Automated fed-batch culture of recombinant Saccharomyces cerevisiae based on on-line monitored maximum substrate uptake rate

A novel method is developed for on-line monitoring of maximum substrate uptake rate (MSUR) for automated control of substrate feeding in aerobic fed-batch cultures. The control scheme depends on real-time measurements of dissolved oxygen (DO) following a process perturbation: the feeding is interrupted momentarily and the instance of complete depletion of substrate is detected by the rise in DO. Immediately, a known amount of substrate is pulsed into the fermenter. The DO level declines, but soon rises (the 'second' rise) as the added pulse is exhausted. The interval between the instances of decline and the 'second' rise of DO, and the known quantity of pulse, are used to compute the MSUR. The feeding then resumes at less than or equal to MSUR. In production of glucoamylase ( 1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) by recombinant Saccharomyces cerevisiae, the MSUR-based feeding assured maximum substrate utilization rate, but suppressed formation of ethanol. This approach enabled rapid production of recombinant glucoamylase and enhanced yield. In addition, the extent of substrate limitation could be quantified. A 100% degree of substrate limitation (DSL) was close to optimal for maximizing the enzyme productivity. The MS UR monitoring allowed some starvation of the cells for potential enhancement of the recombinant plasmid stability in certain situations. (C) 1998 Elsevier Science S.A. All rights reserved.