Regulation by fungal endophyte of Rhodiola crenulata from enzyme genes to metabolites based on combination of transcriptome and metabolome.

BACKGROUND: The contents of some its crucial metabolites tend to decrease when Rhodiola crenulata is cultured at low altitude. Interestingly, it was found that an endophyte, Phialocephala fortinii, could alleviate this problem. RESULTS: There were 16 151 differential genes including 14 706 up-regulated and 1445 down-regulated unigenes with significant differences (P < 0.05), and a total of 1432 metabolites exhibited statistically significant (P < 0.05) metabolic differences comprising 27 different marker metabolites which showed highly significant values of VIP > 5 and P < 0.01. Results highlight differential regulation of 20 enzymatic genes that are involved in the biosynthesis of five different marker metabolites including acetaldehyde, homocysteine, cyclopropylamine, 1-pyrrolinium and halistanol sulfate. CONCLUSIONS: The positive physiological effect of P. fortinii on R. crenulata encompasses differential regulation in carbohydrate metabolism, lipid metabolism and secondary metabolite synthesis. © 2020 Society of Chemical Industry.

Identification of Glutathione Peroxidase (GPX) Gene Family in Rhodiola crenulata and Gene Expression Analysis under Stress Conditions.

Glutathione peroxidases (GPXs) are important enzymes in the glutathione-ascorbate cycle for catalyzing the reduction of H2O2 or organic hydroperoxides to water. GPXs play an essential role in plant growth and development by participating in photosynthesis, respiration, and stress tolerance. Rhodiola crenulata is a popular traditional Chinese medicinal plant which displays an extreme energy of tolerance to harsh alpine climate. The GPXs gene family might provide R. crenulata for extensively tolerance to environment stimulus. In this study, five GPX genes were isolated from R. crenulata. The protein amino acid sequences were analyzed by bioinformation softwares with the results that RcGPXs gene sequences contained three conserve cysteine residues, and the subcellular location predication were in the chloroplast, endoplasmic reticulum, or cytoplasm. Five RcGPXs members presented spatial and temporal specific expression with higher levels in young and green organs. And the expression patterns of RcGPXs in response to stresses or plant hormones were investigated by quantitative real-time PCR. In addition, the putative interaction proteins of RcGPXs were obtained by yeast two-hybrid with the results that RcGPXs could physically interact with specific proteins of multiple pathways like transcription factor, calmodulin, thioredoxin, and abscisic acid signal pathway. These results showed the regulation mechanism of RcGPXs were complicated and they were necessary for R. crenulata to adapt to the treacherous weather in highland.

De novo biosynthesis of Gastrodin in Escherichia coli.

Gastrodin, a phenolic glycoside, is the key ingredient of Gastrodia elata, a notable herbal plant that has been used to treat various conditions in oriental countries for centuries. Gastrodin is extensively used clinically for its sedative, hypnotic, anticonvulsive and neuroprotective properties in China. Gastrodin is usually produced by plant extraction or chemical synthesis, which has many disadvantages. Herein, we report unprecedented microbial synthesis of gastrodin via an artificial pathway. A Nocardia carboxylic acid reductase, endogenous alcohol dehydrogenases and a Rhodiola glycosyltransferase UGT73B6 transformed 4-hydroxybenzoic acid, an intermediate of ubiquinone biosynthesis, into gastrodin in Escherichia coli. Pathway genes were overexpressed to enhance metabolic flux toward precursor 4-hydroxybenzyl alcohol. Furthermore, the catalytic properties of the UGT73B6 toward phenolic alcohols were improved through directed evolution. The finally engineered strain produced 545mgl(-1) gastrodin in 48h. This work creates a new route to produce gastrodin, instead of plant extractions and chemical synthesis.

Engineering an Ancestral Glycosyltransferase for Biosynthesis of 2-Phenylethyl-ß-d-Glucopyranoside and Salidroside.

Phenylethanoid glycosides (PhGs) are naturally occurring glycosides derived from plants with various biological activities. Glycosyltransferases catalyze the production of PhGs from phenylethanols via a transglycosylation reaction. The low activity and stability of glycosyltransferase limit its industrial application. An ancestral glycosyltransferase, UGTAn85, with heat resistance, alkali resistance, and high stability was resurrected using ancestral sequence reconstruction technology. This enzyme can efficiently convert phenylethanols to PhGs. The optimal reaction temperature and pH for UGTAn85 were found to be 70 °C and pH 10.0, respectively. This study employed a combination of structure-guided rational design and co-evolution analysis to enhance its catalytic activity. Potential mutation sites were identified through computer-aided design, including homology modeling, molecular docking, Rosetta dock design, molecular dynamics simulation, and co-evolution analysis. By targeted mutagenesis, the UGTAn85 mutant Q23E/N65D exhibited a 2.2-fold increase in enzyme activity (11.85 U/mg) and elevated affinity (Km = 0.11 mM) for 2-phenylethanol compared to UGTAn85. Following a fed-batch reaction, 36.16 g/L 2-phenylethyl-ß-d-glucopyranoside and 51.49 g/L salidroside could be produced within 24 h, respectively. The findings in this study provide a new perspective on enhancing the stability and activity of glycosyltransferases, as well as a potential biocatalyst for the industrial production of PhGs.

A tyrosine decarboxylase catalyzes the initial reaction of the salidroside biosynthesis pathway in Rhodiola sachalinensis.

Salidroside, the 8-O-ß-D-glucoside of tyrosol, is the main bioactive component of Rhodiola species and is found mainly in the plant roots. It is well known that glucosylation of tyrosol is the final step in the biosynthesis of salidroside; however, the biosynthetic pathway of tyrosol and its regulation are less well understood. A summary of the results of related studies revealed that the precursor of tyrosol might be tyramine, which is synthesized from tyrosine. In this study, a cDNA clone encoding tyrosine decarboxylase (TyrDC) was isolated from Rhodiola sachalinensis A. Bor using rapid amplification of cDNA ends. The resulting cDNA was designated RsTyrDC. RNA gel-blot analysis revealed that the predominant sites of expression in plants are the roots and high levels of transcripts are also found in callus tissue culture. Functional analysis revealed that tyrosine was best substrate of recombinant RsTyrDC. The over-expression of the sense-RsTyrDC resulted in a marked increase of tyrosol and salidroside content, but the levels of tyrosol and salidroside were 274 and 412%, respectively, lower in the antisense-RsTyrDC transformed lines than those in the controls. The data presented here provide in vitro and in vivo evidence that the RsTyrDC can regulate the tyrosol and salidroside biosynthesis, and the RsTyrDC is most likely to have an important function in the initial reaction of the salidroside biosynthesis pathway in R. sachalinensis.

Identification of a novel UDP-glycosyltransferase gene from Rhodiola rosea and its expression during biotransformation of upstream precursors in callus culture.

Roseroot (Rhodiola rosea L.) is a medicinal plant with adaptogenic properties and several pharmaceutically important metabolites. In this study, a full length cDNA encoding a UDPG gene of roseroot was identified, cloned and characterized. Its ORF (1425 bp) was transferred into E. coli, where the expression of the recombinant enzyme was confirmed. To monitor the enzyme activity, 3 precursors (tyramine, 4-hydroxyphenylpyruvate & tyrosol) of salidroside biosynthesis pathway were added to roseroot callus cultures and samples were harvested after 1, 6, 12, 24, 48 & 96 h. Along with the controls (without precursor feeding), each sample was subjected to HPLC and qRT-PCR for phytochemical and relative UDP-glycosyltransferase gene expression analysis, respectively. The HPLC analysis showed that the salidroside content significantly increased; reaching 0.5% of the callus dry weight (26-fold higher than the control) after 96 h when 2 mM tyrosol was given to the media. The expression of the UDP-glycosyltransferase increased significantly being the highest at 12 h after the feeding. The effect of tyramine and 4-hydroxyphenylpyruvate was not as pronounced as of tyrosol. Here, we introduce a R. rosea specific UDPG gene and its expression pattern after biotransformation of intermediate precursors in in vitro roseroot callus cultures.

Effects of overexpression of endogenous phenylalanine ammonia-lyase (PALrs1) on accumulation of salidroside in Rhodiola sachalinensis.

Salidroside, a novel effective adaptogenic drug extracted from the medicinal plant Rhodiola sachalinensis A. Bor, can be derived from phenylalanine or tyrosine. Due to the scarcity of R. sachalinensis and its low yield of salidroside, there is great interest in enhancing production of salidroside by the plant. In this study, a cDNA clone encoding phenylalanine ammonia-lyase (PAL) was isolated from R. sachalinensis using rapid amplification of cDNA ends. The resulting cDNA was designated PALrs1. It is 2407-bp long and encodes 710 deduced amino acid residues. Southern blot analysis of genomic DNA indicated that the PAL gene family is composed of three to five genes in the R. sachalinensis genome. Northern blot analysis revealed that transcripts of PALrs1 were present in calli, leaves and stems, but expression in roots was very low. The PALrs1 under the 35S promoter with double-enhancer sequences from CaMV-Omega and TMV-Omega fragments was transferred into R. sachalinensis via Agrobacterium tumefaciens. PCR and PCR-Southern blot confirmed that the PALrs1 gene had been integrated into the genome of transgenic plants. Northern blot analysis revealed that the PALrs1 gene had been expressed at the transcriptional level. High-performance liquid chromatography indicated that overexpression of the PALrs1 gene resulted in a 3.3-fold increase in p-coumaric acid content, as expected. In contrast, levels of tyrosol and salidroside were 4.7-fold and 7.7-fold, respectively, lower in PALrs1 transgenic plants than in controls. Furthermore, overexpression of the PALrs1 gene resulted in a 2.6-fold decrease in tyrosine content. These data suggest that overexpression of the PALrs1 gene and accumulation of p-coumaric acid did not facilitate tyrosol biosynthesis; tyrosol, as a phenylethanoid derivative, is not derived from phenylalanine; and reduced availability of tyrosine most likely resulted in a large reduction in tyrosol biosynthesis and accumulation of salidroside.

A Novel UDP-Glycosyltransferase of Rhodiola crenulata Converts Tyrosol to Specifically Produce Icariside D2.

Rhodiola crenulata is a Tibetan native herbal plant belonging to the family of Crassulaceae, which produces the pharmaceutical icariside D2 with the activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. In this study, we functionally characterized a novel UDP-glycosyltransferase (RcUGT1) that converted tyrosol to specifically produce icariside D2 from R. crenulata at molecular and biochemical levels. RcUGT1 was highly expressed in flowers and roots, while the icariside D2 content was much higher in stems than that in other organs, suggesting the potential translocation of icariside D2 from flowers and roots to stems. The high production of icariside D2 in stems provided a reasonable suggestion to farmers to harvest stems instead of roots for icariside D2 production. Enzymatic assays of recombinant RcUGT1 indicated that it converted tyrosol to specifically form icariside D2, with the values of Km 0.97±0.10 mM, Vmax 286±8.26 pKat/mg, Kcat 0.01552 s-1, and Kcat/Km 159.55 s-1 M-1. Functional identification of RcUGT1 facilitated the icariside D2 production through metabolic engineering in plants or synthetic biology in microbes.

[Differentiation among populations of the Rhodiola iremelica Boriss. (Grassulaceae). in the Southern Urals].

Isoenzyme markers and polyacrylamide gel electrophoresis have been used to study the genetic structure of populations of Rhodiola iremelica Boriss. (Grassulaceae), a Southern Ural endemic protected by the state and included in the Red Data Book of Bashkortostan Republic. A relatively large genetic variation at the species level has been found. The subdivision among populations (F(ST) = 0.115) is higher than in most cross-pollination angiosperms. No consistent pattern has been observed in the spatial distribution of its genetic variation. The relatively high differentiation among samples of R. iremelica characterized by small effective population sizes, may be accounted for by genetic drift, inbreeding, and a restricted gene flow. To preserve the population gene pool, in situ protection of the species in nature is insufficient. It seems advisable to create synthetic populations ex situ and reintroduce them into nature.

Production of three phenylethanoids, tyrosol, hydroxytyrosol, and salidroside, using plant genes expressing in Escherichia coli.

Polyphenols, which include phenolic acids, flavonoids, stilbenes, and phenylethanoids, are generally known as useful antioxidants. Tyrosol, hydroxytyrosol, and salidroside are typical phenylethanoids. Phenylethanoids are found in plants such as olive, green tea, and Rhodiola and have various biological activities, including the prevention of cardiovascular diseases, cancer, and brain damage. We used Escherichia coli to synthesize three phenylethanoids, tyrosol, hydroxytyrosol, and salidroside. To synthesize tyrosol, the aromatic aldehyde synthase (AAS) was expressed in E. coli. Hydroxytyrosol was synthesized using E. coli harboring AAS and HpaBC, which encodes hydroxylase. In order to synthesize salidroside, 12 uridine diphosphate-dependent glycosyltransferases (UGTs) were screened and UGT85A1 was found to convert tyrosol to salidroside. Using E. coli harboring AAS and UGT85A1, salidroside was synthesized. Through the optimization of these three E. coli strains, we were able to synthesize 531 mg/L tyrosol, 208 mg/L hydroxytyrosol, and 288 mg/L salidroside, respectively.

Fermentative Production of Phenolic Glucosides by Escherichia coli with an Engineered Glucosyltransferase from Rhodiola sachalinensis.

Three rosmarinic acid analogs produced by recombinant Escherichia coli, two xanthones from fungi and honokiol from plants, were explored as the substrates of E. coli harboring a glucosyltransferase mutant UGT73B6FS to generate phenolic glucosides. Six new and two known compounds were isolated from the fermentation broth of the recombinant strain of the feeding experiments, and the compounds were identified by spectroscopy. The biotransformation of rosmarinic acid analogs and xanthones into corresponding glucosides was presented for the first time. This study not only demonstrated the substrate flexibility of the glucosyltransferase mutant UGT73B6FS toward aromatic alcohols but also provided an effective and economical method to produce phenolic glucosides by fermentation circumventing the use of expensive precursor UDP-glucose.

Production of salidroside in metabolically engineered Escherichia coli.

Salidroside (1) is the most important bioactive component of Rhodiola (also called as "Tibetan Ginseng"), which is a valuable medicinal herb exhibiting several adaptogenic properties. Due to the inefficiency of plant extraction and chemical synthesis, the supply of salidroside (1) is currently limited. Herein, we achieved unprecedented biosynthesis of salidroside (1) from glucose in a microorganism. First, the pyruvate decarboxylase ARO10 and endogenous alcohol dehydrogenases were recruited to convert 4-hydroxyphenylpyruvate (2), an intermediate of L-tyrosine pathway, to tyrosol (3) in Escherichia coli. Subsequently, tyrosol production was improved by overexpressing the pathway genes, and by eliminating competing pathways and feedback inhibition. Finally, by introducing Rhodiola-derived glycosyltransferase UGT73B6 into the above-mentioned recombinant strain, salidroside (1) was produced with a titer of 56.9 mg/L. Interestingly, the Rhodiola-derived glycosyltransferase, UGT73B6, also catalyzed the attachment of glucose to the phenol position of tyrosol (3) to form icariside D2 (4), which was not reported in any previous literatures.

Characterization of glycosyltransferases responsible for salidroside biosynthesis in Rhodiola sachalinensis.

Salidroside, the 8-O-ß-D-glucoside of tyrosol, is a novel adaptogenic drug extracted from the medicinal plant Rhodiola sachalinensis A. Bor. Due to the scarcity of R. sachalinensis and its low yield of salidroside, there is great interest in enhancing production of salidroside by biotechnological manipulations. In this study, two putative UDP-glycosyltransferase (UGT) cDNAs, UGT72B14 and UGT74R1, were isolated from roots and cultured cells of methyl jasmonate (MeJA)-treated R. sachalinensis, respectively. The level of sequence identity between their deduced amino acid sequences was ca. 20%. RNA gel-blot analysis established that UGT72B14 transcripts were more abundant in roots, and UGT74R1 was highly expressed in the calli, but not in roots. Functional analysis indicated that recombinant UGT72B14 had the highest level of activity for salidroside production, and that the catalytic efficiency (Vmax/Km) of UGT72B14 was 620% higher than that of UGT74R1. The salidroside contents of the UGT72B14 and UGT74R1 transgenic hairy root lines of R. sachalinensis were also ∼420% and ∼50% higher than the controls, respectively. UGT72B14 transcripts were mainly detected in roots, and UGT72B14 had the highest level of activity for salidroside production in vitro and in vivo.

Isolation and genotype-dependent, organ-specific expression analysis of a Rhodiola rosea cDNA encoding tyrosine decarboxylase.

Tyrosine decarboxylase (TyrDC) is an important enzyme in the secondary metabolism of several plant species, and was hypothesized to play a key role in the biosynthesis of salidroside, a pharmacologically valuable compound of roseroot. A 1520bp cDNA was cloned and sequenced, and turned out to contain an ORF of 963bp, which encodes a protein of 320 amino acids. The expression of the gene was studied by real-time PCR from leaves and roots of both high and low salidroside producer genotype of roseroot. The gene expression analysis showed the gene to be expressed in leaves as well as in roots; however, the expression was significantly higher in roots, which coincides with the fact that salidroside accumulates preferentially in the underground parts of the plant. The expression was also higher in the line accumulating high levels of salidroside, compared to the line with lower salidroside content. The difference in the expression intensity suggests a decisive role for this enzyme in the salidroside biosynthesis.