AbstractWe have developed a yeast Pichia pastoris system for the high-level expression of recombinant Rhizopus oryzae lipase (ROL), which is a potentially effective catalyst in the solvent-free production of biodiesel fuel. In the glycerol fed-batch phase, the combination of the dissolved-oxygen-stat and gradient-control glycerol feeding strategies resulted in a higher cell biomass in the P. pastoris culture, with shorter feed times. In the methanol fed-batch phase, a constant methanol concentration of 0.3-0.5% (v/v) was found to be optimal for high ROL activity, maximum protein concentration, and a maximum biomass. Cells were grown at 30 °C and induced by methanol at 22 °C and a pH of 6.0 with the addition of 0.5% (w/v) casein, which reduced the proteolytic degradation of ROL. Using a combined glycerol step-increasing, on-line methanol feeding strategy and these reducing proteolytic degradation methods, a maximum biomass of 96 g DCW/l, a maximum concentration of 2.0 g/L, and an activity of 1302.2 U/mL ROL were attained. The simple culture process is the highest level of ROL expression reported in P. pastoris. Our results strongly demonstrate that the efficiency of the recombinant ROL expression is directly dependent on feed strategies, lower induction temperature, maintaining the proper pH, and the addition of casein.