Parallel evolution of UbiA superfamily proteins into aromatic O-prenyltransferases in plants.

Plants produce ∼300 aromatic compounds enzymatically linked to prenyl side chains via C-O bonds. These O-prenylated aromatic compounds have been found in taxonomically distant plant taxa, with some of them being beneficial or detrimental to human health. Although their O-prenyl moieties often play crucial roles in the biological activities of these compounds, no plant gene encoding an aromatic O-prenyltransferase (O-PT) has been isolated to date. This study describes the isolation of an aromatic O-PT gene, CpPT1, belonging to the UbiA superfamily, from grapefruit (Citrus × paradisi, Rutaceae). This gene was shown responsible for the biosynthesis of O-prenylated coumarin derivatives that alter drug pharmacokinetics in the human body. Another coumarin O-PT gene encoding a protein of the same family was identified in Angelica keiskei, an apiaceous medicinal plant containing pharmaceutically active O-prenylated coumarins. Phylogenetic analysis of these O-PTs suggested that aromatic O-prenylation activity evolved independently from the same ancestral gene in these distant plant taxa. These findings shed light on understanding the evolution of plant secondary (specialized) metabolites via the UbiA superfamily.

Convergent evolution of the UbiA prenyltransferase family underlies the independent acquisition of furanocoumarins in plants.

Furanocoumarins (FCs) are plant-specialized metabolites with potent allelochemical properties. The distribution of FCs is scattered with a chemotaxonomical tendency towards four distant families with highly similar FC pathways. The mechanism by which this pathway emerged and spread in plants has not been elucidated. Furanocoumarin biosynthesis was investigated in Ficus carica (fig, Moraceae), focusing on the first committed reaction catalysed by an umbelliferone dimethylallyltransferase (UDT). Comparative RNA-seq analysis among latexes of different fig organs led to the identification of a UDT. The phylogenetic relationship of this UDT to previously reported Apiaceae UDTs was evaluated. The expression pattern of F. carica prenyltransferase 1 (FcPT1) was related to the FC contents in different latexes. Enzymatic characterization demonstrated that one of the main functions of FcPT1 is UDT activity. Phylogenetic analysis suggested that FcPT1 and Apiaceae UDTs are derived from distinct ancestors, although they both belong to the UbiA superfamily. These findings are supported by significant differences in the related gene structures. This report describes the identification of FcPT1 involved in FC biosynthesis in fig and provides new insights into multiple origins of the FC pathway and, more broadly, into the adaptation of plants to their environments.

Isolation of Artemisia capillaris membrane-bound di-prenyltransferase for phenylpropanoids and redesign of artepillin C in yeast.

Plants produce various prenylated phenolic metabolites, including flavonoids, phloroglucinols, and coumarins, many of which have multiple prenyl moieties and display various biological activities. Prenylated phenylpropanes, such as artepillin C (3,5-diprenyl-p-coumaric acid), exhibit a broad range of pharmaceutical effects. To date, however, no prenyltransferases (PTs) involved in the biosynthesis of phenylpropanes and no plant enzymes that introduce multiple prenyl residues to native substrates with different regio-specificities have been identified. This study describes the isolation from Artemisia capillaris of a phenylpropane-specific PT gene, AcPT1, belonging to UbiA superfamily. This gene encodes a membrane-bound enzyme, which accepts p-coumaric acid as its specific substrate and transfers two prenyl residues stepwise to yield artepillin C. These findings provide novel insights into the molecular evolution of this gene family, contributing to the chemical diversification of plant specialized metabolites. These results also enabled the design of a yeast platform for the synthetic biology of artepillin C.

Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Eschscholzia californica cells.

Benzophenanthridine alkaloids, such as sanguinarine, are produced from reticuline, a common intermediate in benzylisoquinoline alkaloid biosynthesis, via protopine. Four cytochrome P450s are involved in the biosynthesis of sanguinarine from reticuline; i.e. cheilanthifoline synthase (CYP719A5; EC 1.14.21.2.), stylopine synthase (CYP719A2/A3; EC 1.14.21.1.), N-methylstylopine hydroxylase (MSH) and protopine 6-hydroxylase (P6H; EC 1.14.13.55.). In this study, a cDNA of P6H was isolated from cultured Eschscholzia californica cells, based on an integrated analysis of metabolites and transcript expression profiles of transgenic cells with Coptis japonica scoulerine-9-O-methyltransferase. Using the full-length candidate cDNA for P6H (CYP82N2v2), recombinant protein was produced in Saccharomyces cerevisiae for characterization. The microsomal fraction containing recombinant CYP82N2v2 showed typical reduced CO-difference spectra of P450, and production of dihydrosanguinarine and dihydrochelerythrine from protopine and allocryptopine, respectively. Further characterization of the substrate-specificity of CYP82N2v2 indicated that 6-hydroxylation played a role in the reaction.

Molecular cloning of an O-methyltransferase from adventitious roots of Carapichea ipecacuanha.

Carapichea ipecacuanha produces 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.

Molecular characterization of O-methyltransferases involved in isoquinoline alkaloid biosynthesis in Coptis japonica.

O-Methyltransferases, which catalyze the production of small molecules in plants, play a crucial role in determining biosynthetic pathways in secondary metabolism because of their strict substrate specificity. Using three O-methyltransferase (OMT) cDNAs that are involved in berberine biosynthesis, we investigated the structure that was essential for this substrate specificity and the possibility of creating a chimeric enzyme with novel substrate specificity. Since each OMT has a relatively well-conserved C-terminal putative S-adenosyl-L-methionine-binding domain, we first exchanged the N-terminal halves of different OMTs. Among the 6 combinations that we tested for creating chimeric OMTs, 5 constructs produced detectable amounts of recombinant proteins, and only one of these with an N-terminal half of 6-OMT and a C-terminal half of 4'-OMT (64'-OMT) showed methylation activity with isoquinoline alkaloids as a substrate. Further enzymological analysis of 64'-OMT reaction product indicated that 64'-OMT retained the regio-specificity of 6-OMT. Further examination of the N-terminal region of 64'-OMT showed that about 90 amino acid residues in the N-terminal half were critical for reaction specificity. The creation of OMTs with novel reactivity is discussed.

Over-expression of rate-limiting enzymes to improve alkaloid productivity.

Benzylisoquinoline alkaloids are one of the most important groups of secondary metabolites and include the economically important analgesic morphine and the antimicrobial agent berberine. To improve the productivity of these alkaloids, we investigated the effects of putative rate-limiting step enzymes in alkaloid biosynthesis. We constructed several over-expression vectors for biosynthetic enzymes and introduced them into cultured California poppy, a model isoquinoline alkaloid-producing plant. HPLC/LC-MS analysis of transgenic cells revealed that these enzymes varied in their ability to increase alkaloid production. We describe the use of a rate-limiting step gene to improve alkaloid productivity.

Metabolic diversification of benzylisoquinoline alkaloid biosynthesis through the introduction of a branch pathway in Eschscholzia californica.

Higher plants produce a diverse array of secondary metabolites. These chemicals are synthesized from simple precursors through multistep reactions. To understand how plant cells developed such a complicated metabolism, we examined the plasticity of benzyl isoquinoline alkaloid biosynthesis in transgenic Eschscholzia californica cells with the ectopic expression of Coptis japonica scoulerine-9-O-methyltransferase (CjSMT). CjSMT catalyzes the O-methylation of scoulerine to produce tetrahydrocolumbamine (THC) in berberine biosynthesis and is not involved in benzophenanthridine alkaloid biosynthesis in E. californica. While a preliminary characterization confirmed that columbamine (oxidized product of THC) was produced in transgenic E. californica cells, many newly found peaks were not identified. Here, we report the identification of novel products, including allocryptopine and 10-hydroxychelerythrine. This result indicates that CjSMT reaction products were further converted by endogenous enzymes to produce double O-methylated compounds instead of a methylenedioxy ring at the 7,8-position of the original benzophenanthridine alkaloids. Further metabolite profiling revealed the enhanced diversification of the alkaloid profile in transgenic cells. Metabolic plasticity and the enzymes involved in metabolic diversity are discussed.

Microbial production of plant benzylisoquinoline alkaloids.

Benzylisoquinoline alkaloids, such as the analgesic compounds morphine and codeine, and the antibacterial agents berberine, palmatine, and magnoflorine, are synthesized from tyrosine in the Papaveraceae, Berberidaceae, Ranunculaceae, Magnoliaceae, and many other plant families. It is difficult to produce alkaloids on a large scale under the strict control of secondary metabolism in plants, and they are too complex for cost-effective chemical synthesis. By using a system that combines microbial and plant enzymes to produce desired benzylisoquinoline alkaloids, we synthesized (S)-reticuline, the key intermediate in benzylisoquinoline alkaloid biosynthesis, from dopamine by crude enzymes from transgenic Escherichia coli. The final yield of (S)-reticuline was 55 mg/liter within 1 h. Furthermore, we synthesized an aporphine alkaloid, magnoflorine, or a protoberberine alkaloid, scoulerine, from dopamine via reticuline by using different combination cultures of transgenic E. coli and Saccharomyces cerevisiae cells. The final yields of magnoflorine and scoulerine were 7.2 and 8.3 mg/liter culture medium. These results indicate that microbial systems that incorporate plant genes cannot only enable the mass production of scarce benzylisoquinoline alkaloids but may also open up pathways for the production of novel benzylisoquinoline alkaloids.

Metabolic engineering in isoquinoline alkaloid biosynthesis.

Higher plants produce diverse classes of metabolites. Metabolic engineering offers tremendous potential to improve the production and quality of these chemicals. This report summarizes the possibility of using metabolic engineering in benzylisoquinoline alkaloid biosynthesis. Benzylisoquinoline alkaloids, such as morphine, sanguinarine, and berberine, are synthesized from tyrosine via reticuline in Magnoliaceae, Ranunculaceae, Berberidaceae, Papaveraceae, and many other species. The early pathway from tyrosine to reticuline is common among many plant species, whereas there is more diversity in late pathways. This review describes several strategies to improve the yield and quality of benzylisoquinoline alkaloids. First, the overexpression of a rate-limiting enzyme in an early pathway to increase the overall alkaloid yield is discussed. Second, the introduction of a new branch into the pathway has been shown to produce novel metabolites. Finally, the possibility of accumulating a pathway intermediate by the knock-down of a key step is examined. Further metabolic modification is also discussed, since the latter two modifications may lead to the production of novel compound(s) from an accumulated intermediate through metabolic activation. These metabolic changes could be further modified to increase chemical diversity through somatic variation in cell culture. Besides this direct metabolic engineering with isolated biosynthetic genes, the regulation of biosynthetic activity with transcription factors and/or with reconstruction of the entire biosynthesis will also be discussed for the next generation of metabolite production.