DESIGN OF EXPRESSION SYSTEMS FOR METABOLIC ENGINEERING - COORDINATED SYNTHESIS AND DEGRADATION OF GLYCOGEN

In metabolic engineering, systems which allow coordinated control of t wo metabolic pathways can be useful. We designed two expression system s and demonstrated their application by coordinating glycogen synthesi s and degradation. The first expression vector pMSW2 expressed the gly cogen synthesis genes in one operon and the glycogen degradation gene in a separate, coordinately regulated operon. The plasmid was designed to switch off expression of the first operon and activate expression of the second operon on addition of IPTG. As an alternative means to c ontrol glycogen synthesis and degradation pathways, we constructed exp ression vector pGTSD100, which contains the native Escherichia coli gl ycogen synthesis and degradation operon under control of the tac promo ter. Both expression vectors work successfully to control the net synt hesis and degradation of glycogen. in cultures of the E. coli strain T A3476 carrying the plasmid pMSW2, before the addition of IPTG, glycoge n continued to accumulate in the culture. About three hours after IPTG was added, glycogen levels began to decrease. When no IPTG was added to cultures of TA3476:pMSW2, glycogen accumulated in the cells as befo re but the rate of degradation of glycogen was much lower. When IPTG w as added to TA3476:pMSW2, the total cell protein at the end of batch c ultivation was approximately 15% higher compared to cultures without I PTG addition. The extra biomass was formed during the glycogen degrada tion phase. (C) 1997 John Wiley & Sons, Inc.