Analysis of metabolic flux in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing human-like collagen in fed-batch culture |
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Authors: | Yan E Luo Dai D Fan Long A Shang Hui J Shi Xiao X Ma Yu Mi Gui F Zhao |
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Institution: | (1) Department of Chemical Engineering, Northwest University/Shaanxi Source and Application chemical Engineering Center, Xi’an, 710068, China;(2) Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University), Ministry of Education, Xi’an, 710068, China;(3) College of Biological and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo, 315100, China;(4) Department of Reproductive Biology, Shanghai Institute of Planned Parenthood Research, Shanghai, 200032, China |
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Abstract: | Metabolic flux distributions of recombinant Escherichia coli BL21 expressing human-like collagen were determined by means of a stoichiometric network and metabolic balancing. At the
batch growth stage, the fluxes of the pentose phosphate pathway were higher than the fluxes of the fed-batch growth phase
and the production stage. After the temperature was increased, there was a substantially elevated energy demand for synthesizing
human-like collagen and heat-shock proteins, which resulted in changes in metabolic fluxes. The activities of the Embden-Meyerhof-Parnas
pathway and the tricarboxylic acid cycle were significantly enhanced, leading to a reduction in the fluxes of the pentose
phosphate pathway and other anabolic pathways. The temperature upshift also caused an increase in NADPH production by isocitrate
dehydrogenase in the tricarboxylic acid cycle. The metabolic model predicted the involvement of a transhydrogenase that generates
additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. These data indicated that the maintenance
energy for cellular activity increased with the increase in biomass in fed-batch culture, and that cell growth and synthesis
of human-like collagen could clearly represent the changes in metabolic fluxes. At the production stage, more NADPH was used
to synthesize human-like collagen than for maintaining cellular activity, cell growth, and cell propagation.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | Fed-batch culture Human-like collagen Metabolic flux analysis Recombinant Escherichia coli |
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