Co-expression Of ?-glucosidase And Laccase In Trichoderma Reesei By Random Insertion With Enhanced Filter Paper Activity And Expression Of Aflatoxin Degrading Enzyme In Trichoderma Reesei And Aspergillus Niger

Trichoderma reesei is famous for the strong ability to secrete cellulase and has been studied for cellulose degrading system as well as used in the industrial prudction for a long time.However,it has been demonstrated that ?-glucosidase(short for BGL)is the bottleneck of the cellulase system and the component ligin in biomass can not be degraded by this strain.In order to solve this problem,Rut-C30 was modified with enhanced BGL activity to balance the cellulase system and enzyme laccase(short for LAC)for lignin degradation.Initially,the binary vector p1300-PTrb2 was constructed to express T.reesei bgll/bgl2 under the control of Ppki and T-nos terminator.Random insertion was performed via Agrobacterium tumefaciens-mediated transformation(short for ATMT).A total of 353 mutants were obtained,and 34PTrb2 was exceptionally stable with increased filter paper activity(short for FPA)and BGL activity after screening for extracellular enzyme activity.Subsequently,34PTrb2 was used as host and inserted the laccase gene from Foms lignosus,with Pgpd followed by cellobiohydrolase signal peptide ss and T-nos as terminator.Several mutants successfully expressed LAC with stable activity of approximately 0.13 U/mL.The FPA of mutant 15Gsslac was increased by 8.10%compared with that of 34PTrb2,which showed 40.48%higher activity than that of the host Rut-C30.The cellulase and biomass converting rate of 15Gsslac showed 4.4 times and 3.6 times higher than Rut-C30,respectively.Therefore,15Gsslac is a potential strain for saccharification of lignin-containing biomass materials.It has been demonstrated that using T.reesei and Aspergillus niger to pretreat animal feed can increase the digestibility of the feed.Aflatoxin(short for AFT)is extremely toxic as well as hard to decompose in animal body.In this work,T.reesei and A.niger were also transgenicly modified with aflatoxin degrading enzyme gene(short for atdz).The binary vectors of p1300-Gssatdz and p1300-Cssatdz were constructed to express Armillariella tabescens ATDZ under the control of Pgpd-ss/Pcbh1-ss.However,the enzyme ATDZ did not show any activity towards AFT,which might be caused by condon preference in different strains.But we provided one method to pretreat animal feed,which may become one direction to optimize the enzyme system.