Metabolic engineering of plant secondary metabolite pathways for the production of fine chemicals

The technology of large-scale plant cell culture is feasible for the industrial production of plant-derived fine chemicals. Due to low or no productivity of the desired compounds the economy is only in a few cases favorable. Various approaches are studied to increase yields, these encompass screening and selection of high producing cell lines, media optimization, elicitation, culturing of differentiated cells (organ cultures), immobilization. In recent years metabolic engineering has opened a new promising perspectives for improved production in a plant or plant cell culture.     

Metabolic engineering for the production of plant isoquinoline alkaloids

Several plant isoquinoline alkaloids (PIAs) possess powerful pharmaceutical and biotechnological properties. Thus, PIA metabolism and its fascinating molecules, including morphine, colchicine and galanthamine, have attracted the attention of both the industry and researchers involved in plant science, biochemistry, chemical bioengineering and medicine. Currently, access and availability of high‐value PIAs [commercialized (e.g. galanthamine) or not (e.g. narciclasine)] is limited by low concentration in nature, lack of cultivation or geographic access, seasonal production and risk of overharvesting wild plant species. Nevertheless, most commercial PIAs are still extracted from plant sources. Efforts to improve the production of PIA have largely been impaired by the lack of knowledge on PIA metabolism. With the development and integration of next‐generation sequencing technologies, high‐throughput proteomics and metabolomics analyses and bioinformatics, systems biology was used to unravel metabolic pathways allowing the use of metabolic engineering and synthetic biology approaches to increase production of valuable PIAs. Metabolic engineering provides opportunity to overcome issues related to restricted availability, diversification and productivity of plant alkaloids. Engineered plant, plant cells and microbial cell cultures can act as biofactories by offering their metabolic machinery for the purpose of optimizing the conditions and increasing the productivity of a specific alkaloid. In this article, is presented an update on the production of PIA in engineered plant, plant cell cultures and heterologous micro‐organisms.     

Engineering the plant cell factory for secondary metabolite production

Plant secondary metabolism is very important for traits such as flower color, flavor of food, and resistance against pests and diseases. Moreover, it is the source of many fine chemicals such as drugs, dyes, flavors, and fragrances. It is thus of interest to be able to engineer the secondary metabolite production of the plant cell factory, e.g. to produce more of a fine chemical, to produce less of a toxic compound, or even to make new compounds, Engineering of plant secondary metabolism is feasible nowadays, but it requires knowledge of the biosynthetic pathways involved. To increase secondary metabolite production different strategies can be followed, such as overcoming rate limiting steps, reducing flux through competitive pathways, reducing catabolism and overexpression of regulatory genes. For this purpose genes of plant origin can be overexpressed, but also microbial genes have been used successfully. Overexpression of plant genes in microorganisms is another approach, which might be of interest for bioconversion of readily available precursors into valuable fine chemicals. Several examples will be given to illustrate these various approaches. The constraints of metabolic engineering of the plant cell factory will also be discussed. Our limited knowledge of secondary metabolite pathways and the genes involved is one of the main bottlenecks. This revised version was published online in August 2006 with corrections to the Cover Date.     

植物代谢工程的研究现状

植物代谢工程是一个很有发展前景的新兴学科。它可通过多种方法对植物的代谢流进行改造,如加速限制步骤的反应,改变分叉代谢途径的流向,构建代谢旁路,引入转录调节因子、信号因子、植物激素合成基因,扩展和构建新的代谢途径等方法进行。并取得了一些有意义的研究结果。     

植物类胡萝卜素代谢工程与应用

类胡萝卜素是人类所需要的重要营养成分之一,不仅具有抗氧化、预防肿瘤和心血管等疾病的作用,而且还是人体合成维生素A的前体。全球大约有280万～330万学龄前儿童出现维生素缺乏（vitaminAdeficiency,VAD）的临床症状;近2亿儿童处于半缺乏状态。通过对植物类胡萝卜素生物合成途径的解析,以及对参与这一代谢过程的酶及其调控机制的深入了解,目前已经可以通过基因工程在主要农作物中组织特异性地促进类胡萝卜素的合成与积累。从理论上已经可以利用转基因植物来减少VAD的出现。该文简要回顾近年来这一领域的研究进展。     

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。     

Metabolic engineering and mechanical investigation of enhanced plant autoluminescence

The fungal bioluminescence pathway (FBP) was identified from glowing fungi, which releases self-sustained visible green luminescence. However, weak bioluminescence limits the potential application of the bioluminescence system. Here, we screened and characterized a C3′H1 (4-coumaroyl shikimate/quinate 3′-hydroxylase) gene from Brassica napus, which efficiently converts p-coumaroyl shikimate to caffeic acid and hispidin. Simultaneous expression of BnC3′H1 and NPGA (null-pigment mutant in A. nidulans) produces more caffeic acid and hispidin as the natural precursor of luciferin and significantly intensifies the original fungal bioluminescence pathway (oFBP). Thus, we successfully created enhanced FBP (eFBP) plants emitting 3 × 10¹¹ photons/min/cm², sufficient to illuminate its surroundings and visualize words clearly in the dark. The glowing plants provide sustainable and bio-renewable illumination for the naked eyes, and manifest distinct responses to diverse environmental conditions via caffeic acid biosynthesis pathway. Importantly, we revealed that the biosynthesis of caffeic acid and hispidin in eFBP plants derived from the sugar pathway, and the inhibitors of the energy production system significantly reduced the luminescence signal rapidly from eFBP plants, suggesting that the FBP system coupled with the luciferin metabolic flux functions in an energy-driven way. These findings lay the groundwork for genetically creating stronger eFBP plants and developing more powerful biological tools with the FBP system.     

Glycosyltransferases: key players involved in the modification of plant secondary metabolites

Glycosyltransferases are members of the multigene superfamily in plants that can transfer single or multiple activated sugars to a range of plant molecules, resulting in the glycosylation of plant compounds. Although the activities of many glycosyltransferases and their products have been recognized for a long time, only in recent years were some glycosyltransferase genes identified and a few functionally characterized in detail. Glycosylation is thought to be one of the most important modification reactions towards plant secondary metabolites, and plays a key role in maintaining cell homeostasis, thus likely participating in the regulation of plant growth, development and in defense responses to stress environments. With advances in plant genome projects and the development of novel technologies in analyzing gene function, significant progress could be made in gaining new insights into the properties and precise biological roles of plant secondary product glycosyltransferases, and the new knowledge will have extensive application prospects in the catalytic synthesis of glycoconjugates and metabolic engineering of crops. In this review, we summarize the current research, highlighting the possible biological roles, of plant secondary metabolite glycosyltransferases and discuss their potential applications as well as aspects to be further studied in the near future.     

Glycosyltransferases:key players involved in the modification of plant secondary metabolites

Glycosyltransferases are members of the multigene superfamily in plants that can transfer single or multiple activated sugars to a range of plant molecules,resulting in the glycosylation of plant compounds.Although the activities of many glycosyltransferases and their products have been recognized for a long time,only in recent years were some glycosyltransferase genes identified and a few functionally characterized in detail.Glycosylation is thought to be one of the most important modification reactions towards plant secondary metabolites,and plays a key role in maintaining cell homeostasis,thus likely participating in the regulation of plant growth,development and in defense responses to stress environments.With advances in plant genome projects and the development of novel technologies in analyzing gene function,significant progress could be made in gaining new insights into the properties and precise biological roles of plant secondary product glycosyltransferases,and the new knowledge will have extensive application prospects in the catalytic synthesis of glycoconjugates and metabolic engineering of crops.In this review,we summarize the current research,highlighting the possible biological roles,of plant secondary metabolite glycosyltransferases and discuss their potential applications as well as aspects to be further studied in the near future.     

L-苯丙氨酸生产的代谢工程研究

L-苯丙氨酸是一种重要的食品和医药中间体。工业上一般采用酶法和发酵法来生产L-苯丙氨酸。代谢工程的兴起,使得更加理性的改造菌株成为可能,这更加促进了发酵法的广泛应用。主要介绍了代谢工程在L-苯丙氨酸生产菌的改造中的应用情况,其中涉及苯丙氨酸生物合成途径中相关基因及其酶的调控、中央代谢途径的改造和芳香族氨基酸生物合成支路的修饰。并探讨了将来的发展前景。     

细胞色素P450与植物的次生代谢

细胞色素Ｐ４５０是动植物及微生物体内的一类重要的混合功能的血红素氧化还原酶类，是一个基因超家族。它可催化多种化学反应，在防御生物免受外界侵害方面有重要作用。植物Ｐ４５０广泛参与次生代谢产生有植保素功能的物质和对除草剂、杀虫剂等外毒素的生物转化解毒代谢。许多植物Ｐ４５０已被分离纯化，更多的植物Ｐ４５０的基因被克隆和外源表达。对植物Ｐ４５０的表达调控也取得了一些进展，但在抗虫和抗除草剂的农作物基因工程方面尚在起步阶段。     

Metabolic engineering of plants for artemisinin synthesis

Artemisinin, a natural compound from Artemisia annua, is highly effective in treating drug-resistant malaria. Because chemical synthesis of this natural terpenoid is not economically feasible, its only source remains as the native plant which produces only small quantities of it, resulting in a supply that is far short of demand. Extensive efforts have been invested in metabolic engineering for the biosynthesis of artemisinin precursors in microbes. However, the production of artemisinin itself has only been achieved in plants. Since, A. annua possesses only poorly developed genetic resources for traditional breeders, molecular breeding is the best alternative. In this review, we describe the efforts taken to enhance artemisinin production in A. annua via transgenesis and advocate metabolic engineering of the complete functional artemisinin metabolic pathway in heterologous plants. In both cases, we emphasize the need to apply state-of-the-art synthetic biology approaches to ensure successful biosynthesis of the drug.     

代谢工程：一项不断发展的菌株改造技术

对代谢工程的发展进行了简要回顾,分析了代谢工程发展的推动力,重点评述了本期专栏发表的12篇代谢工程和细胞工厂方面的论文。     

Metabolic engineering of Torulopsis glabrata for malate production

The yeast Torulopsis glabrata CCTCC M202019, which is used for industrial pyruvate production, was chosen to explore the suitability of engineering this multi-vitamin auxotrophic yeast for increased malate production. Various metabolic engineering strategies were used to manipulate carbon flux from pyruvate to malate: (i) overexpression of pyruvate carboxylase and malate dehydrogenase; (ii) identification of the bottleneck in malate production by model iNX804; (iii) simultaneous overexpression of genes RoPYC, RoMDH and SpMAE1. Using these strategies, 8.5 g L^–1 malate was accumulated in the engineered strain T.G-PMS, which was about 10-fold greater than that of the control strain T.G-26. The results presented here suggest that T. glabrata CCTCC M202019 is a promising candidate for industrial malate production.     

代谢工程在微生物法生产番茄红素中的应用

番茄红素作为强抗氧化剂因具有防癌与抗癌等多种生理功能而广受关注。本文综述了代谢工程在微生物法生产番茄红素中的应用,主要讨论了代谢工程方法在扩展构建新的代谢流、增强番茄红素合成代谢流,阻断竞争支路来提高番茄红素代谢流等方面的应用。     

代谢工程与重组大肠杆菌的发酵

利用代谢工程可以在重组大肠杆菌的改良中减少代谢副产物乙酸的累积,优化代谢系统,利于重组蛋白质的高表达以及重组菌的高密度发酵。应用代谢工程改良重组大肠杆菌主要包括阻断乙酸产生的主要途径、限制糖酵解途径上的碳代谢流、将过量的丙酮酸转化为其它低毒的副产物以及对碳代谢流进行分流等几个方面的工作。     

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.     

昆虫对植物次生物质的代谢适应机制及其对昆虫抗药性的意义

植物次生物质(plant secondary metabolites)对昆虫的取食行为、生长发育及繁殖可以产生不利影响,甚至对昆虫可以产生毒杀作用。为了应对植物次生物质的不利影响,昆虫通过对植物次生物质忌避取食、解毒代谢等多种机制,而对寄主植物产生适应性。其中,昆虫的解毒代谢酶包括昆虫细胞色素P450酶系（P450s）及谷胱甘肽硫转移酶（GSTs）等,在昆虫对植物次生物质的解毒代谢及对寄主植物的适应性中发挥了重要作用。昆虫的解毒酶系统不仅可以代谢植物次生物质,还可能代谢化学杀虫剂,因而昆虫对寄主植物的适应性与其对杀虫剂的耐药性甚至抗药性密切相关。昆虫细胞色素P450s和GSTs等代谢解毒酶活性及相关基因的表达可以被植物次生物质影响,这不仅使昆虫对寄主植物的防御产生了适应性,还影响了昆虫对杀虫剂的解毒代谢,因而改变昆虫的耐药性或抗药性。掌握昆虫对植物次生物质的代谢适应机制及其在昆虫抗药性中的作用,对于明确昆虫的抗药性机制具有重要的参考意义。本文综述了植物次生物质对昆虫的影响、昆虫对寄主植物次生物质的代谢机制、昆虫对植物次生物质的代谢适应性对昆虫耐药性及抗药性的影响等方面的研究进展。     

Metabolic engineering strategies for butanol production in Escherichia coli

The global market of butanol is increasing due to its growing applications as solvent, flavoring agent, and chemical precursor of several other compounds. Recently, the superior properties of n-butanol as a biofuel over ethanol have stimulated even more interest. (Bio)butanol is natively produced together with ethanol and acetone by Clostridium species through acetone-butanol-ethanol fermentation, at noncompetitive, low titers compared to petrochemical production. Different butanol production pathways have been expressed in Escherichia coli, a more accessible host compared to Clostridium species, to improve butanol titers and rates. The bioproduction of butanol is here reviewed from a historical and theoretical perspective. All tested rational metabolic engineering strategies in E. coli to increase butanol titers are reviewed: manipulation of central carbon metabolism, elimination of competing pathways, cofactor balancing, development of new pathways, expression of homologous enzymes, consumption of different substrates, and molecular biology strategies. The progress in the field of metabolic modeling and pathway generation algorithms and their potential application to butanol production are also summarized here. The main goals are to gather all the strategies, evaluate the respective progress obtained, identify, and exploit the outstanding challenges.