Metabolic engineering of aromatic group amino acid pathway in Bacillus subtilis for L-phenylalanine production

Ozcelik, IS
Çalık, Pınar
Calik, G
Ozdamar, TH
Metabolic control sites in the aromatic group amino acid pathway (AAAP) of Bacillus subtilis for L-Phenylalanine (Phe) overproduction were determined; and aiming pathway flux amplification, by cloning the flux controlling gene aroH to a multi-copy plasmid, the impact of single gene cloning on pathway flux distributions were investigated. The branch-point metabolites E4P and PEP+E4P supplied in vitro, enhanced Phe production and well defined perturbations were achieved on the AAAP reactions. The intracellular reaction rate distributions calculated by a mass flux balance-based stoichiometric model for B. subtilis based on the metabolic reaction network that contains 184 metabolites and 232 reaction fluxes using time profiles of glucose, dry cell, organic and amino acids, revealed that induction by E4P influenced the pathway reactions via chorismate and prephanate towards Phe by increasing the flux values. The reaction catalysed by chorismate mutase (R96) has the lowest flux value among the preceding reactions of the pathway that reduces the proceeding reaction rates towards Phe. Thus, R96 is predicted to be the primary rate-limiting step among the Phe pathway reactions. On the basis of the strategy designed, the cloning of aroH gene encoding chorismate mutase was achieved first onto the E.coli plasmid pUC19, and its expression was demonstrated to compare its performance. Thereafter, aroH was sub-cloned onto the multi-copy plasmid pMK4 and transferred into host B.subtilis mutants. The performance of r-E.coli carrying pUC19::aroH was lower than the r-B.subtilis strains carrying the pMK4::aroH; and, the highest Phe production was obtained with r-B. subtilis 1A263. The comparison of the calculated intracellular fluxes of r-B. subtilis 1A263 with that of the wild type revealed that the flux of the first reaction of the AAAP was 1.2-fold, but the engineered R96 was 7.2-fold higher in r-B.subtilis 1A263.


Bioreaction network flux analysis for human protein producing Bacillus subtilis based on genome-scale model
ÖZDAMAR, HASAN TUNÇER; Senturk, Birguel; YILMAZ, OĞUZ; KOCABAŞ, PINAR; Calik, Guezide; Çalık, Pınar (Elsevier BV, 2010-01-01)
To interpret the effect of human protein gene, e.g., human growth hormone gene hGH, on the intracellular bioreaction network of Bacillus subtilis, the intracellular reaction fluxes were calculated by solving the mass-balance-based genome-scale mathematical model, at pseudo-steady state by using bioreactor data. The bioreaction network consists of 1340 reactions including 990 metabolites. Reaction fluxes in B. subtilis carrying pMK4::pre(subC)::hGH (rBsHGH) and B. subtilis carrying merely pMK4 (rBsP) were ca...
Bioreactor operation parameters as tools for metabolic regulations in fermentation processes: influence of pH conditions
Çalık, Pınar; Calik, G; Ozdamar, TH (Elsevier BV, 2003-02-01)
The influence of controlled- and uncontrolled-pH conditions together with the initial pH on the product and by-product distributions and oxygen transfer characteristics, whereupon the process rate limitations in relation to the intracellular reaction rates were investigated in serine alkaline protease (SAP) fermentation process by recombinant Bacillus licheniformis carrying pHV1431::subC on a defined medium with the sole carbon source glucose in the pH range of 6.80-7.25 in batch bioreactors. Although the s...
Utilization of pretreated molasses for serine alkaline protease production with recombinant Bacillus species
Calik, G; Pehlivan, N; Kalender, N; Ozdamar, TH; Çalık, Pınar (Informa UK Limited, 2003-05-01)
Recombinant Bacillus amyloliquefaciens, Bacillus cereus, Bacillus subtilis , and Bacillus licheniformis were used for the production of serine alkaline protease (SAP) utilizing chemically and/or physically pretreated molasses. The highest enzyme activity was obtained with r- Bacillus subtilis , with the complex medium involving physically treated molasses having 20 kg m(-3) initial sucrose concentration in small-scale, agitation- and heating rate-controlled bioreactors at t=63 h. Effects of oxygen transfer ...
Effect of Alkyl Derivatization on Several Properties of N-Halamine Antimicrobial Siloxane Coatings
Kocer, Hasan B.; Akdağ, Akın; Ren, Xuehong; Broughton, R. M.; Worley, S. D.; Huang, T. S. (American Chemical Society (ACS), 2008-10-01)
Variation of alkyl substitution at position 5 on the hydantoin ring of a series of N-halamine siloxane derivatives has been employed to better understand the biocidal activities of these compounds for use in preparing antimicrobial coatings. The alkyl derivatization of the hydantoin ring at its 5 position, while an essentially constant chlorine loading is maintained at the I position, has shown that there is little dependence of the antimicrobial efficacy against Escherichia coli O157:H7 on the alkyl chain ...
PH influences intracellular reaction network of beta-lactamase producing Bacillus licheniformis
Çalık, Pınar (Elsevier BV, 2007-09-01)
Effects of pH on metabolic flux distributions in P-lactamase producing Bacillus licheniformis were investigated using metabolic flux analysis, at pH(C) = 6.5, 7.0 and 7.5 controlled-pH conditions. The bioprocess was divided into three periods: Period I (0 < t < 6 h) is the growth phase where beta-lactamase synthesis starts; Period II (6 < t < 8 h) is the transition phase; Period III (8 < t < 12 h) is the stationary phase for the cell-growth where beta-lactamase synthesis increases. The central pathways were...
Citation Formats
I. Ozcelik, P. Çalık, G. Calik, and T. Ozdamar, “Metabolic engineering of aromatic group amino acid pathway in Bacillus subtilis for L-phenylalanine production,” CHEMICAL ENGINEERING SCIENCE, pp. 5019–5026, 2004, Accessed: 00, 2020. [Online]. Available: