Date

2007-09-01

Author

Guzide, Calik
Yasemin, Demirci
Pinar, Calik
Ozdamar, Tuncer H.

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Rate limiting reactions in the aromatic-group amino acid pathway (AAAP) in Bacillus subtilis for l-phenylalanine (Phe) production were determined as reported elsewhere (Özçelik-Şenver et al., 2004). The biochemical reactions in the AAAP start with the reaction using phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) synthesised in the glycolysis pathway and the PPP, respectively, leading to the formation of Phe via chorismate by branching at prephenate. The branch-point metabolites E4P supplied in vitro, enhanced Phe production and well defined perturbations were achieved on the AAAP reactions. E4P did not affect the cell growth significantly, probably because of its low concentrations; nevertheless, E4P increased Phe production. The experimental program enable us to conclude that the phosphorylated compounds, i.e., E4P, in vitro can be transferred into B. subtilis by an irreversible transport process. Further, on the basis of the bioreaction network, the model that contains 184 metabolites and 232 reaction fluxes has been set up and, the metabolic fluxes through the central carbon pathways toward the AAAP in wild-type B. subtilis were calculated. The transport of the phosphorylated compound E4P into B. subtilis implemented the expected influence on the fluxes of the AAAP reactions. By interpreting the related results we conclude that: (i) as E4P increased all the reaction rates toward Phe, aroA gene encoding the enzyme DAHP synthase (EC 2.5.1.54) for the first reaction (R89) of aromatic-group amino acids; (ii) as the flux value was the lowest, aroH gene encoding the enzyme chorismate mutase (EC 5.4.99.5) for R96; were predicted to be the metabolic engineering sites for the Phe overproduction. On the basis of the metabolic engineering strategy designed to overcome the limitation of the reactions catalysed by DAHP synthase and chorismate mutase, and to determine the magnitude of the increase in the yield and selectivity in l-phenylalanine production, aroA and aroH genes encoding, respectively, DAHP synthase and chorismate mutase were cloned onto an E. coli plasmid. Cloning of aroA and aroH genes encoding respectively DAHP synthase and chorismate mutase, were achieved first onto pUC19 and its expression was demonstrated to compare its performance. Thereafter, the genes were sub-cloned onto a multicopy Bacillus-E. coli shuttle vector pMK4 and transferred to B. subtilis. The performance of r-E. coli strains carrying pUC19::aroA and pUC19::aroH were lower than the r-B. subtilis strains carrying pMK4::aroA and pMK4::aroH, since E. coli form three separate DAHP synthases, each subject to feedback inhibition by a different aromatic amino acid. l-Phenylalanine synthesis and excretion performance of r-B. subtilis strains were compared on a defined medium with sole carbon source glucose at T = 37 °C, pH0 6.8, and the oxygen transfer condition of Qo/VR = 0.5 vvm, N = 500 min−1 in batch-bioreactors. The r-B. subtilis carrying pMK4::aroA that is the metabolic engineering product for the reaction catalysed by DAHP synthase, increased Phe production ca. seven-fold; whereas, the r-B. subtilis carrying pMK4::aroH that is metabolic engineering product for the reaction catalysed by chorismate mutase, increased Phe production ca. three-fold. The diversions in the biochemical reaction network and the influence of the metabolic engineering products, i.e., pathway flux amplifications, will also be presented.

Subject Keywords

Biotechnology, Applied Microbiology and Biotechnology, General Medicine

URI

https://hdl.handle.net/11511/67332

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Department of Chemical Engineering, Conference / Seminar