Molecular Engineering And High-yield Production Of Trypsin From Streptomyces Griseus

Streptomyces griseus(S.griseus)trypsin(EC 3.4.21.4)is an alkaline protease and has stringent cleavage specificity at the carboxyl terminal of arginine and lysine.It possesses enzymatic properties similar to bovine trypsin and has great potential applications in the leather processing,bioethanol,detergent,and pharmaceutical industry.In past years,S.griseus trypsin(SGT)had been investigating in different microbial hosts,such as Escherichia coli(E.coli),Bacillus subtilis(B.subtilis),Streptomyces,and Pichia pastoris(P.pastoris).However,the heterologous expression of SGT is still hindered by four critical bottlenecks,(1)low yield in microbial host;(2)SGT trypsinogen can’t auto-activate;(3)the active trypsin cleaves;(4)the problem from the synergistic defect of substrate specificity and catalytical efficiency.In this work,the auto-activation expression strategy of SGT was established by the modification of propeptide,for high-yield production of trypsin.Moreover,the self-degradation site of trypsin was systematically analyzed,and the stability of trypsin was enhanced by protein engineering.Then,the site-directed mutagenesis was applied to improve the selectivity and catalytic activity of trypsin to the lysine substrate.Finally,the production of trypsin was further improved by modular regulating the endogenous protein of Pichia pastoris.The main results are as follows:(1)The expression level of trypsin was increased by adopting the hydrophobic propeptide FVEF at N-terminus.We mutated the Y12 site of YVEF of Exmt(R145I)to screen the EIF(F12VEF)mutant from eight designed mutants.And,the protein expression level reached 50.9 mg·L-1(85.36±1.41 U·mL-1)and the catalytical activity of EIF mutant increased by 1.36-fold with BAPNA as the substrate.Using N-terminal sequencing and western blot analysis,we validated that the FVEF peptide was cleaved during the expression procession.Interestingly,the FVEF peptide facilitated the zymogen SGT inverting to active SGT in P.pastoris,when fused between the native propeptide of SGT trypsinogen and mature polypeptide.(2)The high-yield production of trypsin was engineered by auto-activation strategy and signal peptide engineering method.Screening from three types of auto-catalytical peptides,three propeptides,Dm(LLPQLDGR),Ha(APFDDDDK)and Btc(FPVDDDDK)exhibited increased activities by 81.84%,99.81%,and 105.64%,respectively.Next,by fusing artificial peptide at the N-terminal,the mutant tbcf showed 1.36-fold increase of trypsin activity(136.65±2.81 U·mL-1).Furthermore,an artificial secretory signal peptide αMFH was designed,by screening a library of signal peptides used in P.pastoris,optimizing the secondary structure,and modifying the density of positive charge in C-terminal.Finally,the trypsin activity was increased by 57.96%(26.38±1.61 U·mL-1)in flask culture,and further reached 177.85±2.83U·mL-1(3-L fermentor)and 222.44±2.83 U·mL-1(15-L fermenter).(3)The stability and production of trypsin were improved by engineering the autolysis residues.A mutant tbcf(K101 A)with improved stability was obtained with alanine scanning method.And,the activity of tbcf(K101 A)was increased by 1.54-fold(40.57±2.97 U·mL-1).Moreover,the other five autolysis sensitive sites R21,R32,K122,R153,and R201,were identified from the hydrolysates of tbcf(K101A).Consequently,the recombination mutation K101A/R201V increased the trypsin activity to 60.85±2.42 U·mL-1 in flask culture.Consequently,the molecular dynamics simulation indicated K101A/R201V mutations shortened the distance between D102 and H57.So,the catalytical activity(kcat/Km value)of tbcf(K101A,R201V)increased by 1.24-fold.And,the trypsin activity increased to 549.18±4.27 U·mL-1,which was 2.41-fold higher than the parent strain.The production of trypsin was increased with modularized regulation on the secretory pipeline of trypsin in P.pastoris.The modularized endogenous proteins were overexpressed in GS115-tbcf(K101A,R201V)strain.The UBC1,SEC 1 and SSO2 protein increased the trypsin activity by 31.6%(80.09±2.08 U·mL-1),24.1%(75.54±2.16 U·mL-1)and 41.5%(86.11±1.21 U·mL-1),respectively.The co-overexpression of both UBC1 and SSO2 increased the trypsin activity to 109.25±4.76 U·mL-1 in flask culture and 689.47±6.78 U·mL-1 in the 3-L fermentor.(4)The preference and catalytical activity for lysine substrate were improved by engineering T190,L89,and Q90 sites.Firstly,two antibiotic selection plasmids including pPARS(geneticinR)and pZARS(zeocinR)and a histidine auxotroph selection plasmid pHARS were constructed with autonomous replication sequence pan-ARS_WT.Then,the stability and expression efficiency of plasmids were analyzed by expressing the reporter protein EGFP and trypsin.The plasmid pZARS afforded efficient expression of trypsin,with the supplement of 75 mg·L-1 zeocin in growth media.Based on tbcf(K101A,R201V)expressed by plasmid,its substrate preference and catalytical activity were engineered.Finally,the combined mutation L89Y/Q90R/T190Q afforded a higher preference to lysine substrate(increase 4.16-fold)and increased catalytical activity(2.20 fold).On the other hand,the L89Y/Q90R/T190N mutation led to the highest preference to lysine substrate(increased by 13.00-fold).