Effects of substrate feed rates on heterologous protein expression by Pichia pastoris in DO-stat fed-batch fermentation

摘要

Effects of regulated substrate feed on the cell growth an recombinant protein expression by the pichia system were studied by applying either constant feed rates or variable feed rates in DO-stat fed-batch fermentation.Chimeria alpha-amylase (Amy1A/3D)that consists of 159 aa of one isozyme (Amyl A)of rice alpha-amylases followed by 251 aa of another (Amy3D)was used as a model protein.In the glycerol fed-batch(alcohol oxidase I derepression)phase,a step-decreasign glycerol feed control alowed a high growth rate (0.086 h~-1)and high cell density (97.2g DCW1~-1).In the induction phase,methanol feed rate had a significant effect on the specific expression rate of Amy1A/3D.A constant feed rate of 1.0 ml 1~-1 min~-1 was found to be optimum for a high apecific expression rate of Amy1A/3D (0.091 mg g DCW~-1 h~-1).Under this condition,allowing a higher specific methanol consumption rate,a maximum concentration of 340mg1~-1 of Amy 1A/3D and the expression yield to 2.16 mg Amy1A/3Dg-methanol~-1 were attained.This result strongly demonstrated that the efficiency of recombinant protein expression was directly dependent on methanol utilization in Pichia System.