Biosynthesis of gibberellins in Gibberella fujikuroi: biomolecular aspects

Gibberellins (GAs) are a large family of isoprenoid plant hormones, some of which are bioactive growth regulators, controlling seed germination, stem elongation, and flowering. The rice pathogen Gibberella fujikuroi (mating population C) is able to produce large amounts of GAs, especially the bioactive compounds gibberellic acid (GA₃) and its precursors, GA₄ and GA₇. The main steps of the biosynthetic pathway have long been established from the identification of intermediates in wild-type G. fujikuroi and mutant strains. However, the genetics of the fungus have been rather under-developed, and molecular genetic studies of the GA pathway started just recently. The progress in researching GA biosynthesis in the last 2 years resulted primarily from development of the molecular tools, e.g. transformation systems for the fungus, and cloning the genes encoding GA biosynthesis enzymes, such as the bifunctional ent-copalyl diphosphate/kaurene synthase and several cytochrome P450 monooxygenases. The availability of these genes opened new horizons both for detailed study of the pathway and the regulation mechanisms at the molecular level, and for modern strain improvement programs. This review gives a short overview of the well-known physiological and biochemical studies and concentrates mainly on the new molecular genetic data from GA research, including new information on the regulation of GA biosynthesis. Received: 15 February 1999 / Received revision: 16 April 1999 / Accepted: 16 April 1999     

Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology

Gibberellins (GAs) constitute a large family of tetracyclic diterpenoid carboxylic acids, some members of which function as growth hormones in higher plants. As well as being phytohormones, GAs are also present in some fungi and bacteria. In recent years, GA biosynthetic genes from Fusarium fujikuroi and Arabidopsis thaliana have been cloned and well characterised. Although higher plants and the fungus both produce structurally identical GAs, there are important differences indicating that GA biosynthetic pathways have evolved independently in higher plants and fungi. The fact that horizontal gene transfer of GA genes from the plant to the fungus can be excluded, and that GA genes are obviously missing in closely related Fusarium species, raises the question of the origin of fungal GA biosynthetic genes. Besides characterisation of F. fujikuroi GA pathway genes, much progress has been made in the molecular analysis of regulatory mechanisms, especially the nitrogen metabolite repression controlling fungal GA biosynthesis. Basic research in this field has been shown to have an impact on biotechnology. Cloning of genes, construction of knock-out mutants, gene amplification, and regulation studies at the molecular level are powerful tools for improvement of production strains. Besides increased yields of the final product, GA₃, it is now possible to produce intermediates of the GA biosynthetic pathway, such as ent-kaurene, ent-kaurenoic acid, and GA₁₄, in high amounts using different knock-out mutants. This review concentrates mainly on the fungal biosynthetic pathway, the genes and enzymes involved, the regulation network, the biotechnological relevance of recent studies, and on evolutionary aspects of GA biosynthetic genes.     

Biosynthesis of phloroglucinol compounds in microorganisms—review

Phloroglucinol derivatives are a major class of secondary metabolites of wide occurrence in biological systems. In the bacteria kingdom, these compounds can only be synthesized by some species of Pseudomonads. Pseudomonas spp. could produce 2,4-diacetylphloroglucinol (DAPG) that plays an important role in the biological control of many plant pathogens. In this review, we summarize knowledge about synthesis of phloroglucinol compounds based on the DAPG biosynthetic pathway. Recent advances that have been made in understanding phloroglucinol compound biosynthesis and regulation are highlighted. From these studies, researchers have identified the biosynthesis pathway of DAPG. Most of the genes involved in the biosynthesis pathway have been cloned and characterized. Additionally, heterologous systems of the model microorganism Escherichia coli are constructed to produce phloroglucinol. Although further work is still required, a full understanding of phloroglucinol compound biosynthesis is almost within reach. This review also suggests new directions and attempts to gain some insights for better understanding of the biosynthesis and regulation of DAPG. The combination of traditional biochemistry and molecular biology with new systems biology and synthetic biology tools will provide a better view of phloroglucinol compound biosynthesis and a greater potential of microbial production.     

高等植物赤霉素2-氧化酶基因的克隆、表达及其调控

赤霉素(GA)是一类重要的植物激素,对高等植物整个生命周期的生长发育起关键作用。调控赤霉素生物合成和代谢途径中的关键酶基因的表达可以控制植物体内赤霉素的含量。GA2-氧化酶是调节赤霉素合成和代谢的关键酶之一,使活性GA失活。本文主要对GA2-氧化酶基因的克隆、表达调控及其在植物基因工程中的应用等方面进行综述,为通过基因工程技术调控植物体内活性赤霉素的含量从而得到改良品种提供思路。     

Diversity,regulation, and evolution of the gibberellin biosynthetic pathway in fungi compared to plants and bacteria

Bioactive gibberellins (GAs) are diterpene plant hormones that are biosynthesized through complex pathways and control diverse aspects of growth and development. GAs were first isolated as metabolites of a fungal rice pathogen, Gibberella fujikuroi, since renamed Fusarium fujikuroi. Although higher plants and the fungus produce structurally identical GAs, significant differences in their GA pathways, enzymes involved and gene regulation became apparent with the identification of GA biosynthetic genes in Arabidopsis thaliana and F. fujikuroi. Recent identifications of GA biosynthetic gene clusters in two other fungi, Phaeosphaeria spp. and Sphaceloma manihoticola, and the high conservation of GA cluster organization in these distantly related fungal species indicate that fungi evolved GA and other diterpene biosynthetic pathways independently from plants. Furthermore, the occurrence of GAs and recent identification of the first GA biosynthetic genes in the bacterium Bradyrhizobium japonicum make it possible to study evolution of GA pathways in general.In this review, we summarize our current understanding of the GA biosynthesis pathway, specifically the genes and enzymes involved as well as gene regulation and localization in the genomes of different fungi and compare it with that in higher and lower plants and bacteria.     

Catharanthus terpenoid indole alkaloids: biosynthesis and regulation

Catharanthus roseus is still the only source for the powerful antitumour drugs vinblastine and vincristine. Some other pharmaceutical compounds from this plant, ajmalicine and serpentine are also of economical importance. Although C. roseus has been studied extensively and was subject of numerous publications, a full characterization of its alkaloid pathway is not yet achieved. Here we review some of the recent work done on this plant. Most of the work focussed on early steps of the pathway, particularly the discovery of the 2-C-methyl-d-erythritol 4-phosphate (MEP)-pathway leading to terpenoids. Both mevalonate and MEP pathways are utilized by plants with apparent cross-talk between them across different compartments. Many genes of the early steps in Catharanthus alkaloid pathway have been cloned and overexpressed to improve the biosynthesis. Research on the late steps in the pathway resulted in cloning of several genes. Enzymes and genes involved in indole alkaloid biosynthesis and various aspects of their localization and regulation are discussed. Much progress has been made at alkaloid regulatory level. Feeding precursors, growth regulators treatments and metabolic engineering are good tools to increase productivity of terpenoid indole alkaloids. But still our knowledge of the late steps in the Catharanthus alkaloid pathway and the genes involved is limited.     

Gibberellin biosynthesis and the regulation of plant development

Gibberellins (GAs) form a large family of plant growth substances with distinct functions during the whole life cycle of higher plants. The rate of GA biosynthesis and catabolism determines how the GA hormone pool occurs in plants in a tissue and developmentally regulated manner. With the availability of genes coding for GA biosynthetic enzymes, our understanding has improved dramatically of how GA plant hormones regulate and integrate a wide range of growth and developmental processes. This review focuses on two plant systems, pumpkin and Arabidopsis, which have added significantly to our understanding of GA biosynthesis and its regulation. In addition, we present models for regulation of GA biosynthesis in transgenic plants, and discuss their suitability for altering plant growth and development.     

A Novel Approach to Enhancing Ganoderic Acid Production by Ganoderma lucidum Using Apoptosis Induction

Ganoderma lucidum is one of most widely used herbal medicine and functional food in Asia, and ganoderic acids (GAs) are its active ingredients. Regulation of GA biosynthesis and enhancing GA production are critical to using G. lucidum as a medicine. However, regulation of GA biosynthesis by various signaling remains poorly understood. This study investigated the role of apoptosis signaling on GA biosynthesis and presented a novel approach, namely apoptosis induction, to increasing GA production. Aspirin was able to induce cell apoptosis in G. lucidum, which was identified by terminal deoxynucleotidyl transferase mediated dUPT nick end labeling assay positive staining and a condensed nuclear morphology. The maximum induction of lanosta-7,9(11), 24-trien-3α-01-26-oic acid (ganoderic acid 24, GA24) production and total GA production by aspirin were 2.7-fold and 2.8-fold, respectively, after 1 day. Significantly lower levels of GA 24 and total GAs were obtained after regular fungal culture for 1.5 months. ROS accumulation and phosphorylation of Hog-1 kinase, a putative homolog of MAPK p38 in mammals, occurred after aspirin treatment indicating that both factors may be involved in GA biosynthetic regulation. However, aspirin also reduced expression of the squalene synthase and lanosterol synthase coding genes, suggesting that these genes are not critical for GA induction. To the best of our knowledge, this is the first report showing that GA biosynthesis is linked to fungal apoptosis and provides a new approach to enhancing secondary metabolite production in fungi.     

Phospholipid biosynthesis in mammalian cells.

Identification of the genes and gene products involved in the biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine has lagged behind that in many other fields because of difficulties encountered in purifying the respective proteins. Nevertheless, most of these genes have now been identified. In this review article, we have highlighted important new findings on the individual enzymes and the corresponding genes of phosphatidylcholine synthesis via its two major biosynthetic pathways: the CDP-choline pathway and the methylation pathway. We also review recent studies on phosphatidylethanolamine biosynthesis by two pathways: the CDP-ethanolamine pathway, which is active in the endoplasmic reticulum, and the phosphatidylserine decarboxylase pathway, which operates in mitochondria. Finally, the two base-exchange enzymes, phosphatidylserine synthase-1 and phosphatidylserine synthase-2, that synthesize phosphatidylserine in mammalian cells are also discussed.     

Transformation ofAspergillus flavus to study aflatoxin biosynthesis

Aflatoxin contamination of agricultural commodities continues to be a serious problem in the United States. Breeding for resistant genotypes has been unsuccessful and detoxification of food sources is not economically feasible. New strategies for control may become apparent once more is known about the biosynthesis and regulation of aflatoxin. Although the biosynthetic pathway of aflatoxin has been extensively studied, little is known about the regulation of the individual steps in the pathway. We have developed a genetic transformation system forAspergillus flavus that provides a new and expedient approach to studying the biosynthesis of aflatoxin and its regulation. Through the use of this genetic transformation system, genes for aflatoxin biosynthesis can be identified and isolated by the complementation of aflatoxin negative mutants. In this paper we discuss molecular strategies for studying the regulation and biosynthesis of aflatoxin.     

Role of gibberellins during arbuscular mycorrhizal formation in tomato: new insights revealed by endogenous quantification and genetic analysis of their metabolism in mycorrhizal roots

Gibberellins (GAs) are key regulators of plant growth and development and recent studies suggest also a role during arbuscular mycorrhizal (AM) formation. Here, complementary approaches have been used to obtain a clearer picture that correlates AM fungal development inside roots with GA metabolism. An extensive analysis of genes associated with GA metabolism as well as a quantification of GA content in roots was made. Application of GA₃ and its biosynthesis inhibitor prohexadione calcium (PrCa) combined with a GA‐constitutive response mutant (procera) were used to determine whether fungal colonization is altered by the level of these hormones or by changes in the GA‐signaling pathway. The increased levels of specific GAs from the 13‐hydroxylation pathway in mycorrhizal roots correlate closely with the increased expression of genes coding enzymes from the GA biosynthetic trail. The imbalance of GAs in tomato roots caused by exogenous applications of GA₃ or PrCa affects arbuscules in both negative and positive ways, respectively. In addition, procera plants were adversely affected by the mycorrhization process. Our findings demonstrate that an imbalance in favor of an increased amount of GAs negatively affects the frequency of mycorrhization and particularly the arbuscular abundance in tomato mycorrhizal roots and the results point out that AM formation is associated with a change in the 13‐hydroxylation pathway of GAs.     

植物类胡萝卜素生物合成及其相关基因在基因工程中的应用

近年来类胡萝卜素生物合成基因的分离与功能鉴定,为应用基因工程技术改变植物体内类胡萝卜素成份和提高类胡萝卜素含量提供了新的基因资源.有关类胡萝卜素合成的生物化学及其在体内调控研究的新进展,使通过遗传操作调控植物体内类胡萝卜素生物合成途径成为可能.该文综述了类胡萝卜素生物合成途径及其相关基因的研究现状,并结合作者的工作介绍了应用转基因技术改变植物体内类胡萝卜素成份与含量的最新成功的事例.     

植物赤霉素生物合成和信号传导的分子生物学

赤霉素 (GAs)在植物的种子萌发、茎的伸长和花的发育等许多方面起着非常重要的作用。最近几年 ,对GA生物合成及其信号传导途径相关基因的研究取得了惊人的进展。这些进展促进了对其生物合成及其信号传导途径的认识。GA生物合成相关基因的表达受到多种内源和外源因子的调控 ,其中研究较多的是发育阶段、激素水平和光信号等内源及环境因子的调控。GA信号传导通常处于抑制状态 ,GA信号通过去抑制作用激活该传导途径而促进GA刺激植物生长和发育。     

Dissection of patulin biosynthesis,spatial control and regulation mechanism in Penicillium expansum

The patulin biosynthesis is one of model pathways in an understanding of secondary metabolite biology and network novelties in fungi. However, molecular regulation mechanism of patulin biosynthesis and contribution of each gene related to the different catalytic enzymes in the biochemical steps of the pathway remain largely unknown in fungi. In this study, the genetic components of patulin biosynthetic pathway were systematically dissected in Penicillium expansum, which is an important fungal pathogen and patulin producer in harvested fruits and vegetables. Our results revealed that all the 15 genes in the cluster are involved in patulin biosynthesis. Proteins encoded by those genes are compartmentalized in various subcellular locations, including cytosol, nucleus, vacuole, endoplasmic reticulum, plasma membrane and cell wall. The subcellular localizations of some proteins, such as PatE and PatH, are required for the patulin production. Further, the functions of eight enzymes in the 10-step patulin biosynthetic pathway were verified in P. expansum. Moreover, velvet family proteins, VeA, VelB and VelC, were proved to be involved in the regulation of patulin biosynthesis, but not VosA. These findings provide a thorough understanding of the biosynthesis pathway, spatial control and regulation mechanism of patulin in fungi.