Transient expression and flux changes during a shift from high to low riboflavin production in continuous cultures ofBacillus subtilis

Abstract

At the onset of glucose-limited continuous cultures, riboflavin production in recombinant Bacillus subtilis declines significantly within 3 generations. This phenomenon was specific to riboflavin production and was not correlated with any other physiological parameter. Physiological analyses excluded genetic degeneration or co-metabolism of previously generated overflow metabolites as possible causes for the riboflavin transients. By developing a novel method for (13)C-based metabolic flux analysis under non-steady-state conditions, we showed that the pentose precursors of riboflavin were exclusively synthesized via the non-oxidative pentose-phosphate (PP) pathway as long as riboflavin production was high. The complete redirection of carbon flux to the oxidative branch of the PP pathway was achieved at unaltered PP pathway gene expression and correlated with the declining riboflavin production. With the possible exception of a slight down-regulation of the purine biosynthesis pathway, genome-wide expression analysis indicated that transcriptional regulation was not responsible for the production decline.

Metabolic flux analysis in a nonstationary system: Fed-batch fermentation of a high yielding strain of E. coli producing 1,3-propanediol

“…Note that the estimated fluxes depend on the flux history of the sampled biomass, and thus should be seen as biomass-averaged fluxes. Iwatani et al (Iwatani et al, 2007) measured the labeling of free intracellular and biomass amino acids to account for this effect, and Zamboni et al (Zamboni et al, 2005) calculated mass isotopomer distributions for each time interval prior to flux estimation. An alternative and more rigorous method would be to perform fully dynamic flux analysis, i.e.…”

Metabolic Characterization of Escherichia coli Strains Adapted to Growth on Lactate

“…The model system consists of 26 reactions and is thus underdetermined when only material balance constraints are conducted for 21 metabolites in the model network. Analysis of 13 C isotopomer distribution of intermediates was therefore employed to provide additional useful constraints for fast flux determination through the quantification of the relative contributions of individual pathways to the formation of target metabolites (12,26,33).…”

“…Recently, 13 C isotopomer-based flux analyses have been successfully applied to investigate metabolic responses and underlying mechanisms in various prokaryotic and eukaryotic systems (4,9,12,26,29,33). We have previously studied the metabolic flux states for several gene deletion mutants of Escherichia coli that evolved on glucose (16,20).…”

Dynamic flux responses in riboflavin overproducing Bacillus subtilis to increasing glucose limitation in fed‐batch culture

“…First, isotopic steady state is assumed when the measured FL reaches the value of the labeled substrate mixture. Second, only two species are considered for the intracellular amino acid pools, naturally labeled and continuously produced 13 C labeled amino acids that possess the isotopic steady state 13 C labeling patterns after label addition (Zamboni et al, 2005a). The measured 13 C labeling pattern is hence a superposition of both species.…”

“…The measured 13 C labeling pattern is hence a superposition of both species. Consequently the fraction of washed-in 13 C labeled amino acids ( f labeled ) is represented by the FL according to Equation (2), with FL Sub representing the fractional labeling of the substrate and FL Mean the measured average FL of the used fragments of the respective amino acid (Zamboni et al, 2005a). In our procedure, the fraction f labeled is calculated individually for each measured amino acid…”