Biosynthetic Insights into p-Hydroxybenzoic Acid-Derived Benzopyrans in Piper gaudichaudianum

Abstract

Piper gaudichaudianum Kunth (Piperaceae) accumulates gaudichaudianic acid, a prenylated benzopyran, as its major component. Interestingly, this trypanocidal compound occurs as a racemic mixture. Herein, transcriptomic investigations of Piper gaudichaudianum using the RNAseq approach are reported, and from the analysis of the transcripts expressed it was possible to propose a complete biosynthetic pathway for the production of gaudichaudianic acid, including the steps that originate its precursor, p-hydroxybenzoic acid. Peperomia obtusifolia (L.) A. Dietr. (Piperaceae) also accumulates racemic benzopyrans, however, its chromanes originate from the polyketide pathway, while the chromenes from Piper derives from the shikimate pathway. Recent transcriptomic and proteomic studies of the former species did not identify polyketide synthases involved in the production of the benzopyran moiety, but revealed the expression of tocopherol cyclase, which may be responsible for the cyclization of the 3,4-dihydro-2H-pyran ring. The analysis of the enzymes involved in the secondary metabolism of Piper gaudichaudianum and the comparison with the data previously obtained from Peperomia obtusifolia can provide valuable information on how these compounds are biosynthesized.Keywords: Piperaceae, chromene, transcriptome, RNA-seq, de novo assembly, biosynthesis IntroductionChromanes and chromenes, which are secondary metabolites commonly found in some species from Piper and Peperomia genera (Piperaceae), are characterized by the presence of a benzopyran ring with various levels of oxidation. [1][2][3] This nucleus is considered a privileged structure that is very common in bioactive natural products, such as coumarins, flavones, tocopherols (vitamin E) and tetrahydrocannabinoids. 4,5 The benzopyran moieties, 2H-chromenes and chromanes, isolated from Piper gaudichaudianum and Peperomia obtusifolia, respectively, have been demonstrated as potent trypanocidal compounds. 6,7 Curiously, both classes of compounds occur as racemic mixtures in these species, though their formation follows two distinct biosynthetic routes. 6,8,9 In Piper gaudichaudianum, chromenes originate from the shikimate pathway and use p-hydroxybenzoic acid (p-HBA) as a precursor. 10 In the case of Peperomia obtusifolia, chromanes are formed through the polyketide pathway and use orsellinic acid as a precursor. 1,11 Additionally, the formation of both classes of metabolites involves the condensation of an aromatic unit (p-HBA or orsellinic acid) and an isoprene unit (dimethylallyl pyrophosphate, isopentenyl pyrophosphate or geranyl pyrophosphate), followed by cyclization that gives rise to the benzopyran ring. During cyclization, the carbon atom at C-2 becomes a stereogenic center and, different from other benzopyrans such as vitamin E, both enantiomers are biosynthesized (Figure 1). Thus, the study of the proteins and genes possibly responsible for the biosynthesis of benzopyrans in these species, as well as the comparison between them, may provide new insights into how...

Advances and Prospects of Phenolic Acids Production, Biorefinery and Analysis

“…Here, the phenylalanine, as well as chorismic, salicylic, and gallic acids, are synthesised [ 45 , 46 ], whereas other hydroxycinnamic and hydroxybenzoic acids are synthesised in the cytosol [ 47 ]. It is thought that hydroxybenzoic acids can be produced from structurally analogous hydroxycinnamic acids in coenzyme A (CoA) dependant ( β -oxidative) or CoA-independent (non- β -oxidative) pathways or the combination of both them [ 48 , 49 , 50 , 51 , 52 ], which has been determined as occurring in peroxisomes [ 47 ] and mitochondria [ 53 ]. However, salicylic acid can also be synthesised in a few other ways: directly from either benzoic acid by benzoic acid 2-oxygenase or isochorismate by isochorismoyl-glutamate synthase and through the spontaneous or non-spontaneous (by isochorismoyl-glutamate A pyruvoyl-glutamate lyase) splitting of the isochorismoyl-glutamate A [ 54 , 55 ].…”

Assessment and Partial Characterization of Candidate Genes in Dihydrochalcone and Arbutin Biosynthesis in an Apple–Pear Hybrid by De Novo Transcriptome Assembly

“…Regarding arbutin biosynthesis, isorhamnetin-3- O -rutinoside, rutin, and arbutin showed a strong significant positive correlation (0.88–0.96), which may reflect that these metabolites are characteristically found in pears and the apple–pear hybrid. , The final step of the nonoxidative pathways leading to benzaldehyde and benzoic acid is carried out by aldehyde oxidases and/or dehydrogenases, and they have been proposed as intermediate compounds for 4-hydroxybenzoic acid biosynthesis, ,, the direct proposed precursor of hydroquinone (Figure ). Using protein domains identified in the characterized 4-hydroxybenzaldehyde dehydrogenase, we obtained several putative aldehyde dehydrogenases (ADH), and no candidate had the hallmark PTHR42986 domain annotated as benzaldehyde dehydrogenase.…”

“…Among genes selected for arbutin involvement, we tested a gene annotated as acyl-coenzyme A thioesterase (ACoAT-like) exhibiting a 4-hydroxybenzoyl-CoA thioesterase domain, which catalyzes the final step in the biosynthesis of 4hydroxybenzoate in Pseudomonas CBS-3, 50 as a potential HBT involved in arbutin synthesis by an oxidative pathway as previously proposed. 18 However, there was no significant correlation between ACoAT-like and metabolite accumulation patterns in the pear and apple−pear hybrid (Figure 5B). The unlikely involvement of this gene in arbutin biosynthesis is in agreement with results obtained by in vivo feeding experiments with specific inhibitors carried out in calli of Daucus carota, supporting a CoA-independent route of 4-hydroxybenzoic acid biosynthesis that would not require HBT action from 4hydroxybenzyl-CoA to 4-hydroxybenzoic acid.…”

“…The first committed step toward DHCs in apples has been postulated to involve a double-bond reductase (DBR), catalyzing the reduction of 4coumaroyl-CoA to 4-dihydrocoumaroyl-CoA, which would be condensed with three molecules of malonyl-CoA to phloretin by chalcone synthase (CHS) and further glucosylated by a phloretin-2′-O-UDP-glucosyltransferase (Ph-2′-OGT, named PGT1) to produce phloridzin, the characteristic phloretin-2′-O-glucoside highly accumulated in M. × domestica. 6 The direct arbutin-precursor hydroquinone would require the formation of 4-hydroxybenzoic acid by 4-hydroxybenzaldehyde dehydrogenase (HBD), which could be, in turn, the result of 4hydroxybenzaldehyde or 4-hydroxybenzyl-CoA or directly converted from shikimic acid by chorismate-pyruvate lyase (UbiC), as reported in bacteria 18 (Figure 1). Synthesis of 4hydroxybenzaldehyde from 4-coumaric acid or 4-coumaroyl-CoA would depend on 4-hydroxybenzaldehyde synthase (HBS) and 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL), respectively.…”

Determinación de los cambios inducidos por un elicitor biótico en el perfil metabólico y actividad enzimática de cultivos in vitro de Piper cumanense

“…El sitio activo de la enzima se caracteriza por la presencia de un grupo prostético 3,5- Existe evidencia genética que demuestra la participación de la enzima PAL en la biosíntesis de compuestos con actividades biológicas, por ejemplo, mediante el análisis de transcriptoma se identificaron las principales enzimas implicadas en la biosíntesis del ácido gaudichaudianico, principal benzopirano aislado de Piper gaudichaudianum y con actividad tripanocida y antifúngica, en este trabajo demostraron que las vías metabólicas de su biosíntesis implican una serie de múltiples y complejas reacciones enzimáticas en las que la L-fenilalanina es un precursor primario (Batista et al, 2018). En otro estudio, realizaron un análisis completo del transcriptoma de Calophyllum brasiliense donde identificaron homólogos de todas las enzimas requeridas para la formación de L-fenilalanina a través de la ruta del ácido shikímico de la cual se lleva a cabo la síntesis de umbeliferona, una hidroxicumarina con actividad antifúngica, antibacteriana y utilizada como protector solar (Gómez-Robledo et al, 2016).…”