We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l(-1) when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.

Increased expression of recombinant mini-proinsulin in Pichia pastoris in 2.5 l bioreactors was achieved by increasing the cultivation pH from 5.1 to 6.3, by decreasing the temperature from 28 to 22 degrees C, and by periodical addition of ammonium sulfate and EDTA to the culture broth. Using this procedure, mini-proinsulin reached nearly 0.3 g l(-1) in the culture supernatant after 160 h of growth.

An unstructured model based on mass balance equations for a recombinant methylotrophic yeast Pichia pastoris MutS (Methanol Utilization Slow) strain expressing the mini-proinsulin (MPI), was successfully established in quasi-steady state fed-batch fermentations with varying total quantity of biomass in a 7 l fermenter. The model describes the relationships between the total biomass and induction time, both in the batch and fed-batch phases. In addition, good correlations were obtained when the total quantity of MPI was correlated with the total biomass, demonstrating that the product of interest is associated with growth in the methanol phase. The parameters of the fermentation model obtained are similar to those reported by other researchers.