Study On The Properties,Protein Engineering And Application Of Novel AA9 LPMOs

In recent years,a new family of enzymes that synergizes with cellulase has been discovered in cellulose-degrading filamentous fungus.They catalyze the cleavage of crystalline cellulose and assist cellulase to degrade cellulose,via an oxidative mechanism.They are effective in facilitating the enzymatic saccharification of lignocellulose and can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass.These enzymes were termed lytic polysaccharide monooxygenases(LPMOs)and auxiliary activity family 9(AA9).Trichoderma reesei is an important host for industrial cellulase production.However,the number and content of AA9 LPMOs in the extracellular protein of T.reesei is relatively low.Although there are reports on AA9 LPMOs,which have been shown to exhibit significant synergy with cellulase from T.reesei,it is urgent to indentify AA9 LPMOs possessing high activity,high thermostability and synergism with T.reesei-derived cellulase.In addition,one of the difficulties in improving T.reesei cellulase production by gene manipulation is lacking of efficient genome editing systems.The study in this dissertation is divided into three parts.1)Searching more efficient novel AA9 LPMOs based on the known AA9 LPMOs;2)Protein engineering of novel AA9 LPMOs;3)Construction of efficient genome editing systems in T.reesei.1)Identification and characterization of two novel AA9 LPMOsIn recent years,although there are many reports on AA9 LPMOs,it is still urgent to find efficient and thermostable AA9 LPMOs that have the broad potential for industrial applications.In this study,two novel AA9 LPMOs from Talaromyces cellulolyticus and Neosartorya fischeri called TcAA9A and NfAA9A were identified.TcAA9A or NfAA9A was mixed with phosphoric acid swollen cellulose(PASC),xylan,starch and chitin.It was found that they only degraded PASC.By analyzing the oxidation products of PASC,TcAA9A and NfAA9A were categorized into Type 3.The LPMO activity was investigated spectrophotometrically using 2,6-dimethoxyphenol(2,6-DMP)as the substrate.The results suggested that TcAA9A,not NfAA9A,showed higher activity than TaAA9A(from Thermoascus aurantiacus).Furthermore,TcAA9A,not NfAA9A,showed a greater synergistic effect than TaAA9A in combination with cellulase from Trichoderma reesei,when using Avicel or delignined corncob residues(DCCR)as the substrate.The thermostability of AA9 LPMOs from TrTcAA9A,TrTaAA9A,or parent strain was also investigated.The results suggested that LPMO activity of the enzyme solutions was the maximum at a temperature of 40? for TrTaAA9A or parent strain and 40-45? for TrTcAA9A.At 45?,LPMO activity of the enzyme solution from TrTcAA9A remained over 70%,while the activities of TrTaAA9A and the parent strain were 35%and 31%after incubation for 48h.These results proved that the optimum temperature of TcAA9A was higher than that of TaAA9A.And TcAA9A exhibited excellent thermostable characteristics at 45?compared with TaAA9A.As the saccharification requires several days,thermostability of AA9 LPMOs is an important factor when screening AA9 LPMOs for enzyme cocktail with cellulase.The discovery of TcAA9A offers an innovative solution for improving the activity of cellulase cocktails.These results indicate the TcAA9A has the promising potential for industrial utilization.2)Study on the protein engineering of two novel AA9 LPMOs and mechanism of improving the activity of AA9 LPMOsTcAA9A or NfAA9A has 64 or 120 more amino acids in the C-terminus in comparison with that of TaAA9A.However,it was not identify possible function of C-terminus through sequence homology analysis against NCBI database.Then,it was decided to delete the C-terminus and obtained truncated-TcAA9A and truncated-NfAA9A.It was suggested that truncated-TcAA9A or truncated-NfAA9A belongs to Type 3.Comparing to TcAA9A and NfAA9A,the substrate specificity of truncated-TcAA9A and truncated-NfAA9A did not change.The surface charges of TcAA9A,truncated-TcAA9A,NfAA9A and truncated-NfAA9A were analysed.There was no difference on the surface charges between TcAA9A and truncated-TcAA9A;however,the surface charges near catalytic site of the truncated-NfAA9A were more positive than those of NfAA9A.The synergism of TcAA9A with cellulase from T.reesei was unchanged after truncation,but truncated-NfAA9A exhibited better synergism with cellulase from T.reesei than NfAA9A and TaAA9A.Site-directed mutagenesis of amino acids of truncated-NfAA9A showed that the positive surface charges near catalytic site possibly contributed to elevate the synergism of truncated-NfAA9A.This strategy is different from previous reports.It provides an innovative solution for improving enzyme cocktail activity for lignocellulosic degradation as well as a better understanding of protein engineering AA9 LPMOs to improve synergism with cellulase from T.reesei.3)Construction of efficient genome editing systems for T.reeseiThe lack of selective markers has been a key problem for gene editing in T.reesei.To resolve this problem,a novel genetic manipulation system with the Cre-loxP system and Tet-on system in T.reesei was constructed.However,it was suggested that the Tet-on system is not suitable for T.reesei.Then,an attempt was made to construct the clustered regularly interspaced short palindromic repeats(CRISPR)-Cpf1/Cas9 system in T.reesei,but the experimental results suggested that cpf1 without codon usage optimization may not be expressed in T.reesei and the structure of sgRNA,Cas9 proteins,or the complex formed by single guide RNA(sgRNA)and Cas9 may be disrupted during protoplast transformation process.Although a marker free or marker recycle genome editing system for T.reesei was successfully established,these attempts have laid the foundation for subsequent efforts at constructing an efficient gene editing platform in T.reesei.