Berberine Biosynthesis Research Articles

Molecular cloning and characterization of methylenedioxy bridge‐forming enzymes involved in stylopine biosynthesis in Eschscholzia californica

(S)-stylopine is an important intermediate in the biosynthesis of benzophenanthridine alkaloids, such as sanguinarine. Stylopine biosynthesis involves the sequential formation of two methylenedioxy bridges. Although the methylenedioxy bridge-forming P450 (CYP719) involved in berberine biosynthesis has been cloned from Coptis japonica[Ikezawa N, Tanaka M, Nagayoshi M, Shinkyo R, Sakaki T, Inouye K & Sato F (2003) J Biol Chem278, 38557-38565], no information is available regarding the genes for methylenedioxy bridge-forming enzymes in stylopine biosynthesis. Two cytochrome P450 cDNAs involved in stylopine biosynthesis were isolated using degenerate primers designed for C. japonica CYP719 from cultured Eschscholzia californica cells. Heterologous expression in Saccharomyces cerevisiae showed that both CYP719A2 and CYP719A3 had stylopine synthase activity to catalyze methylenedioxy bridge-formation from cheilanthifoline to stylopine, but not cheilanthifoline synthase activity to convert scoulerine to cheilanthifoline. Functional differences and expression patterns of CYP719A2 and CYP719A3 were examined to investigate their physiological roles in stylopine biosynthesis. Enzymatic analysis showed that CYP719A2 had high substrate affinity only toward (R,S)-cheilanthifoline, whereas CYP719A3 had high affinity toward three similar substrates (R,S)-cheilanthifoline, (S)-scoulerine, and (S)-tetrahydrocolumbamine. An expression analysis in E. californica plant tissues showed that CYP719A2 and CYP719A3 exhibited expression patterns similar to those of three stylopine biosynthetic genes (CYP80B1, berberine bridge enzyme, and S-adenosyl-l-methionine : 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase), whereas the specific expression of CYP719A3 in root was notable. Treatment of E. californica seedlings with methyl jasmonate resulted in the coordinated induction of CYP719A2 and CYP719A3 genes. The physiological roles of CYP719A2 and CYP719A3 in stylopine biosynthesis are discussed.

Identification of a WRKY Protein as a Transcriptional Regulator of Benzylisoquinoline Alkaloid Biosynthesis in Coptis japonica

Selected cultured Coptis japonica cells produce a large amount of the benzylisoquinoline alkaloid berberine. Previous studies have suggested that berberine productivity is controlled at the transcript level of biosynthetic genes. We have identified a regulator of transcription in berberine biosynthesis using functional genomics with a transient RNA interference (RNAi) and overexpression of the candidate gene. The 24 primary candidate clones were selected from 1,014 expressed sequence tags (ESTs) that were obtained from a C. japonica cell line producing high levels of berberine. Further characterization of the expression profiles of these ESTs suggested that five ESTs would be good candidates as regulators of berberine production. A newly developed transient RNAi system with C. japonica protoplasts indicated that double-stranded RNA of an EST clone significantly reduced the level of transcripts of 3'-hydroxy N-methylcoclaurine 4'-O-methyltransferase. Sequence analysis showed that this EST encoded a group-II WRKY, and we named it CjWRKY1. When the effects of double-stranded RNA of the CjWRKY1 gene were examined in detail, a marked reduction in the transcripts of all genes involved in berberine biosynthesis was detected, whereas little effect was found in the transcript levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and chorismate mutase (CM) that are associated with primary metabolism. Ectopic expression of CjWRKY1 cDNA in C. japonica protoplasts clearly increased the level of transcripts of all berberine biosynthetic genes examined compared with control treatment, whereas the levels of GAPDH and CM were not affected. The functional role of CjWRKY1 as a specific and comprehensive regulator of berberine biosynthesis is discussed.

Molecular Cloning and Characterization of CYP719, a Methylenedioxy Bridge-forming Enzyme That Belongs to a Novel P450 Family, from cultured Coptis japonica Cells

Two cytochrome P450 (P450) cDNAs involved in the biosynthesis of berberine, an antimicrobial benzylisoquinoline alkaloid, were isolated from cultured Coptis japonica cells and characterized. A sequence analysis showed that one C. japonica P450 (designated CYP719) belonged to a novel P450 family. Further, heterologous expression in yeast confirmed that it had the same activity as a methylenedioxy bridge-forming enzyme (canadine synthase), which catalyzes the conversion of (S)-tetrahydrocolumbamine ((S)-THC) to (S)-tetrahydroberberine ((S)-THB, (S)-canadine). The other P450 (designated CYP80B2) showed high homology to California poppy (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B1), which converts (S)-N-methylcoclaurine to (S)-3'-hydroxy-N-methylcoclaurine. Recombinant CYP719 showed typical P450 properties as well as high substrate affinity and specificity for (S)-THC. (S)Scoulerine was not a substrate of CYP719, indicating that some other P450, e.g. (S)-cheilanthifoline synthase, is needed in (S)-stylopine biosynthesis. All of the berberine biosynthetic genes, including CYP719 and CYP80B2, were highly expressed in selected cultured C. japonica cells and moderately expressed in root, which suggests coordinated regulation of the expression of biosynthetic genes.

Application of vanadate-induced nucleotide trapping to plant cells for detection of ABC proteins.

The vanadate-induced nucleotide trapping technique, which has been conventionally used to characterize mammalian ATP-binding cassette (ABC) proteins, was applied to berberine-producing plant cell cultures, Thalictrum minus and Coptis japonica. One membrane protein at ca. 180 kDa was photoaffinity-labeled with 8-azido-[alpha-(32)P]ATP in the T. minus cells in the presence of vanadate, which was specifically induced by the addition of benzyladenine in a similar manner as the induction of berberine biosynthesis in these cell cultures, whereas three bands were observed in the C. japonica cells in the size region between 120 and 150 kDa corresponding to full-sized ABC protein. The benzyladenine-induced band in T. minus showed properties similar to those of human MDR1, including the recognition of berberine, which suggests that the ABC protein detected in T. minus takes this endogenous alkaloid as a putative substrate for transport. This is the first application of this technique to plant cells.

Immunocytological localization of two enzymes involved in berberine biosynthesis.

Using post-embedding immunogold techniques the cytological localization of the two branchpoint enzymes of isoquinoline biosynthesis, berberine bridge enzyme (BBE) and (S)-tetrahydroprotoberberine oxidase (STOX), was demonstrated. Electron-microscopic examination revealed their exclusive compartmentation within vesicles. After these vesicles have fused with the central vacuole, they release their contents, resulting in a characteristic electron-dense precipitate at the tonoplast. Vesicles of similar structure could be identified in young meristematic tissues of roots or shoots from different Berberis species and Papaver somniferum L. The appearance of electron-dense osmiophilic material is strictly correlated with the alkaloid content of the tissue. Immunocytological staining of P. somniferum tissue with antibodies directed against BBE led to a characteristic labeling of electron-dense aggregates in idioblasts that are not connected to the laticifer system. This localization demonstrates the strictly cytological separation of benzophenanthridine and morphine biosyntheses within this plant.

Formula omitted]: Norcoclaurine 6- O-methyltransferase from Thalictrum minus cell cultures

S- Adenosyl- l-methionine (SAM): norcoclaurine 6- O-methyltransferase (NCMT), which controls berberine biosynthesis in Thalictrum minus cell cultures, was purified to homogeneity. The purified NCMT had a native M r of 72000 consisting of two subunits of 37000 each, a pH optimum of 9.0 and a p I of 4.3. The enzyme did not require Mg 2+ for activation, but was strongly inhibited by divalent cations of heavy metals and by SH reagents. Kinetic studies indicated a sequential mechanism with K m values of 56 μM for norcoclaurine and 260 μM for SAM. S- Adenosyl- l-homocysteine (SAH) was a potent inhibitor of NCMT and its K i value for SAH vs SAM was 89 μM.

Molecular Cloning and Characterization of S-Adenosyl-L-Methionine:Scoulerine-9-O-Methyltransferase from Cultured Cells of Coptis japonica

S-Adenosyl-L-methionine:scoulerine-9-O-methyltransferase (SMT) catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionine to the 9-hydroxyl group of scoulerine during the biosynthesis of berberine. We have isolated functionally active cDNA clones (pCJSMTs) from a cDNA library prepared from cultured cells of Coptis japonica. The longest cDNA insert (pCJSMT1) had an open reading frame that encoded 351 amino acids, but the calculated molecular mass (38,364 Da) of the deduced product was slightly lower than the experimentally determined molecular mass of purified SMT. Rapid amplification of the 5' end of the cDNA indicated that the full-length cDNA of SMT consisted of 1,458 nucleotides that encoded 381 amino acids. When the full-length cDNA was expressed in E. coli, the molecular mass of the expressed SMT was greater than that of native SMT in Coptis cells. This result suggests that SMT might be produced in a pre-mature form and processed post-translationally. SMT was also found to exhibit sequence homology to other O-methyltransferases from plants and N-terminal region of the SMT polypeptide appeared to be necessary for enzymatic activity.

Separation and characterization of cytokinin-inducible ( S)-tetrahydroberberine oxidases controlling berberine biosynthesis in Thalictrum minus cell cultures

Three forms of cytokinin-induced tetrahydroberberine (THB) oxidase from Thalictrum minus cell suspension cultures were individually separated by chromatography using DEAE-TOYOPEARL. Two of them were ( S)-THB-specific oxidases, whereas the third was found to be a non-stereopecific one. The stereospecific oxidases were different from each other not only in the K m value, but also in the time course of activation induced by 6-benzylaminopurine (BAP). The substrate specificity of these oxidases from T. minus cells was more similar to that reported for the ( S)-THB oxidase of Coptis japonica than that for Berberis stolonifera cell cultures.

Induction of a specific methyltransferase activity regulating berberine biosynthesis by cytokinin in Thalictrum minus cell cultures

The effects of cytokinins on the activities of seven enzymes involved in the early steps and one in the last step of berberine biosynthesis in Thalictrum minus var. hypoleucum cell cultures were examined. Of these enzymes, norcoclaurine- O-methyltransferase (NCMT) was found to be markedly activated especially by 6-benzylaminopurine (BAP), reaching the highest level (34.2 pkat mg −1 protein) six days after administration of the cytokinin. Furthermore, berberine production increased in proportion to the NCMT activity or to the BAP concentration. These results suggest that the induction of berberine production by cytokinin in T. minus cell cultures is primarily attributable to a remarkable increase in the activity of a specific methyltransferase that catalyses the first methylation of norcoclaurine to yield coclaurine, a principal intermediate in the biosynthesis of protoberberine alkaloids.

Partial purification and modulation of tetrahydroberberine oxidase from Coscinium fenestratum

Tetrahydroberberine oxidase, the enzyme involved in the final step of berberine biosynthesis, was partially purified from the plant tissue and cell cultures of Coscinium fenestratum (wood turmeric). Its kinetics and effects of certain modulators such as metallic ions, co-factors and chelators were studied. The kinetic studies indicated that the K m value of the enzyme is 1 × 10 −6 M. Among the different modulators studied, only CuSO 4 showed enhancement in the enzyme activity. THB oxidase activity was shown to be higher in ‘production medium’ optimised for berberine production when compared to that in the growth medium. Supplementation of copper sulphate in the production medium also showed increased activity of the enzyme along with increase in berberine production compared to that of the control.

Induction of berberine biosynthesis by cytokinins in Thalictrum minus cell suspension cultures

Production of berberine could be induced by adding 6-benzylaminopurine (BAP) to Thalictrum minus cells, cultured in suspension in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), early in the growth cycle. In the presence of BAP, the precursor, L-tyrosine, was rapidly converted into berberine which was then released into the medium, whereas substantial amounts of the intermediates, tyramine and dopamine, accumulated in non-berberine-producing cells grown in the same 2,4-D-containing medium without BAP. These results suggest that BAP activated enzymatic reactions subsequent to the formation of the amines in the biosynthesis of berberine.

Enhancement of berberine production by spermidine in Thalictrum minus cell suspension cultures

Berberine production in cell suspension cultures of Thalictrum minus L. var. hypoleucum Miq. was significantly enhanced by administration of spermidine, whereas other polyamines such as cadaverine, putrescine and spermine were ineffective. The results of experiments indicated that spermidine causes an increase of ethylene generation which is closely associated with activation of berberine biosynthesis.

Transport and Secretion of Natural Products in Plant Cell Cultures

Using the example of berberine in cell cultures of THALICTRUM and BERBERIS species, the transport and secretion of natural products in plant cell cultures is reviewed. Topics discussed are the subcellular localization of berberine biosynthesis, transport of berberine to vacuoles, extracellular release of berberine, mechanism of berberine secretion, uptake and transport of exogenous berberine, and the release of berberine by immobilized cells.

The role of ethylene in berberine production by Thalictrum minus cell suspension cultures

Ethylene regulates berberine production in cell suspension cultures of Thalictrum minus, by activating the biosynthesis of berberine from the key intermediate ( S)-reticuline. In the late stage of culture, however, ethylene promoted the formation of bound polyphenolics, which was associated with cell browning.

Isolation, sequence, and bacterial expression of a cDNA for (S)-tetrahydroberberine oxidase from cultured berberine-producing Coptis japonica cells.

cDNA clones for the (S)-tetrahydroberberine (H4Ber) oxidase of cultured berberine-producing Coptis japonica cells were isolated by screening a C. japonica cDNA library with synthetic nucleotides that can encode the NH2-terminal sequence of this enzyme. Analyses of the nucleotide sequences of the cloned cDNA inserts revealed a 759-base-pair open reading frame that encoded a 253-amino acid polypeptide with a Mr of 27,089 and NH2-terminal and internal sequences identical with those of the (S)-H4Ber oxidase, as determined by microsequencing methods. Escherichia coli were transformed with an expression vector carrying (S)-H4Ber oxidase cDNA. The transformed bacteria were induced to overproduce a 28-kDa protein that reacted with Coptis (S)-H4Ber oxidase-specific antibody. A comparison of the derived amino acid sequence of (S)-H4Ber oxidase with sequences in the protein data base of the Protein Research Foundation showed a marked similarity between (S)-H4Ber oxidase and the NH2-terminal portion of mouse P1-450, which is encoded by a single exon of the mouse P1-450 gene. The availability of cloned cDNA for (S)-H4Ber oxidase allows use of the methods of molecular biology to study the regulation of (S)-H4Ber oxidase gene expression in cultured C. japonica cells in relation to berberine biosynthesis.

Elicitor-induced tyrosine decarboxylase in berberine-synthesizing suspension cultures of Thalictrum rugosum.

Tyrosine decarboxylase (EC 4.1.1.25) was induced in suspension cultures of Thalictrum rugosum by treatment with a yeast glucan elicitor. Maximum induction was observed at a carbohydrate concentration of 0.4 mg/g fresh weight of cells and maximum enzyme activity was reached 20 h after addition of elicitor. The enzyme was inducible in late exponential and early stationary growth phases. A good correlation between induced tyrosine decarboxylase activity and berberine biosynthesis has been established. It is suggested that tyrosine decarboxylase may be a key enzyme between primary and secondary metabolisms in the biosynthesis of norlaudanosoline-derived alkaloids.

Increased secondary product formation in plant cell suspension cultures after treatment with a yeast carbohydrate preparation (elicitor)

A carbohydrate fraction isolated from yeast extract by ethanolic precipitation was used as an elicitor to induce secondary product formation in plant cell suspension cultures. The elicitor preparation is effective in inducing glyceollin isomer synthesis (up to 200 μg glyceollin per g dry wt) in cells of Glycine max and enhancing berberine biosynthesis (up to four-fold) in cells of Thalictrum rugosum. The response of the cell cultures to the elicitor treatment is dependent on the amount of carbohydrate per unit of biomass and on the physiological state of the cells. Cells are optimally induced in late exponential or early stationary growth phases.

Intracellular compartmentation of two enzymes of berberine biosynthesis in plant cell cultures

Out of the eight enzymes involved in the biosynthesis of the isoquinoline alkaloid berberine, at least, two enzymes, berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase, are exclusively located in a vesicle with a specific gravity of ϱ=1.14 g·cm(-3) as shown by direct enzymatic assay as well as immunoelectrophoresis. Electronmicroscopic examination of the enzyme-containing particulate preparation from Berberis wilsoniae var. subcaulialata cultured cells demonstrated that it is composed mainly of membranous vesicles. The protein composition of this preparation reveals the presence of only about 20 separable proteins, of which two major ones are berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase. Incubation of these vesicles with the substrate (S)-reticuline in the presence and absence of S-adenosyl-L-methionine leads to the formation of a red product which was identified as dehydroscoulerine. If the cytoplasmic enzyme S-adenosyl-L-methionine:(S)-scoulerine-9-O-methyltransferase is added to the vesicle preparation in the presence of (S)-reticuline and S-adenosyl-L-methionine, not dehydroscoulerine but columbamine, the immediate precursor of berberine is formed. Some of the quaternary alkaloids are located inside the vesicles; fusion of these vesicles leads to vacuoles containing the quaternary alkaloids. These vesicles are the first highly specific and unique compartment serving only alkaloid biosynthesis; they are found in members of four different plant families and in cell cultures as well as in differentiated tissue.

The biosynthesis of protoberberine and related isoquinoline alkaloids

The biosynthesis of berberine and hydrastine (in Hydrastis canadensis), corydaline and protopine (in Corydalis solida), and ochotensimine and protopine (in C. ochotensis), has been investigated by the administration of [3-14C]-3′,4′-dihydroxyphenylalanine ([3-14C]DOPA). In all cases, incorporation of label was predominantly into the isoquinoline portion of the alkaloid. The role of DOPA in the early stages of isoquinoline alkaloid biosynthesis in these plants is discussed in the light of this and other relevant data. In addition, the later stages of corydaline biosynthesis have been studied by the administration of [9-methoxy-14C]palmatine and -tetrahydropalmatine to C. solida, and the origin of the exocyclic carbons of ochotensimine further verified by feeding [methyl-14C,3H]methionine to C. ochotensis.

Biosynthesis. Part XXII. The origin of chelidonine and of other alkaloids derived from the tetrahydroprotoberberine skeleton

The major benzophenanthridine alkaloid chelidonine (14) is shown to be biosynthesised in Chelidonium majus plants from (S)-reticuline (2)via(S)-scoulerine (4) and (S)-stylopine (7), and evidence consistent with a dihydroisoquinoline intermediate [e.g.(11)] is presented. Similar though less extensive evidence is provided for the same pathway to the aromatic systems, e.g. sanguinarine (13). Protopine (10) is shown to be formed in vivo from (S)-scoulerine (4) and (S)-stylopine (7). Studies have been made of the late biosynthetic stages beyond scoulerine and stylopine and the role of isocorypalmine (6) and canadine (21) in the biosynthesis of narcotine (3) and berberine in the opium poppy has been investigated.The results from 3H labelling at four different sites in the various precursors limit the possible mechanisms which can be considered for the enzymic transformations revealed by this research. Unexpected increases in 3H : 14C ratio (up to 20%) are discussed.Extensive degradations of the labelled alkaloids have been carried out to establish quantitatively the level of radioactivity at the multiple sites. The syntheses for the many specifically labelled substances used in this study are described.