Carotenoid metabolism: biosynthesis, regulation, and beyond

Carotenoids are Indispensable to plants and play a critical role in human nutrition and health. Significant progress has been made in our understanding of carotenoid metabolism in plants. The biosynthetic pathway has been extensively studied.Nearly all the genes encoding the biosynthetic enzymes have been isolated and characterized from various organisms. In recent years, there is an increasing body of work on the signaling pathways and plastid development, which might provide global control of carotenoid biosynthesis and accumulation. Herein, we will highlight recent progress on the biosynthesis,regulation, and metabolic engineering of carotenoids in plants, as well as the future research towards elucidating the regulatory mechanisms and metabolic network that control carotenoid metabolism.     

Carotenoid biotechnology in plants for nutritionally improved foods

Carotenoids participate in light harvesting and are essential for photoprotection in photosynthetic plant tissues. They also furnish non-photosynthetic flowers and fruits with yellow to red colors to attract animals for pollination and dispersal of seeds. Although animals can not synthesize carotenoids de novo , carotenoid-derived products such as retinoids (including vitamin A) are required as visual pigments and signaling molecules. Dietary carotenoids also provide health benefits based on their antioxidant properties. The main pathway for carotenoid biosynthesis in plants and microorganisms has been virtually elucidated in recent years, and some of the identified biosynthetic genes have been successfully used in metabolic engineering approaches to overproduce carotenoids of interest in plants. Alternative approaches that enhance the metabolic flux to carotenoids by upregulating the production of their isoprenoid precursors or interfere with light-mediated regulation of carotenogenesis have been recently shown to result in increased carotenoid levels. Despite spectacular achievements in the metabolic engineering of plant carotenogenesis, much work is still ahead to better understand the regulation of carotenoid biosynthesis and accumulation in plant cells. New genetic and genomic approaches are now in progress to identify regulatory factors that might significantly contribute to improve the nutritional value of plant-derived foods by increasing their carotenoid levels.     

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.     

Cloning and nucleotide sequence of the crtI gene encoding phytoene dehydrogenase from the cyanobacterium Aphanocapsa PCC6714

The membrane-bound phytoene dehydrogenase (PD) is an enzyme in carotenoid biosynthesis which is essential in all microorganisms and plants containing these colored pigments. Despite its key role in the regulation of carotenogenesis, the biochemistry and molecular biology of PD are poorly understood. We have cloned, sequenced and expressed a portion of the PD-encoding gene, crtI, from the blue-green algae Aphanocapsa PCC6714. The gene codes for a 532-amino acids (aa) protein, with a calculated Mr of 52,598 Da. Two regions of the aa sequence share significant homology with PD from the purple bacterium Rhodobacter capsulatus, including a 30-aa region which has been proposed to be specific for dehydrogenases in carotenoid biosynthesis.     

Metabolic engineering towards biotechnological production of carotenoids in microorganisms

Carotenoids are important natural pigments produced by many microorganisms and plants. Traditionally, carotenoids have been used in the feed, food and nutraceutical industries. The recent discoveries of health-related beneficial properties attributed to carotenoids have spurred great interest in the production of structurally diverse carotenoids for pharmaceutical applications. The availability of a considerable number of microbial and plant carotenoid genes that can be functionally expressed in heterologous hosts has opened ways for the production of diverse carotenoid compounds in heterologous systems. In this review, we will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms for improved carotenoid productivity. In addition, we will discuss the application of combinatorial and evolutionary strategies to carotenoid pathway engineering to broaden the diversity of carotenoid structures synthesized in recombinant hosts.     

Pathway engineering strategies for production of beneficial carotenoids in microbial hosts

Carotenoids, such as lycopene, β-carotene, zeaxanthin, canthaxanthin and astaxanthin have many benefits for human health. In addition to the functional role of carotenoids as vitamin A precursors, adequate consumption of carotenoids prevents the development of a variety of serious diseases. Biosynthesis of carotenoids is a complex process and it starts with the common isoprene precursors. Condensation of these precursors and subsequent modifications, by introducing hydroxyl- and keto-groups, leads to the generation of diversified carotenoid structures. To improve carotenoid production, metabolic engineering has been explored in bacteria, yeast, and algae. The success of the pathway engineering effort depends on the host metabolism, specific enzymes used, the enzyme expression levels, and the strategies employed. Despite the difficulty of pathway engineering for carotenoid production, great progress has been made over the past decade. We review metabolic engineering approaches used in a variety of microbial hosts for carotenoid biosynthesis. These advances will greatly expedite our efforts to bring the health benefits of carotenoids and other nutritional compounds to our diet.     

Carotenoid biosynthesis and biotechnological application

A survey is given on the carotenoid biosynthetic pathway leading to beta-carotene and its oxidation products in bacteria and plants. This includes the synthesis of prenyl pyrophosphates via the mevalonate or the 1-deoxyxylulose-5-phosphate pathways as well as the reaction sequences of carotenoid formation and interconversion together with the properties of the enzymes involved. Biotechnological application of this knowledge resulted in the development of heterologous carotenoid production systems using bacteria and fungi with metabolic engineered precursor supply and crop plants with manipulated carotenoid biosynthesis. The recent developments in engineering crops with increased carotenoid contents are covered.     

影响光合细菌类胡萝卜素形成因素的研究

对已初步确认为球形红假单胞菌属的S—1菌株进行了类胡萝卜素形成因素的研究。通过对光照强度、温度、pH、碳源、氮源、生长因子和无机盐成份等培养条件的探讨,找到了适合类胡萝卜素形成的条件,为开发光合细菌类胡萝卜素提供了依据。     

Genetic manipulation of carotenoid biosynthesis: strategies, problems and achievements

Carotenoids, some of which are provitamin A, have a range of diverse biological functions and actions, especially in relation to human health. For example, carotenoids are known to be crucial for normal vision and have been associated with reducing the risk of several degenerative diseases including cancer. The putative advantage of modifying and engineering the carotenoid biosynthetic pathways is obvious: to provide sources for the isolation of desired carotenoids or to generate food plants with increased carotenoid content. This article reviews the studies of carotenoid production in heterologous microorganisms and the engineering of crop plants using manipulated carotenoid biosynthesis.     

Engineering of geranylgeranyl pyrophosphate synthase levels and physiological conditions for enhanced carotenoid and astaxanthin synthesis in <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis>

The basidiomycetous yeast, Xanthophyllomyces dendrorhous, is one of the very few organisms which can be used for biological production of the carotenoid astaxanthin. crtE cDNA has been cloned from this fungus for engineering of the terpenoid pathway. The function of its gene product as a geranylgeranyl pyrophosphate synthase was established. X. dendrorhous was transformed with the crtE cDNA to divert metabolite flow from the sterol pathway towards carotenoid biosynthesis. Transformants were obtained with increased levels of geranylgeranyl pyrophosphate synthase leading to higher carotenoid levels including astaxanthin. Physiological conditions for maximum carotenoid synthesis for wild type and the CrtE transformant were dim light and extra air supply of the shaking culture. These conditions and the transformation with crtE had additive effects and resulted in an 8-fold higher astaxanthin formation as compared to the initial wild type culture without illumination and extra air supply yielding 451 μg/g dry wt within 4 days of growth.     

Genetic engineering of carotenoid formation in tomato fruit and the potential application of systems and synthetic biology approaches

The health benefits conferred by numerous carotenoids have led to attempts to elevate their levels in foodstuffs. Tomato fruit and its products contain the potent antioxidant lycopene and are the predominant source of lycopene in the human diet. In addition, tomato products are an important source of provitamin A (β-carotene). The presence of other health promoting phytochemicals such as tocopherols and flavonoids in tomato has led to tomato and its products being termed a functional food. Over the past decade genetic/metabolic engineering of carotenoid biosynthesis and accumulation has resulted in the generation of transgenic varieties containing high lycopene and β-carotene contents. In achieving this important goal many fundamental lessons have been learnt. Most notably is the observation that the endogenous carotenoid pathways in higher plants appear to resist engineered changes. Typically, this resistance manifests itself through intrinsic regulatory mechanisms that are “silent” until manipulation of the pathway is initiated. These mechanisms may include feedback inhibition, forward feed, metabolite channelling, and counteractive metabolic and cellular perturbations. In the present article we will review progress made in the genetic engineering of carotenoids in tomato fruit, highlighting the limiting regulatory mechanisms that have been observed experimentally. The predictability and efficiency of the present engineering strategies will be questioned and the potential of more Systems and Synthetic Biology approaches to the enhancement of carotenoids will be assessed.     

Metabolic engineering of isoprenoids

The metabolic engineering of natural products has begun to prosper in the past few years due to genomic research and the discovery of biosynthetic genes. While the biosynthetic pathways and genes for some isoprenoids have been known for many years, new pathways have been found and known pathways have been further investigated. In this article, we review the recent advances in metabolic engineering of isoprenoids, focusing on the molecular genetics that affects pathway engineering the most. Examples in mono- sequi-, and diterpenoid synthesis as well as carotenoid production are discussed.     

Retinal biosynthesis in fungi: characterization of the carotenoid oxygenase CarX from Fusarium fujikuroi

The car gene cluster of the ascomycete Fusarium fujikuroi encodes two enzymes responsible for torulene biosynthesis (CarRA and CarB), an opsin-like protein (CarO), and a putative carotenoid cleaving enzyme (CarX). It was presumed that CarX catalyzes the formation of the major carotenoid in F. fujikuroi, neurosporaxanthin, a cleavage product of torulene. However, targeted deletion of carX did not impede neurosporaxanthin biosynthesis. On the contrary, DeltacarX mutants showed a significant increase in the total carotenoid content, indicating an involvement of CarX in the regulation of the pathway. In this work, we investigated the enzymatic activity of CarX. The expression of the enzyme in beta-carotene-accumulating Escherichia coli cells led to the formation of the opsin chromophore retinal. The identity of the product was proven by high-performance liquid chromatography and gas chromatography-mass spectrometry. Subsequent in vitro assays with heterologously expressed and purified CarX confirmed its beta-carotene-cleaving activity and revealed its capability to produce retinal also from other substrates, such as gamma-carotene, torulene, and beta-apo-8'-carotenal. Our data indicate that the occurrence of at least one beta-ionone ring in the substrate is required for the cleavage reaction and that the cleavage site is determined by the distance to the beta-ionone ring. CarX represents the first retinal-synthesizing enzyme reported in the fungal kingdom so far. It seems likely that the formed retinal is involved in the regulation of the carotenoid biosynthetic pathway via a negative feedback mechanism.     

Excitation energy transfer and carotenoid radical cation formation in light harvesting complexes — A theoretical perspective

Light harvesting complexes have been identified in all chlorophyll-based photosynthetic organisms. Their major function is the absorption of light and its transport to the reaction centers, however, they are also involved in excess energy quenching, the so-called non-photochemical quenching (NPQ). In particular, electron transfer and the resulting formation of carotenoid radical cations have recently been discovered to play an important role during NPQ in green plants. Here, the results of our theoretical investigations of carotenoid radical cation formation in the major light harvesting complex LHC-II of green plants are reported. The carotenoids violaxanthin, zeaxanthin and lutein are considered as potential quenchers. In agreement with experimental results, it is shown that zeaxanthin cannot quench isolated LHC-II complexes. Furthermore, subtle structural differences in the two lutein binding pockets lead to substantial differences in the excited state properties of the two luteins. In addition, the formation mechanism of carotenoid radical cations in light harvesting complexes LH2 and LH1 of purple bacteria is studied. Here, the energetic position of the S₁ state of the involved carotenoids neurosporene, spheroidene, spheroidenone and spirilloxanthin seems to determine the occurrence of radical cations in these LHCs upon photo-excitation. An elaborate pump-deplete-probe experiment is suggested to challenge the proposed mechanism.