,limited antibiotic markers limit multiple genetic manipulation in filamentous fungi.Therefore,a system of resistance marker used repeatedly in P.oxalicum is necessary,which will provide an powerful operation platform for efficient genetic engineering of the fungus to improve enzyme production.In order to construct a FLP/FRT site-specific recombination system,a suitable inducible promoter is needed in P.oxalicum.Gene POX00715(Pox Tcu1)encode a homolog of copper permease TCU1 from Trichoderma reesei.Compared with the environment without Cu²⁺,the transcription level of Pox Tcu1in P.oxalicum significantly increased at low concentration of Cu²⁺,while decreased at high concentration,indicating that the promoter P_tcu1was strictly regulated by the concentration of copper.P.oxalicum strain?Pox Ku70::flp-gfp was constructed by homologous recombination method.The FLP-GFP fusion protein was expressed in P.oxalicum?Pox Ku70 under the control of P_tcu1.Microscopic investigation indicated that the FLP-GFP localized in the nucleus.Futhermore,a recombination strain?Pox Ku70::flp/frt(G418)carrying G418 resistance gene was constructed and screened at concentration of 5?M Cu SO₄.The G418 resistance gene was cut in the strain when cultivated at concentration of 0.2?M Cu SO₄.The resultant strain could not grow on PDA plates containing G418.When cultivated on solid medium containing wheat bran plus rice stalk(WR)for 2–4 d,the production of filter paper cellulase(FPase),carboxymethyl cellulase(CMCase)and p-nitrophenyl-?-D-cellobisidase(pNPCase)in?Pox Ku70::flp/frt decreased by 33%–37.3%,25.6%–41.6%and 36%–41%comparedwiththe?Pox Ku70,whileshowingsimilar p-nitrophenyl-?-D-glucosidase production(pNPGase)and xylanase production.The colony size of strain?Pox Ku70::flp/frt was slightly smaller than that of the?Pox Ku70 on solid plates containing soluble starch,Avicel,wheat bran plus Avicel or WR,but having no significant difference on either PDA or glucose.In the previous work,transcription factors Atf1(POX03016),MBF1(POX08292)and Cxr C(POX01387)in P.oxalicum negatively regulated the production of cellulase and xylanase production.Here,we constructed three engineered strains?POX03016::flp/frt,?POX03016?POX08292::flp/frtand?POX03016?POX01387::flp/frt by using copper-responsive FLP/FRT recombination system,using the?Pox Ku70 as the parental strain.Under solid-state fermentation(SSF)condition for 2–4 d,the production of cellulase and xylanase of all constructed mutants above increased to various degrees in comparison with the?Pox Ku70::flp/frt.Among them,mutant?POX03016?POX01387::flp/frt showed the highest increase,with a record of2.4–30.7-fold and 79%–131%.The production of FPase,CMCase,pNPCase,pNPGase and xylanase of the mutant?POX03016?POX01387::flp/frt on the2–4 d increased by 369%–386%,244%–418%,2.44%–418%,219%–30.7%,respectively.The mutant?POX03016?POX01387::flp/frt was consecutively sub-cultured on plates for seven times.No significantly alteration of cellulase and xylanase production occurred among different sub-cultures,showing the genetic stable of the mutant.RT-q PCR analysis showed that transcriptional levels of the key genes encoding plant-biomass-degrading enzymes in the mutant?POX03016?POX01387::flp/frt significantly up-regulated in comparison with those of?Pox Ku70::flp/frt.When cultivated in the solid medium containing wheat bran and rice straw as carbon sources for 24 to 48 h,the transcription level of cellulase genes cbh1,cbh2,eg1,eg2,cel12 and bgl1 in?POX03016?POX01387::flp/frt significantly increased by 10.8-fold,14.5 to16-fold,10.96 to 11.66-fold,1.71 to 6.55-fold,0.59 to 1.4-fold,and 4.86 to29.29-fold,respectively,and transcription of xylanase gene(xyn11A)was upregulated by 71%-135%.In addition,?POX03016?POX01387::flp/frt exhibited similar colonic phenotypes and 26%–35.2%reduced biomass accumulation in comparison with?Pox Ku70::flp/frt.Crude enzymes from mutant?POX03016?POX01387::flp/frt and?Pox Ku70::flp/frt cultivated in solid WR medium for 6 d were used to hydrolyze surgarcane bagasse pretreated by combination of 8.0%Na OH and 2.48%H₂O₂.Compared with that of?Pox Ku70::flp/frt,glucose concentration at 96 h from pretreated sugarcane bagasse hydrolyzed by the crude enzyme from?POX03016?POX01387::flp/frt increased by 12.1%,reaching 23.0 mg/mL.