PHYSIOLOGY AND METABOLIC FLUXES OF WILD-TYPE AND RIBOFLAVIN-PRODUCINGBACILLUS-SUBTILIS

Continuous cultivation in a glucose-limited chemostat was used to dete rmine the growth parameters of wildtype Bacillus subtilis and of a rec ombinant, riboflavin-producing strain. Maintenance coefficients of 0.4 5 and 0.66 mmol of glucose g(-1) h(-1) were determined for the mild-ty pe and recombinant strains, respectively, However, the maximum molar g rowth yield of 82 to 85 g (cell dry weight)/mol of glucose was found t o be almost identical in both strains. A nonlinear relationship betwee n the specific riboflavin production rate and the dilution rate was ob served, revealing a coupling of product formation and growth under str ict substrate-limited conditions, Most prominently, riboflavin formati on completely ceased at specific growth rates below 0.15 h(-1). For mo lecular characterization of B. subtilis, the total amino acid composit ion of the wild type was experimentally determined and the complete bu ilding block requirements for biomass formation were derived, In parti cular, the murein sacculus was found to constitute approximately 9% of B. subtilis biomass, three to fivefold more than in Escherichia coli, Estimation of intracellular metabolic fluxes by a refined mass balanc e approach revealed a substantial, growth rate-dependent flux through the oxidative branch of the pentose phosphate pathway, Furthermore, th is Bur is indicated to be increased in the strain engineered for ribof lavin formation, Glucose catabolism at low growth rates with reduced b iomass yields was supported mainly by the tricarboxylic acid cycle.