Determination of metabolic bottlenecks using reaction engineering principles in serine alkaline protease production by recombinant bacillus sp.

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2004

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Telli, İlkin Ece

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In this study, firstly, bioprocess characteristics for Serine Alkaline Protease (SAP) production, using recombinant Bacillus subtilis carrying pHV1431::subC, were examined. The cell concentration, substrate concentration, SAP activity and SAP synthesis rate profiles demonstrated that the system reaches to a steady state in terms of cell growth and SAP synthesis between t=15-25 h, therefore, this time interval is appropriate to employ both metabolic flux analysis and metabolic control analysis, which apply strictly to steady state systems. After that, three separate perturbations were introduced by addition of aspartate to the production medium at a certain time of the bioprocess. The response of the cells were observed and; by comparing the changes in intracellular reactions of aspartate pathway, Asn, Thr and Ile productions were determined to be the bottlenecks in aspartate pathway and the branchpoints splitting from Asp and AspSa were identified to be weakly rigid branchpoints. Lastly, metabolic control analysis principles were applied to determine the elasticity and flux control coefficients of the simplified aspartate pathway. Aspartate formation reaction and Lys, Thr, Ile, Met producing group share the control of asparagine synthesis. The results revealed that lysine producing branch flux dominates the other branch fluxes, therefore to eliminate bottlenecks and increase SAP production, the activity of the branches leading to the formation of Asn, Thr and Ile should be increased while decreasing the activity of lysine synthesizing branch. This could be achieved either by genetic manipulation or by addition of specific inhibitors or activators to the system.

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Biotechnology.

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http://etd.lib.metu.edu.tr/upload/2/12605227/index.pdf
https://hdl.handle.net/11511/14282

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Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats

İ. E. Telli, “Determination of metabolic bottlenecks using reaction engineering principles in serine alkaline protease production by recombinant bacillus sp.,” M.S. - Master of Science, Middle East Technical University, 2004.