Enhanced production of cis,cis-muconate in a cell-recycle bioreactor

For enhanced production of cis,cis-muconate via biocatalytic conversion from benzoate, a high cell density culture of a mutant Pseudomonas putida strain, BM014 in a cell-recycle bioreactor has been investigated, The volumetric productivity in a continuous bioconversion process was dependent on the biomass concentration, specific growth rate, and environmental conditions including dissolved oxygen tension (DOT) and cis,cis-muconate concentration, Biotransformation was started ire a cell-recycle continuous culture mode far obtaining a high cell density of 40 g/l and then carried out in a fed-batch mode for accumulation of cis,cis-muconate to up to 12 g/l at which induction of catechol 1,2-dioxygenase, a key enzyme catalyzing the rate-determining step in the biosynthetic pathway, was maximized. Since in the biosynthesis of cis,cis-muconate growing cells mere utilized as catalysts, cell growth should be continued, but a low specific growth rate achieved under phosphate-limited conditions was indispensable for higher productivity. When the concentration of phosphate in the feed was lowered from 150 to 10 mM, the productivity could be enhanced three fold, It was also found that cobalt ions markedly increased the specific productivity and that DOT should be kept at above 20% of the saturation level for achieving optimum rates of cell growth and product formation. After a period of fed-batch operation continuous production with cell recycling was attempted with phosphate-limited minimal medium supplemented with cobalt ions, and benzoate medium. Enhanced productivity of 5.5 g/(l.h) and a cis,cis-muconate concentration of 13.5 g/l in the culture broth were obtained and continuous production in a cell-recycle bioreactor mas maintained fur more than 7 d.