Metabolic Engineering of Plant Lipids

Lipid metabolism in plants provides uncommon opportunities for genetic engineering to produce plant oils suited to a variety of end-uses. These opportunities include improvement of food and nutritional value, creating specialty lipids and feedstocks for high-value products and designing custom-made materials for industry. Genetic engineering intervention for production of novel transgenic plants which elaborate the desired product has graduated from academic exercise to commercial possibilities. It is now realized that transgenic crops can serve as biological factories for upscaling production of premium lipids via molecular farming. This review is an attempt at analyzing the status in this field.     

动物细胞代谢工程进展

动物细胞被越来越广泛地用于工业生产,一些现代化企业已采用分子生物学技术,将一些比较重要的基因导入动物细胞,生产具有医用价值的药物。但该技术并未成熟,主要是因为体外培养的细胞,其生长代谢及生理模式都比较复杂,而且细胞的应答机制还受外界因素的影响。因此,采用细胞代谢工程手段,提高体外培养细胞的生长率、产品产率及有效性,成为人们追求的新目标。我们从细胞代谢中心途径、抑制细胞调亡的因素、细胞生长周期的控制及其相关代谢、多基因共表达代谢工程及糖基化代谢工程等方面对代谢工程进行阐述,为动物细胞的培养提供新的思路。     

Key Applications of Plant Metabolic Engineering

Great strides have been made in plant metabolic engineering over the last two decades, with notable success stories including Golden rice. Here, we discuss the field''s progress in addressing four long-standing challenges: creating plants that satisfy their own nitrogen requirement, so reducing or eliminating the need for nitrogen fertilizer; enhancing the nutrient content of crop plants; engineering biofuel feed stocks that harbor easy-to-access fermentable saccharides by incorporating self-destructing lignin; and increasing photosynthetic efficiency. We also look to the future at emerging areas of research in this field.

This article is part of the PLOS Biology Collection “The Promise of Plant Translational Research.”

     

食用植物油脂的代谢工程

植物种子油可提供人类营养所需的多种脂肪酸,也是工业用油的原料之一。文章结合我们对植物种子发育、脂肪酸生物合成途径和大豆油脂遗传改良的研究,重点论述参与脂肪酸合成及其调控的一些关键酶的基因、代谢工程改良植物油脂营养价值的技术策略及其研究进展,分析目前应用油料作物种子作为“生物反应器”规模化生产有重要营养价值和特殊用途的脂肪酸的问题及技术“瓶颈”,讨论未来植物脂肪酸代谢工程主攻方向以及在培育可再生资源和推动人类社会及经济可持续发展中的应用前景。     

植物挥发性物质及其代谢工程

植物挥发性物质在植物之间和植物与昆虫间的化学通讯中起着重要作用.有关这些次生物质的生物合成、代谢调控、生理功能以及与环境相互作用的研究近十多年来取得了重要进展.迄今为止,已经有3 0多种植物挥发性物质的合成酶基因被克隆.这些基因调控着植物萜类、芳香化合物、脂肪酸衍生物这三大类主要挥发性物质的生物合成.由于潜在的应用价值,近几年该领域颇受注目,特别是应用基因工程技术设计植物释放特殊气味物质,诸如特定的驱避剂或者其它控制植物或昆虫行为的特殊气味乃至与人类健康相关的药用气味物质.该文就植物挥发性物质的生物合成、生理和生态功能以及基因工程方面的研究进展作一概述.     

植物糖基转移酶及其在代谢工程中的应用

糖基转移酶催化植物次生代谢物合成,在植物的生长发育过程以及代谢工程应用方面起着重要作用。该文介绍糖基转移酶的基本特性,总结近年来研究植物糖基转移酶基因的克隆与功能分析的方法,详细阐述了微生物中3个C-糖基转移酶基因（UrdGT2、gilGT和iroA）的克隆和功能研究,概括糖基转移酶在代谢工程的研究进展,并展望今后的研究趋势,为植物C-糖基转移酶的生物学功能研究和代谢应用方面提供有益帮助。     

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.     

代谢转基因植物的研究现状与展望

代谢转基因是通过基因工程技术对细胞内的代谢途径进行遗传修饰,进而完成细胞特性改造。代谢修饰转基因植物是一个极具商业前景的领域,在医药、环境、农业等方面已有许多成功应用的实例。综合调控代谢的基因工程策略,讨论了代谢转基因植物的研究现状,我国农业生产中存在的主要问题和代谢转基因技术对我国农业发展的意义和前景。     

参与植物次生代谢调控的转录因子及其在植物次生代谢遗传改良中的应用

转录因子与结构基因的结合,激活合成基因的表达是次生代谢物合成途径启动前的重要分子事件,对植物次生代谢起着十分重要的调节作用。转录因了可激活次牛代谢物合成途径中多个基因协同表达,从而有效启动次生代谢途径。因此,转录因子为揭示植物次生代谢调控机制提供重要工具,转录因子的基因工程可为植物次生代谢的遗传改良提供有效的手段。     

植物代谢工程在活性天然产物制备中的应用

本文分析了目前应用植物代谢工程在提高活性天然产物产量方面的研究成果,讨论了植物代谢工程的应用前景。     

Metabolic Engineering Plant Seeds with Fish Oil-Like Levels of DHA

Background

Omega-3 long-chain (≥C₂₀) polyunsaturated fatty acids (ω3 LC-PUFA) have critical roles in human health and development with studies indicating that deficiencies in these fatty acids can increase the risk or severity of cardiovascular and inflammatory diseases in particular. These fatty acids are predominantly sourced from fish and algal oils, but it is widely recognised that there is an urgent need for an alternative and sustainable source of EPA and DHA. Since the earliest demonstrations of ω3 LC-PUFA engineering there has been good progress in engineering the C₂₀ EPA with seed fatty acid levels similar to that observed in bulk fish oil (∼18%), although undesirable ω6 PUFA levels have also remained high.

Methodology/Principal Findings

The transgenic seed production of the particularly important C₂₂ DHA has been problematic with many attempts resulting in the accumulation of EPA/DPA, but only a few percent of DHA. This study describes the production of up to 15% of the C₂₂ fatty acid DHA in Arabidopsis thaliana seed oil with a high ω3/ω6 ratio. This was achieved using a transgenic pathway to increase the C₁₈ ALA which was then converted to DHA by a microalgal Δ6-desaturase pathway.

Conclusions/Significance

The amount of DHA described in this study exceeds the 12% level at which DHA is generally found in bulk fish oil. This is a breakthrough in the development of sustainable alternative sources of DHA as this technology should be applicable in oilseed crops. One hectare of a Brassica napus crop containing 12% DHA in seed oil would produce as much DHA as approximately 10,000 fish.     

Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1-deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently.With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis path way are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosnthesis are discussed.     

Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1-deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently. With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis pathway are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosynthesis are discussed.     

植物挥发物代谢工程在改良香气品质和植物防御中的应用

挥发物次生代谢在植物繁殖、植物防御和改良食物品质方面发挥着重要作用。近年来,随着参与挥发物生物合成的基因和酶类的鉴定以及代谢途径和调控机理等研究的不断发展和深入,挥发物代谢工程已经具备较高的可行性。应用代谢工程改良花、果实的香气品质以及提高植物防御能力的研究成效显著。主要介绍了这些方面的最新进展,同时也讨论了植物挥发物代谢工程应用存在的问题和挑战以及研究思路。     

代谢工程

代谢工程,也称途径工程,是基因工程一个重要分支,一般是多基因的基因工程,与细胞的基因调控、代谢调控和生化工程密切相关。讨论了代谢工程的应用,包括通过改变代谢流和代谢途径提高产量,改善生产过程,构建新的代谢途径和产生新的代谢产物等。     

Metabolic Engineering of Plant L-Ascorbic Acid Biosynthesis: Recent Trends and Applications

ABSTRACT

Vitamin C (L-ascorbic acid; AsA) is the major soluble antioxidant found in plants and is also an essential component of human nutrition. Although numerous biotechnological methods have been exploited to increase its yield, pressures such as commercial competition and environmental concerns make it urgent to find a new way for industrial production of plant-derived AsA. Engineering plant AsA has now become feasible because of our increased understanding of its biosynthetic pathway. Several possible strategies could be followed to increase AsA production, such as overcoming the rate limiting steps in the biosynthetic pathway, promoting recycling, and reducing catabolism. For these purposes, genes of plant, microbial and animal origins have been successfully used. Several examples will be given to illustrate these various approaches. The existing and potential achievements in increasing AsA production would provide the opportunity for enhancing nutritional quality and stress tolerance of crop plants.     

Metabolic Engineering of Escherichia coli for the Synthesis of the Plant Polyphenol Pinosylvin

Plant polyphenols are of great interest for drug discovery and drug development since many of these compounds have health-promoting activities as treatments against various diseases, such as diabetes, cancer, or heart diseases. However, the limited availability of polyphenols represents a major obstacle to clinical applications that must be overcome. In comparison to the quantities of these compounds obtained by isolation from natural sources or costly chemical synthesis, the microbial production of these compounds could provide sufficient quantities from inexpensive substrates. In this work, we describe the development of an Escherichia coli platform strain for the production of pinosylvin, a stilbene found in the heartwood of pine trees which could aid in the treatment of various cancers and cardiovascular diseases. Initially, several configurations of the three-step biosynthetic pathway to pinosylvin were constructed from a set of two different enzymes for each enzymatic step. After optimization of gene expression and evaluation of different construct environments, low pinosylvin concentrations up to 3 mg/liter could be detected. Analysis of the precursor supply and a comparative analysis of the intracellular pools of pathway intermediates and product identified the limited malonyl coenzyme A (malonyl-CoA) availability and low stilbene synthase activity in the heterologous host to be the main bottlenecks during pinosylvin production. Addition of cerulenin for increasing intracellular malonyl-CoA pools and the in vivo evolution of the stilbene synthase from Pinus strobus for improved activity in E. coli proved to be the keys to elevated product titers. These measures allowed product titers of 70 mg/liter pinosylvin from glucose, which could be further increased to 91 mg/liter by the addition of l-phenylalanine.     

Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertès, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.     

Systems Metabolic Engineering

Cover illustration Special Issue: Systems Metabolic Engineering. Metabolic engineering combines a mix of approaches, including in silico modeling, omics studies, synthetic biology and protein engineering to improve microorganism strains for increased yields and reduced production costs of desirable chemicals. Such an achievement is exemplified on this Special Issue's cover, which shows an electron microscopy image of Corynebacterium glutamicum that has been engineered to produce a sustainable bio-nylon monomer from hemicellulose sugar found in the cell walls of plants. Image provided by Buschke et al.     

Accumulation of Rare Phytosterols in Plant Cells on Treatment with Metabolic Inhibitors and Mevalonic Acid

Root application of the metabolic inhibitors D-ethionine (100µM) and L-ethionine (30 µM), which were expectedto inhibit alkylation of the sterol side chains, merely reducedthe sterol content in the roots of Medicago sativa seedlings.The major sterol was stigmasterol. However, when (3RS)-mevalonicacid (2 mM) was applied together with ethionine, cydoartenol(about 50% of the total sterols) accumulated in the roots. Thehypocotyls of the ethionine-treated seedlings accumulated cholesterol(34% of the total sterols), and mevalonic acid showed no additionaleffect in this case. In a suspension culture of Nicotiana tabacum, the most abundantsterol was campesterol. When cells were treated with buthiobate(100 µM), a potent inhibitor of lanosterol 14-demethylationin yeasts and fungi, obtusifoliol accumulated in the treatedcells (50% of the total sterols). 14-Methylfecosterol also accumulatedin the treated cells. The addition of mevalonic acid (1 mM togetherwith buthiobate increased the obtusifoliol content (63% of thetotal sterols). (Received October 16, 1986; Accepted April 2, 1987)