Background: Plants 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. Results: Here, 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.
Gardenia jasminoides is used in traditional Chinese medicine and has drawn attention as a rich source of crocin, a compound with reported activity against various cancers, depression and cardiovascular disease. However, genetic information on the crocin biosynthetic pathway of G. jasminoides is scarce. In this study, we performed a transcriptome analysis of the leaves, green fruits, and red fruits of G. jasminoides to identify and predict the genes that encode key enzymes responsible for crocin production, compared with Crocus sativus. Twenty-seven putative pathway genes were specifically expressed in the fruits, consistent with the distribution of crocin in G. jasminoides. Twenty-four of these genes were reported for the first time, and a novel CCD4a gene was predicted that encodes carotenoid cleavage dioxygenase leading to crocin synthesis, in contrast to CCD2 of C. sativus. In addition, 6 other candidate genes (ALDH12, ALDH14, UGT94U1, UGT86D1, UGT71H4, and UGT85K18) were predicted to be involved in crocin biosynthesis following phylogenetic analysis and different gene expression profiles. Identifying the genes that encode key enzymes should help elucidate the crocin biosynthesis pathway.
Crocins are highly valuable medicinal compounds for treating human disorders, and they also serve as spices and coloring agents. However, the supply of crocins from plant extractions is insufficient for current demands, and using synthetic biology to produce crocins remains a big challenge. Here, we report the in vivo production of five types of crocins in E. coli with GjUGT94E13 and GjUGT74F8, which are responsible for the glycosylation of crocetin, from the crocin-producing plant Gardenia jasminoides. Subsequently, native UDP-glucose biosynthesis in E. coli is strengthened by the overexpression of pgm and galU. The optimization of catalytic reactions has demonstrated that 50 mM NaH 2 PO 4 −Na 2 HPO 4 buffer (pH 8.0) plus 5% glucose is the best medium to use for the efficient glycosylation of crocetin. In engineered E. coli, the conversion rate of crocin III and crocin V from crocetin (50 mg/L) by the catalysis of GjUGT74F8 was increased to 66.1%, and the conversion rate of five types of crocins from crocetin (50 mg/L) via GjUGT94E13 and GjUGT74F8 was 59.6%, much higher than the catalytic activity of the reported microbial UGTs. This study not only sheds light on the in vivo biosynthesis of crocins in E. coli, but also provides important genetic tools for the de novo synthesis of crocins.
With changes in human dietary patterns, the proportion of high-fat and high-cholesterol foods in the daily diet has increased. As a result, the incidence rate of cholelithiasis is increasing rapidly. Many studies have reported on the crucial role that the intestinal microflora plays in the progression of gallstones. Although the whole herb of Lysimachia christinae, a traditional Chinese medicine, has long been extensively used as a remedy for cholelithiasis in China, its effects on the intestinal microflora remain unknown. Hence, in this study, we investigated the ability of the aqueous extract of L. christinae (LAE) to prevent cholesterol gallstones (CGSs) in model animals by affecting the intestinal microflora. The effects of LAE on body weight, serum lipid profile, visceral organ indexes, and histomorphology were studied in male C57BL/6J mice, which were induced by a lithogenic diet. After the 8-week study, CGSs formation was greatly reduced after LAE treatment. LAE also reduced body weight gain and hyperlipidemia and restored the histomorphological changes. Moreover, the intestinal microflora exhibited significant variation. In the model group fed the lithogenic diet, the abundances of the genera unclassified Porphyromonadaceae, Lactobacillus and Alloprevotella decreased, but in contrast, Akkermansia dramatically increased compared with the control check group, which was fed a normal diet; the administration of LAE reversed these changes. These results imply that L. christinae can be considered an efficient therapy for eliminating CGSs induced by a high-fat and high-cholesterol diet, which may be achieved by influencing the intestinal microflora.
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