Elementary mode analysis: a useful metabolic pathway analysis tool for characterizing cellular metabolism

Elementary mode analysis is a useful metabolic pathway analysis tool to identify the structure of a metabolic network that links the cellular phenotype to the corresponding genotype. The analysis can decompose the intricate metabolic network comprised of highly interconnected reactions into uniquely organized pathways. These pathways consisting of a minimal set of enzymes that can support steady state operation of cellular metabolism represent independent cellular physiological states. Such pathway definition provides a rigorous basis to systematically characterize cellular phenotypes, metabolic network regulation, robustness, and fragility that facilitate understanding of cell physiology and implementation of metabolic engineering strategies. This mini-review aims to overview the development and application of elementary mode analysis as a metabolic pathway analysis tool in studying cell physiology and as a basis of metabolic engineering.     

Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies

For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on (13)CO(2) dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.     

Plant Metabolic Modeling: Achieving New Insight into Metabolism and Metabolic Engineering

Models are used to represent aspects of the real world for specific purposes, and mathematical models have opened up new approaches in studying the behavior and complexity of biological systems. However, modeling is often time-consuming and requires significant computational resources for data development, data analysis, and simulation. Computational modeling has been successfully applied as an aid for metabolic engineering in microorganisms. But such model-based approaches have only recently been extended to plant metabolic engineering, mainly due to greater pathway complexity in plants and their highly compartmentalized cellular structure. Recent progress in plant systems biology and bioinformatics has begun to disentangle this complexity and facilitate the creation of efficient plant metabolic models. This review highlights several aspects of plant metabolic modeling in the context of understanding, predicting and modifying complex plant metabolism. We discuss opportunities for engineering photosynthetic carbon metabolism, sucrose synthesis, and the tricarboxylic acid cycle in leaves and oil synthesis in seeds and the application of metabolic modeling to the study of plant acclimation to the environment. The aim of the review is to offer a current perspective for plant biologists without requiring specialized knowledge of bioinformatics or systems biology.     

植物次生代谢物途径及其研究进展

植物次生代谢是植物在长期进化过程中与环境相互作用的结果,由初生代谢派生。萜类、生物碱类、苯丙烷类为植物次生代谢物的主要类型,其代谢途径多以代谢频道形式存在,具有种属、生长发育期等特异性。从植物次生代谢物的分类、代谢途径及代谢调控基因工程等方面展开论述,重点介绍了次生代谢物的生物合成途径,以及利用基因工程等技术对植物次生代谢途径进行遗传改良等方面的研究进展,为全面认识植物代谢网络、合理定位次生代谢及其关键酶、促进野生植物资源可持续利用等提供理论依据。     

Natural products – modifying metabolite pathways in plants

The diversity of plant natural product (PNP) molecular structures is reflected in the variety of biochemical and genetic pathways that lead to their formation and accumulation. Plant secondary metabolites are important commodities, and include fragrances, colorants, and medicines. Increasing the extractable amount of PNP through plant breeding, or more recently by means of metabolic engineering, is a priority. The prerequisite for any attempt at metabolic engineering is a detailed knowledge of the underlying biosynthetic and regulatory pathways in plants. Over the past few decades, an enormous body of information about the biochemistry and genetics of biosynthetic pathways involved in PNPs production has been generated. In this review, we focus on the three large classes of plant secondary metabolites: terpenoids (or isoprenoids), phenylpropanoids, and alkaloids. All three provide excellent examples of the tremendous efforts undertaken to boost our understanding of biosynthetic pathways, resulting in the first successes in plant metabolic engineering. We further consider what essential information is still missing, and how future research directions could help achieve the rational design of plants as chemical factories for high-value products.     

Production and engineering of terpenoids in plant cell culture

Terpenoids are a diverse class of natural products that have many functions in the plant kingdom and in human health and nutrition. Their chemical diversity has led to the discovery of over 40,000 different structures, with several classes serving as important pharmaceutical agents, including the anticancer agents paclitaxel (Taxol) and terpenoid-derived indole alkaloids. Many terpenoid compounds are found in low yield from natural sources, so plant cell cultures have been investigated as an alternate production strategy. Metabolic engineering of whole plants and plant cell cultures is an effective tool to both increase terpenoid yield and alter terpenoid distribution for desired properties such as enhanced flavor, fragrance or color. Recent advances in defining terpenoid metabolic pathways, particularly in secondary metabolism, enhanced knowledge concerning regulation of terpenoid accumulation, and application of emerging plant systems biology approaches, have enabled metabolic engineering of terpenoid production. This paper reviews the current state of knowledge of terpenoid metabolism, with a special focus on production of important pharmaceutically active secondary metabolic terpenoids in plant cell cultures. Strategies for defining pathways and uncovering rate-influencing steps in global metabolism, and applying this information for successful terpenoid metabolic engineering, are emphasized.     

Lessons learned from metabolic engineering of cyanogenic glucosides

Plants produce a plethora of secondary metabolites which constitute a wealth of potential pharmaceuticals, pro-vitamins, flavours, fragrances, colorants and toxins as well as a source of natural pesticides. Many of these valuable compounds are only synthesized in exotic plant species or in concentrations too low to facilitate commercialization. In some cases their presence constitutes a health hazard and renders the crops unsuitable for consumption. Metabolic engineering is a powerful tool to alter and ameliorate the secondary metabolite composition of crop plants and gain new desired traits. The interplay of a multitude of biosynthetic pathways and the possibility of metabolic cross-talk combined with an incomplete understanding of the regulation of these pathways, explain why metabolic engineering of plant secondary metabolism is still in its infancy and subject to much trial and error. Cyanogenic glucosides are ancient defense compounds that release toxic HCN upon tissue disruption caused e.g. by chewing insects. The committed steps of the cyanogenic glucoside biosynthetic pathway are encoded by three genes. This unique genetic simplicity and the availability of the corresponding cDNAs have given cyanogenic glucosides pioneering status in metabolic engineering of plant secondary metabolism. In this review, lessons learned from metabolic engineering of cyanogenic glucosides in Arabidopsis thaliana (thale cress), Nicotiana tabacum cv Xanthi (tobacco), Manihot esculenta Crantz (cassava) and Lotus japonicus (bird’s foot trefoil) are presented. The importance of metabolic channelling of toxic intermediates as mediated by metabolon formation in avoiding unintended metabolic cross-talk and unwanted pleiotropic effects is emphasized. Likewise, the potential of metabolic engineering of plant secondary metabolism as a tool to elucidate, for example, the impact of secondary metabolites on plant–insect interactions is demonstrated.     

植物次生代谢基因工程研究进展

随着对植物代谢网络日渐全面的认识,应用基因工程技术对植物次生代谢途径进行遗传改良已取得了可喜的进展.对次生代谢途径进行基因修饰的策略包括:导入单个、多个靶基因或一个完整的代谢途径,使宿主植物合成新的目标物质;通过反义RNA和RNA干涉等技术降低靶基因的表达水平,从而抑制竞争性代谢途径,改变代谢流和增加目标物质的含量;对控制多个生物合成基因的转录因子进行修饰,更有效地调控植物次生代谢以提高特定化合物的积累.作者结合对大豆种子异黄酮类代谢调控和基因工程改良的研究,着重介绍了花青素和黄酮类物质、生物碱、萜类化合物和安息香酸衍生物等次生代谢产物生物合成的基因工程研究进展.     

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.     

植物挥发性物质代谢的遗传修饰

植物在生命周期里能合成释放多种多样的挥发性化合物,这些物质在植物生长发育和代谢调控,植物抵御病虫侵害,以及植物与环境信息交流中行使重要功能。在介绍植物挥发性化合物的生理功能、合成途径调控和商业应用的基础上,重点论述应用基因工程调控植物挥发性物质合成的技术策略和研究进展,并讨论了植物挥发性物质遗传修饰存在的问题和发展前景。     

Production of polyhydroxybutyrate by polycistronic expression of bacterial genes in tobacco plastid

Transgenic techniques are used to enhance and improve crop production, and their application to the production of chemical resources in plants has been under investigation. To achieve this latter goal, multiple-gene transformation is required to improve or change plant metabolic pathways; when accomplished by plant nuclear transformation, however, this procedure is costly and time consuming. We succeeded in the metabolic engineering of the tobacco plant by introducing multiple genes within a bacteria-like operon into a plastid genome. A tobacco plastid was transformed with a polycistron consisting of the spectinomycin resistance gene and three bacterial genes for the biosynthesis of the biodegradable polyester, poly[(R)-3-hydroxybutyrate] (PHB), after modification of their ribosome binding sites. DNA and RNA analysis confirmed the insertion of the introduced genes into the plastid genome and their polycistronic expression. As the result, the transplastomic tobacco accumulated PHB in its leaves. The introduced genes and the PHB productivity were maternally inherited, avoiding genetic spread by pollen diffusion, and were maintained stably in the seed progeny. Despite the low PHB productivity, this report demonstrates the feasibility of transplastomic technology for metabolic engineering. This "phyto-fermentation" system can be applied to plant production of various chemical commodities and pharmaceuticals.     

Production of useful secondary metabolites in plants: Functional genomics approaches

The paradigm of biological research has been changed by recent developments in genomics, high-throughput biology, and bioinformatics. Conventional biology often was based on empirical, labor-intensive, and time-consuming methods. In the new paradigm, biological research e is driven by a holistic approach on the basis of rational, automatic, and high-throughput methods. New functional compounds can be discovered by using high-throughput screening systems. Secondary metabolite pathways and the genes involved in those pathways are then determined by studying functional genomics in conjunction with the data-mining tools of bioinformatics. In addition, these advances in metabolic engineering enable researchers to confer new secondary metabolic pathways to crops by transferring three to five, or more, heterologous genes taken from various other species. In the future, engineering for the production of useful compounds will be designed by a set of software tools that allows the user to specify a cell’s genes, proteins, and other molecules, as well as their individual interactions.     

Plant biotechnology and breeding: allied for years to come

Plant metabolic engineering is lagging behind other kinds of genetic manipulation of plants. Creating metabolic pathways or improving their yields requires a better understanding of plant metabolism and of its regulation. Metabolic Control Analysis provides an interpretation of experimental failures and a guide for manipulators. It suggests also that there might be intrinsic limits to raising yields in already abundant products. At present, these limits can be dealt with more effectively by plant breeding.     

Stacking transgenes in forest trees

Huge potential exists for improving plant raw materials and foodstuffs via metabolic engineering. To date, progress has mostly been limited to modulating the expression of single genes of well-studied pathways, such as the lignin biosynthetic pathway, in model species. However, a recent report illustrates a new level of sophistication in metabolic engineering by overexpressing one lignin enzyme while simultaneously suppressing the expression of another lignin gene in a tree, aspen. This novel approach to multi-gene manipulation has succeeded in concurrently improving several wood-quality traits.     

Metabolic engineering of plants: the role of membrane transport.

As plant cells are highly compartmentalized, the entrance and exit points of metabolic pathways frequently involve membrane passages of solutes. Transport proteins are often located in strategic positions to control whole pathways and have to be considered in the development of metabolic engineering strategies. Here, we discuss examples of pathways (in carbohydrate metabolism, amino acid and secondary compound synthesis, and mineral metabolism) in which membrane transport steps are considered to exert major control and in which transport proteins have been employed to manipulate metabolic fluxes.     

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.