Analysis Of The Biosynthetic Pathway Of Acteoside

1.1 Phenylalanine Acteoside Metabolism Pathway

Presently, the general biosynthetic potential metabolic modules of Acteoside have reached a consensus, mainly including the phenylalanine metabolic pathway, the dopamine pathway/tyramine pathway, and the downstream acyl transfer and glycosyl modification cross pathways. 

Studies have shown that intermediates in the synthesis of Acteoside include caffeic acid, caffeoyl-CoA, hydroxytyrosol and hydroxytyrosol glucoside [16-18]. In vitro hydrolysis experiments proved that Acteoside was hydrolyzed into caffeic acid, de-rhamnosyl-Acteoside (mutongphenetoside A) and decaffeoyl-Acteoside under the action of glycoside hydrolase[19-20]. Studies on the effects of temperature, light, pH and other factors on the stability of Acteoside show that Acteoside can be degraded into caffeic acid and decaffeoyl-verbasin in solution [21]. Animal feeding experiments showed that the hydrolyzed products of Acteoside in rat urine were hydroxytyrosol and caffeic acid [22]. In the phenylalanine metabolic pathway, phenylalanine is first converted into cinnamic acid under the action of phenylalanine ammonia lyase (PAL), and cinnamic acid is converted into phenylalanine under the action of cinnamic acid-4-hydroxylase (C4H). The ring C4 is generated into p-coumaric acid, which is then catalyzed by coumaric acid 3-hydroxylase (C3H) to generate caffeic acid [3]. In addition, tyrosine ammonia lyase (TAL) derived from Saccharomyces radiata can convert tyrosine into p-coumaric acid, and p-coumaric acid can be catalyzed by C3H to generate caffeic acid, which provides the source for heterologous synthesis of caffeic acid. a new idea [23].

To know more knowledge about Cistanche Acteoside Function

1.2 Dopamine pathway/tyramine metabolism pathway

The intermediates tyrosol and hydroxytyrosol of dopamine pathway/tyramine pathway are another key precursor for the biosynthesis of Acteoside, and they can generate salidroside or hydroxytyrosol glucoside, the precursor substance of Acteoside, through different pathways. This pathway is an important branching pathway in the biosynthesis of Acteoside (Fig. 2b). Tyrosine generates L-dopa under the action of polyphenol oxidase (PPO)/tyrosine hydroxylase (TH), and dopa decarboxylase (DODC)/tyrosine decarboxylase (TyDC) catalyzes L-dopa. -Dopa produces dopamine, which is then processed by primary amine (copper-containing) oxidase (CuAO) and alcohol dehydrogenase (ALDH) to generate hydroxytyrosol [16-18, 24-25]. In another branch, dopamine is catalyzed by primary amine (copper) oxidase (CuAO)/monoamine oxidase (MAO) to generate 3,4-DHPAA. Vavricka et al.[26] utilized hydroxyphenylacetaldehyde synthase (DHPAAS) derived from Bombyx mori with dual functions of aldehyde synthase and decarboxylase to catalyze L-dopa to 3,4-DHPAA, and then ALDH catalyzed 3,4-DHPAA. produce hydroxytyrosol [24]. The tyramine pathway can provide tyrosol or hydroxytyrosol precursors for the Acteoside biosynthesis pathway. Tyrosine is catalyzed by TyDC to form tyramine, which is oxidized to 4-hydroxyphenylacetaldehyde (4-HPAA) by CuAO/tyramine oxidase (TYO), and 4-HPAA can be converted to 4-hydroxyphenylacetaldehyde reductase by 4-hydroxyphenylacetaldehyde reductase. (4HPAR)/ALDH is reduced to tyrosol, and then tyrosol is catalyzed by tyrosolhydroxylase (TLH) to generate hydroxytyrosol [16-17,24,27-28]. Due to the heterogeneity of TyDC, TYO and ALDH, the researchers put forward the hypothesis of other tyramine synthesis pathways, that is, tyrosine generates tyramine under the decarboxylation of TyDC, and then tyramine generates dopamine under the action of TH, and dopamine is decarboxylated by TH. TYO is oxidized to 3,4-DHPAA, which is then reduced to hydroxytyrosol by ALDH, but the TH in this putative pathway has not been verified [28]. Some scholars have hypothesized that polyphenol oxidase (PPO) can catalyze the generation of dopamine from tyramine and the generation of hydroxytyrosol from salidroside by hydroxylation, but its function has not been verified [16-18]. Rosmarinic acid is a naturally occurring polyphenolic compound, and studies have shown that tyrosine transaminase (TAT) is the first enzyme in the tyrosine-derived pathway in the synthesis of rosmarinic acid, which catalyzes the transamination of tyrosine. 4-Hydroxypyruvate (4-HPPDC) is generated, and then 4-HPAA is generated under the action of 4-hydroxyphenylpyruvate decarboxylase (HPPADC) [29-30]. Torrens-Spence et al. [31] first discovered a pyridoxal phosphate-dependent hydroxyphenylacetaldehyde synthase (4HPAAS), which can directly catalyze the conversion of tyrosine to 4-HPAA. A gene encoding a glycosyltransferase that catalyzes the production of salidroside from tyrosol. In addition, tyrosine can be catalyzed by TYR to produce hydroxytyrosol [25]. The enzymes in these enzymatic crossover pathways all enrich the diversity of the biosynthetic pathways of Acteoside. 1.3 The cross-pathway of acyl transfer and glycosyl modification of Acteoside

From the structural analogs of Acteoside, it can be seen that its center is all glucose, which is esterified with caffeoyl, and the C3 position is modified by rhamnosyl (Fig. 1a), but its acylation and rhamnosylation Molecular catalytic mechanism research has not been reported. From the reverse inference of the hydrolysis, metabolic experiments and chemical structure of Acteoside, there are two potential possibilities for the synthesis of Acteoside[19-22]. First, caffeoyl-CoA and hydroxytyrosol glucoside are subjected to the action of acyltransferase (quinine hydroxycinnamyl transferase, HCT) to generate deltoxoside A, which is converted to rhamnosyltransferase (UDP-rhamnosyltransferase). rhamnose glucosyltransferase, URT) catalyzed further production of verbasoseAnother potential pathway is that hydroxytyrosol produces hydroxytyrosol glucoside under the action of glucosyltransferase, and hydroxytyroyl glucoside produces decaffeyl verbasin under the catalysis of URT. It condenses with caffeoyl-CoA to form Acteoside (Fig. 2c).

2 Efficient regulation strategy of Acteoside synthesis

2.1 Transcriptome mining related to Acteoside synthesis

Transcriptome refers to the sum of mRNAs transcribed by organisms under a specific physiological condition or growth cycle and is an important tool to study gene structure, function and growth cycle. It is an important means of gene expression and the main method of association phenotype research. The application of transcriptome technology provides an effective tool for mining related enzyme genes in the biosynthesis of active components of medicinal plants. There are still many key enzymes in the synthesis pathway of Acteoside unresolved, so the mRNA abundance in cells under specific physiological conditions can be used to describe gene expression levels and infer the abundance of final protein products. Through the analysis of relevant transcriptome data, data related to the synthesis of phenylethanoid glycosides were obtained (Table 1), which can provide ideas for the biosynthesis of Acteoside. 

Cistanche Tubulosa is an important medicinal plant for extracting phenylethanoid glycosides, and the study of its genome and transcriptome data provides a valuable resource for the study of the biosynthetic pathway of Acteoside. Deep transcriptome sequencing of the succulent stems of Cistanche Tubulosa was carried out using the RNA-seq analysis method for Cistanche Tubulosa, and its transcriptome data was obtained for the first time. Through sequence comparison and phylogenetic analysis, the key enzymes linking primary metabolism and secondary metabolism were identified. PAL gene, which is also the first key enzyme in phenylethanoid biosynthesis, and 17 enzyme genes involved in phenylethanoid biosynthesis in rhizomes have been speculated for the first time [32]. The results of exploring the quality differences of Cistanche Tubulosa in different types of deserts by metabolomics and transcriptomics showed that the content of phenylethanoid in saline-alkali soils may be higher due to the up-regulation of the expressions of PAL, ALDH, aspartate aminotransferase (GOT), etc. [33]. The analysis of the synthetic pathway of phenethyl glycoside provides the basis. The Scrophulariaceae plant Rehmannia glutinosa is rich in phenylethanol glycosides. By de novo transcriptome sequencing of the tuberous root of Rehmannia glutinosa, and comparing the KEGG pathway and the experimental pathway, 19 candidates that may be related to the biosynthesis of Acteoside are proposed. gene, which provides a direction for the analysis of the synthesis pathway of Acteoside. Then, the data was centrally verified by RT-PCR, and it was found that the transcriptome of Rehmannia glutinosa encodes prephenate dehydratase (ADT), aspartate aminotransferase (ASP5), GOT2, 4CL, primary amine oxidase (AOC3), C4H, HCT. and C3H genes [34]. Interestingly, the content of Acteoside in the leaves of Rehmannia glutinosa is higher than that in the roots, which may be due to the different expression levels of enzymes in different parts due to the differential expression of genes in specific developmental stages. 

Afterwards, four different cultivars of Rehmannia glutinosa were cultivated with radial stripes and non-radial stripes in tuber roots, and their transcriptome data were analyzed to obtain 223 genes related to Acteoside synthesis. PAL, C4H, C3H, 4CL, TyDC, PPO, CuAO, ALDH, HCT, UGT and other genes were analyzed by transcriptome data [16]. 231 genes involved in the biosynthesis of Acteoside were mined in the transcriptome sequencing of Centantheragrandiflora Benth. 10 genes including PPO and HCT. Among them, CuAO was the most expressed, followed by 4CL, ALDH and UGT, and the least was HCT. Genes involved in the phenylalanine and tyrosine pathways were more abundant in leaves and stems than in roots, especially PAL and PPO genes. And compared with roots, the four genes PAL, C4H, C3H and 4CL in leaves and stems were all up-regulated, indicating that the phenylalanine-derived pathway is highly expressed in the aerial parts [18]. However, the above studies predicted that HCT and UGT catalyzed the downstream enzymatic pathway of caffeoyl-CoA and hydroxytyrosol glucoside to Acteoside through transcriptome studies, but the functions of related genes have not been verified [16,18]. Inducers refer to factors that can induce plants to produce resistance or self-defense and defendin, and can rapidly, specifically and selectively induce the expression of a variety of specific genes in plants, thereby activating specific secondary metabolites [35]. In order to screen the best elicitor that significantly increases the accumulation of Acteoside in the hairy roots of Rehmannia glutinosa, the genetic transformation system of Rehmannia glutinosa mediated by Agrobacterium rhizogenes was used to compare elicitors with different concentrations. It can not only significantly promote the growth of hairy roots but also significantly increase the content of Acteoside. Afterwards, RNA-Seq technology was used to analyze the transcriptome of the hairy roots of Rehmannia glutinosa induced at different times, to find differentially expressed genes under different conditions, and finally to determine PAL, C4H, C3H, 4CL, TyDC, PPO, CuAO, ALDH, HCT, UGT. Isogenic may be involved in Acteoside biosynthesis. Among them, C3H and ALDH are the most abundant and least abundant homologous genes, respectively [17]. In addition, Fatemi et al. [29] evaluated the effect of different concentrations of elicitors such as methyl jasmonate (MeJA) and multi-walled carbon nanotubes (MWCNTs) on the accumulation of rosmarinic acid and the expression levels of genes related to its biosynthetic pathway. The genes of PAL, TAT, 4HPPR and rosmarinic acid synthase (RAS) were identified as key genes, among which TAT has transamination, which provides a new idea for the analysis of the synthesis pathway of Acteoside. Olive (Oleaeuropaea) is a rich source of bioactive polyphenols, which can produce hydroxytyrosol, an important precursor of Acteoside. Genes related to polyphenol biosynthesis in olive fruit and leaves were identified by full-length transcriptome sequencing. Twelve polyphenol compounds were quantified in the experiment, and 106 transcripts of 6 gene families were identified and hydroxytyrosol synthesis The genes related to the synthesis of hydroxytyrosol include PPO, DODC, CuAO, ALDH, and PAR [24]. The results of the above phenethylamine transcriptome study provide a basis for the analysis of the synthesis pathway of Acteoside and the mining of regulatory genes and key enzymes.