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Modelling can be used as an approach to improve fluxes by engineering upon the determined sites
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
(t1/2free enzyme = 38 min; t1/2sol—gel = 197 min) at pH 7.6. The results show that PGA is more stable when incubated in the presence of Tris buffer, as compared to phosphate buffer and the immobilization procedure increases PGA stability. The synthesis/hydrolysis ratio (S/H, mol product per mol hydrolyzed side chain donor formed) is often used as an indicator of the viability of the process in the kinetically controlled synthesis of cephalexin (CEX). Synthesis of CEX in aqueous medium, was performed at 14◦ C at pH 7.2, in 50 mM phosphate buffer and 50 mM Tris buffer using free PGA, phenylglycine methyl ester (D-PGM) as acyl donor at 90 mM and 7-aminodeacetoxycephalosporanic acid (7-ADCA) the limiting substrate at 30 mM. The maximum S/H values were 9.3 and 3.0, respectively, Tris and phosphate buffer, which show that the activity of synthetase is increased in the presence of Tris.
metabolic engineering design directed to: (i) R89 in the AAAP provided ca. 4.14-fold; (ii) R96 provided ca. 2.0-fold; and, (iii) R89 and R96 in concert (by r-B. subtilis carrying pMK4::aroA::aroH) provided ca. 7.5-fold increase, compared to the wild-type. The fluxes from prephenate towards Phe were 0.094, 0.215 and 0.380 mmol g−1 DWh−1 , respectively, for r-B. subtilis carrying pMK4::aroH, r-B. subtilis carrying pMK4::aroA, and r-B. subtilis carrying pMK4::aroA::aroH. Thus, we demonstrate how pathway modelling of B. subtilis can be used as an approach to improve the fluxes by engineering upon the determined sites of the genome.
doi:10.1016/j.nbt.2009.06.414
2.3.033
2.3.032
Recombinant production of a difficult-to-express eukaryotic protein in a bacterial expression system
Modelling can be used as an approach to improve fluxes by engineering upon the determined sites G. Calik ∗ , Y. Demirci, T.H. Ozdamar Ankara University, Ankara, Turkey
We have identified and engineered the rate-limiting sites in the aromatic-amino-acid-pathway (AAAP) of Bacillus subtilis aiming pathway-flux-amplification for phenylalanine (Phe) production. Branch-point metabolites erythrose-4-phosphate (E4P) supplied in vitro transported into the cell and enhanced Phe production ca. sevenfold. On the basis of bioreaction-network of B. subtilis, the mass-flux-balance-based mathematical-model that contains 184 metabolites and 232 reaction-fluxes was set up. Intracellular reaction-fluxes were calculated; whereupon, the rate-limiting reactions for Phe production were determined by comparative analysis of the results. To interpret the related results we conclude that, in turn: (i) as the pulse-response experiments using E4P increased all the reaction rates toward Phe, aroA gene encoding the enzyme DAHP-synthase (EC 2.5.1.54) of the first reaction (R89) of the AAAP; (ii) as the flux value was the lowest, aroH gene encoding the enzyme chorismate-mutase (EC 5.4.99.5) of R96; were predicted as the rate-limiting reactions. To demonstrate influence of the catalysis on the determined sites separately, by single-gene cloning two recombinant B. subtilis, each specific to a single-site wherein carries a recombinant-plasmid having the gene encoding the enzyme of the rate-limiting reaction, that is, pMK4::aroA and pMK4::aroH; and then, to demonstrate the concerted influence of the catalysis on the pathway, by multiple-gene cloning a recombinant-plasmid carrying all the genes encoding the enzymes of the determined rate-limiting reactions, that is, pMK4::aroA::aroH, were constructed. The highest product concentration was obtained by r-B. subtilis carrying pMK4::aroA::aroH (CPhe = 1.5 kg m−3 ). To demonstrate pathway-flux-amplification, intracellular reaction-fluxes for r-B. subtilis carrying pMK4::aroH, r-B. subtilis carrying pMK4::aroA, and r-B. subtilis carrying pMK4::aroA::aroH, and for wild-type B. subtilis were calculated using the data obtained in batch-bioreactor experiments. In Phe production-phase, by the
doi:10.1016/j.nbt.2009.06.415
U. Unzueta Elorza ∗ , J. Corchero, E. Garcia Fruitos, V. Toledo Rubio, R. Mendoza, A. Villaverde Corrales, N. Ferrer Miralles IBB (Institut de biotecnologia y de biomedicina) UAB, SABADELL, Spain
Eukaryotic protein production in bacterial expression systems as E. coli presents several problems, namely lack of post-translational modifications, non-optimized codon usage and difficulty of disulfide bond generation (due to the bacterial reducing cytoplasm). Furthermore, heterologous protein over-expression can generate metabolic overload, as well as saturation of the protein quality system, causing protein aggregation and proteolysis. Genetically engineered bacterial strains have been developed to overcome these bottlenecks. These strains have allowed the production of eukaryotic proteins which are difficult to obtain in wild type strains. Human alpha galactosidase A has been chosen as a model protein because of its high number of non-optimized codons for heterologous expression and the presence of disulfide bonds in its native structure. To prove its expression, our model protein has been produced in different bacterial strains such as BL21(DE3), Rosetta, Origami and Rosetta-gami B (DE3). All these strains are deficient in ‘lon’ and ‘ompT’ proteases. No soluble protein was found in BL21(DE3), Rosetta and Origami strains and all the detected protein was found in the insoluble fraction. However, soluble but proteolyzed protein was found in the Rosetta-gami B (DE3) strain. To improve protein solubility, alpha galactosidase A was fused to glutathione-s-transferase (GST) protein and its expression in Rosetta-gami B (DE3) strain was tested. A great amount of soluble protein was obtained and purified. After further characterization the purified protein was found to be proteolyzed, resulting in a lack of 39 residues in its c-terminal extreme. doi:10.1016/j.nbt.2009.06.416
www.elsevier.com/locate/nbt S121
ABSTRACTS
(t1/2free enzyme = 38 min; t1/2sol—gel = 197 min) at pH 7.6. The results show that PGA is more stable when incubated in the presence of Tris buffer, as compared to phosphate buffer and the immobilization procedure increases PGA stability. The synthesis/hydrolysis ratio (S/H, mol product per mol hydrolyzed side chain donor formed) is often used as an indicator of the viability of the process in the kinetically controlled synthesis of cephalexin (CEX). Synthesis of CEX in aqueous medium, was performed at 14◦ C at pH 7.2, in 50 mM phosphate buffer and 50 mM Tris buffer using free PGA, phenylglycine methyl ester (D-PGM) as acyl donor at 90 mM and 7-aminodeacetoxycephalosporanic acid (7-ADCA) the limiting substrate at 30 mM. The maximum S/H values were 9.3 and 3.0, respectively, Tris and phosphate buffer, which show that the activity of synthetase is increased in the presence of Tris.
metabolic engineering design directed to: (i) R89 in the AAAP provided ca. 4.14-fold; (ii) R96 provided ca. 2.0-fold; and, (iii) R89 and R96 in concert (by r-B. subtilis carrying pMK4::aroA::aroH) provided ca. 7.5-fold increase, compared to the wild-type. The fluxes from prephenate towards Phe were 0.094, 0.215 and 0.380 mmol g−1 DWh−1 , respectively, for r-B. subtilis carrying pMK4::aroH, r-B. subtilis carrying pMK4::aroA, and r-B. subtilis carrying pMK4::aroA::aroH. Thus, we demonstrate how pathway modelling of B. subtilis can be used as an approach to improve the fluxes by engineering upon the determined sites of the genome.
doi:10.1016/j.nbt.2009.06.414
2.3.033
2.3.032
Recombinant production of a difficult-to-express eukaryotic protein in a bacterial expression system
Modelling can be used as an approach to improve fluxes by engineering upon the determined sites G. Calik ∗ , Y. Demirci, T.H. Ozdamar Ankara University, Ankara, Turkey
We have identified and engineered the rate-limiting sites in the aromatic-amino-acid-pathway (AAAP) of Bacillus subtilis aiming pathway-flux-amplification for phenylalanine (Phe) production. Branch-point metabolites erythrose-4-phosphate (E4P) supplied in vitro transported into the cell and enhanced Phe production ca. sevenfold. On the basis of bioreaction-network of B. subtilis, the mass-flux-balance-based mathematical-model that contains 184 metabolites and 232 reaction-fluxes was set up. Intracellular reaction-fluxes were calculated; whereupon, the rate-limiting reactions for Phe production were determined by comparative analysis of the results. To interpret the related results we conclude that, in turn: (i) as the pulse-response experiments using E4P increased all the reaction rates toward Phe, aroA gene encoding the enzyme DAHP-synthase (EC 2.5.1.54) of the first reaction (R89) of the AAAP; (ii) as the flux value was the lowest, aroH gene encoding the enzyme chorismate-mutase (EC 5.4.99.5) of R96; were predicted as the rate-limiting reactions. To demonstrate influence of the catalysis on the determined sites separately, by single-gene cloning two recombinant B. subtilis, each specific to a single-site wherein carries a recombinant-plasmid having the gene encoding the enzyme of the rate-limiting reaction, that is, pMK4::aroA and pMK4::aroH; and then, to demonstrate the concerted influence of the catalysis on the pathway, by multiple-gene cloning a recombinant-plasmid carrying all the genes encoding the enzymes of the determined rate-limiting reactions, that is, pMK4::aroA::aroH, were constructed. The highest product concentration was obtained by r-B. subtilis carrying pMK4::aroA::aroH (CPhe = 1.5 kg m−3 ). To demonstrate pathway-flux-amplification, intracellular reaction-fluxes for r-B. subtilis carrying pMK4::aroH, r-B. subtilis carrying pMK4::aroA, and r-B. subtilis carrying pMK4::aroA::aroH, and for wild-type B. subtilis were calculated using the data obtained in batch-bioreactor experiments. In Phe production-phase, by the
doi:10.1016/j.nbt.2009.06.415
U. Unzueta Elorza ∗ , J. Corchero, E. Garcia Fruitos, V. Toledo Rubio, R. Mendoza, A. Villaverde Corrales, N. Ferrer Miralles IBB (Institut de biotecnologia y de biomedicina) UAB, SABADELL, Spain
Eukaryotic protein production in bacterial expression systems as E. coli presents several problems, namely lack of post-translational modifications, non-optimized codon usage and difficulty of disulfide bond generation (due to the bacterial reducing cytoplasm). Furthermore, heterologous protein over-expression can generate metabolic overload, as well as saturation of the protein quality system, causing protein aggregation and proteolysis. Genetically engineered bacterial strains have been developed to overcome these bottlenecks. These strains have allowed the production of eukaryotic proteins which are difficult to obtain in wild type strains. Human alpha galactosidase A has been chosen as a model protein because of its high number of non-optimized codons for heterologous expression and the presence of disulfide bonds in its native structure. To prove its expression, our model protein has been produced in different bacterial strains such as BL21(DE3), Rosetta, Origami and Rosetta-gami B (DE3). All these strains are deficient in ‘lon’ and ‘ompT’ proteases. No soluble protein was found in BL21(DE3), Rosetta and Origami strains and all the detected protein was found in the insoluble fraction. However, soluble but proteolyzed protein was found in the Rosetta-gami B (DE3) strain. To improve protein solubility, alpha galactosidase A was fused to glutathione-s-transferase (GST) protein and its expression in Rosetta-gami B (DE3) strain was tested. A great amount of soluble protein was obtained and purified. After further characterization the purified protein was found to be proteolyzed, resulting in a lack of 39 residues in its c-terminal extreme. doi:10.1016/j.nbt.2009.06.416
www.elsevier.com/locate/nbt S121