Benzoic Acid Biosynthesis Research Articles

Cytosolic aromatic aldehyde dehydrogenase provides benzoic acid for xanthone biosynthesis in Hypericum

Benzoic acid is a building block of a multitude of well-known plant natural products, such as paclitaxel and cocaine. Its simple chemical structure contrasts with its complex biosynthesis. Hypericum species are rich in polyprenylated benzoic acid-derived xanthones, which have received attention due to their biological impact on human health. The upstream biosynthetic sequence leading to xanthones is still incomplete. To supply benzoic acid for xanthone biosynthesis, Hypericum calycinum cell cultures use the CoA-dependent non-β-oxidative pathway, which starts with peroxisomal cinnamate CoA-ligase (HcCNL). Here, we use the xanthone-producing cell cultures to identify the transcript for benzaldehyde dehydrogenase (HcBD), a pivotal player in the non-β-oxidative pathways. In addition to benzaldehyde, the enzyme efficiently catalyzes the oxidation of trans-cinnamaldehyde in vitro. The enzymatic activity is strictly dependent on the presence of NAD+ as co-factor. HcBD is localized to the cytosol upon ectopic expression of reporter fusion constructs. HcBD oxidizes benzaldehyde, which moves across the peroxisome membrane, to form benzoic acid. Increases in the HcCNL and HcBD transcript levels precede the elicitor-induced xanthone accumulation. The current work addresses a crucial step in the yet incompletely understood CoA-dependent non-β-oxidative route of benzoic acid biosynthesis. Addressing this step may offer a new biotechnological tool to enhance product formation in biofactories.

A promiscuous coenzyme A ligase provides benzoyl-coenzyme A for xanthone biosynthesis in Hypericum.

Benzoic acid-derived compounds, such as polyprenylated benzophenones and xanthones, attract the interest of scientists due to challenging chemical structures and diverse biological activities. The genus Hypericum is of high medicinal value, as exemplified by H. perforatum. It is rich in benzophenone and xanthone derivatives, the biosynthesis of which requires the catalytic activity of benzoate-coenzyme A (benzoate-CoA) ligase (BZL), which activates benzoic acid to benzoyl-CoA. Despite remarkable research so far done on benzoic acid biosynthesis in planta, all previous structural studies of BZL genes and proteins are exclusively related to benzoate-degrading microorganisms. Here, a transcript for a plant acyl-activating enzyme (AAE) was cloned from xanthone-producing Hypericum calycinum cell cultures using transcriptomic resources. An increase in the HcAAE1 transcript level preceded xanthone accumulation after elicitor treatment, as previously observed with other pathway-related genes. Subcellular localization of reporter fusions revealed the dual localization of HcAAE1 to cytosol and peroxisomes owing to a type 2 peroxisomal targeting signal. This result suggests the generation of benzoyl-CoA in Hypericum by the CoA-dependent non-β-oxidative route. A luciferase-based substrate specificity assay and the kinetic characterization indicated that HcAAE1 exhibits promiscuous substrate preference, with benzoic acid being the sole aromatic substrate accepted. Unlike 4-coumarate-CoA ligase and cinnamate-CoA ligase enzymes, HcAAE1 did not accept 4-coumaric and cinnamic acids, respectively. The substrate preference was corroborated by in silico modeling, which indicated valid docking of both benzoic acid and its adenosine monophosphate intermediate in the HcAAE1/BZL active site cavity.

Benzoic acid production via cascade biotransformation and coupled fermentation-biotransformation.

As an important bulk chemical, benzoic acid is currently manufactured from nonrenewable feedstocks under harsh conditions. Although there are natural pathways for biosynthesis of benzoic acid, they are often inefficient and subjected to complex regulation. Here we develop a nonnatural enzyme cascade to efficiently produce benzoic acid from styrene or biogenic L-phenylalanine under mild conditions. By using a modular approach, two whole-cell catalysts Escherichia coli LZ305 and LZ325 are engineered for coexpressing seven and nine enzymes for production of 133-146 mM benzoic acid (16.2-17.8 g/Laq ) with 88-97% conversion via seven- and nine-step cascade biotransformation of styrene and L-phenylalanine, respectively. The seven-step cascade represents a formal high-yielding biocatalytic oxidative cleavage of styrene, and the nine-step cascade showcases the high efficiency of extended nonnatural enzyme cascades. Moreover, to achieve benzoic acid production directly from low-cost renewable glycerol, a novel coupled fermentation-biotransformation process was developed by integration of fermentative production of L-phenylalanine with in situ biotransformation to give 63-70 mM benzoic acid (7.6-8.6 g/Laq ), which is around 20 times higher than the reported value via a natural pathway. The coupled fermentation-biotransformation process could be generally applicable to microbial production of growth-inhibitory or toxic chemicals in high concentrations.

A new enzymatic activity from elicitor-treated pear cell cultures converting trans-cinnamic acid to benzaldehyde.

Cell cultures of Asian pear (Pyrus pyrifolia) are known to produce benzoate-derived biphenyl phytoalexins upon elicitor treatment. Although the downstream pathway for biphenyl phytoalexin biosynthesis is almost known, the upstream route of benzoic acid biosynthesis in pear has not been completely elucidated. In the present work, we report benzaldehyde synthase (BS) activity from yeast extract-treated cell suspension cultures of P. pyrifolia. BS catalyzes the in vitro conversion of trans-cinnamic acid to benzaldehyde using a non-oxidative C2 -side chain cleavage mechanism. The enzyme activity was strictly dependent on the presence of a reducing agent, dithiothreitol being preferred. C2 -side chain shortening of the cinnamic acid backbone resembled the mechanisms catalyzed by 4-hydroxybenzaldehyde synthase (HBS) activity in Vanilla planifolia and salicylaldehyde synthase (SAS) activity in tobacco and apple cell cultures. A basal BS activity was also observed in the non-elicited cell cultures. Upon yeast extract-treatment, a 13-fold increase in BS activity was observed when compared to the non-treated control cells. Moreover, feeding of the cell cultures with trans-cinnamic acid, the substrate for BS, resulted in an enhanced level of noraucuparin, a biphenyl phytoalexin. Comparable accumulation of noraucuparin was observed upon feeding of benzaldehyde, the BS product. The preferred substrate for BS was found to be trans-cinnamic acid, for which the apparent Km and Vmax values were 0.5 mM and 50.7 pkat mg-1 protein, respectively. Our observations indicate the contribution of BS to benzoic acid biosynthesis in Asian pear via the CoA-independent and non-β-oxidative route.

Exploring genes involved in benzoic acid biosynthesis in the Populus davidiana transcriptome and their transcriptional activity upon methyl jasmonate treatment.

Benzoic acids (BAs) are important structural elements in a wide variety of essential compounds and natural products, and play crucial roles in plant fitness. BA is a precursor of diverse benzenoid compounds, including the hormone salicylic acid (SA) and the aglycone moiety of salicin, which is particularly important in the Salicaceae family. The biosynthetic pathways leading to BA formation in plants are largely unknown. Recently, the CoA-dependent β-oxidative BA biosynthesis pathway, which occurs in peroxisomes, has been characterized in petunia. The core of this pathway is cinnamic acid → cinnamoyl-CoA→3-hydroxy-3-phenylpropanoyl-CoA→3-oxo-3-phenylpropanoyl-CoA→benzoyl-CoA. Here, we used 454 pyrosequencing to analyze the transcriptome of Populus davidiana and isolate putative genes involved in BA biosynthesis. De novo assembly generated 57,322 unique sequences, including 15,217 contigs and 42,105 singletons. From the unique sequences, we selected six genes exhibiting high similarity to genes encoding L-phenylalanine ammonia lyase, cinnamate:CoA ligase, cinnamoyl-CoA hydratase-dehydrogenase, 3-ketoacyl-CoA thiolase, benzoyl-CoA:benzyl alcohol O-benzoyltransferase, and benzaldehyde dehydrogenase. Each of these enzymes might be involved in BA biosynthesis. Real-time PCR (qPCR) analysis revealed that these six genes were highly transcribed in the aerial organs of P. davidiana, particularly in leaves. Treating the leaves of in vitro cultured plants with methyl jasmonate (MeJA) strongly enhanced the mRNA accumulation of all 6 genes, and this treatment also clearly enhanced the accumulation of BA, SA, salicyl alcohol, benzyl alcohol, benzyl benzoate, and benzaldehyde but not salicin. Our study shows that P. davidiana may possess a CoA-dependent β-oxidative BA synthesis pathway. We also identified a relationship between the transcription of these genes and the accumulation of benzenoids, including BA and SA, which are highly responsive to the defense signaling molecule (MeJA).

Benzaldehyde dehydrogenase-driven phytoalexin biosynthesis in elicitor-treated Pyrus pyrifolia cell cultures

Pyrus pyrifolia (Asian pear) cell cultures respond to yeast extract (YE) treatment by accumulating benzoate-derived biphenyl phytoalexins, namely, noraucuparin and aucuparin. Biphenyl phytoalexins are defense-marker metabolites of the sub-tribe Malinae of the family Rosaceae. The substrates for biphenyl biosynthesis are benzoyl-CoA and malonyl-CoA, which combine in the presence of biphenyl synthase (BIS) to produce 3,5-dihydroxybiphneyl. In the non-β-oxidative pathway, benzoyl-CoA is directly derived from benzoic acid in a reaction catalyzed by benzoate-CoA ligase (BZL). Although the core β-oxidative pathway of benzoic acid biosynthesis is well-understood, the complete cascade of enzymes and genes involved in the non-β-oxidative pathway at the molecular level is poorly understood. In this study, we report the detection of benzaldehyde dehydrogenase (BD) activity in YE-treated cell cultures of P. pyrifolia. BD catalyzes the conversion of benzaldehyde to benzoic acid. BD and BIS activities were coordinately induced by elicitor treatment, suggesting their involvement in biphenyl metabolism. Changes in phenylalanine ammonia-lyase (PAL) activity preceded the increases in BD and BIS activities. Benzaldehyde was the preferred substrate for BD (Km=52.0μM), with NAD+ being the preferred co-factor (Km=64μM). Our observations indicate the contribution of BD towards biphenyl phytoalexin biosynthesis in the Asian pear.

Methoxybenzaldehydes in Plants:Insight to the Natural Resources, Isolation, Application and Biosynthesis

Methoxybenzaldehydes in plants are one of the important groups of benzoate derivatives. Some of them, exhibiting refreshing fragrance can be used as flavouring ingredients in food and cosmetics. Therefore, they have important roles in food and cosmetic industries. Methoxybenzaldehydes also exhibit significant medicinal properties and thus have certain prospects in pharmaceutical industry. Biosynthesis of benzoic acid in plants has been explored in the last decade. There has been focus on benzaldehyde and methoxybenzaldehyde biosynthesis as well. There have been several studies regarding the biosynthetic route and mechanism of formation of benzaldehyde via benzoic acid from cinnamate and further addition of 'methoxy' group to it in the last few years. Still there are many ambiguities regarding the medicinal properties and biosynthesis of methoxybenzaldehydes. This review highlights the latest advances in fragrant methoxybenzaldehyde research and the knowledge gaps till date. The review also discusses the occurrence of methoxybenzaldehydes in plants, their separation methods, medicinal properties and biosynthesis.

De novo Sequencing and Transcriptome Analysis of Pinellia ternata Identify the Candidate Genes Involved in the Biosynthesis of Benzoic Acid and Ephedrine.

Background: The medicinal herb, Pinellia ternata, is purported to be an anti-emetic with analgesic and sedative effects. Alkaloids are the main biologically active compounds in P. ternata, especially ephedrine that is a phenylpropylamino alkaloid specifically produced by Ephedra and Catha edulis. However, how ephedrine is synthesized in plants is uncertain. Only the phenylalanine ammonia lyase (PAL) and relevant genes in this pathway have been characterized. Genomic information of P. ternata is also unavailable.Results: We analyzed the transcriptome of the tuber of P. ternata with the Illumina HiSeq™ 2000 sequencing platform. 66,813,052 high-quality reads were generated, and these reads were assembled de novo into 89,068 unigenes. Most known genes involved in benzoic acid biosynthesis were identified in the unigene dataset of P. ternata, and the expression patterns of some ephedrine biosynthesis-related genes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Also, 14,468 simple sequence repeats (SSRs) were identified from 12,000 unigenes. Twenty primer pairs for SSRs were randomly selected for the validation of their amplification effect.Conclusion: RNA-seq data was used for the first time to provide a comprehensive gene information on P. ternata at the transcriptional level. These data will advance molecular genetics in this valuable medicinal plant.

Peroxisomal ATP-Binding Cassette Transporter COMATOSE and the Multifunctional Protein ABNORMAL INFLORESCENCE MERISTEM Are Required for the Production of Benzoylated Metabolites in Arabidopsis Seeds

Secondary metabolites derived from benzoic acid (BA) are of central importance in the interactions of plants with pests, pathogens, and symbionts and are potentially important in plant development. Peroxisomal β-oxidation has recently been shown to contribute to BA biosynthesis in plants, but not all of the enzymes involved have been defined. In this report, we demonstrate that the peroxisomal ATP-binding cassette transporter COMATOSE is required for the accumulation of benzoylated secondary metabolites in Arabidopsis (Arabidopsis thaliana) seeds, including benzoylated glucosinolates and substituted hydroxybenzoylcholines. The ABNORMAL INFLORESCENCE MERISTEM protein, one of two multifunctional proteins encoded by Arabidopsis, is essential for the accumulation of these compounds, and MULTIFUNCTIONAL PROTEIN2 contributes to the synthesis of substituted hydroxybenzoylcholines. Of the two major 3-ketoacyl coenzyme A thiolases, KAT2 plays the primary role in BA synthesis. Thus, BA biosynthesis in Arabidopsis employs the same core set of β-oxidation enzymes as in the synthesis of indole-3-acetic acid from indole-3-butyric acid.

Completion of the core β-oxidative pathway of benzoic acid biosynthesis in plants

Despite the importance of benzoic acid (BA) as a precursor for a wide array of primary and secondary metabolites, its biosynthesis in plants has not been fully elucidated. BA formation from phenylalanine requires shortening of the C(3) side chain by two carbon units, which can occur by a non-β-oxidative route and/or a β-oxidative pathway analogous to the catabolism of fatty acids. Enzymes responsible for the first and last reactions of the core BA β-oxidative pathway (cinnamic acid → cinnamoyl-CoA → 3-hydroxy-3-phenylpropanoyl-CoA → 3-oxo-3-phenylpropanoyl-CoA → BA-CoA) have previously been characterized in petunia, a plant with flowers rich in phenylpropanoid/benzenoid volatile compounds. Using a functional genomics approach, we have identified a petunia gene encoding cinnamoyl-CoA hydratase-dehydrogenase (PhCHD), a bifunctional peroxisomal enzyme responsible for two consecutively occurring unexplored intermediate steps in the core BA β-oxidative pathway. PhCHD spatially, developmentally, and temporally coexpresses with known genes in the BA β-oxidative pathway, and correlates with emission of benzenoid volatiles. Kinetic analysis of recombinant PhCHD revealed it most efficiently converts cinnamoyl-CoA to 3-oxo-3-phenylpropanoyl-CoA, thus forming the substrate for the final step in the pathway. Down-regulation of PhCHD expression in petunia flowers resulted in reduced CHD enzyme activity, as well as decreased formation of BA-CoA, BA and their derived volatiles. Moreover, transgenic lines accumulated the PhCHD substrate cinnamoyl-CoA and the upstream pathway intermediate cinnamic acid. Discovery of PhCHD completes the elucidation of the core BA β-oxidative route in plants, and together with the previously characterized CoA-ligase and thiolase enzymes, provides evidence that the whole pathway occurs in peroxisomes.

Contribution of CoA Ligases to Benzenoid Biosynthesis in Petunia Flowers

Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the β-oxidative pathway.

A plant thiolase involved in benzoic acid biosynthesis and volatile benzenoid production

The exact biosynthetic pathways leading to benzoic acid (BA) formation in plants are not known, but labeling experiments indicate the contribution of both beta-oxidative and non-beta-oxidative pathways. In Petunia hybrida BA is a key precursor for the production of volatile benzenoids by its flowers. Using functional genomics, we identified a 3-ketoacyl-CoA thiolase, PhKAT1, which is involved in the benzenoid biosynthetic pathway and the production of BA. PhKAT1 is localised in the peroxisomes, where it is important for the formation of benzoyl-CoA-related compounds. Silencing of PhKAT1 resulted in a major reduction in BA and benzenoid formation, leaving the production of other phenylpropanoid-related volatiles unaffected. During the night, when volatile benzenoid production is highest, it is largely the beta-oxidative pathway that contributes to the formation of BA and benzenoids. Our studies add the benzenoid biosynthetic pathway to the list of pathways in which 3-ketoacyl-CoA thiolases are involved in plants.

Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis

Benzoic acid (BA) is an important building block in a wide spectrum of compounds varying from primary metabolites to secondary products. Benzoic acid biosynthesis from L-phenylalanine requires shortening of the propyl side chain by two carbons, which can occur via a beta-oxidative pathway or a non-beta-oxidative pathway, with benzaldehyde as a key intermediate. The non-beta-oxidative route requires benzaldehyde dehydrogenase (BALDH) to convert benzaldehyde to BA. Using a functional genomic approach, we identified an Antirrhinum majus (snapdragon) BALDH, which exhibits 40% identity to bacterial BALDH. Transcript profiling, biochemical characterization of the purified recombinant protein, molecular homology modeling, in vivo stable isotope labeling, and transient expression in petunia flowers reveal that BALDH is capable of oxidizing benzaldehyde to BA in vivo. GFP localization and immunogold labeling studies show that this biochemical step occurs in the mitochondria, raising a question about the role of subcellular compartmentalization in BA biosynthesis.

Arabidopsis Chy1 null mutants are deficient in benzoic acid‐containing glucosinolates in the seeds

The specific set of reactions that lead to the synthesis of benzoic acid in plants is still unclear, and even the subcellular compartment in which these reactions occur is unknown. Biosynthesis of both vegetative tissues and seeds of Arabidopsis thaliana contain a class of defense compounds termed glucosinolates, but only the seeds synthesize and store high levels of two glucosinolate compounds that contain a benzoic acid moiety. To identify genes involved in the synthesis of benzoic acid (directly or via benzaldehyde) in Arabidopsis, we analysed the levels of benzoylated glucosinolates in several lines that carry mutations in genes with homology to Pseudomonas fluorescens feruloyl-CoA hydratase, an enzyme that converts feruloyl-CoA to vanillin and acetyl-CoA, a reaction analogous to the conversion of cinnamoyl-CoA to benzaldehyde. We show here that mutations in the gene At5g65940, previously shown to encode a peroxisomal protein with beta-hydroxyisobutyryl-CoA hydrolase activity and designated as Chy1, lead to a deficiency of benzoic acid-containing glucosinolates in the seeds. Furthermore, Chy1 exhibits cinnamoyl-CoA hydrolase activity with a K(m) of 2.9 mum. Our findings suggest that at least a part of benzoic acid biosynthesis occurs in the peroxisomes, although the specific pathway that leads to benzoic acid and the specific biochemical role of Chy1 remain unclear.

Benzoic acid biosynthesis in cell cultures of Hypericum androsaemum.

Biosynthesis of benzoic acid from cinnamic acid has been studied in cell cultures of Hypericum androsaemum L. The mechanism underlying side-chain shortening is CoA-dependent and non-beta-oxidative. The enzymes involved are cinnamate:CoA ligase, cinnamoyl-CoA hydratase/lyase and benzaldehyde dehydrogenase. Cinnamate:CoA ligase was separated from benzoate:CoA ligase and 4-coumarate:CoA ligase, which belong to xanthone biosynthesis and general phenylpropanoid metabolism, respectively. Cinnamoyl-CoA hydratase/lyase catalyzes hydration and cleavage of cinnamoyl-CoA to benzaldehyde and acetyl-CoA. Benzaldehyde dehydrogenase finally supplies benzoic acid. In cell cultures of H. androsaemum, benzoic acid is a precursor of xanthones, which accumulate during cell culture growth and after methyl jasmonate treatment. Both the constitutive and the induced accumulations of xanthones were preceded by increases in the activities of all benzoic acid biosynthetic enzymes. Similar changes in activity were observed for phenylalanine ammonia-lyase and the xanthone biosynthetic enzymes benzoate:CoA ligase and benzophenone synthase.

A mechanism of benzoic acid biosynthesis in plants and bacteria that mirrors fatty acid beta-oxidation.

Despite the simple structure and widespread occurrence of benzoic acid, its biosynthesis has resisted complete elucidation. However, recent studies reveal that the conversion of cinnamoyl-CoA into benzoyl-CoA (see scheme; R=Ph) can proceed by a route that resembles fatty acid β-oxidation. Feeding studies and sequence analyses show that both pathways not only proceed with the same stereochemistry, but are also mechanistically related.

3-hydroxy-3-phenylpropanoic acid is an intermediate in the biosynthesis of benzoic acid and salicylic acid but benzaldehyde is not.

Stable-isotope-labelled (2H6, 18O) 3-hydroxy-3-phenylpropanoic acid, a putative intermediate in the biosynthesis of benzoic acid (BA) and salicylic acid (SA) from cinnamic acid, has been synthesized and administered to cucumber (Cucumis sativus L.) and Nicotiana attenuata (Torrey). Analysis of the products by gas chromatography-mass spectrometry revealed incorporation of labelling into BA and SA, but not into benzaldehyde. In a separate experiment, 3-hydroxy-3-phenylpropanoic acid was found to be a metabolite of phenylalanine, itself the primary metabolic precursor of BA and SA. These data suggest that cinnamic acid chain shortening is probably achieved by beta-oxidation, and that the proposed "non-oxidative" pathway of side-chain degradation does not function in the biosynthesis of BA and SA, in cucumber and N. attenuata.

A Plant-like Biosynthesis of Benzoyl-CoA in the Marine Bacterium ‘ Streptomyces maritimus’

The first plant-like biosynthesis of benzoic acid in a prokaryote, the marine actinomycete ‘ Streptomyces maritimus’, has been characterized. Feeding experiments with 2H- and 13C-labeled intermediates revealed that phenylalanine is metabolized to benzoyl-coenzyme A (CoA) by means of a phenylalanine ammonia lyase and subsequent β-oxidation of cinnamoyl-CoA. Benzoyl-CoA serves as the rare starter unit of a type II polyketide synthase producing the structurally novel bacteriostatic polyketides enterocin and the wailupemycins. The results from the feeding study are consistent with the proposed biosynthetic model deduced from the cloned enterocin biosynthesis gene cluster.

The biosynthesis of benzoic acid in Bramley's Seedling apples infected by Nectria galligena Bres

Radioactive benzoic acid was synthesized from glucose- 14C and l-phenylalanine- 14C in apples infected with Nectria galligena and in apples infused with protease extracted from tissue rotted by N. galligena. Phenylalanine ammonia-lyase (PAL) activity was rapidly induced in apples infused with the fungal protease but a lag phase occurred following inoculation with the pathogen. Production of benzoic acid was maximal approximately 3 days after maximum PAL production. Enzymes extracted from necrotic apple tissue converted phenylalanine to cinnamic and benzoic acids. P. expansum, a non-protease producing apple pathogen, did not induce either PAL activity or elicit benzoic acid accumulation in apples.