Crocin Biosynthesis Research Articles

Transcriptome-wide Characterization of Novel WRKY Family Genes Engaged in Crocin Biosynthesis in Crocus sativus

Crocins comprise a family of hydrophilic carotenoids with pharmacological properties that are produced in significant quantities in stigma of Crocus sativus. Although the biosynthesis pathway of crocins has been sufficiently elucidated, there is a paucity of information regarding how transcription factors (TFs) regulate crocin biosynthesis in various stigma developmental stages. WRKY TFs play a role in modulating carotenoid/apocarotenoid metabolism. To provide an overview of the WRKY family in Crocus sativus (CsWRKY) and characterize candidate CsWRKY TFs involved in the biosynthesis of crocins, CsWRKY genes were identified from RNA-sequenced stigma at different developmental stages. A phylogenetic analysis was performed to characterize their evolutionary interrelatedness. A coexpression analysis of CsWRKY genes and crocin biosynthesis-related genes was performed. A quantitative real-time polymerase chain reaction was used to corroborate the expression level of CsWRKY TFs in various tissue and at different developmental stages. A total of 34 CsWRKY TFs were identified from the stigma of C. sativus. The CsWRKY TFs, together with their orthologs from Arabidopsis, were clustered into group I, II, or III following phylogenetic analysis. A correlation analysis revealed that the expressions of the TFs CsWRKY1, CsWRKY2, CsWRKY8, CsWRKY10, CsWRKY15, and CsWRKY28 were strongly related to the expression of crocin biosynthesis-related genes CsBCH, CsCCD2L, CsALDH, and CsUGT. CsWRKY2, CsWRKY15, and CsWRKY28 exhibited identical motifs and were stratified into group IId. Transcript levels of candidate CsWRKY genes were higher in stigma than in other tissues and were proportional to the crocin content.

A novel mutation in non-constitutive lycopene beta cyclase (CstLcyB2a) from Crocus sativus modulates carotenoid/apocarotenoid content, biomass and stress tolerance in plants.

Mutation at A126 in lycopene-β-cyclase of Crocus (CstLcyB2a) sterically hinders its binding of δ-carotene without affecting lycopene binding, thereby diverting metabolic flux towards β-carotene and apocarotenoid biosynthesis. Crocus sativus, commonly known as saffron, has emerged as an important crop for research because of its ability to synthesize unique apocarotenoids such as crocin, picrocrocin and safranal. Metabolic engineering of the carotenoid pathway can prove a beneficial strategy for enhancing the quality of saffron and making it resilient to changing climatic conditions. Here, we demonstrate that introducing a novel mutation at A126 in stigma-specific lycopene-β-cyclase of Crocus (CstLcyB2a) sterically hinders its binding of δ-carotene, but does not affect lycopene binding, thereby diverting metabolic flux towards β-carotene formation. Thus, A126L-CstLcyB2a expression in lycopene-accumulating bacterial strains resulted in enhanced production of β-carotene. Transient expression of A126L-CstLcyB2a in C. sativus stigmas enhanced biosynthesis of crocin. Its stable expression in Nicotiana tabacum enhanced β-branch carotenoids and phyto-hormones such as abscisic acid (ABA) and gibberellic acids (GA's). N. tabacum transgenic lines showed better growth performance and photosynthetic parameters including maximum quantum efficiency (Fv/Fm) and light-saturated capacity of linear electron transport. Exogenous application of hormones and their inhibitors demonstrated that a higher ratio of GA4/ABA has positive effects on biomass of wild-type and transgenic plants. Thus, these findings provide a platform for the development of new-generation crops with improved productivity, quality and stress tolerance.

Metabolic engineering of crocins and picrocrocin apocarotenoids in potato group phureja

AbstractThe stigmas of Crocus sativus accumulate the exclusive apocarotenoids crocin and picrocrocin, which are dried and desiccated to make commercial saffron. In addition to providing characteristic organoleptic qualities, saffron apocarotenoids are valuable compounds in the pharmaceutical and health industries. Previously, we developed Desirée potato tubers enriched with these apocarotenoids which also showed increased potential benefits for human health. In the current study, Solanum tuberosum (S. tuberosum) Group Phureja 01H15 which accumulates high levels of zeaxanthin, was engineered to produce saffron apocarotenoids to increase the levels of these metabolites in potato tubers using a construct namely O6, which contains the CsCCD2L, UGT74AD1, and UGT709G1 genes necessary for the biosynthesis of crocin and picrocrocin under the control of the patatin promoter. Here, we obtained transgenic S. tuberosum Group Phureja 01H15 lines with high concentrations of crocins and picrocrocin (up to 3.648 mg/g DW, 2.345 mg/g DW, respectively), which were up to 10 and 3 times higher than those obtained in the Desirée background, respectively. Furthermore, we performed transcriptome analyses of tubers from Desirée and 01H15 wild type and carrying O6 construct. Differentially expressed gene analysis revealed transcript changes not only on tuber carotenoid and apocarotenoid genes but also in other related pathways, suggesting a possible role in isoprenoid metabolism remodeling. Thus, this heterologous system serves as a robust platform for the production of these valuable metabolites.

Construction of a high-efficiency GjCCD4a mutant and its application for de novo biosynthesis of five crocins in Escherichia coli

Crocins are bioactive natural products that rarely exist in plants. High costs and resource shortage severely limit its development and application. Synthetic biology studies on crocins are of considerable global interest. However, the lack of high-efficiency genetic tools and complex cascade biocatalytic systems have substantially hindered progress in crocin biosynthesis-related research. Based on mutagenesis, a high-efficiency GjCCD4a mutant (N212m) was constructed with a catalytic efficiency that was 25.08-fold higher than that of the wild-type. Solubilized GjCCD4a was expressed via fusion with an MBP tag. Moreover, N212m and ten other genes were introduced into Escherichia coli for the de novo biosynthesis of five crocins. The engineered E57 strain produced crocins III and V with a total yield of 11.50 mg/L, and the E579 strain produced crocins I-V with a total output of 8.43 mg/L at shake-flask level. This study identified a marvelous genetic element (N212m) for crocin biosynthesis and achieved its de novo biosynthesis in E. coli using glucose. This study provides a reference for the large-scale production of five crocins using E. coli cell factories.

Abiotic stress tolerance in pearl millet: Unraveling molecular mechanisms via transcriptomics.

Pearl millet (Pennisetum glaucum (L.)) is a vital cereal crop renowned for its ability to thrive in challenging environmental conditions; however, the molecular mechanisms governing its salt stress tolerance remain poorly understood. To address this gap, next-generation RNA sequencing was conducted to compare gene expression patterns in pearl millet seedlings exposed to salt stress with those grown under normal conditions. Our RNA sequencing analysis focused on shoots from 13-day-old pearl millet plants subjected to either salinity stress (150 mmol of NaCl for 3 days) or thermal stress (50°C for 60 s). Of 36,041 genes examined, 17,271 genes with fold changes ranging from 2.2 to 19.6 were successfully identified. Specifically, 2388 genes were differentially upregulated in response to heat stress, whereas 4327 genes were downregulated. Under salt stress conditions, 2013 genes were upregulated and 4221 genes were downregulated. Transcriptomic analysis revealed four common abiotic KEGG pathways that play crucial roles in the response of pearl millet to salt and heat stress: phenylpropanoid biosynthesis, photosynthesis-antenna proteins, photosynthesis, and plant hormone signal transduction. These metabolic pathways are necessary for pearl millet to withstand and adapt to abiotic stresses caused by salt and heat. Moreover, the pearl millet shoot heat stress group showed specific transcriptomics related to KEEG metabolic pathways such as cytochrome P450, cutin, suberine, and wax biosynthesis, zeatin biosynthesis, crocin biosynthesis, ginsenoside biosynthesis, saponin biosynthesis, and biosynthesis of various plant secondary metabolites. In contrast, pearl millet shoots exposed to salinity stress exhibited transcriptomic changes associated with KEEG metabolic pathways related to carbon fixation in photosynthetic organisms, mismatch repair, and nitrogen metabolism. Our findings underscore the remarkable cross-tolerance of pearl millet to simultaneous salt and heat stress, elucidated through the activation of shared abiotic KEGG pathways. This study emphasizes the pivotal role of transcriptomics analysis in unraveling the molecular responses of pearl millet under abiotic stress conditions.

Research Progress in Heterologous Crocin Production.

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.

Crocus genome reveals the evolutionary origin of crocin biosynthesis

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.

Production of Saffron Apocarotenoids in Nicotiana benthamiana Plants Genome-Edited to Accumulate Zeaxanthin Precursor.

Crocins are glycosylated apocarotenoids with strong coloring power and anti-oxidant, anticancer, and neuro-protective properties. We previously dissected the saffron crocin biosynthesis pathway, and demonstrated that the CsCCD2 enzyme, catalyzing the carotenoid cleavage step, shows a strong preference for the xanthophyll zeaxanthin in vitro and in bacterio. In order to investigate substrate specificity in planta and to establish a plant-based bio-factory system for crocin production, we compared wild-type Nicotiana benthamiana plants, accumulating various xanthophylls together with α- and β-carotene, with genome-edited lines, in which all the xanthophylls normally accumulated in leaves were replaced by a single xanthophyll, zeaxanthin. These plants were used as chassis for the production in leaves of saffron apocarotenoids (crocins, picrocrocin) using two transient expression methods to overexpress CsCCD2: agroinfiltration and inoculation with a viral vector derived from tobacco etch virus (TEV). The results indicated the superior performance of the zeaxanthin-accumulating line and of the use of the viral vector to express CsCCD2. The results also suggested a relaxed substrate specificity of CsCCD2 in planta, cleaving additional carotenoid substrates.

Elucidation of Geniposide and Crocin Accumulation and Their Biosysnthsis-Related Key Enzymes during Gardenia jasminoides Fruit Growth.

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.

Enhanced Production of Apocarotenoids by Salicylic Acid Elicitation in Cell Suspension Cultures of Saffron (Crocus sativus L.)

A cell suspension culture of saffron (Crocus sativus L.) was previously established from style-derived calli to obtain an in vitro system for crocin, an uncommon and valuable water-soluble apocarotenoid, and carotenoid production suitable for future scaling up. To shed more light on the correlation between apocarotenoid biosynthesis and key-gene expression, in this study, SA was used at 0.5 mM concentration to elicit crocin production and the effects on carotenoid production were analyzed after 6, 12, 24, and 48 h. HPLC-DAD analysis was used for total crocin quantification as well as the other carotenoids zeaxanthin, β-carotene and lutein. Quantitative RT-PCR was used to analyze the transcript levels of saffron apocarotenoid biosynthetic key genes PSY (phytoene synthase), BCH1 (β-carotene hydroxylase), and CCD2 (carotenoid cleavage dioxygenase) after SA elicitation. In saffron suspension-cultured cells elicited by SA, the carotenoid biosynthetic pathway was mostly enhanced toward crocin biosynthesis, known to exert strong biological activity and therapeutic effects, rather than lutein or xanthins. SA increased BCH1 and CCD2 gene expression 15.6 and 3.3 times, respectively, compared to the control at 24 h after elicitation. Although a dynamic change of metabolite contents and gene expression was observed during the 48 h time course in response to SA elicitation, the changes of zeaxanthin and crocin were consistent with the regulation of the corresponding genes BCH and CCD2 during the time course. In conclusion, the effects of SA on regulation of gene expression in the apocarotenoid pathway could be successfully applied for the biotechnological production of crocin.

Integrated SMRT and Illumina Sequencing Provide New Insights into Crocin Biosynthesis of Gardenia jasminoides.

Crocins are valuable bioactive components of gardenia fruit, and their biosynthesis and accumulation have attracted widespread interest. Studies have investigated the biosynthesis and accumulation of crocin based on Illumina sequencing, but there is a lack of reports based on full-length transcriptome sequencing. Utilising SMRT sequencing and high-performance liquid chromatography (HPLC), we explored crocin biosynthesis and accumulation in the fruit of Gardenia jasminoides. HPLC analysis showed that crocins specifically exist in fruit and that the content of crocins increases gradually during fruit development. SMRT sequencing generated 46,715 high-quality full-length isoforms, including 5230 novel isoforms that are not present in the G. jasminoides genome. Furthermore, a total of 46 genes and 91 lncRNAs were involved in the biosynthesis and accumulation of crocin. The qRT-PCR indicated that genes involved in crocin biosynthesis reached a peak in the NOV stage. These findings contributed to our understanding of crocin biosynthesis and accumulation.

Metabolic Engineering of Crocin Biosynthesis in Nicotiana Species.

Crocins are high-value soluble pigments that are used as colorants and supplements, their presence in nature is extremely limited and, consequently, the high cost of these metabolites hinders their use by other sectors, such as the pharmaceutical and cosmetic industries. The carotenoid cleavage dioxygenase 2L (CsCCD2L) is the key enzyme in the biosynthetic pathway of crocins in Crocus sativus. In this study, CsCCD2L was introduced into Nicotiana tabacum and Nicotiana glauca for the production of crocins. In addition, a chimeric construct containing the Brevundimonas sp. β-carotene hydroxylase (BrCrtZ), the Arabidopsis thaliana ORANGE mutant gene (AtOrMut), and CsCCD2L was also introduced into N. tabacum. Quantitative and qualitative studies on carotenoids and apocarotenoids in the transgenic plants expressing CsCCD2L alone showed higher crocin level accumulation in N. glauca transgenic plants, reaching almost 400 μg/g DW in leaves, while in N. tabacum 36 μg/g DW was obtained. In contrast, N. tabacum plants coexpressing CsCCD2L, BrCrtZ, and AtOrMut accumulated, 3.5-fold compared to N. tabacum plants only expressing CsCCD2L. Crocins with three and four sugar molecules were the main molecular species in both host systems. Our results demonstrate that the production of saffron apocarotenoids is feasible in engineered Nicotiana species and establishes a basis for the development of strategies that may ultimately lead to the commercial exploitation of these valuable pigments for multiple applications.

Metabolomics integrated with transcriptomics reveals the distribution of iridoid and crocin metabolic flux in Gardenia jasminoides Ellis.

Gardenia jasminoides Ellis (G. jasminoides) fruits are used as a resource for obtaining natural colorants and in traditional Chinese herbal medicine. However, G. jasminoides presents a relatively long flowering period and different ripening periods, so there are significant differences in the accumulation of metabolites in fruits of different colors. In addition, the complete metabolic pathways of iridoidsand crocins, which are used as medicinal composition of G. jasminoides, are poorly understood at present. In this research, we comprehensively compared the transcriptome and metabolites profiles of the developmental stages and locations of iridoid and crocin biosynthesis. A large number of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were detected in four groups of samples, and clear variation in the pattern of metabolite abundance and gene expression were observed among different fruit colors and parts. Geniposide and gardenoside mainly accumulated in the sarcocarp of green fruit (GFS) and the sarcocarp of red fruit (FS), respectively. Crocin mainly accumulated in the peel and sarcocarp of red fruits. In the iridoid pathway, we hypothesized that there was a transport mechanism from the sarcocarp to the peel of G. jasminoides because of the inconsistent expression of G8O, 10-HGO and IS associated with differences in fruit ripening. UGTs play an important role in the biosynthesis of the active components of G. jasminoides. Combined transcriptome and metabonomics analysis showed a negative correlation between the biosynthesis of geniposide and crocin. The redirection of the metabolic flux and the regulation of key enzymes may be the main reasons for the changes in the biosynthesis of iridoid and crocin in G. jasminoides fruit. Our study expended valuable information for functional genomic library and provided new insights for metabolic engineering of secondary metabolite in G. Jasminoides.

Transcriptomic analysis of saffron at different flowering stages using RNA sequencing uncovers cytochrome P450 genes involved in crocin biosynthesis.

Saffron is a well-known Chinese traditional herb, and crocin biosynthesis is related to the yield and quality of saffron. This study aimed to screen differentially expressed genes (DEGs) in saffron at different flowering stages and identify cytochrome P450 (CYP) genes involved in crocin biosynthesis. Saffron samples at different flowering stages were used for RNA sequencing, and DEGs between the samples at three days before the flowering stage (- 3da) and two days after the flowering stage (+ 2da) were screened. Thereafter, significantly differentially expressed CYP genes were identified, and CYP gene expression at different flowering stages and in various tissues of saffron was determined using real-time quantitative polymerase chain reaction (RT-qPCR). After sequencing and analysis, 1508 DEGs between the samples at - 3da and + 2da were identified, including 487 upregulated and 1021 downregulated genes, which were enriched in 16 biological processes, 5 cellular components, 3 molecular functions, and 11 KEGG pathways, including protein processing in endoplasmic reticulum, pentose and glucuronate interconversions, starch and sucrose metabolism, estrogen signaling pathway, and mitogen-activated protein kinase signaling pathway. In addition, 12 significantly differentially expressed CYP genes were identified. The RT-qPCR results showed that CYP76C4, CYP72A15, CYP72A219, CYP97B2, CYP714C2, CYP71A1, CYP94C1, and CYP86A8 were all expressed in the pistils, and CYP72A219, CYP72A15, CYP97B2, CYP71A1, and CYP86A8 were highly expressed in the pistils. Our study established a transcriptome library of saffron and found that CYP72A219, CYP72A15, CYP97B2, CYP71A1, and CYP86A8 may be candidates involved crocin biosynthesis in saffron.

Expression of Genes Involved in the Biosynthesis of Crocin and Safranal in Cell Suspension Culture of Saffron (Crocus sativus)

Saffron possesses important medicinal properties, all of which are due to the secondary metabolites such as crocin, picrocrocin, and safranal. This study aims at investigating biosynthesis of the secondary metabolites during growth of saffron callus cells in a suspension culture, and examining the expression of genes controlling the metabolites. Sterilized saffron corms were cultured in a MS½ medium supplemented by 2-4-D and BAP. The calluses were induced after fourteen days, and then put into the suspension culture after being four times subcultured. From the 8th to 14th day after establishment of the suspension culture, the amount of secondary metabolites was measured by HPLC, and gene expressions were analyzed with real-time PCR for three samples taken once a day at the same time from the suspension. It was found that the amount of secondary metabolites and expression of the corresponding genes (CsLYC, CsBCH and CsGT2) were saturated on the 12th day after establishment of the suspension. For the most effective stimulation of genes and biosynthesis of apocarotenoids, suitable elicitors and precursors should be added within 72 h before saturation of the synthesis rate which happens on the 12th day after establishment of the suspension.

Plant apocarotenoid metabolism utilizes defense mechanisms against reactive carbonyl species and xenobiotics.

Carotenoid levels in plant tissues depend on the relative rates of synthesis and degradation of the molecules in the pathway. While plant carotenoid biosynthesis has been extensively characterized, research on carotenoid degradation and catabolism into apocarotenoids is a relatively novel field. To identify apocarotenoid metabolic processes, we characterized the transcriptome of transgenic Arabidopsis (Arabidopsis thaliana) roots accumulating high levels of β-carotene and, consequently, β-apocarotenoids. Transcriptome analysis revealed feedback regulation on carotenogenic gene transcripts suitable for reducing β-carotene levels, suggesting involvement of specific apocarotenoid signaling molecules originating directly from β-carotene degradation or after secondary enzymatic derivatizations. Enzymes implicated in apocarotenoid modification reactions overlapped with detoxification enzymes of xenobiotics and reactive carbonyl species (RCS), while metabolite analysis excluded lipid stress response, a potential secondary effect of carotenoid accumulation. In agreement with structural similarities between RCS and β-apocarotenoids, RCS detoxification enzymes also converted apocarotenoids derived from β-carotene and from xanthophylls into apocarotenols and apocarotenoic acids in vitro. Moreover, glycosylation and glutathionylation-related processes and translocators were induced. In view of similarities to mechanisms found in crocin biosynthesis and cellular deposition in saffron (Crocus sativus), our data suggest apocarotenoid metabolization, derivatization and compartmentalization as key processes in (apo)carotenoid metabolism in plants.

Research progress on biosynthesis and synthetic biology of crocin

Natural products are important resources for drug discovery and have marvelous roles in the treatment of various diseases, such as malaria, cancers, and viral infections. Crocins are the colored apocarotenoids derived from the stigmas of Crocus sativus and often used as a spice/colorant in the food industry. Pharmacologically, crocins confer protection to the central nervous system (especially in the treatment of Alzheimer’s disease) and prevent cardiovascular/cerebrovascular diseases. Unfortunately, the demand for crocins based on their pharmacological benefits combined with a complex harvesting process of C. sativus has driven the retail price of C. sativus stigmas up to 2000–7000 €/kg. Thus, their expense and limited supply greatly restrict the development and clinical application of crocin-based pharmaceuticals. It is therefore urgent to develop an alternative, sustainable way to bolster the supply of crocins. In recent years, synthetic biology has been widely used to overcome shortages of plant-based natural products, including artemisinic acid, etoposide aglycone, opioids, cannabinoids, breviscapine, and ginsenosides. As such, the genetic tools required for crocin biosynthesis have attracted a great deal of attention. The crocin biosynthetic pathway includes three stages: Stage (I), the precursor geranylgeranyl pyrophosphate (GGPP) is generated by the methylerythritol-4-phosphate (MEP) biosynthetic pathway; stage (II), C40-carotenoids are produced via the carotenoid biosynthetic pathway; stage (III), crocins are synthesized by three key catalytic steps, namely, carotenoid cleavage, aldehyde oxidation, and glucosyl group transfer. A series of studies have successfully decoded the crocin biosynthetic pathway and led to its reconstitution in Saccharomyces cerevisiae and Escherichia coli . Hereby, we summarized the chemical characterization of crocins using spectroscopy and mass spectrometry, and elucidated stage (III) of the crocin biosynthetic pathway. Carotenoid cleavage dioxygenases (CCDs) degrade carotenoids. CsCCD2, CaCCD2 ( Crocus ancyrensis ), BdCCD4.1 ( Buddleja davidii ), BdCCD4.3 and GjCCD4a ( Gardenia jasminoides ) can cleave the 7,8 (7′,8′) double bond of zeaxanthin to yield crocetin dialdehyde. Subsequently, crocetin dialdehyde was oxidized to crocetin by the catalysis of aldehyde dehydrogenases (ALDHs), including CsALDH54788, CsALDH3898, CsALDH20158, CsALDH11367, CsALDH3I1, CsALDH3, and GjALDH2C3. Finally, the biosynthesis of crocins from crocetin was catalyzed by UDP-glycosyltransferases (UGTs), including CsUGT74AD1, GjUGT74F8, GjUGT75L6, GjUGT94E5, GjUGT94E13, Bs-GT ( Bacillus subtilis 168), and Bc-GTA ( Bacillus cereus WQ9-2). And then, we displayed the research progress on the synthetic biology of crocins. With the rapid updating of genetic tools, we have the potential to generate more types of crocins. The in vivo production of crocins is highlighted in this review. The engineered E. coli with GjUGT74F8 and GjUGT94E13 can catalyze crocetin into five types of crocins. Furthermore, we put forward some suggestions that might help to improve the bioproduction of crocins, such as the exploration of evolutionary mechanisms of the key enzymes and the optimization of reaction conditions, etc. Taken together, this review will facilitate the industrial production of crocins and the development of crocin-based pharmaceuticals, providing a theoretical basis and method guidance for the sustainable development of crocin-based traditional Chinese medicine.

Comprehensive transcriptome analyses of different Crocus flower tissues uncover genes involved in crocin biosynthesis

The stigma of Crocus sativus is used in traditional Chinese medicine and has drawn attention as a rich source of crocin, a compound with a reported activity that counters various cancers, depression, and cardiovascular diseases. However, our knowledge of crocin biosynthesis in Crocus is still limited. To identify the genes that encode key enzymes responsible for crocin production, transcriptome analyses of Crocus stigma, petal, and stamen were performed. There were 109 136 unigenes in the three Crocus flower tissues: 10 862 unigenes were expressed explicitly in stigmas. A total of 469 and 335 down-regulated differentially expressed genes in comparison to stigmas were detected in stamens and petals, respectively. There were 290 down-regulated genes in both tissues. The gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes analysis revealed that most genes in the two tissues served a similar function. The results of the differences of gene expression among the three tissues showed that most of the carotenoid pathway genes in stigmas had a high expression which was a benefit for producing zeaxanthin, a substrate of crocin. Fourteen candidate genes were identified in Crocus from the co-down-regulated genes, and the genes were expressed explicitly in stigmas and were predicted to be involved in crocin biosynthesis. Overall, the expressions of genes in the crocin biosynthesis pathway in the three Crocus flower tissues had a positive relationship with their crocin content, and some new candidate genes related to crocin biosynthesis were identified. The results can help elucidate the crocin biosynthesis pathway in Crocus.

Cloning and functional characterization of a carotenoid cleavage dioxygenase 2 gene in safranal and crocin biosynthesis from Freesia hybrida

Safranal and crocin, commonly derived from the oxidative cleavage reaction of zeaxanthin in plants, are two kinds of apocarotenoids with versatile functions, which were only found in limited number of plant species. In this study, both metabolites were detected and varied concomitantly with the expression of carotenoid cleavage dioxygenase (CCD) genes in Freesia hybrida, Red River® and Ambiance cultivars. The newly isolated CCD, denoted here as FhCCD2, was phylogenetically clustered with other reported saffron CCD2s. Besides, ten introns were also observed in the genomic DNA sequence of FhCCD2 and the presence of N-terminal transporter peptide suggested its plastidial sub-localization. Biochemical analysis showed that the FhCCD2 cleaved zeaxanthin at the 7, 8 and 7′, 8’ double bonds to generate intermediates prerequisite for the biosynthesis of safranal and crocin. Further, gene transient expression analysis showed that the promoter of FhCCD2 was functional in Ambiance as well as Red River® cultivars, even with slight variation in their promoter sequence. At present, CCD2 proteins have only been found in Freesia and Crocus genus of Iridaceae family. Phylogenetic and intron position analysis infer that CCD2 perhaps emerged after the intron loss during evolutionary process of CCD1 or their shared ancestry.

Tandem gene duplications drive divergent evolution of caffeine and crocin biosynthetic pathways in plants

BackgroundPlants have evolved a panoply of specialized metabolites that increase their environmental fitness. Two examples are caffeine, a purine psychotropic alkaloid, and crocins, a group of glycosylated apocarotenoid pigments. Both classes of compounds are found in a handful of distantly related plant genera (Coffea, Camellia, Paullinia, and Ilex for caffeine; Crocus, Buddleja, and Gardenia for crocins) wherein they presumably evolved through convergent evolution. The closely related Coffea and Gardenia genera belong to the Rubiaceae family and synthesize, respectively, caffeine and crocins in their fruits.ResultsHere, we report a chromosomal-level genome assembly of Gardenia jasminoides, a crocin-producing species, obtained using Oxford Nanopore sequencing and Hi-C technology. Through genomic and functional assays, we completely deciphered for the first time in any plant the dedicated pathway of crocin biosynthesis. Through comparative analyses with Coffea canephora and other eudicot genomes, we show that Coffea caffeine synthases and the first dedicated gene in the Gardenia crocin pathway, GjCCD4a, evolved through recent tandem gene duplications in the two different genera, respectively. In contrast, genes encoding later steps of the Gardenia crocin pathway, ALDH and UGT, evolved through more ancient gene duplications and were presumably recruited into the crocin biosynthetic pathway only after the evolution of the GjCCD4a gene.ConclusionsThis study shows duplication-based divergent evolution within the coffee family (Rubiaceae) of two characteristic secondary metabolic pathways, caffeine and crocin biosynthesis, from a common ancestor that possessed neither complete pathway. These findings provide significant insights on the role of tandem duplications in the evolution of plant specialized metabolism.