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

在线代谢途径分析系统的开发及其在富养菌生产PHB中的应用

基本信息

批准号：
10650789
负责人：
SHIMIZU Kazuyuki
金额：
$ 2.24万
依托单位：
Kyushu Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

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.

本研究的目的是基于代谢通量分析获得的信息，开发一种有效的聚β-羟基丁酸酯（PHB）生产监测系统。得到了底物消耗速率、氧气消耗速率和PHB产率之间的线性关系，并进一步用于对不同碳源下富营养化Ralstonia eutropha分批补料培养PHB产率的在线估计。我们对产PHB的富营养菌进行了通量分析。需要注意的是，在细胞生长阶段，通过异柠檬酸脱氢酶（ICD）产生的NADPH主要消耗在谷氨酸合成途径上，而在培养后期NH γ - D23 γ - coa浓度较低时，氨基酸合成途径受阻，导致NADPH通过ICD过量产生，并加速了PHB的生物合成，因为NADPH依赖的乙酰乙酰辅酶a还原酶可以为过量的还原等量物提供一个库。在大肠杆菌中克隆了嗜营养性r.h eutropha中参与PHB生物合成的基因，其中3个基因按PHB - c - a - b的顺序形成一个操纵子，分别编码PHB合成酶、β-酮硫酶和nadph依赖性乙酰乙酰辅酶a还原酶。几种大肠杆菌K12衍生物，即HMS174， TA3516 （pta/ack）和DF11 (pgi)，用含有天然phb操纵子的质粒转化。对这些扰动的分析结果表明，细胞内乙酰辅酶a的积累可能不能促进体内PHB的合成。另一方面，由于pgi突变菌株的PHB生物合成可以通过PP途径利用NADPH的过量产生，因此pgi突变菌株在葡萄糖上的生长得以恢复，表明NADPH的过量产生可能能够促进PHB的合成。我们考虑了利用乳酸菌和真嗜酸酵母通过乳酸将糖转化为PHB的混合培养体系，建立了数学模型，并研究了该体系的动力学和控制。