The versatile O-methyltransferase LrOMT catalyzes multiple O-methylation reactions in amaryllidaceae alkaloids biosynthesis.

Amaryllidaceae alkaloids are unique benzylphenethylamine derivatives that comprise of more than 600 members with a huge chemical diversity. Most of them showed interesting bioactivities, for instance, galanthamine (GAL) is clinically used for Alzheimer's disease treatment. All Amaryllidaceae alkaloids had been thought to be derived from 4'-O-methylnorbelladine originated from norbelladine catalyzed by norbelladine 4'-O-methyltransferase (N4OMT). Herein we mined the transcriptome datasets of Lycoris radiata, a GAL-producing plant. LrOMT was cloned, overexpressed in Escherichia coli, and purified to homogeneity. Bioinformatics analysis and enzymatic activity assays revealed that LrOMT is an S-adenosylmethionine-dependent Class I OMT. LrOMT exhibited both para- and meta-O-methylation activities toward norbelladine to give 4'- and 3'-O-methylnorbelladine. Twenty-four analogues, including the proposed biosynthetic intermediates, were introduced to investigate the substrate scope of LrOMT and it showed that the aromatic substrates should have two vicinal hydroxyl groups. The LrOMT-catalyzed O-methylation preference is dependent on the properties of the binding group of the substrates. The transcription levels of LrOMT were positively associated with the accumulation of the Amaryllidaceae alkaloids and the biosynthetic intermediates in L. radiata. The present work revealed that LrOMT catalyzes multiple O-methylation reactions and its characterization will be helpful to uncover novel biosynthetic genes for Amaryllidaceae alkaloids biosynthesis.

De Novo Biosynthesis of p-Coumaric Acid in E. coli with a trans-Cinnamic Acid 4-Hydroxylase from the Amaryllidaceae Plant Lycoris aurea.

p-Coumaric acid is a commercially available phenolcarboxylic acid with a great number of important applications in the nutraceutical, pharmaceutical, material and chemical industries. p-Coumaric acid has been biosynthesized in some engineered microbes, but the potential of the plant CYP450-involved biosynthetic route has not investigated in Escherichia coli. In the present study, a novel trans-cinnamic acid 4-hydroxylase (C4H) encoding the LauC4H gene was isolated from Lycoris aurea (L' Hér.) Herb via rapid amplification of cDNA ends. Then, N-terminal 28 amino acids of LauC4H were characterized, for the subcellular localization, at the endoplasmic reticulum membrane in protoplasts of Arabidopsis thaliana. In E. coli, LauC4H without the N-terminal membrane anchor region was functionally expressed when fused with the redox partner of A. thaliana cytochrome P450 enzyme (CYP450), and was verified to catalyze the trans-cinnamic acid to p-coumaric acid transformation by whole-cell bioconversion, HPLC detection and LC-MS analysis as well. Further, with phenylalanine ammonia-lyase 1 of A. thaliana, p-coumaric acid was de novo biosynthesized from glucose as the sole carbon source via the phenylalanine route in the recombinant E. coli cells. By regulating the level of intracellular NADPH, the production of p-coumaric acid was dramatically improved by 9.18-fold, and achieved with a titer of 156.09 µM in shake flasks. The recombinant cells harboring functional LauC4H afforded a promising chassis for biological production of p-coumaric acid, even other derivatives, via a plant CYP450-involved pathway.

Functional characterization of phenylalanine ammonia-lyase- and cinnamate 4-hydroxylase-encoding genes from Lycoris radiata, a galanthamine-producing plant.

Galanthamine (GAL), the well-known Amaryllidaceae alkaloid, is a clinically used drug for the treatment of Alzheimer's disease. L-Phenylalanine (Phe) and trans-cinnamic acid (CA) were enzymatically transformed into the catechol portion of GAL. Herein, a Phe ammonia-lyase-encoding gene LrPAL3 and a cinnamate 4-hydroxylase-encoding gene LrC4H were cloned from Lycoris radiata, a GAL-producing plant. LrPAL3 was overexpressed in Escherichia coli and purified to homogeneity. LrPAL3 catalyzes the forward deamination conversion of L-Phe into trans-CA. The 3-chloro- and 4-fluoro-L-Phe were deaminated to generate the corresponding 3-chloro- and 4-fluoro-trans-CA by LrPAL3. LrPAL3-catalyzed reverse hydroamination was confirmed by the conversion of trans-CA into L-Phe with exceptional regio- and stereo-selectivity. LrC4H was overexpressed in E. coli with tCamCPR, a cytochrome P450 reductase-encoding gene. LrC4H catalyzes the regioselective para-hydroxylation on trans-CA to form p-coumaric acid. The transcriptional levels of both LrPAL3 and LrC4H were positively associated with the GAL contents within the leaves and flowers of L. radiata, which suggested that their expression and function are co-regulated and involved in the biosynthesis of GAL. The present investigations on the biosynthetic genes of GAL will promote the development of synthetic biology platforms for this kind of important drug via metabolic engineering.

Molecular cloning and characterization of S-adenosylmethionine synthetase gene from Lycoris radiata.

S-adenosylmethionine (SAM) synthetase catalyzes the synthesis of SAM, a molecule important for all cellular organisms. It is also considered to play an important role in salt tolerance of plants. Here, we cloned a Lycoris radiata (L. radiata) SAM synthetase gene LrSAMS to determine its biological function. The gene encodes a protein of 401 amino acids with a calculated molecular weight of 43.9 kDa. Amino acid sequence analysis of the deduced protein LrSAMS reveals high sequence identity to SAM synthetases from other organisms, such as Arabidopsis thaliana and Oryza sativa. The deduced LrSAMS protein contains conserved amino acids residues and sequences motifs that closely related to the function of SAM synthetase. Otherwise, the transcript levels of LrSAMS were significantly induced by NaCl treatment in L. radiata leaves, which implied that LrSAMS might play an important role in tolerance to salt stress in L.radiata. Complete ORF of LrSAMS was inserted into expression vector pET-29a(+) and transformed into Escherichia coli BL21 (DE3). The difference between the growth curve of the transgenic strain and control strain with blank vector showed that over-expressing LrSAMS could provide growth advantage to the engineered strain in high salt concentration.

Molecular cloning and characterization of a phenylalanine ammonia-lyase gene (LrPAL) from Lycoris radiata.

LrPAL is a novel full-length cDNA isolated from Lycoris radiata by degenerate oligonucleotide primer PCR (DOP-PCR), 3'- and 5'-RACE approaches, harbours an open reading frame (ORF) encoding a 708 amino acid product. Sequence alignment showed that the deduced amino acid sequence of LrPAL shared more than 80% identity with other PAL sequences reported in Arabidopsis thaliana and other plants. RT-PCR revealed that LrPAL transcripts were higher in bud flowers and wilting flowers (5 days after blooming) than in blooming flowers. The transcript levels of LrPAL in leaves were significantly induced by methyl jasmonate (MJ) and nitric oxide (NO), and salicylic acid (SA). Similarly, HPLC analysis showed that galantamine (GAL) content was also higher in bud flowers and wilting flowers than in blooming flowers. The GAL content in leaves was significantly induced by MJ and NO, and inhibited by SA. This study enables us to further elucidate the role of LrPAL in the biosynthesis of GAL in Lycoris radiata at a molecular level.

Cloning and heterologous expression of a new 3'-hydroxylase gene from Lycoris radiata.

A full-length cDNA (LC3'H) was obtained from a cDNA library of Lycoris radiata by DOP-PCR (degenerate oligonucleotide primer PCR), 3'race and 5'race methods. Compared with the other reported enzymes from different plants, the deduced amino acid sequence of LC3'H exhibits significant homologies to 3'-hydroxylases that are involved in the caffeic acid biosynthesis. These findings suggest that the new gene is closely related to the biosynthesis of caffeic acid, which is also an important step of the galanthamine biosynthesis in Amaryllidaceae plants.

Detection of hydrolytic activity of trypsin with a fluorescence-chymotryptic peptide on a TLC plate.

To find a new trypsin-like enzyme, a simple assay method of the hydrolysis activity for trypsin has been found. We used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the peptide labeling as a substrate for the trypsin-like peptidase in this study. The peptidase activity of trypsin was detected by using an AQC-chymotryptic peptide (AHP1) obtained from bovine hemoglobin. This showed that the substrate specificity of trypsin-like peptidase was distinguishable from that of the others by this procedure, and the method was used extensively in cases of various trypsin inhibitors with no significant interference from the concomitant.