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Metabolic Characterization and Engineering of Streptomyces lividans Producing Heterologous Proteins
Streptomycetes have become attractive hosts for heterologous protein production due to for instance, their ability to efficiently secrete theirproteins in the extracellular environment. Among these microbes, Streptomyces lividans</>, known to have a reduced restriction-modification barrier, has been used more extensively for heterologous protein production. Hitherto, studies on metabolic characterization and engineering of S. lividans</> for heterologous protein production are limited, and of these, few are performed in (upscale) controlled fermentation environments. Without metabolomics, the metabolic impact/load that heterologous protein production exerts on the central carbon metabolism of S. lividans</> remains loosely understood, and can not easily be relieved.
Therefore, the general objective of this dissertation was to characterize the growth and metabolism of S. lividans</> producing heterologous proteins in (upscale) controlled fermentation environments. The studyis further extended to assess the effect of overexpressing the gene (pck</>) encoding phosphoenolpyruvate carboxykinase on heterologous protein production in S. lividans</>. A genome scale metabolic network model of wild-type S. lividans </>was extended to include mouse tumor necrosis factor-alpha (mTNF-alpha) production. The resulting model matrix was mathematically analyzed to check for its inherent properties and sensitivity to key measurable fluxes. Results obtained showed that model sensitivity to mTNF-alpha was tenfold higher than biomass production, while the latter was at least tenfold more sensitive than the measurable amino acid fluxes. Metabolic characterization of recombinant S. lividans</>, in comparison with the wild-type S. lividans</>, was done to assess metabolic impact based on both extracellular metabolomics and constraint-based modeling. Results obtained showed that the metabolic impact due to heterologous protein production results in lower substrate uptake and slower growth, the former an indication for metabolic channeling.The metabolic impact is widely-distributed in the genome of S. lividans</>, causing a great shift in its performance. Critical exchange fluxes in the recombinant cells or models performance have been identifiedas biomass, mTNF-alpha, histidine, valine and alanine. Finally, based on metabolic engineering techniques, it was revealed that pck overexpression in S. lividans</> TK24 results in up to 1.7-fold increase in hTNF-alpha production. The major findings in this dissertation are an indication of metabolic shifts within the recombinant cell as well as a positive correlation between the explored metabolic engineering approach andheterologous protein production.
Therefore, the general objective of this dissertation was to characterize the growth and metabolism of S. lividans</> producing heterologous proteins in (upscale) controlled fermentation environments. The studyis further extended to assess the effect of overexpressing the gene (pck</>) encoding phosphoenolpyruvate carboxykinase on heterologous protein production in S. lividans</>. A genome scale metabolic network model of wild-type S. lividans </>was extended to include mouse tumor necrosis factor-alpha (mTNF-alpha) production. The resulting model matrix was mathematically analyzed to check for its inherent properties and sensitivity to key measurable fluxes. Results obtained showed that model sensitivity to mTNF-alpha was tenfold higher than biomass production, while the latter was at least tenfold more sensitive than the measurable amino acid fluxes. Metabolic characterization of recombinant S. lividans</>, in comparison with the wild-type S. lividans</>, was done to assess metabolic impact based on both extracellular metabolomics and constraint-based modeling. Results obtained showed that the metabolic impact due to heterologous protein production results in lower substrate uptake and slower growth, the former an indication for metabolic channeling.The metabolic impact is widely-distributed in the genome of S. lividans</>, causing a great shift in its performance. Critical exchange fluxes in the recombinant cells or models performance have been identifiedas biomass, mTNF-alpha, histidine, valine and alanine. Finally, based on metabolic engineering techniques, it was revealed that pck overexpression in S. lividans</> TK24 results in up to 1.7-fold increase in hTNF-alpha production. The major findings in this dissertation are an indication of metabolic shifts within the recombinant cell as well as a positive correlation between the explored metabolic engineering approach andheterologous protein production.
Date:27 Oct 2008 → 18 Dec 2012
Keywords:Metabolic flux analysis
Disciplines:Catalysis and reacting systems engineering, Chemical product design and formulation, General chemical and biochemical engineering, Process engineering, Separation and membrane technologies, Transport phenomena, Other (bio)chemical engineering
Project type:PhD project