代谢工程的发展及其应用

本首先论述了代谢工程的代谢网络理论、代谢分析、节点分析和中心代谢物作用机理等代谢工程理论基础。然后,分析了代谢工程的各种具体设计思路,并以实际例子作了详细说明。另外还对代谢工程的新兴研究方向－逆代谢工程进行了简单说明。     

城市代谢研究评述:内涵与方法

随着城市发展面临的生态问题日益显著,部分研究者试图通过对自然生态系统进行类比来寻求解决途径,城市代谢理论应运而生。当以系统科学来研究城市生态系统时,哲学思想的引入为探索和城市及城市代谢的内涵提供了最原始的桥梁。因此,在介绍城市代谢内涵与研究进展的基础上,融合产业、家庭、社会等多尺度代谢理论,对城市代谢的边界进行扩展,将其分为狭义与广义两类,结合亚里士多德的"四因说"对其质料因(组分)、形式因(结构)、动力因(驱动力)和目的因(功能)进行识别分析,据此将城市代谢研究方法归纳为质料、形式和混合研究方法三类,并提出未来研究的主要动向和解决手段。城市代谢"四因图"可为相关研究者提供参考。     

厦门市城市能源代谢综合分析方法及应用

城市代谢理论目前被认为是系统解析城市病症结的重要切入点。能源是人类活动和城市发展的物质基础, 研究其代谢规律可以揭示城市能源利用过程中存在的问题及其代谢污染物的生态环境效应, 从而为城市能源规划与管理提供科学依据。文章在部门调研数据、统计年鉴数据和文献资料分析的基础上, 提出了一种基于能量流的城市能源代谢综合分析方法。该方法系统分析能源在城市内部的流动过程, 全面核算5 种主要代谢污染物的排放量, 并使用7 个指标对能源代谢进行评价, 进一步使用该方法系统研究了厦门市2009 年能源代谢情况, 结果表明: (1)厦门本地能源稀缺, 对外依存度高, 并且结构较为单一, 主要依赖煤炭和燃油, 分别占能源总量的61%和23%。(2)代谢污染物CO₂、NO₂ 和废热首要来自能源加工转化, SO₂ 和PM2.5 首要来自工业。(3)2009 年能源代谢总效率为0.43 tce·t^-1, 在各部门中服务业及其他的效率最高, 加工转化的效率最低, 代谢效率与能源结构关系密切。(4)文章提出的方法可为城市能源代谢研究提供方法参考。     

蜡状芽胞杆菌群代谢途径的分析和比较

微生物基因组测序和高通量分析方法获得了大量的数据和信息,利用这些信息研究代谢网络成为当前的一个新热点。文章在比较和分析重构代谢网络不同方法的基础上,利用蜡状芽胞杆菌群中已测序的9株蜡状芽胞杆菌、6株炭疽芽胞杆菌、6株苏云金芽胞杆菌基因组,对它们的碳水化合物代谢途径、氨基酸代谢途径和能量代谢途径进行比较与分析,找出它们的共性和特性。这3种菌都存在必需的糖酵解、三羧酸循环、丙氨酸代谢、组氨酸代谢及能量代谢等途径;同时它们还存在特殊的代谢途径,蜡状芽胞杆菌对单糖的利用率较高;炭疽芽胞杆菌的氨基酸降解和转运途径较丰富;苏云金芽胞杆菌中存在催化谷氨酸转化的代谢旁路等。代谢途径的分析为深入研究它们的食物毒素、炭疽毒素和杀虫毒素提供了新思路。     

鸟苷发酵过程代谢流迁移的分析

以典型的代谢控制发酵产品鸟苷为例说明了一种基于过程参数的相关分析来研究发酵过程中代谢流迁移的方法。通过对发酵过程多参数的相关性分析,结合生物合成代谢途径、氨基酸和有机酸积累的分析,确认了发酵过程代谢流向EMP途径的迁移,认为造成这种代谢流迁移的原因可能是过程铵离子积累。在此基础上,通过对过程参数实时检测分析和及时调整EMP和HMP代谢通量使产率提高了35％。      

微生物代谢工程原理与应用

代谢工程是利用分子生物学原理系统分析细胞代谢网络,并通过DNA重组技术和应用分析生物学相关的遗传学手段对细胞进行有精确目标的基因操作,改变微生物原有的代谢或调节系统,实现目的产物代谢活性的提高。代谢工程综合了生物化学、化学工程、数学分析等多学科内容,是当前国内外学者研究热点之一。论述了微生物代谢工程的理论基础及其应用进展和前景。     

代谢流量比率分析及其在代谢工程中的应用

细胞内代谢反应流量在系统理解细胞代谢特性和指导代谢工程改造等方面都起着重要的作用。由于代谢流量难以直接测量得到,在很多情况下通过跟踪稳定同位素在代谢网络中的转移并进行相应的模型计算能有效地定量代谢流量。代谢流量比率分析法能够高度体现系统的生物化学真实性、辨别细胞代谢网络的拓扑结构,并且能够相对简单快速地定量反应速率等,因此受到代谢工程研究者越来越多的重视。以下着重介绍并讨论了利用代谢物同位体分布信息分析关键代谢节点合成途径的流量比率、基于流量比率的代谢流量解析、以及应用于代谢工程等的相关原理、实验测量、数据分析、使用条件等,以期充分发挥代谢流量比率分析法的优势,并将其拓展推广至更多细胞体系的代谢特性阐明和代谢工程改造中去。     

产业生态系统资源代谢分析方法

产业生态系统是由企业群、资源及环境组成的社会-经济-环境复合生态系统。资源代谢是其功能运行的重要保障。资源代谢在时间和空间尺度上的耗竭及阻滞是造成严重生态环境问题的主要原因。根据生态学原理,运用物质流分析手段解析了产业生态系统的物质流、能流及资金流结构,构建了产业生态系统资源代谢分析模型,提出了资源输入-使用-输出-循环共生四方面的资源代谢分析指标体系和基于模糊综合分析的资源代谢问题树分析方法。在此基础上提出了基于循环共生网络结构模型的生态管理模式。以期为产业资源的生态管理提供方法支撑。     

肌苷发酵过程中后期的代谢流节点分析

应用代谢流分析方法,实验测定副产物的积累速率,将数据输入计算机,应用MArllAB软件计算肌苷发酵中后期代谢流分布。通过分析代谢网络中重要的节点,提出了优化肌苷生物合成途径的建议。     

代谢速率调控物种丰富度格局的研究进展

提出生物多样性分布格局的普适性理论和探索其内在形成机制一直是生态学家们研究的焦点之一.到目前为止,已有很多假说被用来解释生物多样性分布规律,但是这些假说的普适性均受到学者们的质疑.最新理论--代谢速率假说以能量相当法则和代谢分形分配网络模型为基础,定量预测了个体及种群生态进化动态过程与群落生物多样性分布格局之间的关系,以及物种丰富度和环境因子之间的关系.代谢速率假说解释了生物多样性的起源问题,也回答了生物多样性如何维持的问题.该文重点综述了代谢生物多样性理论的发展及其相关研究进展.通过和其他假说比较、分析,我们认为随着代谢理论假说的不断发展和完善,代谢生物多样性理论将更具有普适性.同时我们也提出了进一步完善该假说需要解决的一些科学问题.     

Interpretation of metabolic flux maps by limitation potentials and constrained limitation sensitivities

Two new concepts, "Limitation Potential" and "Constraint Limitation Sensitivity" are introduced that use definitions derived from metabolic flux analysis (MFA) and metabolic network analysis (MNA). They are applied to interpret a measured flux distribution in the context of all possible flux distributions and thus combine MFA with MNA. The proposed measures are used to quantify and compare the influence of intracellular fluxes on the production yield. The methods are purely based on the stoichiometry of the network and constraints that are given from irreversible fluxes. In contrast to metabolic control analysis (MCA), within this approach no information about the kinetic mechanisms are needed. A limitation potential (LP) is defined as the reduction of the reachable (theoretical) maximum by a measured flux. Measured fluxes that strongly narrow the reachable maximum are assumed to be limiting as the network has no ability to counterbalance the restriction due to the observed flux. In a second step, the sensitivity of the reduced maximum is regarded. This measure provides information about the necessitated changes to reach higher yields. The methods are applied to interpret the capabilities of a network based on measured fluxes for a L-phenylalanine producer. The strain was examined by a series of experiments and three flux maps of the production phase are analyzed. It can be shown that the reachable yield is drastically reduced by the measured efflux into the TCA cycle, while the oxidative pentose-phosphate pathway only plays a secondary role on the reachable maximum.     

基因组尺度代谢网络自动重构及分析工具研究进展

高通量数据的产出为基因组尺度代谢网络的构建提供了基础,但同时也对网络构建和分析方法的改进提出了挑战。随着数据量的不断增大,耗时耗力的人工构建及分析已经无法满足模型发展的需要,因而各种自动化的方法应运而生。模型构建和分析的自动化不仅能够大幅度提高模型构建和解析的速度,同时对于模型构建和分析方法的标准化和程序化也有着不可替代的作用。文中结合作者的实际研究经验,对基因组尺度代谢网络构建的自动化进程和主要的代谢网络分析工具进行了较为详细的介绍,总结了代谢网络自动重构的流程,并提出了目前面对的主要问题和未来的研究方向。     

Physiology of microbial cells and metabolic engineering

This review is devoted to the problems of the physiology and cell biology of microorganisms in relation to metabolic engineering. The latter is considered as a branch of fundamental and applied biotechnology aimed at controlling microbial metabolism by methods of genetic engineering and classical genetics and based on intimate knowledge of cell metabolism. Attention is also given to the problems associated with the metabolic limitation of microbial biosyntheses, analysis and control of metabolic fluxes, rigidity of metabolic pathways, the role of pleiotropic (global) regulatory systems in the control of metabolic fluxes, and prospects of physiological and evolutionary approaches in metabolic engineering.     

Construction and comparative analysis of two-component system and metabolic network profile based phylogenetic trees

In this study, two-component system (TCS) gene profile and metabolic network gene profile based phylogenetic trees were constructed and compared to each other to evaluate the evolutionary relationship between the bacterial sensing system and metabolism. The gene profiles of the these systems suggested that bacteria employed different evolutionary strategies to optimize the two-component system and metabolic network. In addition, comparative analysis revealed that the TCS based tree showed better family grouping than the metabolic network based tree, which indicated that the TCS and metabolic network have been modified via self-evolution and recruitment methods, respectively.     

A review on metabolic pathway analysis with emphasis on isotope labeling approach

The recent progress on metabolic systems engineering was reviewed based on our recent research results in terms of (1) metabolic signal flow diagram approach, (2) metabolic flux analysis (MFA) in particular with intracellular isotopomer distribution using NMR and/or GC-MS, (3) synthesis and optimization of metabolic flux distribution (MFD), (4) modification of MFD by gene manipulation and by controlling culture environment, (5) metabolic control analysis (MCA), (6) design of metabolic regulation structure, and (7) identification of unknown pathways with isotope tracing by NMR. The main characteristics of metabolic engineering is to treat metabolism as a network or entirety instead of individual reactions. The applications were made for poly-3-hydroxybutyrate (PHB) production usingRalstonia eutropha and recombinantEscherichia coli, lactate production by recombinantSaccharomyces cerevisiae, pyruvate production by vitamin auxotrophic yeastToluropsis glabrata, lysine production usingCorynebacterium glutamicum, and energetic analysis of photosynthesic microorganisms such as Cyanobateria. The characteristics of each approach were reviewed with their applications. The approach based on isotope labeling experiments gives reliable and quantitative results for metabolic flux analysis. It should be recognized that the next stage should be toward the investigation of metabolic flux analysis with gene and protein expressions to uncover the metabolic regulation in relation to genetic modification and/or the change in the culture condition.     

Measuring multiple fluxes through plant metabolic networks

Fluxes through metabolic networks are crucial for cell function, and a knowledge of these fluxes is essential for understanding and manipulating metabolic phenotypes. Labeling provides the key to flux measurement, and in network flux analysis the measurement of multiple fluxes allows a flux map to be superimposed on the metabolic network. The principles and practice of two complementary methods, dynamic and steady-state labeling, are described, emphasizing best practice and illustrating their contribution to network flux analysis with examples taken from the plant and microbial literature. The principal analytical methods for the detection of stable isotopes are also described, as well as the procedures for obtaining flux maps from labeling data. A series of boxes summarizing the key concepts of network flux analysis is provided for convenience.     

应用代谢网络模型解析工业微生物胞内代谢

为了快速、高效地理解工业微生物胞内代谢特征,寻找潜在的代谢工程改造靶点,基因组规模代谢网络模型(GSMM)作为一种系统生物学工具越来越受到人们的关注。文中在回顾GSMM 20年发展历程的基础上,分析了当前GSMM的研究现状,总结了GSMM的构建及分析方法,从预测细胞表型和指导代谢工程两个方面阐述了GSMM在解析工业微生物胞内代谢中的应用,并展望了GSMM未来的发展趋势。     

OP-Synthetic: identification of optimal genetic manipulations for the overproduction of native and non-native metabolites

Constraint-based flux analysis has been widely used in metabolic engineering to predict genetic optimization strategies. These methods seek to find genetic manipulations that maximally couple the desired metabolites with the cellular growth objective. However, such framework does not work well for overproducing chemicals that are not closely correlated with biomass, for example non-native biochemical production by introducing synthetic pathways into heterologous host cells. Here, we present a computational method called OP-Synthetic, which can identify effective manipulations (upregulation, downregulation and deletion of reactions) and produce a step-by-step optimization strategy for the overproduction of indigenous and non-native chemicals. We compared OP-Synthetic with several state-of-the-art computational approaches on the problems of succinate overproduction and N-acetylneuraminic acid synthetic pathway optimization in Escherichia coli. OP-Synthetic showed its advantage for efficiently handling multiple steps optimization problems on genome wide metabolic networks. And more importantly, the optimization strategies predicted by OP-Synthetic have a better match with existing engineered strains, especially for the engineering of synthetic metabolic pathways for non-native chemical production. OP-Synthetic is freely available at:http://bioinfo.au.tsinghua.edu.cn/member/xwwang/OPSynthetic/.