Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis

Continuous cultivation in a glucose-limited chemostat was used to det. the growth parameters of wild-type Bacillus subtilis and of a recombinant, riboflavin-producing strain. Maintenance coeffs. of 0.45 and 0.66 mmol of glucose g-1 h-1 were detd. for the wild-type and recombinant strains, resp. However, the max. molar growth yield of 82 to 85 g (cell dry wt.)/mol of glucose was found to be almost identical in both strains. A nonlinear relationship between the specific riboflavin prodn. rate and the diln. rate was obsd., revealing a coupling of product formation and growth under strict substrate-limited conditions. Most prominently, riboflavin formation completely ceased at specific growth rates below 0.15 h-1. For mol. characterization of B. subtilis, the total amino acid compn. of the wild type was exptl. detd. and the complete building block requirements for biomass formation were derived. In particular, the murein sacculus was found to constitute approx. 9% of B. subtilis biomass, three- to five-fold more than in Escherichia coli. Estn. of intracellular metabolic fluxes by a refined mass balance approach revealed a substantial, growth rate-dependent flux through the oxidative branch of the pentose phosphate pathway. Furthermore, this flux is indicated to be increased in the strain engineered for riboflavin formation. Glucose catabolism at low growth rates with reduced biomass yields was supported mainly by the tricarboxylic acid cycle. [on SciFinder (R)]