Optimization of saponin extraction from the leaves of Panax notoginseng and Panax quinquefolium and evaluation of their antioxidant, antihypertensive, hypoglycemic and anti-inflammatory activities.

Panax notoginseng and Panax quinquefolium are important economic plants that utilize dried roots for medicinal and food dual purposes; there is still insufficient research of their stems and leaves, which also contain triterpenoid saponins. The extraction process was developed with a total saponin content of 12.30 ± 0.34% and 12.19 ± 0.64% for P. notoginseng leaves (PNL) and P. quinquefolium leaves (PQL) extracts, respectively. PNL and PQL saponin extracts showed good antioxidant, antihypertensive, hypoglycemic, and anti-inflammatory properties in vitro and RAW264.7 cells. A total of 699 metabolites were identified in PNL and PQL saponin extracts, with the majority being triterpenoid saponins, flavonoids and amino acids. Fourteen ginsenosides, 18 flavonoids or alkaloids, and 16 amino acids were enriched in both saponin extracts. Overall, the utilization of saponins from medicinal plants PNL and PQL has been developed to facilitate systematic research in the functional food and natural product industries.

Promiscuous Oxidosqualene Cyclases from Neoalsomitra integrifoliola Catalyzing the Formation of Tetracyclic, Pentacyclic, and Heterocyclic Triterpenes.

Six oxidosqualene cyclases (NiOSC1-NiOSC6) from Neoalsomitra integrifoliola were characterized for the biosynthesis of diverse triterpene scaffolds, including tetracyclic and pentacyclic triterpenes from the 2,3-oxidosqualene (1) and oxacyclic triterpenes from the 2,3:22,23-dioxidosqualene (2). NiOSC1 showed high efficiency in the production of naturally rare (20R)-epimers of oxacyclic triterpenes. Mutagenesis results revealed that the NiOSC1-F731G mutant significantly increased the yields of (20R)-epimers compared to the wild type. Homology modeling and molecular docking elucidated the origin of the (20R)-configuration in the epoxide addition step.

Development of a stable genetic transformation system in Erigeron breviscapus: a case study with EbYUC2 in relation to leaf number and flowering time.

MAIN CONCLUSION: A stable genetic transformation system for Erigeron breviscapus was developed. We cloned the EbYUC2 gene and genetically transformed it into Arabidopsis thaliana and E. breviscapus. The leaf number, YUC2 gene expression, and the endogenous auxin content in transgenic plants were significantly increased. Erigeron breviscapus is a prescription drug for the clinical treatment of cardiovascular and cerebrovascular diseases. The rosette leaves have the highest content of the major active compound scutellarin and are an important component in the yield of E. breviscapus. However, little is known about the genes related to the leaf number and flowering time of E. breviscapus. In our previous study, we identified three candidate genes related to the leaf number and flowering of E. breviscapus by combining resequencing data and genome-wide association study (GWAS). However, their specific functions remain to be characterized. In this study, we cloned and transformed the previously identified full-length EbYUC2 gene into Arabidopsis thaliana, developed the first stable genetic transformation system for E. breviscapus, and obtained the transgenic plants overexpressing EbYUC2. Compared with wild-type plants, the transgenic plants showed a significant increase in the number of leaves, which was correlated with the increased expression of EbYUC2. Consistently, the endogenous auxin content, particularly indole-3-acetic acid, in transgenic plants was also significantly increased. These results suggest that EbYUC2 may control the leaf number by regulating auxin biosynthesis, thereby laying a foundation for revealing the molecular mechanism governing the leaf number and flowering time of E. breviscapus.

Functional characterization of genes related to triterpene and flavonoid biosynthesis in Cyclocarya paliurus.

MAIN CONCLUSION: The oxidosqualene cyclases (OSCs) generating triterpenoid skeletons in Cyclocarya paliurus were identified for the first time, and two uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyzing the glycosylation of flavonoids were characterized. Cyclocarya paliurus, a native rare dicotyledonous plant in China, contains an abundance of triterpenoid saponins and flavonoid glycosides that exhibit valuable pharmaceutical effects in preventing hypertension, hyperlipidemia, and diabetes. However, the molecular mechanism explaining the biosynthesis of triterpenoid saponin and flavonoid glycoside in C. paliurus remains unclear. In this study, the triterpene content in different tissues and the expression pattern of genes encoding the key enzymes associated with triterpenoid saponin and flavonoid glycoside biosynthesis were studied using transcriptome and metabolome analysis. The eight upstream oxidosqualene cyclases (OSCs) involved in triterpenoid saponin biosynthesis were functionally characterized, among them CpalOSC6 catalyzed 2,3;22,23-dioxidosqualene to form 3-epicabraleadiol; CpalOSC8 cyclized 2,3-oxidosqualene to generate dammarenediol-II; CpalOSC2 and CpalOSC3 produced ß-amyrin and CpalOSC4 produced cycloartenol, while CpalOSC2-CpalOSC5, CpalOSC7, and CpalOSC8 all produced lanosterol. However, no catalytic product was detected for CpalOSC1. Moreover, two downstream flavonoid uridine diphosphate (UDP)-glycosyltransferases (UGTs) (CpalUGT015 and CpalUGT100) that catalyze the last step of flavonoid glycoside biosynthesis were functionally elucidated. These results uncovered the key genes involved in the biosynthesis of triterpenoid saponins and flavonoid glycosides in C. paliurus that could be applied to produce flavonoid glycosides and key triterpenoid saponins in the future via a synthetic strategy.

Reconstruction of engineered yeast factory for high yield production of ginsenosides Rg3 and Rd.

Panax notoginseng is one of the most valuable traditional Chinese herbs. The main active ingredients, dammarane-type ginsenosides, show multiple pharmacological activities. Recently, the key UDP-dependent glycosyltransferases (UGTs) involved in the biosynthesis of common ginsenosides have been widely studied. However, only a few UGTs that catalyze ginsenoside formation have been reported. This study further investigated the new catalytic function of 10 characterized UGTs from the public database. PnUGT31(PnUGT94B2) and PnUGT53 (PnUGT71B8)exhibited promiscuous sugar-donor specificity of UDP-glucose and UDP-xylose, which could catalyze the glycosylation of C20-OH sites and elongation of the sugar chain at the C3 and/or C20 sites. We further analyzed the expression patterns in P. notoginseng and predicted the catalytic mechanisms of PnUGT31 and PnUGT53 using molecular docking simulations. Moreover, different gene modules were built to increase the yield of ginsenosides in engineered yeast. The metabolic flow of the proginsenediol (PPD) synthetic pathway was enhanced by LPPDS gene modules based on the engineered strain. The resulting yeast was constructed to produce 1.72 g/L PPD in a shaking flask, but cell growth was significantly inhibited. EGH and LKG gene modules were constructed to achieve high-level production of dammarane-type ginsenosides. The production of G-Rg3 controlled by LKG modules increased 3.84 times (254.07 mg/ L), whereas the G-Rd titer reached 56.68 mg/L after 96 h in shaking flask culture under the control of all modules, both of which yielded the highest values for known microbes.

Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis.

Hemsleya chinensis is a Chinese traditional medicinal plant, containing cucurbitacin IIa (CuIIa) and cucurbitacin IIb (CuIIb), both of which have a wide range of pharmacological effects, including antiallergic, anti-inflammatory, and anticancer properties. However, few studies have been explored on the key enzymes that are involved in cucurbitacins biosynthesis in H. chinensis. Oxidosqualene cyclase (OSC) is a vital enzyme for cyclizing 2,3-oxidosqualene and its analogues. Here, a gene encoding the oxidosqualene cyclase of H. chinensis (HcOSC6), catalyzing to produce cucurbitadienol, was used as a template of mutagenesis. With the assistance of AlphaFold2 and molecular docking, we have proposed for the first time to our knowledge the 3D structure of HcOSC6 and its binding features to 2,3-oxidosqualene. Mutagenesis experiments on HcOSC6 generated seventeen different single-point mutants, showing that single-residue changes could affect its activity. Three key amino acid residues of HcOSC6, E246, M261 and D490, were identified as a prominent role in controlling cyclization ability. Our findings not only comprehensively characterize three key residues that are potentially useful for producing cucurbitacins, but also provide insights into the significant role they could play in metabolic engineering.

Biosynthetic pathway of prescription bergenin from Bergenia purpurascens and Ardisia japonica.

Bergenin is a typical carbon glycoside and the primary active ingredient in antitussive drugs widely prescribed for central cough inhibition in China. The bergenin extraction industry relies on the medicinal plant species Bergenia purpurascens and Ardisia japonica as their resources. However, the bergenin biosynthetic pathway in plants remains elusive. In this study, we functionally characterized a shikimate dehydrogenase (SDH), two O-methyltransferases (OMTs), and a C-glycosyltransferase (CGT) involved in bergenin synthesis through bioinformatics analysis, heterologous expression, and enzymatic characterization. We found that BpSDH2 catalyzes the two-step dehydrogenation process of shikimic acid to form gallic acid (GA). BpOMT1 and AjOMT1 facilitate the methylation reaction at the 4-OH position of GA, resulting in the formation of 4-O-methyl gallic acid (4-O-Me-GA). AjCGT1 transfers a glucose moiety to C-2 to generate 2-Glucosyl-4-O-methyl gallic acid (2-Glucosyl-4-O-Me-GA). Bergenin production ultimately occurs in acidic conditions or via dehydration catalyzed by plant dehydratases following a ring-closure reaction. This study for the first time uncovered the biosynthetic pathway of bergenin, paving the way to rational production of bergenin in cell factories via synthetic biology strategies.

RNA-seq analysis reveals key genes associated with seed germination of Fritillaria taipaiensis P.Y.Li by cold stratification.

Seed dormancy is an adaptive strategy for environmental evolution. However, the molecular mechanism of the breaking of seed dormancy at cold temperatures is still unclear, and the genetic regulation of germination initiated by exposure to cold temperature requires further investigation. In the initial phase of the current study, the seed coat characteristics and embryo development of Fritillaria taipaiensis P.Y.Li at different temperatures (0°C, 4°C, 10°C & 25°C) was recorded. The results obtained demonstrated that embryo elongation and the dormancy-breaking was most significantly affected at 4°C. Subsequently, transcriptome analyses of seeds in different states of dormancy, at two stratification temperatures (4°C and 25°C) was performed, combined with weighted gene coexpression network analysis (WGCNA) and metabolomics, to explore the transcriptional regulation of seed germination in F. taipaiensis at the two selected stratification temperatures. The results showed that stratification at the colder temperature (4°C) induced an up-regulation of gene expression involved in gibberellic acid (GA) and auxin biosynthesis and the down-regulation of genes related to the abscisic acid (ABA) biosynthetic pathway. Thereby promoting embryo development and the stimulation of seed germination. Collectively, these data constitute a significant advance in our understanding of the role of cold temperatures in the regulation of seed germination in F. taipaiensis and also provide valuable transcriptomic data for seed dormancy for other non-model plant species.

PanaxGDB: A Comprehensive Platform for Panax.

The genus Panax is a valuable natural medicinal source used worldwide that contains high levels of triterpenoid saponins with extensive pharmacological activities. In past decades, molecular biotechnology and breeding techniques have been respectively used to generate omics data and information on cultivars primarily from Panax ginseng (ginseng), Panax quinquefolium (American ginseng), and Panax notoginseng (Sanqi) to biosynthesize valuable saponins, improve product quality, and conduct cost-controlled cultivation. Although much data have been produced, there are concerns that redundant data might be generated and that relatively scattered data might be overlooked. Therefore, many scientists desire a reliable, comprehensive omics database of the Panax genus that could save time and promote integrated analysis. Therefore, to provide all-inclusive, reliable, and valuable information on the Panax genus, PanaxGDB, an open comprehensive database that integrates data on omics and information on varieties, was established. The database contains information on nearly 600 compounds from 12 Panax species, draft genomic sequences with annotations and gene expression levels, single nucleotide polymorphisms, genome-wide association analysis based on agronomic traits, globally collected germplasm information, summaries, omics data of the Panax genus, and online versatile analytic tools. The Panax genus database will be updated when new data are released to continue serving as a central portal to boost research on the biology and functions of Panax. PanaxGDB is available at: http://panaxGDB.ynau.edu.cn.

[Identification and expression profiling of R2R3-MYB transcription factors in Erigeron breviscapus].

R2 R3-MYB transcription factors are ubiquitous in plants, playing a role in the regulation of plant growth, development, and secondary metabolism. In this paper, the R2 R3-MYB transcription factors were identified by bioinformatics analysis of the genomic data of Erigeron breviscapus, and their gene sequences, structures, physical and chemical properties were analyzed. The functions of R2 R3-MYB transcription factors were predicted by cluster analysis. Meanwhile, the expression patterns of R2 R3-MYB transcription factors in response to hormone treatments were analyzed. A total of 108 R2 R3-MYB transcription factors, named EbMYB1-EbMYB108, were identified from the genome of E. breviscapus. Most of the R2 R3-MYB genes carried 2-4 exons. The phylogenetic tree of MYBs in E. breviscapus and Arabidopsis thaliala was constructed, which classified 234 MYBs into 30 subfamilies. The MYBs in the five MYB subfamilies of A.thaliala were clustered into independent clades, and those in E. breviscapus were clustered into four clades. The transcriptome data showed that MYB genes were differentially expressed in different tissues of E. breviscapus and in response to the treatments with exogenous hormones such as ABA, SA, and GA for different time. The transcription of 13 R2 R3-MYB genes did not change significantly, and the expression patterns of some genes were up-regulated or down-regulated with the extension of hormone treatment time. This study provides a theoretical basis for revealing the mechanisms of R2 R3-MYB transcription factors in regulating the growth and development, stress(hormone) response, and active ingredient accumulation in E. breviscapus.