OVERPRODUCTION OF GLYCOGEN IN ESCHERICHIA-COLI BLOCKED IN THE ACETATEPATHWAY IMPROVES CELL-GROWTH

Excessive production of acetate is a problem frequently encountered in aerobic high-cell-density fermentations of Escherichia coli. Here, we have examined genetic alterations resulting in glycogen overproductio n as a possible means to direct the flux of carbon away from the aceta te pool. Glycogen overaccumulation was achieved either by using a regu latory glgQ mutation or by transforming cells with a plasmid containin g the glycogen biosynthesis genes glgC (encoding ADPG pyrophosphorylas e) and glgA (encoding glycogen synthase) under their native promoter. Both strategies resulted in an approximately five-fold increase in gly cogen levels but had no significant effect on acetate excretion. The g lgC and glgA genes were then placed under the control of the isopropyl -beta-D-thiogalactopyranoside (IPTG) inducible tac promoter, and this construct was used to stimulate glycogen production ina mutant defecti ve in acetate biosynthesis due to deletion of the ack (acetate kinase) and pta (phosphotransacetylase) genes. If glycogen overproduction in the ack pta strain was induced during the late log phase, biomass prod uction increased by 15 to 20% relative to uninduced controls. Glycogen overaccumulation had a significant influence on carbon partitioning: The output of carbon dioxide peaked earlier than in the control strain , and the levels of an unusual fermentation byproduct, pyruvate,were r educed. Exogenous pyruvate was metabolized more rapidly, suggesting hi gher activity of gluconeogenesis or the tricarboxylic acid (TCA) cycle as a result of glycogen overproduction. Potential mechanisms of the o bserved metabolic alterations are discussed. Our results suggest that ack pta mutants overproducing glycogen may be a suitable starting poin t for constructing E. coli strains with improved characteristics in hi gh-cell-density fermentations. (C) 1994 John Wiley and Sons, Inc.