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Kinetic modeling of riboflavin biosynthesis in Bacillus subtilis under production conditions

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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 −1CDW 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).

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Abbreviations

c :

Vector of metabolite concentrations

t :

Simulation time, s

N :

Stoichiometric matrix

r :

Vector of reaction rates

r 1 :

Reaction rate of the GTP cyclohydrolase II activity of RibA, mM s−1

r 2 :

Reaction rate of the 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate deaminase activity of RibG, mM s−1

r 3 :

Reaction rate of the 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate reductase activity of RibG, mM s−1

r 4 :

Reaction rate of the hypothetical phosphatase, mM s−1

r 5 :

Reaction rate of the 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase activity of RibA, mM s−1

r 6 :

Reaction rate of the 6,7-dimethyl-8-ribityllumazine synthase (RibH), mM s−1

r 7 :

Reaction rate of the riboflavin synthase (RibB), mM s−1

r 8 :

Rate of the diffusive outflow of riboflavin, mM s−1

J 3 :

Reaction rate of the 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate reductase activity of RibG at steady state, mM s−1

J 6 :

Reaction rate of the 6,7-dimethyl-8-ribityllumazine synthase (RibH) at steady state, mM s−1

J 7 :

Reaction rate of the riboflavin synthase (RibB) at steady state, mM s−1

k cat :

Turnover number, s−1

K m :

Michaelis–Menten constant, mM

E RibA :

Enzyme concentration of RibA, mM

E RibG :

Enzyme concentration of RibG, mM

E RibH :

Enzyme concentration of RibH, mM

E RibB :

Enzyme concentration of RibB, mM

E RibX,min :

Minimized enzyme concentration of a specific Rib protein X, mM

\( e_{\left[ A \right]}^{{r_{1} }} \) :

Scaled elasticity: sensitivity of the GTP cyclohydrolase II reaction rate to a change in the concentration of A

\( e_{\left[ B \right]}^{{r_{2} }} \) :

Scaled elasticity: sensitivity of the deaminase reaction rate to a change in the concentration of B

\( e_{\left[ C \right]}^{{r_{3} }} \) :

Scaled elasticity: sensitivity of the reductase reaction rate to a change in the concentration of C

\( e_{\left[ F \right]}^{{r_{5} }} \) :

Scaled elasticity: sensitivity of the DHBP synthase reaction rate to a change in the concentration of F

\( e_{\left[ E \right]}^{{r_{6} }} \) :

Scaled elasticity: sensitivity of the lumazine synthase reaction rate to a change in the concentration of E

\( e_{\left[ G \right]}^{{r_{6} }} \) :

Scaled elasticity: sensitivity of the lumazine synthase reaction rate to a change in the concentration of G

\( e_{\left[ H \right]}^{{r_{7} }} \) :

Scaled elasticity: sensitivity of the riboflavin synthase reaction rate to a change in the concentration of H

μ :

Specific growth rate, s−1

A :

GTP concentration, mM

B :

2,5-Diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate concentration, mM

C :

5-Amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate concentration, mM

D :

5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate concentration, mM

E :

5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione concentration, mM

F :

Ribulose 5-phosphate concentration, mM

G :

3,4-Dihydroxy-2-butanone 4-phosphate (DHBP) concentration, mM

H :

6,7-Dimethyl-8-ribityllumazine concentration, mM

R :

Riboflavin concentration, mM

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Acknowledgments

This work was financed by the German Federal Ministry of Education and Research (BMBF) in the context of the NANOKAT Graduate Program (Ref. No. 0316052A). The study benefited from interdisciplinary discussions within the graduate program. The authors would especially like to thank Prof. Dr. M. Mack for his remarkable commitment within the program.

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  1. Institute of Chemical Process Engineering, Mannheim University of Applied Sciences, Paul-Wittsack-Straße 10, 68163, Mannheim, Germany

    Markus Birkenmeier, Susanne Neumann & Thorsten Röder

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  1. Markus Birkenmeier
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  2. Susanne Neumann
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  3. Thorsten Röder
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Correspondence to Thorsten Röder.

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Birkenmeier, M., Neumann, S. & Röder, T. Kinetic modeling of riboflavin biosynthesis in Bacillus subtilis under production conditions. Biotechnol Lett 36, 919–928 (2014). https://doi.org/10.1007/s10529-013-1435-8

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