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。     

红酵母生物合成类胡萝卜素研究进展

类胡萝卜素(carotenoid)是一类呈黄色、橙红色或红色的多烯类化合物,具有预防血管硬化和抑制肿瘤发生,增加宿主免疫力,抗氧化能力和抑制自由基等功能.目前类胡萝卜素已被FAO和WHO等国际组织定为A类营养色素,并在50多个国家获准为营养、色素双重功用的食品添加剂,被广泛应用于保健食品及医药和化妆品工业[1].生产类胡萝卜素可采用提取法、化学合成法和微生物发酵法,其中最有发展前景的是微生物发酵法.目前国内外微生物发酵技术生产天然类胡萝卜素的研究,主要集中在三孢布腊拉霉菌和红酵母等菌种上.利用红酵母生产类胡萝卜素具有营养要求简单,培养周期短,可综合利用等优点,具有很好的应用价值和开发前景.本文主要对红酵母生物合成类胡萝卜素的菌种选育、培养条件及类胡萝卜素的提取检测方法进行了综述.     

胶红酵母利用番茄渣和豆粕为原料发酵生产类胡萝卜素的研究

基于降低微生物类胡萝卜素生产成本的考虑,采用番茄渣、豆粕的纤维素酶酶解产物培养胶红酵母,以单位体积发酵液中的总类胡萝卜素浓度增量作为优化目标,先后运用逐步单因素法和均匀设计法系统性地考查了胶红酵母的总类胡萝卜素产量和增量与各个相关因素之间的关系。实验获得的总类胡萝卜素最大产量以及扣除了番茄渣中的类胡萝卜素含量而计算得到的增量分别为12.25 mg/L和10.25 mg/L。实验结果证明设计的生产工艺能够以较低的成本生产出富含类胡萝卜素的饲料,因而是经济可行的。     

高等植物类胡萝卜素代谢工程研究进展

类胡萝卜素是自然界中种类繁多的天然化合物。植物类胡萝卜素由类异戊二烯碳骨架组成,并含有或没有环氧的、羟基或酮基基团。类胡萝卜素是人类饮食结构中不可缺少的重要成分,具有多种重要的保健功能,并且是安全的食品、饲料和化妆品着色剂。类胡萝卜素植物代谢基因工程的应用旨在增加特殊植物的营养价值、利用植物来生产特殊的类胡萝卜素、提高植物对光氧化的抵抗力及对植物的花色进行修饰等。     

逆境对红酵母发酵生产类胡萝卜素的促进作用

类胡萝卜素广泛存在于细菌、藻类、真菌和植物中,是一类具有重要的生理保健功能的呈黄色、橙红色或红色的多烯类化合物。考察了不同逆境条件,包括低温、低温处理时间、酸处理及高浓度盐处理对促进一株红酵母(Rhodotorulasp.)发酵生产类胡萝卜素的影响,并利用响应面分析方法研究了其交互作用。结果表明,低温,酸处理、高浓度盐的影响以及低温和酸处理、低温和高浓度盐以及低温处理时间和酸处理的交互作用对类胡萝卜素产量的影响均显著,在温度为15℃处理48 h,pH3.5,NaCl浓度为2 mol/L的条件下,类胡萝卜素最高产量达到31.04 mg/L,说明逆境对红酵母发酵生产类胡萝卜素具有促进作用。     

利用微藻培养生产类胡萝卜素的研究进展

类胡萝卜素(包括虾青素、β-胡萝卜素和叶黄素等)的重要功能引起了人们的广泛关注,微藻是生产类胡萝卜素的重要来源。本文综述了虾青素与β-胡萝卜素的合成、功能和生产,以及富含叶黄素藻株的选育和异养培养,并提出了今后的发展方向。     

红酵母简介

红酵母细胞内富含类胡萝卜素和油脂等生物活性成分,色泽诱人,具有较高的生长速率和较强的环境适应性。综述了红酵母在天然类胡萝卜素生物合成、微生物油脂生产、全细胞生物催化、果蔬采后生物防治和功能饲料开发等领域的研究和应用。     

研究发现类胡萝卜素生产必要条件

2020年8月19日PNAS报道,由西班牙农业基因组学研究中心(Centre for Research in Agricultural Genomics,CRAG)和植物分子和细胞生物学研究所(Institute for Plant Molecular and Cellular Biology,IBMCP)的研究者合作发现了一种改善植物生长的有前途的策略,通过八氢番茄红素(phytoene)将叶绿体转化为生产和储存大量类胡萝卜素的原生质体。该研究有望为作物的营养改良以及化妆品、制药和食品加工用类胡萝卜素的可持续生产开辟了新的方法。     

β-胡萝卜素合成的代谢工程研究进展

β-胡萝卜素是自然界中最重要的商业化生产的植物色素之一,具有多种生理功能和生物活性。自上世纪60年代开始,随着系统生物学概念的提出以及对类胡萝卜素合成途径研究的不断深入,系统代谢工程在提高类胡萝卜素产量方面发挥了重要作用。文中在介绍β-胡萝卜素传统生产方法的基础上,重点介绍了如何运用系统代谢工程手段构建β-胡萝卜素高产菌株,并分析了进一步提高工程菌β-胡萝卜素产量所面临的主要问题及可能的解决方案,为β-胡萝卜素的高效生产提供了思路。     

干燥工艺对锁掷酵母中类胡萝卜素的影响

以玉米浆为培养基生产锁掷酵母,对干燥后锁掷酵母的质量进行研究,比较不同干燥方式对锁掷酵母中类胡萝卜素以及干酵母粉亮度值L、红色值a、黄色值b的影响。结果表明:冷冻干燥能较好地保持锁掷酵母中的类胡萝卜素,并且对其酵母粉的L、a、b值影响较小。选择-70℃冷冻干燥,干酵母粉的L、a、b值分别为64.55、18.44和32.71,类胡萝卜素的保持率达到88.87%。     

番茄(Solanum lycopersicum)类胡萝卜素降解关键基因筛选

类胡萝卜素衍生挥发物对提升番茄风味至关重要。为筛选调控类胡萝卜素衍生挥发物合成的关键基因,以90个番茄自交系中香气寡淡的TI4001和香气浓郁的CI1005为材料,分析了番茄类胡萝卜素裂解双加氧酶(SlCCDs)基因在不同组织及不同发育期果实中的表达量,果实不同成熟期类胡萝卜素及其衍生挥发物的含量。发现在7个SlCCDs基因中,SlCCD1A和SlCCD1B基因在番茄果实中表达量最高,且随着果实发育成熟表达量显著升高。果实中类胡萝卜素及其衍生挥发物含量也显著升高。SlCCD1A和SlCCD1B基因表达量与类胡萝卜素及其衍生挥发物含量之间极显著正相关。推测SlCCD1A和SlCCD1B基因是裂解类胡萝卜素合成挥发物的关键基因。     

Carotenoid biosynthesis genes in rice: structural analysis, genome-wide expression profiling and phylogenetic analysis

Carotenoids, important lipid-soluble antioxidants in photosynthetic tissues, are known to be completely absent in rice endosperm. Many studies, involving transgenic manipulations of carotenoid biosynthesis genes, have been performed to get carotenoid-enriched rice grain. Study of genes involved in their biosynthesis can provide further information regarding the abundance/absence of carotenoids in different tissues. We have identified 16 and 34 carotenoid biosynthesis genes in rice and Populus genomes, respectively. A detailed analysis of the domain structure of carotenoid biosynthesis enzymes in rice, Populus and Arabidopsis has shown that highly conserved catalytic domains, along with other domains, are present in these proteins. Phylogenetic analysis of rice genes with Arabidopsis and other characterized carotenoid biosynthesis genes has revealed that homologous genes exist in these plants, and the duplicated gene copies probably adopt new functions. Expression of rice and Populus genes has been analyzed by full-length cDNA- and EST-based expression profiling. In rice, this analysis was complemented by real-time PCR, microarray and signature-based expression profiling, which reveal that carotenoid biosynthesis genes are highly expressed in light-grown tissues, have differential expression pattern during vegetative/reproductive development and are responsive to stress.     

Engineering novel carotenoids in microorganisms

A considerable number of microbial and plant carotenoid biosynthesis genes have been cloned over the past few years. Functional heterologous expression of most of these genes has made it possible to engineer carotenoid biosynthesis in non-carotenogenic E. coli and yeasts. Recently, gene combination and molecular breeding of pathways have been used to produce novel and rare high-value carotenoids.     

Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathways

The biosynthetic pathways of flavonoids and carotenoids have been well established, and the biosynthetic genes have been mostly isolated. Metabolic engineering of their biosynthetic pathways has provided not only novel colored or health-beneficial plants but also excellent models to study the efficacy of such engineering. In order to achieve a specific color by accumulating a corresponding compound, it is necessary to upregulate the pathway leading to the compound and downregulate the competing pathway. The regulation of gene expression has to be optimized in a target crop as well.     

The biosynthesis and nutritional uses of carotenoids

Carotenoids are isoprenoid molecules that are widespread in nature and are typically seen as pigments in fruits, flowers, birds and crustacea. Animals are unable to synthesise carotenoids de novo, and rely upon the diet as a source of these compounds. Over recent years there has been considerable interest in dietary carotenoids with respect to their potential in alleviating age-related diseases in humans. This attention has been mirrored by significant advances in cloning most of the carotenoid genes and in the genetic manipulation of crop plants with the intention of increasing levels in the diet. The aim of this article is to review our current understanding of carotenoid formation, to explain the perceived benefits of carotenoids in the diet and review the efforts that have been made to increase carotenoids in certain crop plants.     

Endosymbiotic bacteria as a source of carotenoids in whiteflies

Although carotenoids serve important biological functions, animals are generally unable to synthesize these pigments and instead obtain them from food. However, many animals, such as sap-feeding insects, may have limited access to carotenoids in their diet, and it was recently shown that aphids have acquired the ability to produce carotenoids by lateral transfer of fungal genes. Whiteflies also contain carotenoids but show no evidence of the fungus-derived genes found in aphids. Because many sap-feeding insects harbour intracellular bacteria, it has long been hypothesized that these endosymbionts could serve as an alternative source of carotenoid biosynthesis. We sequenced the genome of the obligate bacterial endosymbiont Portiera from the whitefly Bemisia tabaci. The genome exhibits typical signatures of obligate endosymbionts in sap-feeding insects, including extensive size reduction (358.2 kb) and enrichment for genes involved in essential amino acid biosynthesis. Unlike other sequenced insect endosymbionts, however, Portiera has bacterial homologues of the fungal carotenoid biosynthesis genes in aphids. Therefore, related lineages of sap-feeding insects appear to have convergently acquired the same functional trait by distinct evolutionary mechanisms—bacterial endosymbiosis versus fungal lateral gene transfer.     

Two genes encoding new carotenoid-modifying enzymes in the green sulfur bacterium Chlorobium tepidum

The green sulfur bacterium Chlorobium tepidum produces chlorobactene as its primary carotenoid. Small amounts of chlorobactene are hydroxylated by the enzyme CrtC and then glucosylated and acylated to produce chlorobactene glucoside laurate. The genes encoding the enzymes responsible for these modifications of chlorobactene, CT1987, and CT0967, have been identified by comparative genomics, and these genes were insertionally inactivated in C. tepidum to verify their predicted function. The gene encoding chlorobactene glucosyltransferase (CT1987) has been named cruC, and the gene encoding chlorobactene lauroyltransferase (CT0967) has been named cruD. Homologs of these genes are found in the genomes of all sequenced green sulfur bacteria and filamentous anoxygenic phototrophs as well as in the genomes of several nonphotosynthetic bacteria that produce similarly modified carotenoids. The other bacteria in which these genes are found are not closely related to green sulfur bacteria or to one another. This suggests that the ability to synthesize modified carotenoids has been a frequently transferred trait.     

The biotechnological potential and design of novel carotenoids by gene combination in Escherichia coli.

Carotenoids are antioxidants with considerable pharmaceutical potential. More than 600 carotenoid structures are known but their availability is limited owing to practical difficulties associated with chemical synthesis and isolation from microorganisms or plant tissue. To overcome some of these problems, heterologous expression of carotenoid genes in Escherichia coli can be used for the synthesis of rare derivatives or even of novel carotenoids. Novel and rare carotenoids can be obtained by combining carotenoid genes from different host species in E. coli.     

A relationship between carotenoid accumulation and the distribution of species of the fungus Neurospora in Spain

The ascomycete fungus Neurospora is present in many parts of the world, in particular in tropical and subtropical areas, where it is found growing on recently burned vegetation. We have sampled the Neurospora population across Spain. The sampling sites were located in the region of Galicia (northwestern corner of the Iberian peninsula), the province of Cáceres, the city of Seville, and the two major islands of the Canary Islands archipelago (Tenerife and Gran Canaria, west coast of Africa). The sites covered a latitude interval between 27.88° and 42.74°. We have identified wild-type strains of N. discreta, N. tetrasperma, N. crassa, and N. sitophila and the frequency of each species varied from site to site. It has been shown that after exposure to light Neurospora accumulates the orange carotenoid neurosporaxanthin, presumably for protection from UV radiation. We have found that each Neurospora species accumulates a different amount of carotenoids after exposure to light, but these differences did not correlate with the expression of the carotenogenic genes al-1 or al-2. The accumulation of carotenoids in Neurospora shows a correlation with latitude, as Neurospora strains isolated from lower latitudes accumulate more carotenoids than strains isolated from higher latitudes. Since regions of low latitude receive high UV irradiation we propose that the increased carotenoid accumulation may protect Neurospora from high UV exposure. In support of this hypothesis, we have found that N. crassa, the species that accumulates more carotenoids, is more resistant to UV radiation than N. discreta or N. tetrasperma. The photoprotection provided by carotenoids and the capability to accumulate different amounts of carotenoids may be responsible, at least in part, for the distribution of Neurospora species that we have observed across a range of latitudes.     

Introduction of new carotenoids into the bacterial photosynthetic apparatus by combining the carotenoid biosynthetic pathways of Erwinia herbicola and Rhodobacter sphaeroides.

Carotenoids have two major functions in bacterial photosynthesis, photoprotection and accessory light harvesting. The genes encoding many carotenoid biosynthetic pathways have now been mapped and cloned in several different species, and the availability of cloned genes which encode the biosynthesis of carotenoids not found in the photosynthetic genus Rhodobacter opens up the possibility of introducing a wider range of foreign carotenoids into the bacterial photosynthetic apparatus than would normally be available by producing mutants of the native biosynthetic pathway. For example, the crt genes from Erwinia herbicola, a gram-negative nonphotosynthetic bacterium which produces carotenoids in the sequence of phytoene, lycopene, beta-carotene, beta-cryptoxanthin, zeaxanthin, and zeaxanthin glucosides, are clustered within a 12.8-kb region and have been mapped and partially sequenced. In this paper, part of the E. herbicola crt cluster has been excised and expressed in various crt strains of Rhodobacter sphaeroides. This has produced light-harvesting complexes with a novel carotenoid composition, in which the foreign carotenoids such as beta-carotene function successfully in light harvesting. The outcome of the combination of the crt genes in R. sphaeroides with those from E. herbicola has, in some cases, resulted in an interesting rerouting of the expected biosynthetic sequence, which has also provided insights into how the various enzymes of the carotenoid biosynthetic pathway might interact. Clearly this approach has considerable potential for studies on the control and organization of carotenoid biosynthesis, as well as providing novel pigment-protein complexes for functional studies.