The Codon Optimization And Expression Of The Sea Cucumber I-type Lysozyme Gene In Pichia Pastoris

Lysozyme is also called lytic enzyme,which was discovered by Fleming in 1922 for the first time.Currently,the c-type lysozyme has been commercialized which is directly extracted from egg white.But the c-type lysozyme not only has high production cost and low yield,but also has a narrow antibacterial spectrum.The inhibitory effect of the c-type lysozyme on gram-negative bacteria is also limited.Therefore,an i-type lysozyme from the sea cucumber tissue was obtained by gene identification and expression in our lab previous study.The experimental data showed that the i-type lysozyme has broad antimicrobial spectra which could effectively inhibit both gram-positive bacteria and gram-negative bacteria.It also found that the i-type lysozyme contains glycosidase and isopeptidase activities.Therefore,it deserves further study and has great application value.Recent progress in genetic engineering makes it possible that some yeast cells have been used as bioreactors to be efficient expression of exogenous genes,especially the Pasteur Pichia pastoris expression system.The promoter of the alcohol oxidase I(AOX?)gene in P.pastoris could be efficiently induced and strongly initiated.Thus it is suitable for high level expression of foreign genes.Another advantage is that P.pastoris has a strong preference for aerobic growth that could be achieved by high density cell culture.Therefore,it is useful to conduct large-scale industrial production.It is proved by research that the codon preferences of P.pastoris and the mRNA structure of target protein can affect the expression of target protein.In this study,the gene sequence of the i-type lysozyme from the sea cucumber was optimized based on these principles.Firstly,codons in the i-type lysozyme gene which are less than 50%of codon preference in P.pastoris were changed to higher preference codons.Secondly,the mRNA structure of the i-type lysozyme was predicted by bioinformatics software and optimized the gene sequence by opening the stem-loop structure in mRNA to reduce the free energy without affecting the codon preference.Finally,a tag containing 6 histidines was designed to add on the N-terminal site of the optimized gene sequence coding the i-type lysozyme.The optimized gene sequence of the i-type lysozyme was synthesized by the Takara Biotechnology(Dalian,China),and the enzyme was called opt-SjLys.The above synthesized gene was ligated into the pMD18-T to construct the cloning plasmid pMD18-T-opt-SjLys.Next the plasmid digested by EcoR?and Not?was ligated into the expression vector pPIC9K to construct the recombinant expression plasmid pPIC9K-opt-SjLys.Then the large fragment of Bgl?-linearized pPIC9K-opt-SjLys was transferred into the P.pastoris GS115 by electroporation for integrating the target gene into the chromosome of P.pastoris.Next,spreading the transformation liquid on the MD plates was done to screen the His~+transformants.Furthermore,transformants with the high copy numbers were selected using the YPD medium containing 1 mg/ml and 4 mg/ml of G418.These recombinant strains were inoculated into the YPD liquid medium and induced with 1%methanol for 96 h,respectively.After induction,the culture samples were centrifuged and the supernatants were analyzed by SDS-PAGE to assay the effect of expression of the i-type lysozyme protein.As a result,4 recombinant strains were obtained that could efficiently express the purpose protein in this experiment.Further study was done by 5 L fermentor in order to test the ability of the lysozyme production by fermentation using a recombinant strain HS3-1 which was selected and optimized in our previous study.The initial medium is basal salt medium(BSM).The glycerol fed-batch phase was initiated by feeding 50%glycerol containing 1.2%PTM1 trace salt solution at the rate of 18.15 mL per h per liter culture broth for a period of 5 h.The methanol fed-batch phase was initiated by feeding 100%methanol containing 1.2%PTM1 trace salt solution,and the methanol feeding rate was controlled by the online methanol sensor to maintain the residual methanol concentration at 1%.The optimal process parameters was confirmed by adjusting the fermentation process.When the pH 6.0,the temperature is 30?,the initial DO kept around 30%,800~1000 rpm,the concentration of methanol is 1%,the induction time is 96 h,the expression quantity of lysozyme is the highest,and the bacteriostatic activity of fermenting by fermentor is higher than the flask.This experiment has constructed the pichia pastoris genetic engineering bacteria which highly expressed i-type lysozyme from sea cucumber successfully,the enzyme activity of lysozyme that fermenting in flask is 8.79 U/ml,the enzyme activity of lysozyme that fermenting in fermentor is 455.07 U/ml,after purification by Ni~+column the enzyme activity reaches 1833.33 U/ml,providing theoretical basis for subsequent larger amplification cultivation.