Biosynthesis of p-Hydroxybenzoate from p-Coumarate and p-Coumaroyl-Coenzyme A in Cell-Free Extracts of Lithospermum erythrorhizon Cell Cultures

Loscher, Ralf; Heide, Lutz

doi:10.1104/pp.106.1.271

Cited by 100 publications

(53 citation statements)

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“…Radioisotope studies with Lithospermum erythrorhizon suggest that this compound is derived from the CoA ester of p-hydroxycinnamic acid (pHCA-CoA) through a b-oxidation-like mechanism (Loscher and Heide, 1994). However, earlier studies with the same plant species (Yazaki et al, 1991) and carrot (Daucus carota) cell cultures (Schnitzler et al, 1992) supported a cleavage mechanism that occurs via intermediacy of p-hydroxybenzaldehyde.…”

mentioning

confidence: 80%

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

et al. 2004

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p-Hydroxybenzoic acid (pHBA) is the major monomer in liquid crystal polymers. In this study, the Escherichia coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated into the tobacco (Nicotiana tabacum) chloroplast genome under the control of the light-regulated psbA 5# untranslated region. CPL catalyzes the direct conversion of chorismate, an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids, to pHBA and pyruvate. The leaf content of pHBA glucose conjugates in fully mature T 1 plants exposed to continuous light (total pooled material) varied between 13% and 18% dry weight, while the oldest leaves had levels as high as 26.5% dry weight. The latter value is 50-fold higher than the best value reported for nuclear-transformed tobacco plants expressing a chloroplast-targeted version of CPL. Despite the massive diversion of chorismate to pHBA, the plastid-transformed plants and control plants were indistinguishable. The highest CPL enzyme activity in pooled leaf material from adult T 1 plants was 50,783 pkat/mg of protein, which is equivalent to approximately 35% of the total soluble protein and approximately 250 times higher than the highest reported value for nuclear transformation. These experiments demonstrate that the current limitation for pHBA production in nucleartransformed plants is CPL enzyme activity, and that the process becomes substrate-limited only when the enzyme is present at very high levels in the compartment of interest, such as the case with plastid transformation. Integration of CPL into the chloroplast genome provides a dramatic demonstration of the high-flux potential of the shikimate pathway for chorismate biosynthesis, and could prove to be a cost-effective route to pHBA. Moreover, exploiting this strategy to create an artificial metabolic sink for chorismate could provide new insight on regulation of the plant shikimate pathway and its complex interactions with downstream branches of secondary metabolism, which is currently poorly understood.All plants normally produce p-hydroxybenzoic acid (pHBA), albeit usually in small quantities. Radioisotope studies with Lithospermum erythrorhizon suggest that this compound is derived from the CoA ester of p-hydroxycinnamic acid (pHCA-CoA) through a b-oxidation-like mechanism (Loscher and Heide, 1994). However, earlier studies with the same plant species (Yazaki et al., 1991) and carrot (Daucus carota) cell cultures (Schnitzler et al., 1992) supported a cleavage mechanism that occurs via intermediacy of p-hydroxybenzaldehyde. Notwithstanding our current ignorance of the detailed plant biosynthetic pathway, a number of studies have shown that it is possible to dramatically elevate pHBA levels in plants through metabolic engineering. Indeed, two different ''singleenzyme'' pathways have been described, both involving microbial proteins that have no known plant counterparts. One of these enzymes is chorismate pyruvate-lyase (CPL). Its substrate is chorismate, an important branch po...

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mentioning

confidence: 80%

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

et al. 2004

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“…This pathway involves the activation of p-coumaric acid by the action of the enzyme 4-coumarate-CoA ligase (4CL), leading to its thioester with subsequent chain-shortening into p-hydroxybenzoyl-CoA in a reaction mechanism analogous to that of NAD-dependent β-oxidation of fatty acids. 37,38 The operation of this pathway is supported by in vitro enzymatic activity assays using cell-free extracts from Lithospermum erythrorhizon cell cultures. 37 However, this conversion requires steps not fully elucidated yet, i.e., those corresponding to the hydration, dehydrogenation, and thiolation of the β-oxidative cycle followed by the final hydrolysis of the 4-hydroxybenzoyl-CoA thioester.…”

Section: Candidate Genes Involved In Benzopyran Biosynthesismentioning

confidence: 99%

“…37,38 The operation of this pathway is supported by in vitro enzymatic activity assays using cell-free extracts from Lithospermum erythrorhizon cell cultures. 37 However, this conversion requires steps not fully elucidated yet, i.e., those corresponding to the hydration, dehydrogenation, and thiolation of the β-oxidative cycle followed by the final hydrolysis of the 4-hydroxybenzoyl-CoA thioester. 33 A Petunia gene encoding the bifunctional peroxisomal enzyme cinnamoyl-CoA hydratase-dehydrogenase (CHD), which is responsible for the initial two-step conversion of cinnamoyl-CoA into benzoic acid, was identified by a functional genomics approach and has been shown to be active with p-coumaroyl-CoA ( Figure S4, Supplementary Information).…”

Section: Candidate Genes Involved In Benzopyran Biosynthesismentioning

confidence: 99%

“…40 The second route involves a nonoxidative pathway for the conversion of p-coumaric acid or p-coumaroyl-CoA to p-hydroxybenzaldehyde in a retro-aldol reaction with no co-factor requirement. 37,38,41 A 4-hydroxybenzaldehyde synthase (HBS) and 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) catalyzes the penultimate step of p-HBA biosynthesis by performing the phenylpropanoid side-chain cleavage of p-coumaric acid and p-coumaroyl-CoA, respectively. 31,41 Then, the biosynthesis of p-HBA proceeds via p-hydroxybenzaldehyde in a reaction catalyzed by the enzyme 4-hydroxybenzaldehyde dehydrogenase.…”

Section: Candidate Genes Involved In Benzopyran Biosynthesismentioning

confidence: 99%

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

Batista¹,

Batista²,

Souza-Moreira³

et al. 2017

J. Braz. Chem. Soc.

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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...

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“…The existence of a novel pathway for the biosynthesis of 4-hydroxybenzoate from tyrosine has been was proposed by Booth et al (Booth, Masri et al 1960) in 1960 in mammals ( Figure 4). Evidence in support of this pathway has recently been obtained in higher plants (Loscher and Heide 1994). However, no genes have been associated with these enzymes in any organism.…”

mentioning

confidence: 99%

An integrative approach to energy, carbon, and redox metabolism in the cyanobacterium Synechocystis sp. PCC 6803

Overbeek¹,

Fonstein²,

Osterman³

et al. 2005

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Biosynthesis of p-Hydroxybenzoate from p-Coumarate and p-Coumaroyl-Coenzyme A in Cell-Free Extracts of Lithospermum erythrorhizon Cell Cultures

Cited by 100 publications

References 19 publications

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

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

An integrative approach to energy, carbon, and redox metabolism in the cyanobacterium Synechocystis sp. PCC 6803

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