Optimization Of Human Lysozyme Expression In Pichia Pastoris

Human lysozyme（h LYZ）is a novel type of antimicrobial compounds,and its mechanism of action is different from traditional antibiotics.h LYZ does not cause bacteria to develop drug resistance,and because of its extremely high safety,it has received extensive attention from various industries.In this paper,Pichia pastoris is used to secrete and express h LYZ,and the main research results are as follows:（1）In this study,the DNA sequence encoding h LYZ was optimized and artificially synthesized based on the Pichia pastoris codon preference.The h LYZ gene sequence was inserted into the plasmid p PIC9K to obtain the recombinant plasmid p PIC9K-h LYZ,and the constitutive promoter P_GAP was used to replace the inducible promoter P_AOX1 in p PIC9K-h LYZ to obtain the recombinant plasmid p PIC9K-P_GAP-h LYZ.The recombinant plasmids containing different promoters were inserted into the genome of Pichia pastoris GS115 respectively.The inducible promoter P_AOX1 and the constitutive promoter P_GAP were used to secrete and express h LYZ.The secretion and expression of h LYZ in the fermentation supernatant of Pichia pastoris was verified by SDS-PAGE and Western blot,and the activity of h LYZ in the fermentation supernatant was detected using Micrococcus lysodeikticus as an indicator.Then the fermentation conditions were optimized in shaking flasks.The results showed that the optimal fermentation conditions of the inducible promoter P_AOX1 for h LYZ production were 26℃,p H 6.0,and the maximum enzymatic activity reached 16,400±700 U·m L^-1 at 96 h after methanol induction.The optimum conditions for the production of h LYZ by the constitutive promoter P_GAPfermentation were 28℃,p H 6.0,and the enzyme activity reached the highest at 48 h after using glucose as the carbon source,but only reached 310±17.3 U·m L^-1,Therefore,the inducible promoter P_AOX1 was used to produce h LYZ in the following study.（2）In this study,a combination of multiple strategies was used to improve the secretion efficiency and enzymatic activity of h LYZ.To optimize the secretion signal peptide of h LYZ,ten signal peptides were used to replace theαsignal peptide in p PIC9K-h LYZ.Among them,the HFBI signal peptide（Hss）could increase the secretase activity of h LYZ to 24,880±1034.6 U·m L^-1,which was 1.51 times of the original strain.On the basis of using the best signal peptide,CRISPR/Cas9 gene editing technology was adopted to knock out the vacuolar sorting receptor VPS10-1/2 gene in Pichia pastoris.As a result,two strains with single gene defect and one strain with double gene defect were constructed.Compared with other strains,the strain with the knockout of the VPS10-1 gene exhibited the greatest increase in the secretion efficiency of h LYZ,and its enzymatic activity was increased to59,080±3040 U·m L^-1,which was 3.6 times of the original strain.Finally,the effects of ten Pichia pastoris endogenous chaperones/transcription factors on the secretion efficiency and enzymatic activity of h LYZ.Two chaperones,Ero1p and Pdi1p,were co-expressed to increase the secretion efficiency and enzymatic activity of h LYZ,and the secretase activity of h LYZ increased to88,780±4830 U·m L^-1,which was 5.41 times of the original strain.（3）In order to reduce the production cost of h LYZ,ROMY medium was obtained by optimizing the medium of Pichia pastoris,Then,the secretion and expression of h LYZ was improved by optimizing the initial cell concentration of methanol flow using a 5 L fermenter,and the highest enzyme activity in the 5 L fermenter reached 352,000±16696.5 U·m L^-1,the protein content reached3.18 mg·m L^-1.This study laid the foundation for the industrial production of h LYZ.