Biochemical characterization of tyrosine aminotransferase and enhancement of salidroside production by suppressing tyrosine aminotransferase in Rhodiola crenulata

https://doi.org/10.1016/j.indcrop.2021.114075Get rights and content

Highlights

  • A tyrosine aminotransferase gene (RcTAT) was cloned from Rhodiola crenulata.

  • RcTAT catalyzes the formation of 4-hydroxyphenylpyruvate from tyrosine.

  • Suppression of RcTAT significantly enhances the salidroside production.

Abstract

Rhodiola crenulata is a traditional Tibetan medicinal plant mainly distributed in high altitude areas of Tibet. As the main extract of R. crenulata, salidroside has important medicinal activities in enhancing the body’s immunity and resisting microwave radiation by influencing cell metabolism. Tyrosine is the starting amino acid precursor for salidroside biosynthesis. As an aromatic amino acid, tyrosine participates not only in salidroside biosynthesis, but also in other metabolite biosynthesis which might compete against salidroside biosynthetic pathway. In this study, we functionally identified a tyrosine aminotransferase in R. crenulata (RcTAT) that could convert tyrosine into 4-hydroxyphenylpyruvate (4-HPP). The Km and Vmax values of RcTAT for tyrosine were respectively 0.21 mM and 0.17 μmol/min.mg under 9.0 pH and 39 °C. RNAi-mediated suppression of RcTAT expression markedly increased salidroside production in hairy root culture of R. crenulata. In summary, RcTAT is a key gene involved in the metabolic pathway that competes against salidroside biosynthesis, and suppressing its expression is a promising way to elevate salidroside production in planta.

Introduction

Rhodiola crenulata, belonging to Rosaceae, mainly grows in alpine meadows and rock crevices at an altitude of 3500∼5000 m in Tibet (Fig. 1A) (Chan, 2012). Although Rhodiola crenulata grows in a harsh environment with poor soil, strong ultraviolet radiation and insufficient oxygen, it has a special ability to adapt to drought and cold, making it a very rare Tibetan medicinal plant. In addition, Rhodiola crenulata has high medicinal and nutritional values, with the reputation of “Snow Ginseng” and “Plant Gold” in the folk (Zhao et al., 2012). In the market, Rhodiola crenulata is used as raw material to produce a wide variety of drugs with remarkable curative effects, and also used to produce wine, drinks, tea and other kinds of food. The wonderful values of R. crenulata make the demand for it increase continuously (Yang et al., 2012). Salidroside is the representative active pharmaceutical ingredient in R. crenulata (Lei et al., 2004), which not only has the effect on enhancing immunity, lowering blood sugar, maintaining cardiovascular stability, protecting liver injury in clinical practice, but also has anti-hypoxia, anti-aging, anti-radiation, anti-fatigue and other resistance effects (Xue et al., 2019). Rhodiola crenulata is the main plant source of salidroside. Due to the limitation of its special growth environment and the continuous consumption of wild R. crenulata resources, the supply of salidroside will be in short in the future. Therefore, it is urgent to find new strategies to elevate salidroside production (Kaminaga et al., 2004).

The biosynthesis of salidroside has been well elucidated in Rhodiola species. The chemical structural formula of salidroside is tyrosol 8-O-β-d-glucoside (C14H20O7). Tyrosine is the starting precursor for salidroside biosynthesis, and there are three enzymes involved in the salidroside biosynthetic pathway, including 4-hydroxyphenylacetaldehyde synthase (4-HPAAS), 4-hydroxyphenylacetaldehyde reductase (4-HPAR) and tyrosol:UDP-glucose 8-O-glucosyltransferase (T8GT) in sequence (Fig. 1B). The 4-HPAAS enzyme catalyzes tyrosine to generate 4-hydroxyphenyl acetaldehyde (4-HPAA), which is then reduced to form tyrosol under the catalysis of 4-HPAR. Finally the glucosyltransferase (T8GT) converts tyrosol to salidroside (Torrens-Spence et al., 2018). According to previous research, 4-HPAA is involved not only in salidroside biosynthesis, but also in the biosynthesis of benzylisoquinoline alkaloids (BIAs) (Hagel and Facchini, 2013). Identification of the genes involved in salidroside biosynthesis provided candidates for engineering salidroside production in planta. Overexpression of these genes is often used to promote the production of salidroside. The 4-HPAAS gene of R. crenulata, formerly named RcTyDC, was overexpressed to promote the production of tyrosol and salidroside in hairy root cultures of R. crenulata (Lan et al., 2013). Besides overexpression, suppression of genes involved in other competitive metabolic branches is an alternative method to enhance the target metabolite production. For example, artemisinin and other sesquiterpenes, such as β-caryophyllene and β-farnesene, share the same 15-carbon precursor (farnesyl diphosphate, FPP), suggesting that reduced biosynthesis of β-caryophyllene or β-farnesene might facilitate artemisinin production. When genes responsible for β-caryophyllene and β-farnesene biosynthesis were respectively suppressed by RNA interference (RNAi) technology, more FPP flux went into artemisinin biosynthesis and hence the artemisinin production was markedly elevated in Artemisia annua (Lv et al., 2016).

In salidroside biosynthesis, tyrosine is the starting precursor. As an aromatic amino acid, tyrosine participates not only in salidroside biosynthesis, but also in the biosynthesis of other metabolites, such as tocopherols, plastoquinone and ubiquinone that are rich in plants (Xu et al., 2020). Obviously, the biosynthetic pathways of tyrosine-derived metabolites compete against salidroside biosynthesis. Therefore, salidroside production might be enhanced through reducing tyrosine’s conversion to non-salidroside metabolites. Tyrosine aminotransferase (TAT) is an enzyme widely present in organisms, which transfers the amino group from tyrosine to the carbonyl group of α-ketoglutarate (Hudson and Prabhu, 2010), generating 4-hydroxyphenylpyruvate (4-HPP) and glutamate. Then 4-HPP goes into the biosynthetic pathways of tocopherols, plastoquinone and ubiquinone (Fig. 1B) (Xu et al., 2020). So, suppression of TAT will be an alternative method to enhance salidroside production in planta. In this study, it is reported that the TAT gene from R. crenulata (RcTAT) was functionally identified and the production of salidroside was increased by suppressing RcTAT in hairy root cultures of R. crenulata for the first time.

Section snippets

Plant materials and chemicals

The plant material of R. crenulata was formally granted by the Tibet Science and Technology Commission (Lhasa, Tibet, China) in July 2011 (Liao et al., 2018). The R. crenulata seeds used in the experiment were collected in September 2016 from a 5000-meter-high mountain in Lang County, Tibet. They were germinated and cultivated in our laboratory. Large and plump R. crenulata seeds were chosen and dried naturally, and then stored at 4 °C for 3 weeks. Then the seeds were soaked in sterile water

Isolation and characterization of RcTAT

The 1574-bp full-length cDNA of RcTAT was isolated by RACE (Fig. 2A). Its coding region is 1236-bp long, which encodes 411 amino acids (Fig. 2B). The calculated protein molecular weight is 45.3 kDa and the theoretical isoelectric point is 8.93. The prediction of the conserved domain indicated that RcTAT includes a pyridoxal phosphate binding site (SLSKRWLVPGWRLG) of the aminotransferase family (Huang et al., 2008), and it also belongs to the tyrosine aminotransferase subfamily, according to the

Conclusion

In this study, the enzyme RcTAT was identified and characterized from R. crenulata that catalyzes the conversion of tyrosine into 4-HPP, which competes for a common substrate tyrosine with the enzymes in salidroside biosynthetic pathway. The repression of RcTAT expression enhanced salidroside biosynthesis, suggesting that the biosynthesis of target metabolites can be increased through attenuating its competitive metabolic pathways.

CRediT authorship contribution statement

Xuechao Liu: Conceptualization, Investigation, Formal analysis, Data curation, Visualization, Writing – original draft, Writing – review & editing. Yueli Tang: Formal analysis, Validation, Writing – original draft. Junlan Zeng: Conceptualization, Investigation, Formal analysis, Data curation. Jianbo Qin: Resources. Min Lin: Resources, Supervision. Min Chen: Resources. Zhihua Liao: Conceptualization, Funding acquisition, Supervision, Project administration, Writing – review & editing. Xiaozhong

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgments

We thank the support of the NSFC project (Grant NO. 81660628) and the Forth National Survey of Traditional Chinese Medicine Resources, Chinese or Tibet Medicinal Resources Investigation in Tibet Autonomous Region (Grant NO. 20191217-540124, 20191223-540126, and 20200501-542329).

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