| Lysozyme cleaves the ?-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan of bacterial cell wall,resulting in bacterial cell lysis.It is a highly safe drug,feed and food additive.Currently,the lysozyme on the market is mainly extracted from egg white.Human lysozyme(hLYZ)has high biological activity and thermal stability compared with that from egg white.However,the use of human lysozyme is restricted because of its limited source,purification cost and other factors.The construction of engineered strains to produce recombinant hLYZ is an effective means to solve the problem.In this research,the effects of co-expression of molecular chaperone and transcription factor,and gene dosage on hLYZ expression were studied in order to obtain the engineered P.pastoris with high hLYZ expression.(1)Based on the gene sequences of Kar2p,EROlp and Haclp in GenBank,primers were designed and recombinant vectors pG418-Kar2p,pG418-ERO1p and p9K-Haclp were constructed.The recombinant vectors were transferred into KM71-pPIC-hLYZ-3 genome by electroporation.The Engineered strains KM71-pPIC-hLYZ-Kar2p-5,KM71-pPIC-hLYZ-ERO1p-7 and KM71-pPIC-hLYZ-Hac1p-11,co-expressing molecular chaperone Kar2p,ERO1p and transcription factor Hac1p with hLYZ,respectively,were screened by G418 resistance.KM71-pPIC-HLYZ-3,KM71-pPIC-hLYZ-Kar2p-5,KM71-pPIC-hLYZ-ERO1p-7 and KM71-pPIC-hLYZ-Haclp-11 were induced by methanol for 168h in shake flask,and the hLYZ expression levels were analyzed.The total amount of extracellular protein secreted by the four strains arrived at 361.02±11.97?g/mL,518.99±17.92?g/mL,339.68±28.69?g/mL and 440.35±26.95?g/mL,respectively.The total enzyme activity of the four strains arrived at 40400±1898.13U/mL,48240±4145.87U/mL,37480±2493.15U/mL and 528000±3259.84U/mL,respectively.Analysis of the extracellular total protein and lysozyme activity showed that EROlp co-expression had no significant effect on hLYZ expression and Kar2p co-expression had a significant effect on hLYZ expression.The extracellular protein and the lysozyme activity of strain KM71-pPIC-hLYZ-Kar2p were higher than that of strain KM71-pPIC-hLYZ,which were increased by 43.76%and 19.41%,respectively.Results also showed that Haclp co-expression could promote hLYZ production.Compared with the control strain KM71-pPIC-hLYZ-3,the extracellular protein and the lysozyme activity of strain KM71-pPIC-hLYZ-Haclp were increased by 21.97%and 30.69%,respectively.(2)Starting from plasmid pPIC-hLYZ(containing 1 copy of hLYZ expression cassette),a series of recombinant vectors,pPIC-2hLYZH,pPIC-4hLYZH and pPIC-6hLYZH,with 2,4 and 6 hLYZ expression cassette,respectively,were constructed through using same tail enzymes BamH ? and Bgl ?.The linearized recombinant plasmids pPIC-hLYZ,pPIC-2hLYZH,pPIC-4hLYZH and pPIC-6hLYZH were transferred into Pichia pastoris KM71 by electroporation.As a result,recombinant strains KM71-pPIC-hLYZ-8,KM71-pPIC-hLYZ-3,KM71-pPIC-2hLYZH-5,KM71-pPIC-4hLYZH-3 and KM71-pPIC-6hLYZH-4 were obtained,containing 1,3,2,4,and 6 copies of hLYZ expression cassette,respectively.Strains KM71-pPICZaA,KM71-pPIC-hLYZ-8,KM71-pPIC-hLYZ-3,KM71-pPIC-2hLYZH-5,KM71-pPIC-4hLYZH-3 and KM71-pPIC-6hLYZH-4 were induced by methanol for 168h,and the hLYZ expression levels were analyzed.The total amount of extracellular protein secreted by the six strains arrived at 69.09±11.31?g/mL,178.62±32.52?g/mL,212.32±7.00?g/mL,353.68±12.77pg/mL,436.99±26.08?g/mL and 334.02±11.97?g/mL,respectively.The total enzyme activity by the six strains arrived at 0±0/mL,23700±339.41U/mL,490000±1175.76U/mL,30200±565.69U/mL,61900±2036.47U/mL and 57000±2899.93U/mL,respectively.Analysis of the extracellular total protein and lysozyme activity showed that strain KM71-pPIC-4hLYZH-3(containing 4 copies of hLYZ expression cassette)had the highest extracellular protein and lysozyme activity,which were increased by 1.45 times and 1.61 times,respectively,compared with KM71-pPIC-hLYZ-8(containing 1 copy of hLYZ expression cassette).Results also showed that the extracellular total protein and the lysozyme activity of strain KM71-pPIC-6hLYZH-4 were 23.6%lower and 7.92%lower than that of strain KM71-pPIC-4hLYZH-3,respectively.(3)The linearized recombinant vector p9K-Haclp was transfreed into KM71-pPIC-4hL YZH-3 genome by electroporation.The engineered strain KM71-pPIC-4hLYZH-Haclp-8 was obtained,co-expressing the transcription factor Haclp and human lysozyme.Strains KM71-pPIC-4hLYZH-Haclp-8 was induced by methanol for 168h.The total amount of extracellular protein secreted by strains KM71-pPIC-hLYZ-8,KM71-pPIC-4hLYZH-3 and KM71-pPIC-4hL YZH-Haclp-8 arrived at 196.51±5.25?g/mL,453.59±7.08?g/mL and 517.82±4.19?g/mL,respectively.The enzyme activitiy of fermentation supernatant arrived at 23200±1064.64U/mL,64800±2372.75U/mL and 78600±1134.95U/mL,respectively.Analysis of the extracellular total protein and the lysozyme activity showed that,compared with KM71-pPIC-4hLYZ-3,the extracellular protein and lysozyme activity of strain KM71-pPIC-4hLYZH-Haclp-8 were increased by 14.16%and 21.30%,respectively.Compared with KM71-pPIC-hLYZ-3,the extracellular total protein and the lysozyme activity of strain KM71-pPIC-4hLYZH-Hac1p-8 was higher,which was increased by 2.64 times and 3.39 times,respectively.This research showed that the hLYZ expression could significantly be enhanced through optimizing gene dosage and co-expressing transcription factor Haclp in P.pastoris KM71. |
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