Promoting Photosynthetic Production of Dammarenediol-II in Chlamydomonas reinhardtii via Gene Loading and Culture Optimization.

Ginsenosides are major bioactive compounds found in Panax ginseng that exhibit various pharmaceutical properties. Dammarenediol-II, the nucleus of dammarane-type ginsenosides, is a promising candidate for pharmacologically active triterpenes. Dammarenediol-II synthase (DDS) cyclizes 2,3-oxidosqualene to produce dammarenediol-II. Based on the native terpenoids synthetic pathway, a dammarane-type ginsenosides synthetic pathway was established in Chlamydomonas reinhardtii by introducing P. ginseng PgDDS, CYP450 enzyme (PgCYP716A47), or/and Arabidopsis thaliana NADPH-cytochrome P450 reductase gene (AtCPR), which is responsible for producing dammarane-type ginsenosides. To enhance productivity, strategies such as "gene loading" and "culture optimizing" were employed. Multiple copies of transgene expression cassettes were introduced into the genome to increase the expression of the key rate-limiting enzyme gene, PgDDS, significantly improving the titer of dammarenediol-II to approximately 0.2 mg/L. Following the culture optimization in an opt2 medium supplemented with 1.5 mM methyl jasmonate under a light:dark regimen, the titer of dammarenediol-II increased more than 13-fold to approximately 2.6 mg/L. The C. reinhardtii strains engineered in this study constitute a good platform for the further production of ginsenosides in microalgae.

Molecular Cloning and Functional Characterization of Oxidosqualene Cyclases from Panax vietnamensis.

Panax vietnamensis is a valuable medicinal resource with promising preclinical applications. Ginsenosides, which are triterpenoids, are the primary active components in P. vietnamensis. Oxidosqualene cyclases (OSCs) catalyze the formation of the basic skeleton of triterpenes from 2,3-oxidosqualene, which is a crucial step in the biosynthesis of triterpenoids. The OSCs involved in triterpenoid biosynthesis in P. vietnamensis have not yet been characterized. Four OSC genes (PvOSC1-4) were cloned from P. vietnamensis and functionally characterized via heterologous expression in yeast. Transgenic yeast expressing PvOSC1, PvOSC3, and PvOSC4 produced the corresponding products ß-amyrin, cycloartenol, and dammarenediol-II, respectively. PvOSC1, PvOSC3, and PvOSC4 are monofunctional OSCs. In this study, we characterized three PvOSC genes, providing a better understanding of the biosynthesis of triterpenoids in P. vietnamensis and the multiple choices of plant OSCs for metabolic engineering in yeast and other hosts.

Integrated Metabolomic and Transcriptomic Analysis and Identification of Dammarenediol-II Synthase Involved in Saponin Biosynthesis in Gynostemma longipes.

Gynostemma longipes contains an abundance of dammarane-type ginsenosides and gypenosides that exhibit extensive pharmacological activities. Increasing attention has been paid to the elucidation of cytochrome P450 monooxygenases (CYPs) and UDP-dependent glycosyltransferases (UGTs) that participate downstream of ginsenoside biosynthesis in the Panax genus. However, information on oxidosqualene cyclases (OSCs), the upstream genes responsible for the biosynthesis of different skeletons of ginsenoside and gypenosides, is rarely reported. Here, an integrative study of the metabolome and the transcriptome in the leaf, stolon, and rattan was conducted and the function of GlOSC1 was demonstrated. In total, 46 triterpenes were detected and found to be highly abundant in the stolon, whereas gene expression analysis indicated that the upstream OSC genes responsible for saponin skeleton biosynthesis were highly expressed in the leaf. These findings indicated that the saponin skeletons were mainly biosynthesized in the leaf by OSCs, and subsequently transferred to the stolon via CYPs and UGTs biosynthesis to form various ginsenoside and gypenosides. Additionally, a new dammarane-II synthase (DDS), GlOSC1, was identified by bioinformatics analysis, yeast expression assay, and enzyme assays. The results of the liquid chromatography-mass spectrometry (LC-MS) analysis proved that GlOSC1 could catalyze 2,3-oxidosqualene to form dammarenediol-II via cyclization. This work uncovered the biosynthetic mechanism of dammarenediol-II, an important starting substrate for ginsenoside and gypenosides biosynthesis, and may achieve the increased yield of valuable ginsenosides and gypenosides produced under excess substrate in a yeast cell factory through synthetic biology strategy.

Ginsenosides in Panax genus and their biosynthesis.

Ginsenosides are a series of glycosylated triterpenoids which belong to protopanaxadiol (PPD)-, protopanaxatriol (PPT)-, ocotillol (OCT)- and oleanane (OA)-type saponins known as active compounds of Panax genus. They are accumulated in plant roots, stems, leaves, and flowers. The content and composition of ginsenosides are varied in different ginseng species, and in different parts of a certain plant. In this review, we summarized the representative saponins structures, their distributions and the contents in nearly 20 Panax species, and updated the biosynthetic pathways of ginsenosides focusing on enzymes responsible for structural diversified ginsenoside biosynthesis. We also emphasized the transcription factors in ginsenoside biosynthesis and non-coding RNAs in the growth of Panax genus plants, and highlighted the current three major biotechnological applications for ginsenosides production. This review covered advances in the past four decades, providing more clues for chemical discrimination and assessment on certain ginseng plants, new perspectives for rational evaluation and utilization of ginseng resource, and potential strategies for production of specific ginsenosides.

Metabolomes and transcriptomes revealed the saponin distribution in root tissues of Panax quinquefolius and Panax notoginseng.

BACKGROUND: Panax quinquefolius and Panax notoginseng are widely used and well known for their pharmacological effects. As main pharmacological components, saponins have different distribution patterns in the root tissues of Panax plants. METHODS: In this study, the representative ginsenosides were detected and quantified by desorption electrospray ionization mass spectrometry and high-performance liquid chromatography analysis to demonstrate saponin distribution in the root tissues of P. quinquefolius and P. notoginseng, and saponin metabolite profiles were analyzed by metabolomes to obtain the biomarkers of different root tissues. Finally, the transcriptome analysis was performed to demonstrate the molecular mechanisms of saponin distribution by gene profiles. RESULTS: There was saponin distribution in the root tissues differed between P. quinquefolius and P. notoginseng. Eight-eight and 24 potential biomarkers were detected by metabolome analysis, and a total of 340 and 122 transcripts involved in saponin synthesis that were positively correlated with the saponin contents (R > 0.6, P < 0.05) in the root tissues of P. quinquefolius and P. notoginseng, respectively. Among them, GDPS1, CYP51, CYP64, and UGT11 were significantly correlated with the contents of Rg1, Re, Rc, Rb2, and Rd in P. quinquefolius. UGT255 was markedly related to the content of R1; CYP74, CYP89, CYP100, CYP103, CYP109, and UGT190 were markedly correlated with the Rd content in P. notoginseng. CONCLUSIONS: These results provided the visual and quantitative profiles of and confirmed the pivotal transcripts of CYPs and UGTs regulating the saponin distribution in the root tissues of P. quinquefolius and P. notoginseng.

Genetically modified rice produces ginsenoside aglycone (protopanaxadiol).

MAIN CONCLUSION: Protopanaxadiol is dammarane-type tetracyclic triterpene sapogenin found in ginseng and has a high medicinal values. We successfully constructed transgenic rice producing protopanaxadiol by introducing the ginseng PgDDS and CYP716A47 genes in this crop plant. Protopanaxadiol (PPD), an aglycone of ginsenosides, possesses pleiotropic anticarcinogenesis activities in many cancers. Here, we constructed transgenic rice overexpressing the Panax ginseng dammarenediol-II synthase gene (PgDDS) and protopanaxadiol synthase gene (CYP716A47) driven by a rice endosperm-specific α-globulin promoter. Among more than 50 independent lines, five transgenic lines were selected. The introduction of the genes in the T1 generation of the transgenic lines was confirmed by genomic PCR. The expression of the introduced genes in T2 seeds was confirmed by qPCR. Methanol extracts of transgenic rice grains were analyzed by LC/MS to detect the production of PPD and dammarenediol-II (DD). The production of both PPD and DD was identified not only by comparing the retention times but also mass fraction patterns of authentic PPD and DD standards. The mean concentrations of PPD and DD in rice grains were 16.4 and 4.5 µg/g dry weight, respectively. The invention of genetically engineered rice grains producing PPD and DD can be applied to rice breeding to reinforce new medicinal values.

Integrated metabolomic and transcriptomic analyses revealed the distribution of saponins in Panax notoginseng.

Panax notoginseng is famous for its important therapeutic effects. Saponins are bioactive compounds found in different parts and developmental stages of P. notoginseng plants. Thus, it is urgently to study saponins distribution in different parts and growth ages of P. notoginseng plants. In this study, potential biomarkers were found, and their chemical characteristic differences were revealed through metabolomic analysis. High-performance liquid chromatography data indicated the higher content of saponins (i.e., Rg1, Re, Rd, and Rb1) in the underground parts than that in the aerial parts. 20(S)-Protopanaxadiol saponins were mainly distributed in the aerial parts. Additionally, the total saponin content in the 3-year-old P. notoginseng plant (188.0 mg/g) was 1.4-fold higher than that in 2-year-old plant (130.5 mg/g). The transcriptomic analysis indicated the tissue-specific transcription expression of genes, namely, PnFPS, PnSS, PnSE1, PnSE2, and PnDS, which encoded critical synthases in saponin biosyntheses. These genes showed similar expression patterns among the parts of P. notoginseng plants. The expression levels of these genes in the flowers and leaves were 5.2fold higher than that in the roots and fibrils. These results suggested that saponins might be actively synthesized in the aerial parts and transformed to the underground parts. This study provides insights into the chemical and genetic characteristics of P. notoginseng to facilitate the synthesis of its secondary metabolites and a scientific basis for appropriate collection and rational use of this plant.

Construction of efficient yeast cell factories for production of ginsenosides precursor dammarenediol-II / 药学学报

Dammarenediol-Ⅱ is an important precursor in the biosynthesis pathway of ginsenosides which are the main active components of Panax quinquefolius and Panax ginseng. For constructing a dammarenediol- Ⅱ-producing cell factory, the triterpenoid precursors of yeast are improved significantly by the modular pathway engineering strategy on the basis of an MVA optimized strain. The strain overexpressing Salvia miltiorrhiza SmFPS and Arabidopsis thaliana AtSQS2 could yield 67.4 mg·g−1 squalene, accounting for about 6.74% of cell dry weight. In our further work, an Arabidopsis thaliana 2,3-oxidosqualene synthase AtSQE2 was found to be able to increase the downstream lanosterol yield by 22-fold, reaching 47.9 mg·g−1. Then, regulating dammarenediol-Ⅱ synthase gene expression, using anti-sense RNA technology for regulation of ERG7 in the ergosterol pathway, and optimizing fermentation process were successively performed. Finally, the synthesis flux of triterpenes was increased to 10 g·L−1 for the first time, and we constructed an efficient cell factory that can produce 15 g·L−1 dammarenediol-Ⅱ, which lays a solid foundation of industrial synthesis of dammarane-type ginsenosides.

Integrated metabolomic and transcriptomic analyses revealed the distribution of saponins in

is famous for its important therapeutic effects. Saponins are bioactive compounds found in different parts and developmental stages of plants. Thus, it is urgently to study saponins distribution in different parts and growth ages of plants. In this study, potential biomarkers were found, and their chemical characteristic differences were revealed through metabolomic analysis. High-performance liquid chromatography data indicated the higher content of saponins (, Rg1, Re, Rd, and Rb1) in the underground parts than that in the aerial parts. 20()-Protopanaxadiol saponins were mainly distributed in the aerial parts. Additionally, the total saponin content in the 3-year-old plant (188.0 mg/g) was 1.4-fold higher than that in 2-year-old plant (130.5 mg/g). The transcriptomic analysis indicated the tissue-specific transcription expression of genes, namely, , , , , and , which encoded critical synthases in saponin biosyntheses. These genes showed similar expression patterns among the parts of plants. The expression levels of these genes in the flowers and leaves were 5.2fold higher than that in the roots and fibrils. These results suggested that saponins might be actively synthesized in the aerial parts and transformed to the underground parts. This study provides insights into the chemical and genetic characteristics of to facilitate the synthesis of its secondary metabolites and a scientific basis for appropriate collection and rational use of this plant.

Enhancing Biosynthesis of a Ginsenoside Precursor by Self-Assembly of Two Key Enzymes in Pichia pastoris.

Ginsenosides from the edible and medicinal plant ginseng have demonstrated various pharmacological activities. However, producing ginsenoside efficiently remains a challenge. Engineering metabolic pathways through protein assembly in yeast is a promising way for ginsenoside production. In the biosynthetic pathway of ginsenosides, dammarenediol-II synthase and squalene epoxidase are two key enzymes that determine the production rate of the dammarane-type ginsenoside precursor dammarenediol-II. In this work, a strategy to enhance the biosynthesis of dammarenediol-II in Pichia pastoris was developed by the self-assembly of the two key enzymes via protein-protein interaction. After being modified by interacting proteins, the two enzymes were successfully co-localized, resulting in a 2.1-fold enhancement in dammarenediol-II yields.

Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae.

Ginsenosides, the major bioactive components of Panax ginseng, are regarded as promising high-value pharmaceutical compounds. In ginseng, ginsenosides are produced from their precursor protopanaxadiol. Recently, an artificial biosynthetic pathway of protopanaxadiol was built in Saccharomyces cerevisiae by introducing a P. ginseng dammarenediol-II synthase, a P. ginseng cytochrome P450-type protopanaxadiol synthase (PPDS), and a Arabidopsis thaliana NADPH-cytochrome P450 reductase (ATR1). In this engineered yeast strain, however, the low metabolic flux through PPDS resulted in a low productivity of protopanaxadiol. Moreover, health of the yeast cells was significantly affected by reactive oxygen species released by the pool coupling between PPDS and ATR1. To overcome the obstacles in protopanaxadiol production, PPDS was modified through transmembrane domain truncation and self-sufficient PPDS-ATR1 fusion construction in this study. The fusion enzymes conferred approximately 4.5-fold increase in catalytic activity, and 71.1% increase in protopanaxadiol production compared with PPDS and ATR1 co-expression. Our in vivo experiment indicated that the engineered yeast carrying fusion protein effectively converted 96.8% of dammarenediol-II into protopanaxadiol. Protopanaxadiol production in a 5 L bioreactor in fed-batch fermentation reached 1436.6 mg/L. Our study not only improved protopanaxadiol production in yeast, but also provided a generic method to improve activities of plant cytochrome P450 monooxygenases. This method is promising to be applied to other P450 systems in yeast. Biotechnol. Bioeng. 2016;113: 1787-1795. © 2016 Wiley Periodicals, Inc.

Heterologous biosynthesis of triterpenoid dammarenediol-II in engineered Escherichia coli.

OBJECTIVES: To achieve heterologous biosynthesis of dammarenediol-II, which is the precursor of dammarane-type tetracyclic ginsenosides, by reconstituting the 2,3-oxidosqualene-derived triterpenoid biosynthetic pathway in Escherichia coli. RESULTS: By the strategy of synthetic biology, dammarenediol-II biosynthetic pathway was reconstituted in E. coli by co-expression of squalene synthase (SS), squalene epoxidase (SE), NADPH-cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae, and SE from Methylococcus capsulatus (McSE), NADPH-cytochrome P450 reductase (CPR) from Arabidopsis thaliana. Sequences of transmembrane domains were truncated if necessary in each of the genes. Different sources of SE/CPR combinations were tested, during which two CPRs were detected to be new reductase partners of McSE. When the gene encoding dammarenediol-II synthase was co-expressed with the 2,3-oxidosqualene expression modules, dammarenediol-II was detected and the production was 8.63 mg l(-1) in E. coli under the shake-flask conditions. CONCLUSIONS: Two E. coli chassis for production of dammarenediol-II were established which could be potentially applied in other triterpenoid production in E. coli when different oxidosqualene cyclases (OSCs) introduced into the system.

Metabolic engineering of Pichia pastoris for the production of dammarenediol-II.

Dammarenediol-II is the nucleus of dammarane-type ginsenosides, which are a group of active triterpenoids exhibiting various pharmacological activities. Based on the native triterpene synthetic pathway, a dammarenediol-II synthetic pathway was established in Pichia pastoris by introducing a dammarenediol-II synthase gene (PgDDS) from Panax ginseng, which is responsible for the cyclization of 2,3-oxidosqualene to dammarenediol-II in this study. To enhance productivity, a strategy of "increasing supply and reducing competitive consumption of 2,3-oxidosqualene" was used. To increase the supply of 2,3-oxidosqualene, we augmented expression of the ERG1 gene, which is responsible for 2,3-oxidosqualene synthesis. This significantly improved the yield of dammarenediol-II over 6.7-fold, from 0.030mg/g dry cell weight (DCW) to 0.203mg/g DCW. Subsequently, to reduce competition for 2,3-oxidosqualene from ergosterol biosynthesis without affecting the normal growth of P. pastoris, we targeted the ERG7gene, which is responsible for conversion of 2,3-oxidosqualene to lanosterol. This gene was downregulated by replacing its native promoter with a thiamine-repressible promoter, using a marker-recycling and gene-targeting Cre- lox71/66 system developed for P. pastoris herein. The yield of dammarenediol-II was further increased more than 3.6-fold, to 0.736mg/g DCW. Furthermore, the direct addition of 0.5g/L squalene into the culture medium further enhanced the yield of dammarenediol-II to 1.073mg/g DCW, which was 37.5-fold higher than the yield from the strain with the PgDDS gene introduction only. The P. pastoris strains engineered in this study constitute a good platform for further production of ginsenosides in Pichia species.

Production of dammarane-type sapogenins in rice by expressing the dammarenediol-II synthase gene from Panax ginseng C.A. Mey.

Ginsenosides are the main active ingredients in Chinese medicinal ginseng; 2,3-oxidosqualene is a precursor metabolite to ginsenosides that is present in rice. Because rice lacks a key rate-limiting enzyme (dammarenediol-II synthase, DS), rice cannot synthesize dammarane-type ginsenosides. In this study, the ginseng (Panax ginseng CA Mey.) DS gene (GenBank: AB265170.1) was transformed into rice using agrobacterium, and 64 rice transgenic plants were produced. The Transfer-DNA (T-DNA) insertion sites in homozygous lines of the T2 generation were determined by using high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) and differed in all tested lines. One to two copies of the T-DNA were present in each transformant, and real-time PCR and Western blotting showed that the transformed DS gene could be transcribed and highly expressed. High performance liquid chromatography (HPLC) analysis showed that the dammarane-type sapogenin 20(S)-protopanaxadiol (PPD) content was 0.35-0.59 mg/g dw and the dammarane-type sapogenin 20(S)-protopanaxatriol (PPT) content was 0.23-0.43 mg/g dw in the transgenic rice. LC/MS analysis confirmed production of PPD and PPT. These results indicate that a new "ginseng rice" germplasm containing dammarane-type sapogenins has been successfully developed by transforming the ginseng DS gene into rice.

Dammarenediol-II Prevents VEGF-Mediated Microvascular Permeability in Diabetic Mice.

Diabetic retinopathy is a major diabetic complication predominantly caused by vascular endothelial growth factor (VEGF)-induced vascular permeability in the retina; however, treatments targeting glycemic control have not been successful. Here, we investigated the protective effect of dammarenediol-II, a precursor of triterpenoid saponin biosynthesis, on VEGF-induced vascular leakage using human umbilical vein endothelial cells (HUVECs) and diabetic mice. We overproduced the compound in transgenic tobacco expressing Panax ginseng dammarenediol-II synthase gene and purified using column chromatography. Analysis of the purified compound using a gas chromatography-mass spectrometry system revealed identical retention time and fragmentation pattern to those of authentic standard dammarenediol-II. Dammarenediol-II inhibited VEGF-induced intracellular reactive oxygen species generation, but it had no effect on the levels of intracellular Ca(2+) in HUVECs. We also found that dammarenediol-II inhibited VEGF-induced stress fiber formation and vascular endothelial-cadherin disruption, both of which play critical roles in modulating endothelial permeability. Notably, microvascular leakage in the retina of diabetic mice was successfully inhibited by intravitreal dammarenediol-II injection. Our results suggest that the natural drug dammarenediol-II may have the ability to prevent diabetic microvascular complications, including diabetic retinopathy.

Expression profiling of the triterpene saponin biosynthesis genes FPS, SS, SE, and DS in the medicinal plant Panax notoginseng.

Panax notoginseng (Burk) F. H. Chen, an economically significant medicinal plant with hemostatic and health tonic activities, has been used in Traditional Chinese Medicine (TCM) for more than 3,000 years. Triterpene saponins are responsible for most of the pharmacological activities of P. notoginseng. Here, we cloned five cDNA sequences encoding the key enzymes involved in triterpene saponin biosynthesis, namely, PnFPS, PnSS, PnSE1, PnSE2, and PnDS, and analyzed the conserved domains and phylogenetics of their corresponding proteins. Their organ-specific expression patterns in four-year-old P. notoginseng were detected by real-time PCR, showing that they were all most highly expressed in flowers. In addition, four of the genes, excluding PnSE2, were upregulated in leaves following stimulation with methyl jasmonate. This study is the first comprehensive analysis of the expression patterns of pivotal genes for triterpene saponin biosynthesis in P. notoginseng and provides a basis to further elucidate the molecular mechanism for the biosynthesis of these medically important compounds.

Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides.

Ginsenosides are the primary bioactive components of ginseng, which is a popular medicinal herb and exhibits diverse pharmacological activities. Protopanaxadiol is the aglycon of several dammarane-type ginsenosides, which also has anticancer activity. For microbial production of protopanaxadiol, dammarenediol-II synthase and protopanaxadiol synthase genes of Panax ginseng, together with a NADPH-cytochrome P450 reductase gene of Arabidopsis thaliana, were introduced into Saccharomyces cerevisiae, resulting in production of 0.05 mg/g DCW protopanaxadiol. Increasing squalene and 2,3-oxidosqualene supplies through overexpressing truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, farnesyl diphosphate synthase, squalene synthase and 2,3-oxidosqualene synthase genes, together with increasing protopanaxadiol synthase activity through codon optimization, led to 262-fold increase of protopanaxadiol production. Finally, using two-phase extractive fermentation resulted in production of 8.40 mg/g DCW protopanaxadiol (1189 mg/L), together with 10.94 mg/g DCW dammarenediol-II (1548 mg/L). The yeast strains engineered in this work can serve as the basis for creating an alternative way for production of ginsenosides in place of extraction from plant sources.