Novel Strategies For Induction Expression Of Heterologous Protein In A New Type Of Recombinant Pichia Pastoris

The methylotrophic yeast Pichia pastoris is one of the most widely used expression system for heterologous expression of various recombinant proteins.The popularity of this expression system mainly attributes to its ability of strongly and strictly controlled methanol-induced alcohol oxidase 1 promoter(PAOX1)to produce foreign proteins at high levels.However,methanol is potentially hazardous for its toxicity and flammability in the large-scale fermentation industry.The toxic properties of methanol limit the application range of Pichia pastoris expression system.Moreover,methanol catabolism requires huge oxygen coupled with considerable heat release.The need for high oxygen supply and heat removal increases the production cost and causes difficulties in scale-up when heat exchange and oxygen transfer capacities are low.Our laboratory has been working to develop a new expression system of P.pastoris based on methanol-free AOX1 promoter.In the previous work.PAOX1 activity could be detected in △mig1△mig2-Mit1 in glycerol medium and in △mig1△mig2△nrg1-Mit1 in glucose and glycerol medium respectively.In this study,the relationship between the maximum specific growth rates in glucose and glycerol medium was WT>△mig1△mig2-Mit1>△mig1△mig2△nrg1-Mit1 in both flask culture and fed-batch fermentation.The strains△mig1△mig2-Mit1 and △mig1△mig2△nrg1-Mit1 were cultured in fed-batch mode,and the final cell concentration could reach the one at which the expression of the exogenous protein was induced.This gave an evidence that they were suitable as hosts for heterologous protein expression.Then,PAOX1 expression cassette with gene coded for human insulin precursor(IP)was transferred into the strains △mig1△mig2-Mit1 and △mig1△mig2△nrg1-Mit1.Finally,a high expression strain of △mig1△mig2△nrg1*-Mit1-IP(MF1-IP)was obtained,which could express IP with high level in glycerol medium.It was found that the expression of PAOX1 was hardly detected when the glucose concentration was higher than 2%in △mig1△mig2△nrg1*-Mit1.In fed-batch fermentation,the concentration of glucose in the fermentation medium higher than 20 g/L could inhibit the expression of PAOX1.Using these characteristics,a novel fermentation process similar to the traditional methanol induction three-step fermentation process of Pichia pastoris was developed using glucose as growth carbon source and glycerol as induction carbon source.The parameters of fermentation process(dissolved oxygen.pH.induction wet weight and glycerol feeding rate)were then optimized.The results showed that the optimum conditions for the induction of PAOX1 expression were in the dissolved oxygen of 30%～50%,induction pH of 3.5,induction biomass of 200 g/L(WCW)and glycerol feeding rate of 15.5 ml/(h·L broth).Compared with the fermentation of MF1-IP in glycerol and that of WT-IP in methanol,the fermentation period was reduced by 16 h and the production of IP reached a high level of 2.46 g/L.Moreover,the shift from glucose to glycerol in the MF1-IP became much easier to control than that from glycerol to methanol in the WT-IP during the fermentation process.The total oxygen consumption of the MF1-IP was 51.5%lower than that of the WT-IP during the whole fermentation process.The total heat evolution of the MF1-IP reached 81 kJ/L fermentation broth,51.5%lower than that of the WT-IP.The results of the calculation of OUR and kLa well as well chemostat experiment showed that the decrease of mass transfer efficiency in the late induction period of MF1-IP was resulted in the high viscosity by high cell concentration.And viscosity could be controlled using semi-continuous feeding method.In addition.a fermentation process using low concentration of glucose as the induction carbon source was established.It was found that the faster the feeding rate of the strategy,the faster the growth rate of the cells and the faster the generation rate of the insulin precursor.The maximal expression of IP reached at 36 h,but the expression level was only 1.28 g/L.The strategy is more suitable for the situation that the product needs to be obtained quickly.At the same time,it was found that the maximal specific growth rate of MF1 strain in methanol medium was much lower than Mut+ wild type strain,but slightly higher than that of Muts wild type strain.This is related to the genetic engineering of cells.MF1-IP strain was cultured to a suitable biomass when the glucose concentration was above 20 g/L followed by the expression of insulin precursor induced by methanol with different strategies.The expression of insulin precursor induced by three different methanol feeding rates in MF1-IP strains was all higher than that induced by methanol in WT-IP strain.When the methanol feeding rate was 6.67 ml/(h·L broth),the expression of insulin precursor of MF1-IP strain reached 6.69 g/L supernatant,which was 59%higher than the WT-IP strain.When the expression level was converted into fermentation broth,3.55 g/L broth was obtained,which was 137%higher than the WT-IP.The maximum specific IP production rate of MF1-IP strain was 3.8 x 10-4 g/(g·h),which was 77%higher than the WT-IP strain.The oxygen consumption rate and the rate of heat evolution of MF1-IP during induction was also lower than that of WT-IP,and the highest oxygen consumption rate and heat evolution rate were 18.3%and 37.7%lower than the WT-IP,respectivly.Thus,the knockout of the carbon source repressors Migl,Mig2,Nrgl and the overexpression of the methanol activator Mitl enhanced the expression of the AOX1 promoter in Pichia pastoris in methanol.