Heterologous Expression Of Antibacterial Peptides And Enzymes In Yeast

The overuse and abuse of antibiotics have lead to the emergence of a large number of drug-resistance bacteria,even super drug-resistant bacteria.These bacteria have threatened human health severely.So,seeking for more safe and effective antibiotics as well as the alternatives of antibiotics that do not readily result in the bacterial drug resistance is urgent for human health as well as the production of health foods and animal feeding.The antimicrobial peptides are the natural antibiotics which synthesized by some varieties of organisms.These compounds possess good stability,have non toxic and side effects to human and animals,and do not result in the bacterial drug resistance.They have thus great potential for replacing some current antibiotics.The aim of this study was to construct the recombined expression vectors containing the antibacterial peptide genes and antibacterial enzyme genes,and further to realize the heterologous expression of the genes through transformation of the plasmids into Pichia pastoris and Kluyveromyces lactis.Our study also provides the technological data for practical uses of antibacterial peptides and enzymes.This thesis reports the main results on the study of the heterologous expression of several antibacterial peptide and glucose oxidase genes in the cells of Pichia pastoris and Kluyveromyces lactis.1.The synthetic gene of antimicrobial peptide AP,a good antimicrobial peptide for gram negative bacteria,was recombined into the vector pPICAP,and transformed into the cells of Pichia pastoris SMD1168.After selection,we obtained a strain,designated as AP26,which had a strong ability to inhibit the growth of Escherichia coli.The 72 h cultures of this strain had the greatest bacteriostatic activity,indicating the highest expression of antimicrobial peptide AP at 72 h of the incubation.Further experiment demonstrated that,after 72 h of the incubation,the decrease in the bacteriostatic activity was due to the loss of recombined plasmids in the cells of P.pastoris SMD1168.The LC-MS analysis identified that the antimicrobial peptide AP was exactly expressed in the cultures of the recombined P.pastoris.Furthermore,after mutagenesis of the strain AP26 using nitrosoguanidine(NTG),we selected a mutant of recombined P.pastoris SMD1168 strain,designated as APmu4,which had the highest expression level of 1.7×109 U/L of antimicrobial peptide AP.The culture conditions of the strain APmu4 were optimized and the result obtained showed that p H 7.5,the inoculum size of 50 g/L,the YDFMY medium with higher salt concentration,and yeast extract addition could enhance the secretion of antibacterial peptide AP by APmu4.2.The vector pPICAB containing the gene of antimicrobial peptide AB,which is a broad spectrum antibacterial peptide,was constructed and transformed into the cells of Pichia pastoris SMD1168.The recombinant strain showed strong inhibitory effect on E.coli.LC-MS experiment demonstrated that this recombinant P.pastoris strain successfully expressed antibacterial peptide AB.Furthermore,this recombinant strain was mutated using NTG mutagenesis and a mutant strain that can over-express antibacterial peptide AB was screened and assigned as ABN97.Moreover,the recombinant vector containing hemoglobin gene vgb of oscillatoria,was constructed and transformed into the cells of strain ABN97.The strain ABN97 vgb that had great ability to produce the antibacterial peptide AB was consequently screened,which had the highest production of 4.53×108 U/L of antibacterial peptide AB.The maximal 14-fold production of antibacterial peptide AB by strain ABN97 vgb was obtained in the YDFMY medium with higher salt concentration,pH 6.0,the inoculum size of 50 g/L,and yeast extract addition.3.We successfully constructed the recombinant plasmid pPICPle in which a gene of plectasin was recombined,which is a good antibacterial peptide for Gram positive bacteria.The plasmid pPICPle was transformed and the plectasin was exactly expressed in the recombinant strains of P.pastoris.The recombinant strain was further mutated using NTG mutagenesis and then a 1.93-fold expression of plectasin strain relative to the control strain was screened and assigned as Ple100.Further experiment showed that the YDFMY medium higher salt concentration with addition of yeast extract could enhance the production of plectasin by the mutant Ple100 strain.4.We constructed the integrated vector pLACPSI and episomal vector pKDUPSI,both containing the gene of antibacterial peptide PSI,and introduced them into Kluyveromyces lactis KlSEL1.The recombinant strain was obtained after screening,in which the antibacterial peptide PSI was successfully expressed.The result of SDS-PAGE electrophoresis identified the exactly expression of antibacterial peptide PSI in the fermentation broth of the recombinant strain after purification using the methods of ammonium sulfate precipitation and cation exchange column.The results from the fermentation condition optimization of the recombinant strain showed that neutral or alkaline pH,YDFMY medium with higher salt level and the addition of yeast extract benefited the production of antibacterial peptide PSI by the recombinant strain.The antibacterial peptide PSI purified from the fermentation bruth of the recombinant strain showed a strong inhibitory effect on the growth of Verticillium dahliae,Fusarfum tricinctum,Alternaria alternata,Botrytis cinerea,and Colletotrichum truncatum.In addition,this antibacterial peptide PSI was spread on the surface of peaches and strawberries to examine whether it can be used in the preservation of fruit and vegetable.The result showed that the antibacterial peptide PSI has the potential to become a new fruits and vegetables preservative.5.Glucose oxidase(GOX)has been widely applied in food,medicine and other industries.We introduced the GOX gene into the vectors and constructed the integrated vector p LACGOX and episomal vector pKDUGOX.These two vectors were introduced into K.lactis KlSEL1,and a recombined strain that could over-express the GOX was obtained after screening.The result obtained from flask-shaking fermentation showed that the GOX activity in the fermentation broth of the recombined strain was 70?7 kU/L.The SDS-PAGE result confirmed the GOX that was secreted into the fermentation broth by the recombined strain.The influence factors of GOX enzyme activity were studied.The recombinant GOX showed the highest activity at pH 4.0 and high stability at 37?,whereas,the high salt concentration in YPGal medium suppressed the enzyme activity.