Optimization Of Fermentation Condition Of Glycerol Transporter 1 Deletion P.pastoris And Identification Of Glycerol Transporter 2

Pichia pastoris is one of the most successful eukaryotic expression systems for the production of recombinant proteins,which is based on the the very strong alcohol oxidase I(AOX1)promoter.However,the methanol-inducible promoter Paox1 is usually strongly repressed by glycerol and glucose.However,methanol will have a negative effect on itself,which is not conducive to high-density fermentation.One GT1-defective strain named ?GT1,constructed in previous works,could relieve the repression of glycerol on methanol metabolism.In this study,the mutant strain ?GT1 was used as the host cell and the fermentation process was optimized in the shake flask stage to construct a new Pichia pastoris expression system.The identification and characterization of a glycerol transporter GT2 in P.pastoris is described.The main results are as follows:(1)EGFP green fluorescent protein was used as the reporter protein to explore the effects of different concentrations of methanol,glycerol and glycerol and methanol mixture with different proportions on the activity of AOX1 promoter.In 0.25% glycerol / 1% methanol mixed carbon source,?GT1-EGFP was able to grow well and make full use of methanol to the expression of foreign protein.(2)The culture condition parameters(temperature,initial pH,media volume)were optimized to maximize the yield of foreign protein using a design of experiments strategy according to the response surface methodology(RSM).The result is that in the optimum conditions(initial pH=7,temperature=26 ?,media volume=50 mL/500 m L)the specific fluorescence values of ?GT1 cultured at the time of 48 h,72 h and 96 h were 79%,74% and 28%,which are higher than those of X-33 under the traditional culture conditions;total fluorescence values are 82%?98%?67%,which are higher than those of X-33.(3)HSA human serum protein was used as reporter protein to verify the reliability of the new expression system.At the time point 48 h,72 h and 96 h,the amount of HSA protein of ?GT1 in the optimized culture mode was 60%?71%?85.5%,which are higher than those of the wild strain in the traditional culture mode.(4)Glyceol Transporter 2,a homologue of P.pastoris GT1,was identified.The growth and the ability to transport glycerol of ?GT2 is hampered.The activity of the AOX1 promoter was significantly improved in ?GT2 compared with wild type under glycerol and methanol mixed carbon sources.Pichia pastoris ?GT2(GT2-deficiency)strain partially relieves the repressive effect of glycerol on the AOX1 promoter.In this study,the new P.pastoris expression was successfully applied to the efficient expression of exogenous proteins,and the glycerol transporter GT2 gene was involved in the glycerol derepression effect of the AOX1 promoter,which was useful for the development of the new P.pastoris expression system.