Cloning and characterization of a tyrosine decarboxylase involved in the biosynthesis of galanthamine inLycoris aurea

Abstract

Background Galanthamine, one kind of Amaryllidaceae alkaloid extracted from the Lycoris species, is used in the treatment of Alzheimer’s disease. In regards to medical and economic importance, the biosynthesis and regulatory mechanism of the secondary metabolites in Lycoris remain uninvestigated. Methods BLAST was used to identify the sequence of tyrosine decarboxylase in the transcriptome of Lycoris aurea (L’Hér) Herb. The enzyme activity of this TYDC was determined by using heterologous expressed protein in the Escherichia coli cells. The related productive contents of tyramine were detected using High Performance Liquid Chromatography (HPLC). According to the available micro RNA sequencing profiles and degradome database of L. aurea, microRNA396 were isolated, which targets to LaTYDC1 and RNA Ligase-Mediated-Rapid Amplification of cDNA Ends (RLM-RACE) were used to confirm the cleavage. The expression levels of miR396 and LaTYDC1 were measured using a quantitative real-time polymerase chain reaction (qRT-PCR). Results LaTYDC1 was mainly expressed in root, bulb, leaf and flower fitting the models for galanthamine accumulation. This decarboxylase efficiently catalyzes tyrosine to tyramine conversion. Under methyl jasmonate (MeJA) treatment, the expression of LaTYDC1 and the content of tyramine sharply increase. The use of RLM-RACE confirms that miR396 promotes the degradation of LaTYDC1 mRNA. Under MeJA treatment, the expression of miR396 was suppressed while the expression level of LaTYDC1 sharply increased. Following the increase of the miR396 transcriptional level, LaTYDC1 was significantly repressed. Conclusion LaTYDC1 participates in the biosynthesis of galanthamine, and is regulated by miR396. This finding also provides genetic strategy for improving the yield of galanthamine in the future.

Biosynthesis and Biological Activities of Newly Discovered Amaryllidaceae Alkaloids

“…The enzyme responsible for tyramine biosynthesis is the tyrosine decarboxylase (TYDC) ( Figure 2). Two gene transcript variants of TYDC, named TYDC1 and TYDC2, were identified from the transcriptome of different Amaryllidaceae species including N. pseudonarcissus [41], Narcissus papyraceus [42], Lycoris radiata [43], and L. aureus [44].…”

Identification, molecular characterization and expression of JAZ genes in Lycoris aurea

“…Herb, is an ornamentally and medicinally important perennial herbaceous plant, belonging to the Amaryllidaceae family, from which there were a variety of Amaryllidaceae alkaloids found to exhibit medicinal values [22]. Previous studies of L. aurea have mainly focused on karyotype [23][24][25], physiological analysis [26,27], chemical composition [28][29][30], medicinal usage [31,32], and molecular aspects for gene cloning [33][34][35][36]. For example, several enzymes involved in Amaryllidaceae alkaloids biosynthesis, including cinnamate 4-hydroxylase [34], tyrosine decarboxylase [35] and norbelladine 4'-O-methyltransferase [36] have been identified.…”

“…Previous studies of L. aurea have mainly focused on karyotype [23][24][25], physiological analysis [26,27], chemical composition [28][29][30], medicinal usage [31,32], and molecular aspects for gene cloning [33][34][35][36]. For example, several enzymes involved in Amaryllidaceae alkaloids biosynthesis, including cinnamate 4-hydroxylase [34], tyrosine decarboxylase [35] and norbelladine 4'-O-methyltransferase [36] have been identified. Besides, although the induction effects of exogenous methyl jasmonate (MeJA) treatments on Amaryllidaceae alkaloids accumulation has been reported [37,38], little is known about the possible transcriptional or post-transcriptional regulation mechanism of this biological process in L. aurea.…”

Recent Progress in Amaryllidaceae Biotechnology

“…TYDC is an important enzyme, which catalyzes the production of tyramine from L-tyrosine [ 68 ]. It has been found that two transcript variants of TYDC are present in Amaryllidaceae alkaloid producing plants [ 59 , 65 , 66 , 69 , 70 ]. The phylogenetic analysis showed that Amaryllidaceae TYDCs can be divided into two main clusters: “TYDC1” and “TYDC2” [ 59 ].…”

“…Recently, TYDC1 from Lycoris aurea (L’Hér) Herb. was cloned in Escherichia coli BL21 (DE3) and the expressed enzyme was characterized [ 69 ]. The specialized enzymes involved in stage three of Amaryllidaceae alkaloid biosynthesis are norbelladine synthase (NBS), noroxomaritidine reductase (NR) and norbelladine 4′- O -methyltransferase (N4OMT) ( Figure 2 ).…”