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.     

Supply of precursors for carotenoid biosynthesis in plants

Carotenoids are isoprenoids of industrial and nutritional interest produced by all photosynthetic organisms, including plants. Too often, the metabolic engineering of plant carotenogenesis has been obstructed by our limited knowledge on how the endogenous pathway interacts with other related metabolic pathways, particularly with those involved in the production of isoprenoid precursors. However, recent discoveries are providing new insights into this field. All isoprenoids derive from prenyl diphosphate precursors. In the case of carotenoids, these precursors are produced predominantly by the methylerythritol 4-phosphate (MEP) pathway in plants. This review focuses on the progress in our understanding of how manipulation of the MEP pathway impacts carotenoid biosynthesis and on the discoveries underlining the central importance of coordinating the supply of MEP-derived precursors with the biosynthesis of carotenoids and other derived isoprenoids.     

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.     

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.     

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 carotenoid accumulation by creating a metabolic sink

Carotenoids are highly beneficial for human nutrition and health because they provide essential nutrients and important antioxidants in our diets. However, many food crops, especially the major staple crops contain only trace to low amounts of carotenoids. Although significant progress has been made in developing food crops rich in carotenoids by altering the expression of carotenoid biosynthetic genes, in many cases it has proved to be difficult to reach the desired levels of carotenoid enrichment. The recent identification and characterization of a novel gene mutation in cauliflower reveals that creating a metabolic sink to sequester carotenoids is an important mechanism to control carotenoid accumulation in plants. The successful demonstration of increased carotenoid accumulation in association with the formation of sink structures in transgenic crops offers a new and alternative approach to increase carotenoid content. Manipulation of the formation of metabolic sink along with the catalytic activity of the pathway may represent a promising strategy for maximally improving the nutritional quality of food crops.     

Metabolic engineering for the microbial production of carotenoids and related products with a focus on the rare C50 carotenoids

Carotenoids, a subfamily of terpenoids, are yellow- to red-colored pigments synthesized by plants, fungi, algae, and bacteria. They are ubiquitous in nature and take over crucial roles in many biological processes as for example photosynthesis, vision, and the quenching of free radicals and singlet oxygen. Due to their color and their potential beneficial effects on human health, carotenoids receive increasing attention. Carotenoids can be classified due to the length of their carbon backbone. Most carotenoids have a C40 backbone, but also C30 and C50 carotenoids are known. All carotenoids are derived from isopentenyl pyrophosphate (IPP) as a common precursor. Pathways leading to IPP as well as metabolic engineering of IPP synthesis and C40 carotenoid production have been reviewed expertly elsewhere. Since C50 carotenoids are synthesized from the C40 carotenoid lycopene, we will summarize common strategies for optimizing lycopene production and we will focus our review on the characteristics, biosynthesis, glycosylation, and overproduction of C50 carotenoids.     

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。     

Making extra room for carotenoids in plant cells: New opportunities for biofortification

Plant carotenoids are essential for photosynthesis and photoprotection and provide colors in the yellow to red range to non-photosynthetic organs such as petals and ripe fruits. They are also the precursors of biologically active molecules not only in plants (including hormones and retrograde signals) but also in animals (including retinoids such as vitamin A). A carotenoid-rich diet has been associated with improved health and cognitive capacity in humans, whereas the use of carotenoids as natural pigments is widespread in the agrofood and cosmetic industries. The nutritional and economic relevance of carotenoids has spurred a large number of biotechnological strategies to enrich plant tissues with carotenoids. Most of such approaches to alter carotenoid contents in plants have been focused on manipulating their biosynthesis or degradation, whereas improving carotenoid sink capacity in plant tissues has received much less attention. Our knowledge on the molecular mechanisms influencing carotenoid storage in plants has substantially grown in the last years, opening new opportunities for carotenoid biofortification. Here we will review these advances with a particular focus on those creating extra room for carotenoids in plant cells either by promoting the differentiation of carotenoid-sequestering structures within plastids or by transferring carotenoid production to the cytosol.     

Nutritious crops producing multiple carotenoids--a metabolic balancing act

Plants and microbes produce multiple carotenoid pigments with important nutritional roles in animals. By unraveling the basis of carotenoid biosynthesis it has become possible to modulate the key metabolic steps in plants and thus increase the nutritional value of staple crops, such as rice (Oryza sativa), maize (Zea mays) and potato (Solanum tuberosum). Multigene engineering has been used to modify three different metabolic pathways simultaneously, producing maize seeds with higher levels of carotenoids, folate and ascorbate. This strategy may allow the development of nutritionally enhanced staples providing adequate amounts of several unrelated nutrients. By focusing on different steps in the carotenoid biosynthesis pathway, it is also possible to generate plants with enhanced levels of several nutritionally-beneficial carotenoid molecules simultaneously.     

Analysis of metabolites in the terpenoid pathway of Catharanthus roseus cell suspensions

In Catharanthus roseus cell cultures, the monoterpenoid pathway has been shown to be a limiting factor in terpenoid indole alkaloid (TIA) production. This could be due to competition at the level of isopentenyl diphosphate::dimethylallyl diphosphate (C5) which leads to the biosynthesis of different terpenoid groups. For future engineering of the terpenoid pathway, chemical characterization of C. roseus cell cultures is a necessity. Therefore, in this study nine C. roseus cell suspension lines were characterized by analyzing the levels of the major terpenoids derived from different biosynthetic pathways which may compete for the same precursors; TIA (monoterpenoid, C10), carotenoids (tetraterpenoid, C40), and sterols (triterpenoid, C30). Among the cell lines, CRPP (S) was the most promising TIA-producing cell line which provided more TIA [24 μmol g^?1 dry weight (DW)] than carotenoids (15 μmol g^?1 DW) and sterols (2 μmol g^?1 DW). However, when considering the distribution of the isopentenyl-precursor (C5), the carotenoids which assemble from 8× C5 represent twofold more C5-units (122 μmol g^?1 DW) than the TIA in this cell line. In the CRPP (G), A12A2 (G), and A12A2 (S) cell lines, the C5 distribution was predominant toward carotenoid biosynthesis as well, resulting in a relatively high accumulation of carotenoids. The geranylgeranyl diphosphate (C20) pathway toward carotenoid production is therefore considered competitive toward TIA biosynthesis. For channeling more precursors to the TIA, the branch point for C10 and C20 seems an interesting target for metabolic engineering. Using principal component analysis of the chromatographic data, we characterized the cell lines chemically based on their metabolite levels. The information on the metabolic composition of C. roseus cell cultures is useful for developing strategies to engineer the metabolic pathways and for selection of cell lines for future studies.     

Progress on molecular breeding and metabolic engineering of biosynthesis pathways of C(30), C(35), C(40), C(45), C(50) carotenoids

At least 700 natural carotenoids have been characterized; they can be classified into C(30), C(40) and C(50) subfamilies. The first step of C(40) pathway is the combination of two molecules of geranylgeranyl pyrophosphate to synthesize phytoene by phytoene synthase (CrtB or PSY). Most natural carotenoids originate from different types and levels of desaturation by phytoene desaturase (CrtI or PDS+ZDS), cyclization by lycopene cyclase (CrtY or LYC) and other modifications by different modifying enzyme (CrtA, CrtU, CrtZ or BCH, CrtX, CrtO, etc.) of this C(40) backbone. The first step of C(30) pathway is the combination of two molecules of FDP to synthesize diapophytoene by diapophytoene synthase (CrtM). But natural C(30) pathway only goes through a few steps of desaturation to form diaponeurosporene by diapophytoene desaturase (CrtN). Natural C(50) carotenoid decaprenoxanthin is synthesized starting from the C(40) carotenoid lycopene by the addition of 2 C(5) units. Concerned the importance of carotenoids, more and more attention has been concentrated on achieving novel carotenoids. The method being used successfully is to construct carotenoids biosynthesis pathways by metabolic engineering. The strategy of metabolic engineering is to engineer a small number of stringent upstream enzymes (CrtB, CrtI, CrtY, CrtM, or CrtN), then use a lot of promiscuous downstream enzymes to obtain large number of novel carotenoids. Two key enzymes phytoene desaturase (CrtI(m)) and lycopene cyclase (CrtY(m)) have been modified and used with a series of downstream modifying enzymes with broad substrate specificity, such as monooxygenase (CrtA), carotene desaturase (CrtU), carotene hydroxylase (CrtZ), zeaxanthin glycosylase (CrtX) and carotene ketolase (CrtO) to extend successfully natural C(30) and C(40) pathways in E. coli. Existing C(30) synthase CrtM to synthesize carotenoids with different chain length have been engineered and a series of novel carotenoids have been achieved using downstream modifying enzymes. C(35) carotenoid biosynthesis pathway has been constructed in E. coli as described. C(45) and C(50) carotenoid biosynthesis pathways have also been constructed in E. coli, but it is still necessary to extend these two pathways. Those novel acyclic or cyclic carotenoids have a potential ability to protect against photooxidation and radical-mediated peroxidation reactions which makes them interesting pharmaceutical candidates.     

Structure and function of the genes involved in the biosynthesis of carotenoids in the mucorales

Carotenoids are widely distributed natural pigments which are in an increasing demand by the market, due to their applications in the human food, animal feed, cosmetics, and pharmaceutical industries. Although more than 600 carotenoids have been identified in nature, only a few are industrially important (β-carotene, astaxanthin, lutein or lycopene). To date chemical processes manufacture most of the carotenoid production, but the interest for carotenoids of biological origin is growing since there is an increased public concern over the safety of artificial food colorants. Although much interest and effort has been devoted to the use of biological sources for industrially important carotenoids, only the production of biological β-carotene and astaxanthin has been reported. Among fungi, several Mucorales strains, particularlyBlakeslea trispora, have been used to develop fermentation process for the production of β-carotene on almost competitive cost-price levels. Similarly, the basidiomycetous yeastXanthophyllomyces dendrorhous (the perfect state ofPhaffia rhodozyma), has been proposed as a promising source of astaxanthin. This paper focuses on recent findings on the fungal pathways for carotenoid production, especially the structure and function of the genes involved in the biosynthesis of carotenoids in the Mucorales. An outlook of the possibilities of an increased industrial production of carotenoids, based on metabolic engineering of fungi for carotenoid content and composition, is also discussed.     

An update on microbial carotenoid production: application of recent metabolic engineering tools

Carotenoids are ubiquitous pigments synthesized by plants, fungi, algae, and bacteria. Industrially, carotenoids are used in pharmaceuticals, neutraceuticals, and animal feed additives, as well as colorants in cosmetics and foods. Scientific interest in dietary carotenoids has increased in recent years because of their beneficial effects on human health, such as lowering the risk of cancer and enhancement of immune system function, which are attributed to their antioxidant potential. The availability of carotenoid genes from carotenogenic microbes has made possible the synthesis of carotenoids in non-carotenogenic microbes. The increasing interest in microbial sources of carotenoid is related to consumer preferences for natural additives and the potential cost effectiveness of creating carotenoids via microbial biotechnology. In this review, we will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms with particular focus on the potential of Escherichia coli for improved carotenoid productivity. Amitabha Das and Sang-Hwal Yoon contributed equally to this work.     

Targeted transcriptomic and metabolic profiling reveals temporal bottlenecks in the maize carotenoid pathway that may be addressed by multigene engineering

Carotenoids are a diverse group of tetraterpenoid pigments found in plants, fungi, bacteria and some animals. They play vital roles in plants and provide important health benefits to mammals, including humans. We previously reported the creation of a diverse population of transgenic maize plants expressing various carotenogenic gene combinations and exhibiting distinct metabolic phenotypes. Here we performed an in‐depth targeted mRNA and metabolomic analysis of the pathway to characterize the specific impact of five carotenogenic transgenes and their interactions with 12 endogenous genes in four transgenic lines representing distinct genotypes and phenotypes. We reconstructed the temporal profile of the carotenoid pathway during endosperm development at the mRNA and metabolic levels (for total and individual carotenoids), and investigated the impact of transgene expression on the endogenous pathway. These studies enabled us to investigate the extent of any interactions between the introduced transgenic and native partial carotenoid pathways during maize endosperm development. Importantly, we developed a theoretical model that explains these interactions, and our results suggest genetic intervention points that may allow the maize endosperm carotenoid pathway to be engineered in a more effective and predictable manner.     

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 microbial cell factories for biosynthesis of isoprenoid molecules: beyond lycopene

The isoprenoid superfamily of compounds holds great potential for delivering commercial therapeutics, neutraceuticals and fine chemicals. As such, it has attracted widespread attention and prompted research aimed at metabolic engineering of the pathway for isoprenoid overproduction. The carotenoids in particular, because of their convenient colorimetric screening properties, have facilitated the investigation of new tools for pathway optimization. Because all isoprenoids share common metabolic precursors, genetic platforms resulting from work with carotenoids can be applied to the biosynthesis of other valuable products. In this review we summarize the many tools and methods that have been developed for isoprenoid pathway engineering, and the potential of these technologies for producing other molecules of this family, especially terpenoids.     

A multicarotenoid beadlet for human nutrition - proof of concept of in vitro timed release

Since the 1980's when the predominate focus of study and use of carotenoids in human nutritional formulations was solely on beta-carotene, there has been a steady increase in research aimed to understand the role of a wide variety of carotenoids in human health. This work has increasingly demonstrated the benefits of a number of carotenoids, and there has been a corresponding increase in the number of carotenoids provided in nutritional supplements (multicarotenoids). Numerous published observations in both human and animal studies suggest significant interaction and competition between various carotenoids during absorption and metabolism, resulting in the inhibition of uptake of one over the other. This competition has the end result of reducing the beneficial effects of the inhibited carotenoid. To limit such competition and maximize carotenoid uptakes, a layered beadlet was designed to release a defined ratio of carotenoids sequentially. Preliminary dissolution testing is presented showing the release profile in simulated digestive conditions of a combination of beta-carotene, alpha carotene, lutein, zeaxanthin, lycopene and astaxanthin derived from natural sources. Comparison is made to an immediate release beadlet formulation using the same combination of carotenoids. These results will be used to guide proof of concept clinical testing for effectiveness in humans.