Design of expression systems for metabolic engineering: Coordinated synthesis and degradation of glycogen

Abstract

In metabolic engineering, systems which allow coordinated control of two metabolic pathways can be useful. We designed two expression systems and demonstrated their application by coordinating glycogen synthesis and degradation. The first expression vector pMSW2 expressed the glycogen 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 control glycogen synthesis and degradation pathways, we constructed expression vector pGTSD100, which contains the native Escherichia coli glycogen synthesis and degradation operon under control of the tac promoter. Both expression vectors work successfully to control the net synthesis and degradation of glycogen. In cultures of the E. coli strain TA3476 carrying the plasmid pMSW2, before the addition of IPTG, glycogen 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 before but the rate of degradation of glycogen was much lower. When IPTG was added to TA3476:pMSW2, the total cell protein at the end of batch cultivation was approximately 15% higher compared to cultures without IPTG addition. The extra biomass was formed during the glycogen degradation phase. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 419-426, 1997.