类胡萝卜素合成的相关基因及其基因工程

类胡萝卜素具有多种生物功能,尤其在保护人类健康方面起着重要的作用,如它们是合成维生素A的前体,能够增强人体免疫力和具有防癌抗癌的功效。人体自身不能合成类胡萝卜素,必须通过外界摄入;但类胡萝卜素在许多植物中含量较低,并且很难用化学方法合成。随着类胡萝卜素生物合成途径的阐明及其相关基因的克隆,运用基因工程手段调控类胡萝卜素的生物合成已成为可能。本文综述了微生物和高等植物类胡萝卜素生物合成途径中相关基因的克隆,以及运用这些基因通过异源微生物生产类胡萝卜素和提高作物类胡萝卜素含量的基因工程研究进展。     

利用基因工程改良植物类胡萝卜素的合成

类胡萝卜素(carotenoids)是植物体内存在的一类重要天然色素物质的总称,其生物功能广泛.类胡萝卜素是人类饮食结构中重要的组成部分,并且在医药、化妆品及食品与饲料工业等方面扮演着重要的角色.类胡萝卜素还是一些维生素合成的前体,具有多种保健功能,可以提高人体免疫力,并具有抗癌的功效.目前有大量关于提高植物体内类胡萝卜素含量及定向改变其种类的研究,对类胡萝卜素代谢途径及其调控的理解是这些研究的基础.主要阐述植物天然类胡萝卜素的生物合成及利用基因工程对类胡萝卜素改良的研究进展.     

Occurrence and biosynthesis of carotenoids in phytoplankton

Naturally occurring carotenoids are important sources of antioxidants, anti-cancer compounds and anti-inflammatory agents and there is thus considerable market demand for their pharmaceutical applications. Carotenoids are widely distributed in marine and freshwater organisms including microalgae, phytoplankton, crustaceans and fish, as well as in terrestrial plants and birds. Recently, phytoplankton-derived carotenoids have received much attention due to their abundance, rapid rate of biosynthesis and unique composition. The carotenoids that accumulate in particular phytoplankton phyla are synthesized by specific enzymes and play unique physiological roles. This review focuses on studies related to the occurrence of carotenoids in different phytoplankton phyla and the molecular aspects of their biosynthesis. Recent biotechnological advances in the isolation and characterization of some representative carotenoid synthases in phytoplankton are also discussed.     

Dietary carotenoid levels affect carotenoid and retinoid allocation in female Chinook salmon Oncorhynchus tshawytscha

This study examined the effect of dietary carotenoid availability on carotenoid and retinoid concentrations in the flesh, plasma, skin and eggs of female Chinook salmon Oncorhynchus tshawytscha. Carotenoid concentrations in all tissues were closely related to dietary availability. Early in the breeding season, carotenoids were stored primarily in the muscle, with a flesh carotenoid concentration of 9·9 µg g^?1 in fish fed a high carotenoid diet compared with 1·9 µg g^?1 in fish fed a low carotenoid diet. During the breeding season, carotenoid reserves were mobilized predominantly to the eggs and also to the skin. By the end of the breeding season, carotenoid concentrations in the eggs were 17·9 µg g^?1 in fish fed a high carotenoid diet and 3·9 µg g^?1 in fish fed a low carotenoid diet. Conversely, egg retinoid concentrations were only c. 20% lower in fish fed a low v. high carotenoid diet, which suggests that retinoid concentrations were not limited by the availability of carotenoid precursors. Egg carotenoid concentrations were not correlated with either skin carotenoid concentration or colouration, which suggests that female carotenoid displays are not a reliable signal that males can use to evaluate egg carotenoid resources.     

Testosterone treatment can increase circulating carotenoids but does not affect yellow carotenoid‐based plumage colour in blue tits Cyanistes caeruleus

A number of mechanisms are responsible for producing the variation in natural colours, and these need not act in isolation. A recent hypothesis states that carotenoid‐based coloration, in addition to carotenoid availability, is also enhanced by elevated levels of circulating testosterone (T). This has only been tested for carotenoid‐coloured bare parts in birds. We performed an experimental manipulation of T levels and examined the effects on the yellow carotenoid‐based breast plumage in captive yearling blue tits Cyanistes caeruleus, of which half received a diet supplemented with carotenoids. T treatment resulted in elevated plasma T compared to controls and carotenoid supplementation strongly increased plasma carotenoid levels. T treatment resulted in an additional increase in plasma carotenoid levels but only in the carotenoid‐supplemented males. Carotenoid supplementation resulted in more intense breast colour (carotenoid chroma), as expected. However, there was no effect of testosterone on plumage coloration at either dietary carotenoid level. Our results suggest that T can cause an increase in plasma carotenoid concentration, but that this does not necessarily lead to improved carotenoid‐based plumage coloration.     

Plasma carotenoid concentrations of incubating American kestrels (Falco sparverius) show annual,seasonal, and individual variation and explain reproductive outcome

In wild birds, the proximate and ultimate factors that affect circulating carotenoid concentrations remain poorly understood. We studied variation in plasma carotenoid concentrations across several scales: annual, seasonal, pair, territory and individual, and evaluated whether plasma carotenoid concentrations explained reproductive outcome of wild American kestrels (Falco sparverius). We sampled plasma carotenoid concentrations of 99 female and 80 male incubating kestrels from April to June in 2008 to 2012. Plasma carotenoid concentrations were explained by an interaction between year and sex, date, and random effects for pair and individual identity. In general, plasma carotenoid concentrations of males were significantly higher than females, but this depended on year. Within a breeding season, earlier nesting kestrels had higher carotenoid concentrations than later nesting kestrels, a pattern that is coincident with seasonal trends in local fitness. Pair and individual identity explained variation in carotenoid concentrations suggesting that carotenoid concentrations of mated birds were correlated, and some individuals consistently maintained higher carotenoid levels than others. Male carotenoid concentrations were positively associated with number of young fledged per pair. These results are consistent with the hypothesis that higher quality individuals have higher carotenoid levels compared to lower quality individuals, despite annual variations in carotenoid availability.     

Effect of Dietary Carotenoid Supplementation on Food Intake and Immune Function in a Songbird with no Carotenoid Coloration

Studies of ornamental carotenoid coloration suggest that animals may have evolved specialized mechanisms for maximizing color expression and advertising their potential worth as a mate. For example, when given a choice of foods, many carotenoid‐pigmented fishes and birds select the more colorful, presumably carotenoid‐rich foods, and then accumulate these pigments at high levels in both the integument and systemically, in order to boost their immune system and hence directly advertise their health state with their colors. The majority of animals, however, do not exhibit sexually selected carotenoid coloration, which raises the question of whether they still optimize pigment intake and allocation in ways that boost endogenous accumulation and health. We tested the effect of carotenoid supplementation on food intake, carotenoid accumulation in blood, and both innate and adaptive immunity in male society finches (Lonchura domestica) – a non‐carotenoid‐colored estrildid finch relative of the zebra finch (Taeniopygia guttata; a species in which males do display sexually attractive and health‐revealing carotenoid color). Males fed a carotenoid‐rich diet for 2 wk did not consume more food than control males. Still, consumption of the carotenoid‐rich diet for 2 wk significantly elevated circulating levels of carotenoids in blood in male society finches, yielding the potential for immune enhancement. In fact, carotenoid‐enriched finches performed significantly better than control birds in our assay of constitutive innate immunity (bacterial‐killing activity of whole blood), although not in our test of inducible adaptive immunity (response to a mitogenic challenge with phytohemagglutinin). These results suggest that affinities for carotenoid‐rich foods may be particular to species with sexually selected carotenoid pigmentation, but that, as in humans and other mammals (e.g. mice, rats) without carotenoid color, the immune‐boosting action of carotenoids is conserved regardless of the strength of sexual selection on pigment use.     

Energy transfer between the carotenoid and the bacteriochlorophyll within the B-800-850 light-harvesting pigment-protein complex of Rhodopseudomonas sphaeroides

Energy transfer between carotenoid and bacteriochlorophyll has been studied in isolated B-800-850 antenna pigment-protein complexes from different strains of Rhodopseudomonas sphaeroides which contain different types of carotenoid. Singlet-singlet energy transfer from the carotenoid to the bacteriochlorophyll is efficient (75-100%) and is rather insensitive to carotenoid type, over the range of carotenoids tested. The yield of carotenoid triplets is low (2-15%) but this arises from a low yield of bacteriochlorophyll triplet formation rather than from an inefficient triplet-triplet exchange reaction. The rate of the triplet-triplet exchange reaction between the bacteriochlorophyll and the carotenoid is fast (Ktt greater than or equal to 1.4 . 10(8) S-1) and also relatively independent of the type of carotenoid present.     

产氢红杆菌类胡萝卜素突变株的诱变和筛选

用紫外线照射和氯化锂夹层平板培养法对产氢红杆菌(Rhodobacter sp. R7)进行复合诱变, 分离获得了一株产氢效率提高的类胡萝卜素突变株R726。该突变株在表观特征、光谱学特征、色谱特征、生长和产氢性能等方面与出发菌株有明显不同, 但16S rDNA序列一致。R726菌株有 550 nm类胡萝卜素特征性吸收峰, 类胡萝卜素组成上比出发菌株少一黄色类胡萝卜素组分, 生长和产氢性能均高于出发菌株, 产氢效率比出发菌株提高了33.3%, 类胡萝卜素含量比出发株提高了53.8%。     

CAROTENOID COMPOSITION OF MARINE RED ALGAE1

Photosynthetic organisms possess carotenoids that function either as accessory, photoprotective, or structural pigments. Therefore, the carotenoid profile provides information about certain photoacclimation and photoprotection responses. Carotenoids are also important chemosystematic markers because specific enzymes mediate each step of carotenoid biosynthesis. For red algae, diverse and often contradictory carotenoid compositions have been reported. As a consequence, it is difficult to infer the physiological importance of carotenoids in Rhodophyta. To characterize the relationship between carotenoid composition, rhodophycean phylogeny, and the presence of potentially photoprotective pigments, we analyzed the carotenoid composition of 65 subtropical species from 12 orders and 18 rhodophyte families. Our results showed that red algae do not present a unique carotenoid profile. However, a common profile was observed up to the level of order, with exception of the Ceramiales and the Corallinales. The main difference between profiles is related to the xanthophyll that represents the major carotenoid. In some species lutein is the major carotenoid while in others it is substituted by zeaxanthin or antheraxanthin. The presence of this epoxy carotenoid together with the presence of violaxanthin that are xanthophyll cycle (XC)‐related pigments was found in four of the 12 analyzed orders. The carotenoid pigment profiles are discussed in relation to Rhodophyta phylogeny, and it is suggested that the xanthophyll cycle‐related pigments appeared early in the evolution of eukaryotic phototrophs.     

Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5

Transgenic tomato plants expressing antisense RNA to a ripening-related cDNA clone (pTOM5) had yellow ripening fruit and pale coloured flowers. Carotenoid levels in fruit of these plants were reduced by up to 97%. In order to determine the step of carotenoid biosynthesis which was blocked, a cell-free system active in the synthesis of carotenoid intermediates was prepared. Incubations with radiolabelled carotenoid precursors led to the identification of the block between GGDP and phytoene. Analysis of carotenoids in different tissues of transgenic and control plants indicated that although ripe fruit and flower carotenoid levels were reduced in the modified plants, leaf carotenoid levels were not decreased. This implies that the pTOM5 gene product is not involved in carotenoid synthesis in the leaf.     

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.     

产氢红杆菌类胡萝卜素突变株的诱变和筛选

用紫外线照射和氯化锂夹层平板培养法对产氢红杆菌(Rhodobacter sp.R7)进行复合诱变,分离获得了一株产氢效率提高的类胡萝卜素突变株R726.该突变株在表观特征、光谱学特征、色谱特征、生长和产氢性能等方面与出发菌株有明显不同,但16S rDNA序列一致.R726菌株有550 nm类胡萝卜素特征性吸收峰,类胡萝卜素组成上比出发菌株少一黄色类胡萝卜素组分,生长和产氢性能均高于出发菌株,产氢效率比出发菌株提高了33.3%,类胡萝卜素含量比出发株提高了53.8%.     

Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein

In order to enhance the carotenoid content of potato tubers, transgenic potato plants have been produced expressing an Erwinia uredovora crtB gene encoding phytoene synthase, specifically in the tuber of Solanum tuberosum L. cultivar Desiree which normally produces tubers containing c. 5.6 microg carotenoid g(-1) DW and also in Solanum phureja L. cv. Mayan Gold which has a tuber carotenoid content of typically 20 microg carotenoid g(-1) DW. In developing tubers of transgenic crtB Desiree lines, carotenoid levels reached 35 microg carotenoid g(-1) DW and the balance of carotenoids changed radically compared with controls: beta-carotene levels in the transgenic tubers reached c. 11 microg g(-1) DW, whereas control tubers contained negligible amounts and lutein accumulated to a level 19-fold higher than empty-vector transformed controls. The crtB gene was also transformed into S. phureja (cv. Mayan Gold), again resulting in an increase in total carotenoid content to 78 microg carotenoid g(-1) DW in the most affected transgenic line. In these tubers, the major carotenoids were violaxanthin, lutein, antheraxanthin, and beta-carotene. No increases in expression levels of the major carotenoid biosynthetic genes could be detected in the transgenic tubers, despite the large increase in carotenoid accumulation. Microarray analysis was used to identify a number of genes that were consistently up- or down-regulated in transgenic crtB tubers compared with empty vector controls. The implications of these data from a nutritional standpoint and for further modifications of tuber carotenoid content are discussed.     

Comparison of carotenoid content,gene expression and enzyme levels in tomato (Lycopersicon esculentum) leaves

Physiological conditions which lead to changes in total carotenoid content in tomato plantlets were identified. Carotenoid levels were found to increase after the onset of a dark period during a normal 24 h cycle. This rapid initial increase is followed by a steady decrease in carotenoid content throughout the night. A decrease in the expression of several carotenogenic genes, namely pds, zds (carotenoid desaturases) and ptox (plastid terminal oxidase), was observed following the removal of the light (when carotenoid content is at its highest). An increase in gene expression was observed before the return to light for pds and zds (when carotenoid levels were at their lowest), or following the return to light for ptox. The phytoene desaturation inhibitor norflurazon leads to a decrease coloured carotenoid content and, in the light, this correlated with pds and zds gene induction. In the dark, norflurazon treatment led to only a weak decrease in carotenoid content and only a small increase in pds and zds gene expression. The striking absence of phytoene accumulation under norflurazon treatment in the dark suggests a down-regulation of carotenoid formation in darkness However, prolonged dark conditions, or treatment with photosynthetic inhibitors, surprisingly led to higher carotenoid levels, which correlated with decreased expression of most examined genes. In addition to light, which acts in a complex way on carotenoid accumulation and gene expression, our results are best explained by a regulatory effect of carotenoid levels on the expression of several biosynthetic genes. In addition, monitoring of protein amounts for phytoene desaturase and plastid terminal oxidase (which sometimes do not correlate with gene expression) indicate an even more complex regulatory pattern.     

Accumulation and bioavailability of dietary carotenoids in vegetable crops

Carotenoids are lipid-soluble pigments found in many vegetable crops that are reported to have the health benefits of cancer and eye disease reduction when consumed in the diet. Research shows that environmental and genetic factors can significantly influence carotenoid concentrations in vegetable crops, and that changing cultural management strategies could be advantageous, resulting in increased vegetable carotenoid concentrations. Improvements in vegetable carotenoid levels have been achieved using traditional breeding methods and molecular transformations to stimulate biosynthetic pathways. Postharvest and processing activities can alter carotenoid chemistry, and ultimately affect bioavailability. Bioavailability data emphasize the importance of carotenoid enhancement in vegetable crops and the need to characterize potential changes in carotenoid composition during cultivation, storage and processing before consumer purchase.