Search

ABSTRACTBerberine (BBR) is a protoberberine alkaloid extracted from plants such as Coptis japonica(Ranunculaceae). In a previous report, we demonstrated the existence of a 11-hydroxylation pathway employed by BBR-utilizing bacteria for metabolism of BBR. In the present study, we report the identification of the genes brhA, brhB, and brhCas encoding a multicomponent BBR 11-hydroxylase in Burkholderiasp. strain CJ1. BrhA is belonging to the Rieske non-heme iron oxygenase (RO) family, a class of enzymes known to catalyze the first step in bacterial aromatic-ring hydroxylation. We further demonstrate that BrhA activity requires BrhB (ferredoxin reductase) and BrhC (ferredoxin) as electron transport chain components. A BLAST search revealed that BrhA exhibits 38% and 33% sequence identity to dicamba O-demethylase (DdmC; AY786443) and chloroacetanilide herbicides N-dealkylase (CndA; KJ461679), respectively. To our knowledge, this work represents the first report of a bacterial oxygenase catalyzing the metabolism of a polycyclic aromatic-ring alkaloid.Abbreviations:BBR: berberine; D-BBR: demethyleneberberine; H-BBR: 11-hydroxyberberine; HD-BBR: 11-hydroxydemethyleneberberine; HDBA: 2-hydroxy-3,4-dimethoxybenzeneacetic acid; PAL: palmatine; H-PAL: 11-hydroxypalmatine; BRU: berberrubine; Fd: ferredoxin; FdR: ferredoxin reductase; ETC: electron transport chain

Based on the finding of a novel berberine (BBR)-utilizing bacterium, Rhodococcussp. strain BD7100, we investigated the degradation of BBR and its analog berberrubine (BRU). Resting cells of BD7100 demethylenated BBR and BRU, yielding benzeneacetic acid analogs. Isolation of benzeneacetic acid analogs suggested that BD7100 degraded the isoquinoline ring of the protoberberine skeleton. This work represents the first report of cleavage of protoberberine skeleton by a microorganism.

Carapichea ipecacuanhaproduces various emetine-type alkaloids, known as ipecac alkaloids, which have long been used as expectorants, emetics, and amebicides. In this study, we isolated an O-methyltransferase cDNA from this medicinal plant. The encoded protein (CiOMT1) showed 98% sequence identity to IpeOMT2, which catalyzes the 7′-O-methylation of 7′-O-demethylcephaeline to form cephaeline at the penultimate step of emetine biosynthesis (Nomura and Kutchan, J. Biol. Chem., 285, 7722–7738 (2010)). Recombinant CiOMT1 showed both 7′-O-methylation and 6′-O-methylation activities at the last two steps of emetine biosynthesis. This indicates that small differences in amino acid residues are responsible for distinct regional methylation specificities between IpeOMT2 and CiOMT1, and that CiOMT1 might contribute to two sequential O-methylation steps from 7′-O-demethylcephaeline to emetine.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the formation of homogentisate from 4-hydroxyphenylpyruvate and O2. In plants, HPPD has been identified as a molecular target for herbicides. We report the isolation and characterization of a cDNA encoding a HPPD from cultured Coptis japonicacells. Recombinant CjHPPD showed significantly higher half-maximum inhibitory concentration (IC50) values for the HPPD-inhibiting herbicide destosyl pyrazolate than other plant HPPDs.

RNAi (RNA interference, RNA silencing) is a powerful tool for functional genomics, but the construction of an RNAi vector(s) and the establishment of stable transformants are time-consuming and laborious. Here we report the transient RNAi of endogenous biosynthetic genes involved in isoquinoline alkaloid biosynthesis in Coptis japonicaprotoplasts. Double stranded (ds) RNA fragments of various lengths prepared from several different positions of the coding sequence of scoulerine 9-O-methyltransferase (SMT) were introduced into C. japonicaprotoplasts by polyethylene glycol-mediated transformation, and their effects were monitored by reverse transcription-polymerase chain reaction. Substantial silencing of SMT gene expression was obtained by the introduction of these SMT dsRNAs. A significant reduction in SMT protein levels was also observed. The potentials of this transient RNAi system to evaluate the functions of biosynthetic genes in Coptisalkaloid research are discussed.

RNA interference with double-stranded RNA is a new method for the study of gene function in various organisms. In this report, we show that an inverted repeat of a short (103-bp) 3′-untranslated sequence of an isogene, 1A, of psbP genes, encoded by a small multigene family of four genes (1A, 2AF, 3F, and 5B) in Nicotiana tabacum, can specifically suppress the expression of psbP isogenes 1A and 5B with a 3′-untranslated sequence similar to a transcribed double-stranded RNA. The expression of other psbP isogenes, 2AF and 3F, was not affected, although the coding sequences of the psbP family genes are highly conserved. Consistent with this observation, small interfering RNAs were detected for the 3′-untranslated sequence used for the inverted-repeat transgene, and not for the coding sequence. These results suggest that double-stranded RNA having a 3′-untranslated sequence could be useful for an isogene-specific RNA interference of the family genes in Nicotiana tabacum.

Three genes encoding phosphoenolpyruvate carboxylase were isolated from Nicotiana sylvestrisand designated Nsppc1-3. Sequencing of nucleotides showed that the coding sequence and deduced amino acid sequences were highly conserved among the genes, but sequences for noncoding regions including introns and 5′-flanking regions were not conserved. Analysis of the transcript level of the genes by a combination of reverse transcriptase-PCR and restriction fragment polymorphism showed mostNsppc1in the leaves, stems, roots, and cultured cells of N. sylvestris. β-Glucuronidase activity was detected histochemically in mesophyll cells in leaves, lateral buds, and vascular bundles in roots of transgenic tobacco harboring a chimeric construct of the Nsppc1promoter and the gene for β-glucuronidase. Deletion analysis indicated the presence of a silencer-like element for basal expression in the promoter region of Nsppc1.

Benzylisoquinoline alkaloids (BIAs) are pharmaceutically important compounds. We have previously devised a reticuline (BIA) production method from dopamine by using Escherichia coli; however, its productivity was relatively low (33 µM, 11 mg/L). We report here, by fine-tuning the method, higher reticuline productivity of 165 µM(54 mg/L), increasing the conversion efficiency by 8-fold. These results are important for developing an efficient route to fermentative reticuline production.

We clarified important roles of ArabidopsisTCP1, a member of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) transcription factors, in the longitudinal elongation of petioles, rosette leaves and inflorescent stems. We found that the promoter of TCP1was active in the cotyledonary petioles and the distal part of the expanding leaves, as well as the midrib region and the petiole.

Coclaurine N-methyltransferase from Coptis japonicacatalyzes the N-methylation of coclaurine as well as simple tetrahydroisoquinoline. We examined the possibility of converting 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline into its N-methylated product using transgenic Escherichia coli, which expressed recombinant coclaurine N-methyltransferase, without the addition of a methyl-group donor. Transgenic E. colisuccessfully N-methylated the substrate added to the medium and excreted the product. Limitation of bioconversion by the supply of methyl-group donor is discussed.