DEVELOPMENT OF ON-LINE METABOLIC PATHWAY ANALYSIS SYSTEM AND ITS APPLICATION TO PHB PRODUCTION BY R.eutropha

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The objective of this study was to develop an effective monitoring system for the production of poly-β-hydroxybutyrate (PHB) based on the information obtained from metabolic flux analysis. A linear relationship among substrate consumption rate, oxygen consumption rate, and PHB production rate was obtained and further used for the on-line estimation of PHB production in fed-batch culture of Ralstonia eutropha on various carbon sources.We made flux analysis for efficient production of PHB using R. eutropha. It should be noted that NADPH generated via isocitrate dehydrogenase (ICD) was mainly consumed in glutamic acid synthesis pathway during cell growth phase, while the block in the amino acid synthetic pathway in late culture phase where NHィイD23ィエD2 concentration was low results in the overproduction of NADPH through ICD and accelerates the biosynthesis or PHB since NADPH-dependent acetoacetyl-CoA reductase can provide a sink for excess reducing equivalents.The genes involved in PHB biosynthesis in R.eutropha were cloned in Escherichia coli, where three genes form an operon in the order of phb-C-A-B, coding for PHB synthase,β-ketothiolase, and NADPH-dependent acetoacetyl-CoA reductase, respectively. Several E.coli K12 derivatives, namely, HMS174, TA3516 (pta/ack), and DF11 (pgi), were transformed with a plasmid which contains the native phb operon. Results from the analyses of these perturbations indicate that intracellular buildup of acetyl-CoA may not be able to promote PHB synthesis in vivo. On the other hand, since the biosynthesis or PHB in the pgi mutant strain can utilize the NADPH overproduction through the PP pathway, the growth of the pgi mutant on glucose was recovered, indicating that the overproduction of NADPH might be able to enhance PHB synthesisWe considered the mixed culture system for converting sugars to PHB via lactate using lactic acid bacteria and R.eutropha, developed a mathematical model, and studied the dynamics and control of the system.