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Metabolic analysis of the synthesis of high levels of intracellular human SOD in Saccharomyces cerevisiae rhSOD 2060 411 SGA122.
Biotechnol Bioeng. 2003 Apr 20; 82(2):152-69.BB

Abstract

The synthesis of human superoxide dismutase (SOD) in batch cultures of a Saccharomyces cerevisiae strain using a glucose-limited minimal medium was studied through metabolic flux analysis. A stoichiometric model was built, which included 78 reactions, according to metabolic pathways operative in these strains during respirofermentative and oxidative metabolism. It allowed calculation of the distribution of metabolic fluxes during diauxic growth on glucose and ethanol. Fermentation profiles and metabolic fluxes were analyzed at different phases of diauxic growth for the recombinant strain (P+) and for its wild type (P-). The synthesis of SOD by the strain P+ resulted in a decrease in specific growth rate of 34 and 54% (growth on glucose and ethanol respectively) in comparison to the wild type. Both strains exhibited similar flux of glucose consumption and ethanol synthesis but important differences in carbon distribution with biomass/substrate yields and ATP production 50% higher in P-. A higher contribution of fermentative metabolism, with 64% of the energy produced at the phosphorylation level, was observed during SOD production. The flux of precursors to amino acids and nucleotides was higher in the recombinant strain, in agreement with the higher total RNA and protein levels. Lower specific growth rates in strain P+ appear to be related to the decrease in the rate of synthesis of nonrecombinant protein, as well as a decrease in the activities of the pentose phosphate (PP) pathway and TCA cycle. A very different way of entry into the stationary phase was observed for each strain: in the wild-type strain most metabolic fluxes decreased and fluxes related to energy reserve synthesis increased, while in the P+ strain the flux of 22 reactions (including PP pathway and amino acids biosynthesis) related to SOD production increased their fluxes. Changes in SOD production rates at different physiological states appear to be related to the differences in building blocks availability between respirofermentative and oxidative metabolism. Using the present expression system, ideal conditions for SOD synthesis are represented by either active growth during respirofermentative metabolism or transition from a growing to a nongrowing state. An increase in SOD flux could be achieved using an expression system nonassociated to growth and potentially eliminating part of the metabolic burden.

Authors+Show Affiliations

Gonzalez R
Department of Chemical Engineering, Millennium Institute for Advanced Studies In Cell Biology and Biotechnology, University of Chile, Beauchef 861, Santiago, Chile.
Andrews BA
No affiliation info available
Molitor J
No affiliation info available
Asenjo JA
No affiliation info available

MeSH

Adenosine TriphosphateComputer SimulationEnergy MetabolismEthanolGlucoseHumansModels, BiologicalMultienzyme ComplexesOxidation-ReductionRecombinant ProteinsSaccharomyces cerevisiaeSpecies SpecificitySuperoxide Dismutase

Pub Type(s)

Comparative Study
Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't
Validation Study

Language

eng

PubMed ID

12584757

Citation

Gonzalez, Ramon, et al. "Metabolic Analysis of the Synthesis of High Levels of Intracellular Human SOD in Saccharomyces Cerevisiae rhSOD 2060 411 SGA122." Biotechnology and Bioengineering, vol. 82, no. 2, 2003, pp. 152-69.
Gonzalez R, Andrews BA, Molitor J, et al. Metabolic analysis of the synthesis of high levels of intracellular human SOD in Saccharomyces cerevisiae rhSOD 2060 411 SGA122. Biotechnol Bioeng. 2003;82(2):152-69.
Gonzalez, R., Andrews, B. A., Molitor, J., & Asenjo, J. A. (2003). Metabolic analysis of the synthesis of high levels of intracellular human SOD in Saccharomyces cerevisiae rhSOD 2060 411 SGA122. Biotechnology and Bioengineering, 82(2), 152-69.
Gonzalez R, et al. Metabolic Analysis of the Synthesis of High Levels of Intracellular Human SOD in Saccharomyces Cerevisiae rhSOD 2060 411 SGA122. Biotechnol Bioeng. 2003 Apr 20;82(2):152-69. PubMed PMID: 12584757.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Metabolic analysis of the synthesis of high levels of intracellular human SOD in Saccharomyces cerevisiae rhSOD 2060 411 SGA122. AU - Gonzalez,Ramon, AU - Andrews,Barbara A, AU - Molitor,Julia, AU - Asenjo,Juan A, PY - 2003/2/14/pubmed PY - 2003/9/27/medline PY - 2003/2/14/entrez SP - 152 EP - 69 JF - Biotechnology and bioengineering JO - Biotechnol Bioeng VL - 82 IS - 2 N2 - The synthesis of human superoxide dismutase (SOD) in batch cultures of a Saccharomyces cerevisiae strain using a glucose-limited minimal medium was studied through metabolic flux analysis. A stoichiometric model was built, which included 78 reactions, according to metabolic pathways operative in these strains during respirofermentative and oxidative metabolism. It allowed calculation of the distribution of metabolic fluxes during diauxic growth on glucose and ethanol. Fermentation profiles and metabolic fluxes were analyzed at different phases of diauxic growth for the recombinant strain (P+) and for its wild type (P-). The synthesis of SOD by the strain P+ resulted in a decrease in specific growth rate of 34 and 54% (growth on glucose and ethanol respectively) in comparison to the wild type. Both strains exhibited similar flux of glucose consumption and ethanol synthesis but important differences in carbon distribution with biomass/substrate yields and ATP production 50% higher in P-. A higher contribution of fermentative metabolism, with 64% of the energy produced at the phosphorylation level, was observed during SOD production. The flux of precursors to amino acids and nucleotides was higher in the recombinant strain, in agreement with the higher total RNA and protein levels. Lower specific growth rates in strain P+ appear to be related to the decrease in the rate of synthesis of nonrecombinant protein, as well as a decrease in the activities of the pentose phosphate (PP) pathway and TCA cycle. A very different way of entry into the stationary phase was observed for each strain: in the wild-type strain most metabolic fluxes decreased and fluxes related to energy reserve synthesis increased, while in the P+ strain the flux of 22 reactions (including PP pathway and amino acids biosynthesis) related to SOD production increased their fluxes. Changes in SOD production rates at different physiological states appear to be related to the differences in building blocks availability between respirofermentative and oxidative metabolism. Using the present expression system, ideal conditions for SOD synthesis are represented by either active growth during respirofermentative metabolism or transition from a growing to a nongrowing state. An increase in SOD flux could be achieved using an expression system nonassociated to growth and potentially eliminating part of the metabolic burden. SN - 0006-3592 UR - https://www.unboundmedicine.com/medline/citation/12584757/Metabolic_analysis_of_the_synthesis_of_high_levels_of_intracellular_human_SOD_in_Saccharomyces_cerevisiae_rhSOD_2060_411_SGA122_ DB - PRIME DP - Unbound Medicine ER -