Expression Of Rhizomucor Miehei Lipase RML In Protease-deficient PichiaPink System

Rhizomucor miehei lipase?RML?is widely used in papermaking,food,leather production,biodiesel and other industries due to its high catalytic specificity at 1,3 sites.P.pastoris expression system is recognized as a powerful tool for large-scale protein expression,which has the advantages of short expression cycle,high expression,easy industrialization and high-density culture,as well as post-translational modification of exogenous proteins by eukaryotic expression system,such as glycosylation and phosphorylation.However,there are two drawbacks in the expression of RML in P.pastoris:?1?RML is degraded by yeast vacuolar protease in both secretion expression and surface display,which is aggravated by high-density fermentation of P.pastoris.?2?the activity of RML secreted or displayed on the surface is not high enough to meet the needs of industrial production.Based on this,the commercial protease-deficient P.pastoris was used as host cells to effectively improve the degradation of RML.The secretion and display of RML were further enhanced by means of genetic engineering,such as signal peptide and anchoring protein,which laid a solid foundation for its theoretical research and industrial application.The specific research contents and results are as follows:?1?In this paper,nine potential endogenous signal peptides from P.pastoris secretome were predicted by online software signalP4.1:FLO10,CPR5,PRY2,DSE4,NUP145,MSB2,SSP120,FRE2 and FLO9.At the same time,eight commonly used P.pastoris exogenous signal peptides were screened.The secretory effects of those signal peptides on enhanced green fluorescent protein?EGFP?and RML in protease-deficient PichiaPink expression system were investigeted,using signal peptide of Saccharomyces cerevisiae alpha-mating factor??-MF?as control.The results showed that:compared with?-MF,endogenous signal peptide FLO10,PRY2,DSE4,MSB2,SSP120,FRE2 could effectively mediate EGFP secretion,and PRY2 mediated the highest level of EGFP secretion,which was 2.15 times that of?-MF.Endogenous signal peptide FLO10,PRY2,DSE4,FRE2 could effectively mediate RML secretion,and FLO10 mediated the highest level of RML secretion,which was 1.50times that of?-MF.The secretion of EGFP and RML by exogenous signal peptide was not as good as that of?-MF.?2?Using GPI cell wall protein GCW21 of P.pastoris as anchoring protein,the surface display of lipase RML in protease-deficient PichiaPink expression system was realized.A protease-deficient strain GS115-ade2?-pep4?/pPink-HC-RML-GCW21 containing high copy plasmid of pPink-HC was screened.The lipase activity at shake flask fermentation level was the highest,reaching 121.72 U/g,which was higher than that of non-knockout protease strain GS115-ade2/pPink-HC-RML-GCW21 increased by 51.35%,which was 46.7%higher than that of GS115/PHKA-RML-GCW21 constructed in the laboratory.The optimum substrate for surface display of RML was C₈,the optimum temperature was 45?,and the optimum pH was 8.0.It was also found that Li⁺,Na⁺,K⁺,Mg²⁺could activate the activity of RML.