Jinxin Li

Fungal elicitors enhance ginsenosides biosynthesis, expression of functional genes as well as signal molecules accumulation in adventitious roots of Panax ginseng C. A. Mey

In this work, we selected three fungi strains (Aspergillus niger, Aspergillus flavus and Aspergillus oryzae) as elicitors prepared from mycelium or fermentation broth to improve ginsenosides production in adventitious roots culture. The results showed that ginsenosides production (29.90±4.67mgg-1) was significantly enhanced upon elicitation with 200mgL-1A. niger elicitor prepared from mycelium, which was 3.52-fold of untreated group. HPLC-ESI-MSn analysis was performed, showing that ginsenoside Rb3 was present after treatment with the A. niger. Furthermore, we found that A. niger significantly enhanced accumulation of Nitric oxide (NO), salicylic acid (SA) and jasmonic acid (JA) involved in plant defense response, and significantly up-regulated the expression of the geranyl diphosphate synthase (GPS), farnesyl diphosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), dammarenediol synthase (DS), Two cytochrome P450 (CYP) genes (CYP716A47 and CYP716A53v2) and three UDP-glycosyltransferases (UGT) genes (UGTAE2, UGT94Q2 and UGTpg100).

Transcriptome profiling shows gene regulation patterns in ginsenoside pathway in response to methyl jasmonate in Panax Quinquefolium adventitious root.

Here, we combine elicitors and transcriptomics to investigate the inducible biosynthesis of the ginsenoside from the Panax quinquefolium. Treatment of P. quinquefolium adventitious root with methyl jasmonate (MJ) results in an increase in ginsenoside content (43.66 mg/g compared to 8.32 mg/g in control group). Therefore, we sequenced the transcriptome of native and MJ treated adventitious root in order to elucidate the key differentially expressed genes (DEGs) in the ginsenoside biosynthetic pathway. Through DEG analysis, we found that 5,759 unigenes were up-regulated and 6,389 unigenes down-regulated in response to MJ treatment. Several defense-related genes (48) were identified, participating in salicylic acid (SA), jasmonic acid (JA), nitric oxide (NO) and abscisic acid (ABA) signal pathway. Additionally, we mapped 72 unigenes to the ginsenoside biosynthetic pathway. Four cytochrome P450s (CYP450) were likely to catalyze hydroxylation at C-16 (c15743_g1, c39772_g1, c55422_g1) and C-30 (c52011_g1) of the triterpene backbone. UDP-xylose synthases (c52571_g3) was selected as the candidate, which was likely to involve in ginsenoside Rb3 biosynthesis.

Advances in ginsenoside biosynthesis and metabolic regulation

In this paper, we reviewed the advances in ginsenoside biosynthesis and metabolic regulation. To begin with, the application of elicitors in the ginsenoside biosynthesis was discussed. Methyl jasmonate (MJ) and analogues have the best effect on accumulation of ginsenoside compared with other elicitors, and few biotic elicitors are applied in Panax genus plants tissue culture. In addition, so far, more than 40 genes encoding ginsenoside biosynthesis related enzymes have been cloned and identified from Panax genus, such as UDP‐glycosyltransferases (UGT) genes UDPG, UGTAE2, UGT94Q2, UGTPg100, and UGTPg1. However, the downstream pathway of the ginsenoside biosynthesis is still not clear. Moreover, some methods have been used to increase the expression of functional genes and ginsenoside content in the ginsenoside synthesis pathway, including elicitors, overexpression, RNAi, and transcription factors. The ginsenoside biosynthesis pathway should be revealed so that ginsenoside contents can be regulated.

Production of Active Compounds in Medicinal Plants: From Plant Tissue Culture to Biosynthesis

Abstract Over past decades plant tissue culture has emerged as an alternative of whole plant cultivation in the production of valuable secondary metabolites. Adventitious roots culture of Panax ginseng and Echinacea purpure has reached the scale of 1-10 kL. Some molecular biological techniques, such as transgenic technology and genetic stability are increasingly used in the studies on plant tissue cultures. The studies on elicitors have deepened into the induction mechanism, including signal molecules, functional genes, and so on. More and more biological elicitors, such as A. niger and yeast are used to increase the active compounds in plant tissue cultures. We also discussed the application of synthetic biology in the studies on biosynthesis of artemisinin, paclitaxel, and tanshinon. The studies on active ingredients biosynthesis of medicinal plants provide unprecedented possibilities to achieve mass production of active ingredients. Plant tissue cultures can not only produce active ingredients but also as experimental materials for biosynthesis. In order to improve the contents of active compounds in medicinal plants, following aspects could be carried out gene interference or gene silencing, gene overexpression, combination with chemical synthesis, application of elicitors, and site-directed mutagenesis of the key enzymes.

Gene expression of glycyrrhizin acid and accumulation of endogenous signaling molecule in Glycyrrhiza uralensis Fisch adventitious roots after Saccharomyces cerevisiae and Meyerozyma guilliermondii applications

This study reports the best culture conditions for roots growth and accumulation of active components by optimizing the parameters. Glycyrrhiza uralensis adventitious roots metabolites were significantly increased after adding Saccharomyces cerevisiae and Meyerozyma guilliermondii. The highest contents of polysaccharide, glycyrrhizic acid, glycyrrhetinic acid, and total flavonoids were obtained in M. guilliermondii group; the content of glycyrrhizic acid was 5.3‐fold higher than the control. In control and treatment groups, 12 compounds were identified by high‐performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS/MS), among which some new compounds have been detected in elicitor groups including 5,7‐dihydroxyflavanone, glycyrrhisoflavanone, licorice saponin J2, uralsaponin B, (3R)‐vestitol, and uralenol. Meyerozyma guilliermondii significantly upregulated the expression of the genes such as 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, farnesyl diphosphate synthase, geranyl diphosphate synthase, squalene synthase, squalene epoxidase, β‐amyrin synthase, and CYP88D6 and CYP72A154. Meanwhile, it increased the biosynthesis of signaling molecules (nitric oxide, salicylic acid, and jasmonic acid) in defense mechanism.

Jasmonic acid and Methyl dihydrojasmonate enhance saponin biosynthesis as well as expression of functional genes in adventitious roots of Panax notoginseng F.H. Chen.

Panax notoginseng, an important herbal medicine, has wide uses for its bioactive compounds and health function. In this work, we compared the content of saponin in cultivation and adventitious root. The total content of saponins in adventitious root (8.48 mg⋅g−1) was found lower than in the native one (3‐year‐old) (34.34 mg⋅g−1). To enhance the content of bioactive compounds, we applied elicitors jasmonic acid (JA) and methyl dihydrojasmonate (MDJ) to the adventitious root culture. It was observed that the highest total content of saponins (71.94 mg⋅g−1) was achieved after treatment with 5 mg⋅L−1 JA, which was 2.09‐fold higher than native roots and 8.45‐fold higher than the control group. The findings from high‐performance liquid chromatography–electrospray ionization–tandem mass spectrometry analysis showed that six new compounds were present after the treatment with the elicitors. Furthermore, we found that JA and MDJ significantly upregulated the expression of the geranyl diphosphate synthase, farnesyl diphosphate synthase, squalene synthase, squalene epoxidase, dammarenediol synthase, and CYP716A47 and CYP716A53v2 (CYP450 enzyme) genes; downregulated the expression of the cycloartenol synthase gene; and increased superoxide dismutase and peroxidase activities.

Protein elicitor isolated from Escherichia coli induced bioactive compound biosynthesis as well as gene expression in Glycyrrhiza uralensis Fisch adventitious roots

This study explored the ability of three rhizobacterial strains (Bacillus subtilis, Penicillium fellutanum and Escherichia coli) to trigger metabolism. The protein fragment of more than 10 kDa significantly increased the metabolite contents in Glycyrrhiza uralensis adventitious roots. The results showed the highest accumulation of total flavonoids (7.59 mg g−1), glycyrrhizic acid (0.29 mg g−1), glycyrrhetinic acid (0.27 mg g−1) and polysaccharide (93.11 mg g−1) by up to 2.27-fold, 2.64-fold, 2.70-fold and 2.32-fold that of control roots, respectively. Besides, the protein fragment of more than 10 kDa significantly activated defense signaling and extremely up-regulated the expression of defense-related genes and functional genes in glycyrrhizic acid and flavonoid biosynthesis. In Glycyrrhiza uralensis adventitious roots, HPLC-ESI-MSn analysis showed that the protein fragment of more than 10 kDa induced the generation of four new compounds over the control group.

Characterization of UDP-glycosyltransferase involved in biosynthesis of ginsenosides Rh1 and Rd and identification of critical conserved amino acid residues for its function

Ginsenosides attract great attention for their bioactivities. However, their contents are low, and many UDP-glycosyltransferases (UGTs) that play crucial roles in the ginsenoside biosynthesis pathways have not been identified, which hinders the biosynthesis of ginsenosides. In this study, we reported that one UDP-glycosyltransferase, UGTPg71A29, from Panax ginseng could glycosylate C20-OH of Rh1 and transfer a glucose moiety to Rd, producing ginsenosides Rg1 and Rb1, respectively. Ectopic expression of UGTPg71A29 in Saccharomyces cerevisiae stably generated Rg1 and Rb1 under its corresponding substrate. Overexpression of UGTPg71A29 in transgenic cells of P. ginseng could significantly enhance the accumulation of Rg1 and Rb1, with their contents of 3.2- and 3.5-fold higher than those in the control, respectively. Homology modeling, molecular dynamics, and mutational analysis revealed the key catalytic site, Gln283, which provided insights into the catalytic mechanism of UGTPg71A29. These results not only provide an efficient enzymatic tool for the synthesis of glycosides but also help achieve large-scale industrial production of glycosides.

LSP1, a Responsive Protein from Meyerozyma guilliermondii, Elicits Defence Response and Improves Glycyrrhizic Acid Biosynthesis in Glycyrrhiza uralensis Fisch Adventitious Roots

This research explored the effects of protein and polysaccharide in Meyerozyma guilliermondii on active compounds in Glycyrrhiza uralensis Fisch adventitious roots. In this study, a responsive protein LSP1 was purified from the Meyerozyma guilliermondii since the excellent induction. The contents of total flavonoids (3.46 mg · g−1), glycyrrhizic acid (0.41 mg · g−1), glycyrrhetinic acid (0.41 mg · g−1), and polysaccharide (94.49 mg · g−1) in adventitious root peaked at LSP1 group, which were 1.6, 3.4, 2.4, 2.0‐fold that of control, respectively. Besides, the responsive protein LSP1 significantly activated the defense signaling, mitogen‐activated protein kinases and extremely up‐regulated the expression of defense‐related genes and functional genes involved in glycyrrhizic acid biosynthesis.