Kinetic modeling of riboflavin biosynthesis in Bacillus subtilis under production conditions

Abstract

To study the network dynamics of the riboflavin biosynthesis pathway and to identify potential bottlenecks in the system, an ordinary differential equation-based model was constructed using available literature data for production strains. The results confirmed that the RibA protein is rate limiting in the pathway. Under the conditions investigated, we determined a potential limiting order of the remaining enzymes under increased RibA concentration (>0.102 mM) and therefore higher riboflavin production (>0.045 mmol g(CDW)(-1) h(-1) and 0.0035 mM s(-1), respectively). The reductase activity of RibG and lumazine synthase (RibH) might be the next most limiting steps. The computational minimization of the enzyme concentrations of the pathway suggested the need for a greater RibH concentration (0.251 mM) compared with the other enzymes (RibG: 0.188 mM, RibB: 0.023 mM).

Factors stimulating riboflavin produced by Lactobacillus plantarum CRL 725 grown in a semi‐defined medium

“…These results confirm a previous study [32] where it was shown that when an excess of GTP and ribulose-5-phosphate are found in culture media, the ribA gene was overexpressed as were the enzymes, GTP cyclohydrolase II, and DHBP synthase, coding by this gene. These same authors also showed that under these conditions, the enzymes coded by FIGURE 3 Comparison of the relative expression levels of rib genes of L. plantarum CRL 725 between late-exponential-phase-cells growing in SSDM and late-exponential-phase-cells growing in SSM at 30°C.…”

“…These results confirm a previous study where it was shown that when an excess of GTP and ribulose‐5‐phosphate are found in culture media, the ribA gene was overexpressed as were the enzymes, GTP cyclohydrolase II, and DHBP synthase, coding by this gene. These same authors also showed that under these conditions, the enzymes coded by ribH (lumazine synthase) and ribG (deaminase and reductase) were not modified, suggesting that they are not affected by external nutrients sources and would constitute non‐modifiable bottleneck steps in riboflavin biosynthesis.…”

Thermodynamic and Probabilistic Metabolic Control Analysis of Riboflavin (Vitamin B2) Biosynthesis in Bacteria

“…Considering the mean values given above, the GTP cyclohydrolase II reaction, and therefore the bifunctional RibA protein of B. subtilis, exhibits the largest average control over the riboflavin synthase flux (mean values of C vnet;7 e 1 and C vnet;7 e RibA are 0.45 and 0.55, respectively). This finding is consistent with our previously published in silico kinetic study [61]. Regarding the experimental findings, it appears that GTP cyclohydrolase II has a special limiting influence on riboflavin production in recombinant bacterial overproducer strains.…”

“…Here, we used the convenience rate law to describe the enzyme kinetics because detailed in vivo enzymatic rate laws with respect to reactants and products are not available [61]. The convenience rate law is a heuristic approximate enzyme kinetic rate law that is used in models of biochemical reaction networks with plausible biological properties [22,29,60].…”

“…Furthermore, two molecules of G are obtained from DHBP synthase flux (reaction 5). Hence, a flux ratio of GTP cyclohydrolase II (reaction 1) to DHBP synthase (reaction 5) of 1:2 mol/mol is essential to maintain the steady state [61]. A deviation of this ratio causes stoichiometrically unbalanced production of E (B, C, and D) and G [1].…”