Effects of regulated substrate feed on the cell growth and 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. Chimeric alpha-amylase (Amy1A/3D) that consists of 159 aa of one isozyme (Amy1A) 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-decreasing glycerol feed control allowed a high growth rate (0.086 h(-1)) and high cell density (97.2 g DCW l(-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 l(-1) min(-1) was found to be optimum for a high specific 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 340 mg l(-1) of Amy1A/3D and the expression yield of 2.16 mg Amy1A/3D g-methanol(-1) were attained. This result strongly demonstrated that the efficiency of recombinant protein expression was directly dependent on methanol utilization in Pichia system. (C) 2003 Elsevier Inc. All rights reserved.