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 μmax) growth rate (0.02/h). Important differences were observed between the lower and higher growth rate cultivation phases in terms of specific production rate (qp) profiles. In all three cases, maximum q p 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 μmax. The best results in terms of VP/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. © 2005 Wiley Periodicals, Inc.
- Fed-batch cultivation
- Formaldehyde dehydrogenase promoter
- Pichia pastoris
- Rhizopus oryzae lipase