A Pichia pastoris strain expressing a Rhizopus oryzae lipase gene under the transcriptional control of the promoter from the P. pastoris formaldehyde dehydrogenase 1 gene (PFLD) was utilized to study the feasibility of this expression system for recombinant protein production using methanol-free fed-batch high cell density cultivations. We have developed a simple and reliable fed-batch strategy using the PFLD system based on the use of methylamine and sorbitol as nitrogen and carbon sources, respectively, for the induction phase. Three different fed-batch fermentations were performed at three different constant growth rates, i.e., at a low growth rate (0.005/h), at an intermediate growth rate of (0.01/h), and at a constant residual sorbitol concentration of 8 g/L, i.e., allowing cells to grow at high (near mu(max)) growth rate (0.02/h). Important differences were observed between the lower and higher growth rate cultivation phases in terms of specific production rate (q(p)) profiles. In all three cases, maximum qp were reached soon after the start of the induction phase; after that maximum, an exponential decrease reaching final values close to zero were observed, except for the cells growing at near pm,, The best results in terms of Y-P/X, productivity and specific productivity were obtained when the microorganism was growing at the highest growth rate. Furthermore, such results were significantly better in relation to those obtained with the PAOX-based system expressing the same protein. (c) 2005 Wiley Periodicals, Inc.