Enhanced production of cis-cis muconic acid in bioreactor fermentations of recombinant Escherichia coli cells

Cis-cis muconic acid (MA) utilized as a raw material for new functional resins, pharmaceuticals and agrichemicals is an unsaturated dicarboxylic acid with six carbon atoms. MA was revealed to be easily converted to adipic acid, a valuable commodity chemical for bio-plastics including nylon 6-6 and polyurethane and polyethylene terephthalate (PET). For development of MA high-yielding recombinant Escherichia coli cells, three additional genes from various sources (i.e., both aroZ gene encoding DHS dehydratase from and aroY gene encoding PCA decarboxylase from K. pneumonia, and catA gene encoding catechol 1,2-dioxygenase from A. calcoaceticus) were introduced into aromatic amino acids auxotrophic mutants by use of plasmid expression vectors with various strong promoters. As a first step to enhanced production of MA by the newly-developed E. coli cells, induction strategy was set up at 5L bioreactor level through determination of inducer kind & amount, induction time and inducing medium composition. Especially, intensive studies were conducted to develop an efficient induction method for overcoming catabolite repression phenomenon caused by high level of residual glucose. Notable was the positive role of glycerol added in the production medium, since it contributed to remarkable enhancement in the MA-biosynthetic capability of the recombinant without causing catabolite repression. In addition, fermentation conditions such as agitation speed, dissolved oxygen level and pH-control were also investigated using the 5L stirred tank bioreactors. Fermentation results from the batch and fed-batch bioreactor operations performed with the recombinant E. coli cells will be presented in this paper as well.