RECOMBINANT BIOPROCESS OPTIMIZATION FOR HETEROLOGOUS PROTEIN-PRODUCTION USING 2-STAGE, CYCLIC FED-BATCH CULTURE

A two-stage, cyclic fed-batch bioprocess was designed, and its perform ance evaluated to improve rice a-amylase productivity by the yeast Yar rowia lipolytica SMY2 (MatA, ade1, ura3, xpr2), ATCC 201847, containin g a replicative plasmid coding for a rice alpha-amlyase. Transcription of the recombinant gene is controlled by the XPR2 promoter. The first stage (or growth stage) was operated in the fed-batch mode, and the g rowth medium, designed to maintain a constant high cell density (i.e., 60 g/l), was fed according to a predetermined and preprogrammed optim al feed rate which, in turn, maintained the specific cell growth rate at an optimal value (i.e., 0.1 h(-1)). Typically, when the volume in t he first stage reached a preset value, a portion of culture broth (i.e ., 55%) was transferred to the second stage (or production stage). The remaining cells in the growth stage were then fed with fresh growth m edium according to the bioprocess control strategy developed, while in duction of alpha-amylase expression and its production was taking plac e in the second stage. The second stage was also operated in the fed-b atch mode, and the production medium designed to maintain a constant h igh cell density and high productivity of heterologous protein was fed at a predetermined and preprogrammed rate, which maintained the speci fic cell growth rate at an optimal level. The volumetric alpha-amylase productivity achieved (1835 units l(-1) h(-1)) from the two-stage, cy clic fed-batch culture process was twofold higher than that of the fed -batch culture process. The genetic stability of the recombinant strai n and the design of optimal media for growth and production stages are also critically important to a successful implementation of the two-s tage, cyclic fed-batch process for production of heterologous protein.