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The crocin biosynthetic pathways in C. sativus and G. jasminoides. The precursors of carotenoids are generated via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The enzymes marked in green are known enzymes from the upstream carotenoid biosynthetic pathway, and those in red and purple are identified from C. sativus and G. jasminoides, respectively. GGPPS, geranylgeranyl diphosphate synthase; PSY, phytoene synthase; PDS, phytoene desaturase; Z-ISO, f-carotene isomerase; ZDS, fcarotene desaturase; CRTISO, carotenoid isomerase; LCYB, lycopene b-cyclase; BHY, b-carotene hydrolase; CCD, carotenoid cleavage dioxygenase; ALDH, aldehyde dehydrogenase; UGT, UDP-glucosyltransferase. Information on these enzymes can be found in Table 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Source publication
Crocins are a group of highly valuable apocarotenoid-derived pigments mainly produced in Crocus sativus stigmas and Gardenia jasminoides fruits, which display great pharmacological activities for human health, such as anticancer, reducing the risk of atherosclerosis, and preventing Alzheimer’s disease. However, traditional sources of crocins are no...
Contexts in source publication
Context 1
... b-D-glucosyl and b-D-gentiobiosyl, and according to the position and numbers of these two glycosyls, crocins could be divided into five forms, crocin-I, crocin-II, crocin-III, crocin-IV, and crocin-Ⅴ. To date, the crocin biosynthetic pathway in plants has been elucidated by transcriptome and genome sequencing of C. sativus and G. jasminoides (Fig. 1) ...
Context 2
... b-D-glucosyl and b-D-gentiobiosyl, and according to the position and numbers of these two glycosyls, crocins could be divided into five forms, crocin-I, crocin-II, crocin-III, crocin-IV, and crocin-Ⅴ. To date, the crocin biosynthetic pathway in plants has been elucidated by transcriptome and genome sequencing of C. sativus and G. jasminoides (Fig. 1) ...
Similar publications
Crocins are a group of highly valuable water-soluble carotenoids that are reported to have many pharmacological activities, such as anticancer properties, and the potential for treating neurodegenerative diseases including Alzheimer’s disease. Crocins are mainly biosynthesized in the stigmas of food–medicine herbs Crocus sativus L. and Gardenia jas...
Citations
... Crocin, also known as crocin A, is a water-soluble carotenoid isolated from the stigma of Crocus sativus L., which has cardioprotective activity [166]. There is experimental evidence that saffron not only alleviates I/R-induced left ventricular dysfunction and MI size, but also reduces the levels of Bax, caspase 3, miR-34a and endoplasmic reticulum stress biomarkers (GRP78 and CHOP), while increasing the levels of Bcl-2, Sirt1, Nrf2 and HO-1. ...
Myocardial ischemia is the leading cause of health loss from cardiovascular disease worldwide. Myocardial ischemia and hypoxia during exercise trigger the risk of sudden exercise death which, in severe cases, will further lead to myocardial infarction. The Nrf2 transcription factor is an important antioxidant regulator that is extensively engaged in biological processes such as oxidative stress, inflammatory response, apoptosis, and mitochondrial malfunction. It has a significant role in the prevention and treatment of several cardiovascular illnesses, since it can control not only the expression of several antioxidant genes, but also the target genes of associated pathological processes. Therefore, targeting Nrf2 will have great potential in the treatment of myocardial ischemic injury. Natural products are widely used to treat myocardial ischemic diseases because of their few side effects. A large number of studies have shown that the Nrf2 transcription factor can be used as an important way for natural products to alleviate myocardial ischemia. However, the specific role and related mechanism of Nrf2 in mediating natural products in the treatment of myocardial ischemia is still unclear. Therefore, this review combs the key role and possible mechanism of Nrf2 in myocardial ischemic injury, and emphatically summarizes the significant role of natural products in treating myocardial ischemic symptoms, thus providing a broad foundation for clinical transformation.
... To find a sustainable way of supplying crocins, researchers have developed various approaches to produce them, including plant cell suspension culture, heterologous biosynthesis, and total synthesis [16,17]. Crocins can be obtained from plant cell culture, but the production is prone to epigenetic silencing and toxic intermediates. ...
Crocins are water-soluble apocarotenoids isolated from the flowers of crocus and gardenia. They exhibit various pharmacological effects, including neuroprotection, anti-inflammatory properties, hepatorenal protection, and anticancer activity. They are often used as coloring and seasoning agents. Due to the limited content of crocins in plants and the high cost of chemical synthesis, the supply of crocins is insufficient to meet current demand. The biosynthetic pathways for crocins have been elucidated to date, which allows the heterologous production of these valuable compounds in microorganisms by fermentation. This review article provides a comprehensive overview of the chemistry, pharmacological activity, biosynthetic pathways, and heterologous production of crocins, aiming to lay the foundation for the large-scale production of these valuable natural products by using engineered microbial cell factories.
... Crocetin and crocin can be synthesized using microbial processes employing microorganisms, including Escherichia coli and Saccharomyces cerevisiae [24][25][26]. Crocetin and crocin production via microbial processes can be divided into two approaches: total microbial synthesis of crocetin and crocin from a raw carbon source, such as glucose, and whole-cell (or enzymatic) biotransformation of crocetin into crocin. However, comprehensive engineering of the crocin pathway in microbial hosts, particularly for crocin-3 and crocin-4, has not been documented yet. ...
Background
Crocin, a glycosylated apocarotenoid pigment predominantly found in saffron, has garnered significant interest in the field of biotechnology for its bioactive properties. Traditional production of crocins and their aglycone, crocetin, typically involves extraction from crocin-producing plants. This study aimed to develop an alternative biosynthetic method for these compounds by engineering the metabolic pathways of zeaxanthin, crocetin, and crocin in Escherichia coli strains.
Results
Employing a series of genetic modifications and the strategic overexpression of key enzymes, we successfully established a complete microbial pathway for synthesizing crocetin and four glycosylated derivatives of crocetin, utilizing glycerol as the primary carbon source. The overexpression of zeaxanthin cleavage dioxygenase and a novel variant of crocetin dialdehyde dehydrogenase resulted in a notable yield of crocetin (34.77 ± 1.03 mg/L). Further optimization involved the overexpression of new types of crocetin and crocin-2 glycosyltransferases, facilitating the production of crocin-1 (6.29 ± 0.19 mg/L), crocin-2 (5.29 ± 0.24 mg/L), crocin-3 (1.48 ± 0.10 mg/L), and crocin-4 (2.72 ± 0.13 mg/L).
Conclusions
This investigation introduces a pioneering and integrated microbial synthesis method for generating crocin and its derivatives, employing glycerol as a sustainable carbon feedstock. The substantial yields achieved highlight the commercial potential of microbial-derived crocins as an eco-friendly alternative to plant extraction methods. The development of these microbial processes not only broadens the scope for crocin production but also suggests significant implications for the exploitation of bioengineered compounds in pharmaceutical and food industries.
... Carotenoids are an important group of pigments found in plants, algae, bacteria, and fungi that can provide color from yellow to red, depending on their cellular accumulation levels, and can also participate in multiple biological functions, such as light harvesting and photoprotection [14,15]. One of the final products of carotenoid metabolism, crocin has been used in cosmetics, dietary supplements, and medicines; in the latter case, it has been used as an anticancer agent, to reduce the risk of atherosclerosis, and to help prevent Alzheimer's disease [5,[16][17][18][19]. Crocin has become known as "red gold" and is reputed to In this study, we review the latest progress on crocin biosynthesis in various host cells, which is a prerequisite for industrial production of crocin in large quantities. ...
... Many botanists are working to improve the breeding and cultivation techniques of C. sativus to increase production. It has been documented that C. sativus shows higher heterozygosity owing to its three homologous chromosomes, thus rendering efforts to alter its genome by traditional plant breeding, which follows Mendelian principles, a major challenge [18,36,37]. The breeding of polyploid plants requires the aggregation of each allele to achieve a state of polyploid homozygosity, but achieving this ideal with C. sativus would require considerable acreage, a great deal of manpower, and significant financial investment over a long period [37]. ...
... A comparatively high yield of crocins can be produced by chemical synthesis, but this approach suffers from the formation of many unwanted side products that make purifying the synthetic crocins complicated. Contamination with such side products represents a high risk when using such crocins as medicine or food ingredients [18,21,22]. Different biotechnological approaches, such as tissue culture and genetic engineering, have been applied as alternative bio-sustainable resources for the production of crocins [53,54]. ...
Crocin is one of the most valuable components of the Chinese medicinal plant Crocus sativus and is widely used in the food, cosmetics, and pharmaceutical industries. Traditional planting of C. sativus is unable to fulfill the increasing demand for crocin in the global market, however, such that researchers have turned their attention to the heterologous production of crocin in a variety of hosts. At present, there are reports of successful heterologous production of crocin in Escherichia coli, Saccharomyces cerevisiae, microalgae, and plants that do not naturally produce crocin. Of these, the microalga Dunaliella salina, which produces high levels of β-carotene, the substrate for crocin biosynthesis, is worthy of attention. This article describes the biosynthesis of crocin, compares the features of each heterologous host, and clarifies the requirements for efficient production of crocin in microalgae.
... Using k-means clustering, all the expressed genes were clustered into 48 clusters based on their expression profiles in different Fig. S21). The functional enrichment for clusters 3,5,13,15,17,20,34, and 43 that contained significantly or specifically expressed genes in stigmas showed that the terpenoid and carotenoid biosynthetic genes are significantly enriched (Supporting Information Fig. S22, Tables S19 and S20). In these clusters, 85 candidate transcription factors were further identified with FPKM >10. ...
Crocus sativus (saffron) is a globally autumn-flowering plant, and its stigmas are the most expensive spice and valuable herb medicine. Crocus specialized metabolites, crocins, are biosynthesized in distant species, Gardenia (eudicot) and Crocus (monocot), and the evolution of crocin biosynthesis remains poorly understood. With the chromosome-level Crocus genome assembly, we revealed that two rounds of lineage-specific whole genome triplication occurred, contributing important roles in the production of carotenoids and apocarotenoids. According to the kingdom-wide identification, phylogenetic analysis, and functional assays of carotenoid cleavage dioxygenases (CCDs), we deduced that the duplication, site positive selection, and neofunctionalization of Crocus-specific CCD2 from CCD1 members are responsible for the crocin biosynthesis. In addition, site mutation of CsCCD2 revealed the key amino acids, including I143, L146, R161, E181, T259, and S292 related to the catalytic activity of zeaxanthin cleavage. Our study provides important insights into the origin and evolution of plant specialized metabolites, which are derived by duplication events of biosynthetic genes.
... Crocins, a class of highly valuable apocarotenoids derived primarily from saffron, have significant pharmacological activity for treating human disorders [28]. CsCCD2 is the rate-limiting enzyme involved in the biosynthetic pathway of crocins in saffron. ...
Crocins are important natural products predominantly obtained from the stigma of saffron, and that can be utilized as a medicinal compound, spice, and colorant with significant promise in the pharmaceutical, food, and cosmetic industries. Carotenoid cleavage dioxygenase 2 (CsCCD2) is a crucial limiting enzyme that has been reported to be responsible for the cleavage of zeaxanthin in the crocin biosynthetic pathway. However, the catalytic activity of CsCCD2 on β-carotene/lycopene remains elusive, and the soluble expression of CsCCD2 remains a big challenge. In this study, we reported the functional characteristics of CsCCD2, that can catalyze not only zeaxanthin cleavage but also β-carotene and lycopene cleavage. The molecular basis of the divergent functionality of CsCCD2 was elucidated using bioinformatic analysis and truncation studies. The protein expression optimization results demonstrated that the use of a maltose-binding protein (MBP) tag and the optimization of the induction conditions resulted in the production of more soluble protein. Correspondingly, the catalytic efficiency of soluble CsCCD2 was higher than that of the insoluble one, and the results further validated its functional verification. This study not only broadened the substrate profile of CsCCD2, but also achieved the soluble expression of CsCCD2. It provides a firm platform for CsCCD2 crystal structure resolution and facilitates the synthesis of crocetin and crocins.
... Various pharmacological activities of crocin are of great significance to human health. It has the potential to anti-tumor, anti-inflammation, reduce atherosclerosis and prevent neurological diseases (7,15). Crocin is possible to prevent and treat thrombus because of its effects on promoting the growth and differentiation of endothelial cells (16). ...
... Over the years, the biosynthetic pathway of crocin in Crocus sativus L. and Gardenia jasminoides has been elucidated (Figure 1) [14][15][16]. Three molecules of isopentenyl diphosphate (IPP) react with dimethylallyl diphosphate (DMAPP), which are derived from the mevalonic acid (MVA) pathway or the meth-ylerythritol 4-phosphate (MEP) pathway to generate geranyl-geranyl diphosphate (GGPP) under the action of geranylgeranyl pyrophosphate synthase (GGPPS) [17]. ...
... In this study, the GjCCD4a we used was derived from G. jasminoides. According to previous studies, carotenoid cleavage dioxygenases from Bixa orellana, B. davidii, C. ancyrensis, and Crocus sativus have the function of cleaving carotenoids to produce crocetin dialdehyde [16]. Lycopene, β-carotene, and zeaxanthin are the main substrates of CCDs, but different CCDs have different substrate preferences. ...
Crocins are a group of highly valuable water-soluble carotenoids that are reported to have many pharmacological activities, such as anticancer properties, and the potential for treating neurodegenerative diseases including Alzheimer’s disease. Crocins are mainly biosynthesized in the stigmas of food–medicine herbs Crocus sativus L. and Gardenia jasminoides fruits. The distribution is narrow in nature and deficient in resources, which are scarce and expensive. Recently, the synthesis of metabolites in the heterologous host has opened up the potential for large-scale and sustainable production of crocins, especially for the main active compounds crocin I and crocin II. In this study, GjCCD4a, GjALDH2C3, GjUGT74F8, and GjUGT94E13 from G. jasminoides fruits were expressed in Nicotiana benthamiana. The highest total content of crocins in T1 generation tobacco can reach 78,362 ng/g FW (fresh weight) and the dry weight is expected to reach 1,058,945 ng/g DW (dry weight). Surprisingly, the primary effective constituents crocin I and crocin II can account for 99% of the total crocins in transgenic plants. The strategy mentioned here provides an alternative platform for the scale-up production of crocin I and crocin II in tobacco.
... In the study of Pan et al., it was found that the synthesis of Geniposide was dominant in the green fruit stage, while the expression level of the key genes in the red fruit stage was very high. It led to the increase of ion abundance of Crocin in pulp [45]. In particular, this study also found that there were differential expressions of Geniposide and Crocin genes in young and middle fruits during the morphological changes of gardenia fruit development. ...
Background
Gardenia jasminoides Ellis is a perennial evergreen shrub of G. jasminoides of Rubiaceae. Geniposide and Crocin are important components in the fruit of G. jasminoides. In addition to being used as medicinal materials, they are also widely used in food, medicine, cosmetics, and other fields. They have high medicinal value, economic value, and ornamental value. However, at present, the utilization rate of G. jasminoides resources is low, mainly focused on germplasm cultivation, primary processing, and clinical pharmacology, and there are few studies on the quality of Gardenia fruit.
Methods and results
Based on transcriptome sequencing and metabolic group analysis, the morphological and structural changes of Gardenia fruit with young fruit, middle fruit, and ripe fruit were analyzed, and the formation mechanism and content changes of Geniposide and Crocin in Gardenia fruit were studied. The content of Geniposide decreased with the development of fruit, so did the expression of the main structural gene GES, G10H, and IS in its synthesis pathway, while the content of Crocin increased with the development of fruit, and the expression of the main structural gene CCD, ALDH, and UGT in its synthesis pathway also increased. The relationship between the morphological structure of G. jasminoides and the accumulation of Geniposide and Crocin was summarized.
Conclusions
This study not only provides a theoretical basis for the mining and utilization of Geniposide and Crocin, but also provides a theoretical basis for genetic background for the identification and cloning of bioactive substances in gardenia fruit in future. At the same time, it provides support for increasing the dual-use value of G. jasminoides and breeding excellent germplasm resources.
... Carotenoids are the most widely distributed class pigments in nature, and their synthesis is regulated by a number of metabolic enzyme genes. Crocin compounds are the products of crocin biosynthesis and are often mediated by the carotenoid pathway in Saffron crocus, Crocus sativus, Buddleja officinalis and other plants [8,41,48]. The key gene-encoding enzymes for crocin synthesis in the upstream pathway, including DN81678_c1_g1_i5 (crtB), DN85606_c3_g3_i1 (PDS), and DN82463_c0_g1_i6 (ZDS), and key genes encoding enzymes for crocin synthesis in the middle-downstream pathway including DN81253_c0_g1_i1 (lcyB), DN79477_c0_g1_i2 (lcyE), DN84511_c2_g4_i1 (LUT), and DN84975_c1_g7_i11 (CCD) were identified, which showed these genes significantly expressed during the G. jasminoides fruit developmental stages. ...
Gardenia jasminoides fruits are extensively grown worldwide, with a large harvest, and its major medicinal ingredients are geniposide and crocins. Research on their accumulation and biosynthsis-related enzymes is rare. In this study, the accumulation of geniposide and crocin of G. jasminoides fruits at different developmental stages were clarified by HPLC. The highest cumulative amount of geniposide was 2.035% during the unripe-fruit period, and the highest content of crocin was 1.098% during the mature-fruit period. Furthermore, transcriptome sequencing was performed. A total of 50 unigenes encoding 4 key enzymes related in geniposide biosynthsis pathways were screened, and 41 unigenes encoding 7 key enzymes in the pathways of crocin were elucidated. It was found that the expression levels of differentially expressed genes of DN67890_c0_g1_i2-encoding GGPS, which is highly related to geniposide biosynthesis, and DN81253_c0_g1_i1-encoding lcyB, DN79477_c0_g1_i2-encoding lcyE, and DN84975_c1_g7_i11-encoding CCD, which are highly related to crocin biosynthesis, were consistent with the accumulation of geniposide and crocin content, respectively. The qRT-PCR results showed that the trends of relative expression were consistent with transcribed genes. This study provides insights for understanding the geniposide and crocin accumulation and biosynthsis during fruit development in G. jasminoides.
