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9-Carbon aldehydes such as (2E)-nonenal, (3Z)-nonenal, and (2E,6Z)-nonadienal are important melon and cucumber fragrance compounds. Currently, these molecules are produced either synthetically, which faces consumer aversion, or through biotransformation using plant-extracted enzymes, which is costly and inefficient. In this study, we constructed a Saccharomyces cerevisiae platform for the whole cell biotransformation of polyunsaturated fatty acids (PUFAs) to 9-carbon aldehydes. Heterologous expression of lipoxygenase (LOX) from Nicotiana benthamiana and hydroperoxide lyase (HPL) from Cucumis melo (melon) in S. cerevisiae enabled the production of (2E)-nonenal from readily available polyunsaturated fatty acid substrates. A 5.5-fold increase in (2E)-nonenal titer was then achieved utilizing genetic and reaction condition enhancement strategies. The highest titer of (2E)-nonenal was more than 0.11 mM, with about 9% yield. This platform can potentially be used to produce a variety of other aldehyde products by customizing with LOX and HPL enzymes of different regio-selectivities. Key points: • Establishment of a S. cerevisiae whole-cell biotransformation platform for cost-efficient, high-yield conversion of PUFAs into high value 9-carbon aldehyde compounds • 5.5-Fold improvement of (2E)-nonenal titer to > 0.11 mM achieved by enhancing reaction conditions and gene expression levels of LOX and HPL [ABSTRACT FROM AUTHOR]

The allene oxide synthase (AOS) and hydroperoxide lyase (HPL) branches of the oxylipin biosynthesis pathway lead to the formation of different products from one substrate—13-hydroperoxy-(9,11,15)-octadecatrienoic acid (13-HPOT). It remains unknown how the distribution of 13-HPOT substrate between these branches is regulated in plants, and how the metabolites of each branch influence the activity of the parallel branch. In this work, the activity of HPL branch in the leaves of bread wheat (Triticum aestivum L.) of the Saratovskaya-60 variety, as well as transgenic plants overexpressing the gene for the biosynthesis of jasmonates from Arabidopsis thalianaAtOPR3 (12-OXOPHYTODIENOATE REDUCTASE 3), was investigated. The studies revealed a high content of HPL metabolites in the leaves, indicating a high activity of the HPL pathway of oxylipin biosynthesis in wheat. Overexpression of the AtOPR3 gene has been shown to lead to an increased content of HPL metabolites in the leaves, primarily cis-3-hexenal, as well as to changes in the profile of volatile compounds of the HPL branch released by damaged plants. After mechanical damage, leaves of transgenic wheat overexpressing AtOPR3 produced more cis-3-hexenol and cis-3-hexenyl acetate, but less trans-2-hexenal compared to nontransgenic plants. Changes in the content of HPL branch metabolites in leaf tissues and the mixture of released volatile compounds were more pronounced in transgenic lines with higher expression of AtOPR3. Thus, it was shown for the first time that genetic modification of the AOS branch of oxylipin biosynthesis leads to a change in the activity of the HPL branch in wheat plants. [ABSTRACT FROM AUTHOR]

The lipoxygenase cascade in plants is a source of oxylipins (oxidized fatty acid derivatives), which play an important role in regulatory processes and formation of plant response to stress factors. Some of the most common enzymes of the lipoxygenase cascade are 13-specific hydroperoxide lyases (HPLs, also called hemiacetal synthases) of the CYP74B subfamily. In this work, we identified and cloned the CYP74B34 gene from carrot (Daucus carota L.) and described the biochemical properties of the corresponding recombinant enzyme. The CYP74B34 enzyme was active towards 9- and 13-hydroperoxides of linoleic (9-HPOD and 13-HPOD, respectively) and α-linolenic (9-HPOT and 13-HPOT, respectively) acids. CYP74B34 specifically converted 9-HPOT and 13-HPOT into aldo acids (HPL products). The transformation of 13-HPOD led to the formation of aldo acids and epoxyalcohols [products of epoxyalcohol synthase (EAS) activity] as major and minor products, respectively. At the same time, conversion of 9-HPOD resulted in the formation of epoxyalcohols as the main products and aldo acids as the minor ones. Therefore, CYP74B34 is the first enzyme with a double HPL/EAS activity described in carrot. The presence of these catalytic activities was confirmed by analysis of the oxylipin profiles for the roots from young seedlings and mature plants. In addition, we substituted amino acid residues in one of the catalytically essential sites of the CYP74B34 and CYP74B33 proteins and investigated the properties of the obtained mutant enzymes. [ABSTRACT FROM AUTHOR]

The aim of this study was to explore the formation of volatile lipid oxidation products by the lipoxygenase (LOX)-hydroperoxide lyase (HPL)-mediated pathway in oat, barley and soy bean. LOX activity was found only in barley and soy bean samples, but the lipase and HPL activity was detected in all samples. HPL showed particularly high activity with 13-hydroperoxides, while the activity was quite low when using 9-hydroperoxides, especially in the oat and barley. The optimum pH for HPL in different samples was similar, i.e., pH 6–7. In this condition, the volatile compounds formed dramatically with aldehydes and furans as the dominant products. Furthermore, a remarkable enzymatic degradation of lipids occurred during the preparation of food models with highly refined rapeseed oil (RO) and rapeseed oil fatty acid (ROFA) emulsions, where the ROFAs were more prone to oxidation than RO. This study shows the significance of lipid-degrading enzymes in plant-food flavour formation.

Short-chain and medium-chain aldehydes and their derivatives, yielded by the enzymes hydroperoxide lyases from fatty acids, are present in many products of plant origin. They are often used as supplements to food to postpone its expiration date and to add a flavor of freshness. Since these compounds can be absorbed by the intestine cells and pass into systemic circulation, it is important to be aware of their influence on human health. In the present study, the potential biological activity of aldehydes and alcohols with chains containing six to nine carbon atoms were assessed. Their proinflammatory activities were tested in the experimental system based on donors' whole blood. It was found that nine-carbon oxylipins stimulated the synthesis of the proinflammatory TNF-α cytokine (tumor necrosis factor alpha), and the stimulation by the aldehydes was weaker than that caused by the alcohols. The oxylipins containing six or eight carbons did not manifest proinflammatory activity. The obtained data may be of help to work out nutritional recommendations for patients suffering from inflammatory diseases. [ABSTRACT FROM AUTHOR]

Biocatalytic oxidation of a fatty acid preparation by enzymes released from a plant extract provided (2E,4E)-deca-2,4-dienal (2,4-DDAL), a valuable natural aroma. The post-reaction mixture contained several other compounds, from which 2,4-DDAL must be recovered. These included the main by-product trans-2-hexenal (2-HAL) and the unreacted substrate composed mainly of linoleic, palmitic, and oleic acids. The adsorption capacity and selectivity of 17 hydrophobic adsorbents were examined in batch equilibrium experiments. The highest binding capacity for 2,4-DDAL was around 30 mg/g. Due to a large excess of fatty acids in the mixture, the total adsorbed amounts of other components analyzed reached the values up to 150 mg/g. In addition, 2-HAL was always adsorbed more strongly than 2,4-DDAL, whereas fatty acids were mostly not adsorbed as effectively. The best-performing adsorbent, AmberLite FPX66, had a selectivity ratio of 6 for 2,4-DDAL with respect to the key impurity and linoleic acid. Ethanol, isopropanol, and acetone were examined as potential desorbents using batch adsorption experiments on AmberLite FPX66 from 2,4-DDAL solutions in organic solvents. Ethanol was found to be the best choice. Based on the batch equilibrium data, a column elution experiment was performed to purify 2,4-DDAL from the biocatalytic reaction mixture using elution with 96% ethanol. [ABSTRACT FROM AUTHOR]

Hydroperoxide lyases (HPLs) catalyze the splitting of 13S-hydroperoxyoctadecadienoic acid (13S-HPODE) into the green note flavor hexanal and 12-oxo-9(Z)-dodecenoic acid, which is not yet used industrially. Here, HPL from Carica papaya (HPLCP) was cloned and functionally expressed in Escherichia coli to investigate synthesis of 12-oxo-9(Z)-dodecenoic acid in detail. To improve the low catalytic activity of full-length HPLCP, the hydrophobic, non-conserved N-terminal sequence was deleted. This enhanced enzyme activity from initial 10 to 40 U/l. With optimization of solubilization buffer, expression media enzyme activity was increased to 2700 U/l. The tetrameric enzyme was produced in a 1.5 l fermenter and enriched by affinity chromatography. The enzyme preparation possesses a slightly acidic pH optimum and a catalytic efficiency (kcat/KM) of 2.73 × 106 s−1·M−1 towards 13S-HPODE. Interestingly, HPLCP-N could be applied for the synthesis of 12-oxo-9(Z)-dodecenoic acid, and 1 mM of 13S-HPODE was transformed in just 10 s with a yield of 90%. At protein concentrations of 10 mg/ml, the slow formation of the 10(E)-isomer traumatin was observed, pointing to a non-enzymatic isomerization process. Bearing this in mind, a one-pot enzyme cascade starting from safflower oil was developed with consecutive addition of Pseudomonas fluorescens lipase, Glycine max lipoxygenase (LOX-1), and HPLCP-N. A yield of 43% was obtained upon fast extraction of the reaction mixtures after 1 min of HPLCP-N reaction. This work provides first insights into an enzyme cascade synthesis of 12-oxo-9(Z)-dodecenoic acid, which may serve as a bifunctional precursor for bio-based polymer synthesis. [ABSTRACT FROM AUTHOR]

The plant lipoxygenase cascade is a source of various regulatory oxylipins that play a role in cell signalling, stress adaptation, and immune response. Recently, we detected an unprecedented 16(S)-lipoxygenase, CsLOX3, in the leaves and fruit pericarp of cucumber (Cucumis sativus L.). In the present work, an array of products biosynthesized through the conversions of α-linolenic acid 16-hydroperoxide (16-HPOT) was detected. Firstly, a prominent 15-hydroxy-9,12-pentadecadienoic acid (Me/TMS) was detected, the product of hydroperoxide lyase (HPL) chain cleavage of 16-HPOT and further reduction of aldehyde 15-oxo-9,12-pentadecadienoic acid to alcohol. Besides, the presence of dicarboxylic acid, 3,6-pentadecadiene-1,15-dioic acid, was deduced from the detection of its catalytic hydrogenation product, pentadecane-1,15-dioic acid. Finally, 12,15-dihydroxypentadecanoic acid (Me/TMS) was detected amongst the hydrogenated products, thus indicating the presence of the parent 12,15-dihydroxy-9,13-pentadecadienoic acid. To confirm the proposed HPL chain cleavage, the 16(S)-HPOT was prepared and incubated with the recombinant cucumber HPL CYP74B6 enzyme. The CYP74B6 possessed high activity towards 16-HPOT. Chain cleavage yields the (9 Z ,12 Z)-15-oxo-9,12-pentadecadienoic acid, undergoing a spontaneous isomerization into (9 Z ,13 E)-15-oxo-9,13-pentadecadienoic acid. Thus, the cucumber plants as well as the recombinant cucumber HPL CYP74B6 possessed unprecedented 16-HPL activity, cleaving 16-HPOT into a C 15 fragment, 15-oxo-9,12-pentadecadienoic acid, and a complementary volatile C 3 fragment, propionic aldehyde. The 16-LOX/16-HPL route of oxylipin biosynthesis presents a novel facet of the plant LOX pathway. The products of 16-hydroperoxide lyase were detected in cucumber leaves and fruit pericarp. Cucumber CYP74B6 and CYP74C1_CS enzymes catalyze the conversion of 16-hydroperoxide of α-linolenic acid resulted in the formation of a C15 and a complementary C3 fragments. [Display omitted] • Novel 16-lipoxygenase/hydroperoxide lyase route was detected in cucumber plants. • Recombinant CYP74B6 and CYP74C1_CS possessed 16-hydroperoxide lyase activity. • Both enzymes cleaved α-linolenate 16-hydroperoxide into C 15 and C 3 fragments. • Detected 16-hydroperoxide lyases produced 15-oxo-9,12-pentadecadienoic acid. [ABSTRACT FROM AUTHOR]

The genome of the neotropical fruit bat Sturnira hondurensis was recently sequenced, revealing an unexpected gene encoding a plant-like protein, CYP74C44, which shares ca. 90% sequence identity with the putative CYP74C of Populus trichocarpa. The preparation and properties of the recombinant CYP74C44 are described in the present work. The CYP74C44 enzyme was found to be active against the 13- and 9-hydroperoxides of linoleic and α-linolenic acids (13-HPOD, 13-HPOT, 9-HPOD, and 9-HPOT, respectively), as well as the 15-hydroperoxide of eicosapentaenoic acid (15-HPEPE). All substrates studied were specifically transformed into chain cleavage products that are typical for hydroperoxide lyases (HPLs). The HPL chain cleavage reaction was validated by the identification of NaBH4-reduced products (Me/TMS) of 15-HPEPE and 13- and 9-hydroperoxides as (all-Z)-14-hydroxy-5,8,11-tetradecatrienoic, (9Z)-12-hydroxy-9-dodecenoic, and 9-hydroxynonanoic acids (Me/TMS), respectively. Thus, CYP74C44 possessed the HPL activity that is typical for the CYP74C subfamily proteins. [ABSTRACT FROM AUTHOR]

Green leaf volatiles impart characteristic aroma and flavour to a variety of natural foods due to their inherent grassy note contributed by aldehydes. Hydroperoxide lyase (HPL) is an enzyme that helps in the cleavage of fatty acid hydroperoxides to short-chain aldehydes and ω-oxo-acids. A tomato hydroperoxide lyase gene was successfully expressed in E. coli BL21 (DE3) cells and used in the subsequent production of (Z)-3-hexenal. Biochemical characterization of the HPL activity exhibited by these whole cells enabled the development of a suitable one-pot reaction process for conversion of the hydroperoxide substrate to the corresponding aldehyde, (Z)-3-hexenal, and finally to (Z)-3-hexenol, a high-value flavour and fragrance ingredient. [ABSTRACT FROM AUTHOR]

Green leaf volatiles (GLVs) consist of six-carbon volatile aldehydes, alcohols, and their esters. They are formed from polyunsaturated fatty acids and are involved in the defense of plants against herbivores and pathogens. GLVs generally have low concentrations in intact healthy plant tissues, but the biosynthetic pathway to form GLVs is quickly activated by mechanical damage to tissues, an event called the GLV-burst. Most seed plants have the ability to implement GLV-burst; however, this potential in non-seed plants has not been extensively researched. In this study, we examined the GLV-burst capacity of monilophytes, lycophytes, and bryophytes, and confirmed that monilophytes and lycophytes showed substantial GLV-burst ability, while bryophytes did not, with a few exceptions. When the genome sequence of a model lycophyte, Selaginella moellendorffii was reviewed, 10 genes were found that showed high similarity with the non-canonical cytochrome P450 enzymes, CYP74s, specialized in oxylipin formation. Recombinant proteins expressed with Escherichia coli showed that one of them had the ability to encode allene oxide synthase, and another encoded hydroperoxide lyase (HPL), preferring linolenic acid 13-hydroperoxide, and it was inferred that this gene was responsible for GLV-burst in S. moellendorffii. Based on the phylogenetic tree constructed with CYP74s of non-seed and seed plants, we hypothesized that HPL was acquired independently in the lycophyte and seed plants through diversification of CYP74 genes.

Green leaf volatiles (GLVs) consist of six-carbon volatile aldehydes, alcohols, and their esters. They are formed from polyunsaturated fatty acids and are involved in the defense of plants against herbivores and pathogens. GLVs generally have low concentrations in intact healthy plant tissues, but the biosynthetic pathway to form GLVs is quickly activated by mechanical damage to tissues, an event called the GLV-burst. Most seed plants have the ability to implement GLV-burst; however, this potential in non-seed plants has not been extensively researched. In this study, we examined the GLV-burst capacity of monilophytes, lycophytes, and bryophytes, and confirmed that monilophytes and lycophytes showed substantial GLV-burst ability, while bryophytes did not, with a few exceptions. When the genome sequence of a model lycophyte, Selaginella moellendorffii was reviewed, 10 genes were found that showed high similarity with the non-canonical cytochrome P450 enzymes, CYP74s, specialized in oxylipin formation. Recombinant proteins expressed with Escherichia coli showed that one of them had the ability to encode allene oxide synthase, and another encoded hydroperoxide lyase (HPL), preferring linolenic acid 13-hydroperoxide, and it was inferred that this gene was responsible for GLV-burst in S. moellendorffii. Based on the phylogenetic tree constructed with CYP74s of non-seed and seed plants, we hypothesized that HPL was acquired independently in the lycophyte and seed plants through diversification of CYP74 genes. [ABSTRACT FROM AUTHOR]

The industrial processing and cold storage of minimally processed tomato juice might produce changes in volatile compounds responsible for the fresh tomato flavour. Many of these changes are related to the activity of the enzymes lipoxygenase (LOX) and hydroperoxide lyase (HPL), which are involved in the production of volatile compounds. The aim of this research was to ascertain changes in these enzymes and the major volatile compounds contributors to the fresh and green aroma attributes (aldehydes, alcohols, and lycopene volatile derivatives) in a refrigerated tomato juice, following a standardized industrial short thermal processing (96 °C, 30 s) and during refrigerated shelf-life storage (4 °C, 22 and 40 days). Results showed that, after the heat treatment, residual LOX and HPL activities were reduced in a 97% and 60%, respectively, compared to homogenized fresh tomatoes. The content of the volatile aldehydes hexanal and (Z)-3-hexenal decreased substantially from homogenized tomatoes (fresh tomato juice) to short thermal processed juice, however, (E)-2-hexenal increased probably by the action of isomerases. The content of the related alcohols, 1-hexenol and (Z)-3-hexenol, was increased with processing. The slight increase of the lycopene derived compound 6-methyl-5-hepten-2-one during storage (from 0.68 to 0.73 mg L−1) could be associated with residual enzymatic activities and lycopene oxidative degradation processes, contributing positively to aroma attributes in the tomato juice. The content of lycopene in this tomato juice (27.25 mg kg−1 FW) only decreased 8% during storage. Minimizing the elapsed time from the initial tomatoes processing and the short thermal treatment is crucial to avoid volatile compounds degradation, mainly the aldehydes as the main contributors to the fresh aroma of tomato juice. The residual LOX and HPL enzymes activities should be studied in every particular case to avoid non-desirable changes in flavor-related compounds that could modify the final quality of the product. [ABSTRACT FROM AUTHOR]

This work aims to propose a pathway for the production of 2(E),4(E)-decadienal from arachidonic acid (ARA) via 11-hydroperoxide eicosanoid (11 hydroperoxyeicosatetraenoic acid, 11-HPETE) through lipoxygenase (LOX) and hydroperoxide lyase (HPL) in the brown alga, Saccharina angustata, by identifying pathway intermediates through three studies. The first study investigated the biogeneration of 2(E),4(E)-decadienal in crude homogenates of fronds (CHF), while the second and third investigated whether ARA is the precursor of the pathway and if 11-HPETE is produced from ARA as an intermediate in 2(E),4(E)-decadienal generation, respectively. The results showed that 2(E),4(E)-decadienal was formed in CHF and its concentration increased after incubation. This finding led to the hypothesis that 2(E),4(E)-decadienal is formed enzymatically via LOX-HPL and consequently its biogeneration was determined. ARA was indicated to be a precursor of the pathway since, after CHF was incubated with ARA as a substrate, the amount of aldehyde increased significantly compared with that produced by CHF without ARA and without incubation. The production of 11-HPETE from ARA was also demonstrated as an intermediate reaction in 2(E),4(E)-decadienal formation via the LOX-HPL pathway. The hydroxy isomer of 11-HPETE was produced, and ARA was incubated with homogenated fronds in combination with glutathione peroxidase and glutathione to control its stability during identification. The compound was identified as 11-HPETE because the purified isomer showed the same retention time when co-injected with the standard using an HPLC technique; moreover, the same indicated mass spectrum was obtained as that of the standard via a GC/GCMS technique. The indicated pathway and the pathway for the production of short chain aldehydes [n-hexanal, 2(E),3(Z)-nonenal and 2(E),4(E)-decadienal] from ARA and linoleic acid through theirs hydroperoxides are proposed. [ABSTRACT FROM AUTHOR]

Environmental stresses induce production of oxylipins synthesized by the two main biosynthetic branches, allene oxide synthase (AOS) and hydroperoxide lyase (HPL). Here, we investigate how waterlogging-mediated alteration of AOS- and HPL-derived metabolic profile results in modulation of central metabolism and ultimately enhanced tolerance to this environmental stress in Arabidopsis thaliana. Waterlogging leads to increased levels of AOS- and HPL-derived metabolites, and studies of genotypes lacking either one or both branches further support the key function of these oxylipins in waterlogging tolerance. Targeted quantitative metabolic profiling revealed oxylipin-dependent alterations in selected primary metabolites, and glycolytic and citric acid cycle intermediates, as well as a prominent shift in sucrose cleavage, hexose activation, the methionine salvage pathway, shikimate pathway, antioxidant system, and energy metabolism in genotypes differing in the presence of one or both functional branches of the oxylipin biosynthesis pathway. Interestingly, despite some distinct metabolic alterations caused specifically by individual branches, overexpression of HPL partially or fully alleviates the majority of altered metabolic profiles observed in AOS-depleted lines. Collectively, these data identify the key role of AOS- and HPL-derived oxylipins in altering central metabolism, and further provide a metabolic platform targeted at identification of gene candidates for enhancing plant tolerance to waterlogging. [ABSTRACT FROM AUTHOR]

Oxygenated membrane fatty acid derivatives termed oxylipins play important roles in plant defense against biotic and abiotic cues. Plants challenged by insect pests, for example, synthesize a blend of different defense compounds that include volatile aldehydes and jasmonic acid (JA), among others. Because all oxylipins are derived from the same pathway, we investigated how their synthesis might be regulated, focusing on two closely related atypical cytochrome P450 enzymes designated CYP74A and CYP74B, respectively, allene oxide synthase (AOS) and hydroperoxide lyase (HPL). These enzymes compete for the same substrate but give rise to different products: the final product of the AOS branch of the oxylipin pathway is JA, while those of the HPL branch comprise volatile aldehydes and alcohols. AOS and HPL are plastid envelope enzymes in Arabidopsis thaliana but accumulate at different locations. Biochemical experiments identified AOS as a constituent of complexes also containing lipoxygenase 2 (LOX2) and allene oxide cyclase (AOC), which catalyze consecutive steps in JA precursor biosynthesis, while excluding the concurrent HPL reaction. Based on published X-ray data, the structure of this complex was modelled and amino acids involved in catalysis and subunit interactions predicted. Genetic studies identified the microRNA 319-regulated clade of TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factor genes and CORONATINE INSENSITIVE 1 (COI1) as controlling JA production through the LOX2-AOS-AOC2 complex. Together, our results define a molecular branch point in oxylipin biosynthesis that allows fine-tuning of the plant's defense machinery in response to biotic and abiotic stimuli. [ABSTRACT FROM AUTHOR]

Bagging is widely practiced to produce high quality and unblemished fruit; however, little is currently known about the effect of bagging on flavor and nutritional quality of cucumber fruits. Here we determined the influence of bagging on fruit quality of cucumber (Cucumis sativus L.) using three genotypes from different geographic groups. Exocarp chlorophyll and carotenoid levels were significantly decreased by bagging, accompanied by color change. Ascorbate content in bagged fruits decreased to some extent, while contents of soluble sugars, starch, and cellulose were comparable with those of control fruits. Compositions related to fruit flavor quality could be enhanced largely through bagging treatment, with elevation of the relative proportion of C6 aldehyde, as well as (E,Z)-2,6-nonadienal/(E)-2-nonenal ratio, and linoleic/α-linolenic acid ratio. Lipoxygenase and hydroperoxide lyase, two key enzymes in the production of volatiles, displayed distinctive transcript expression patterns and trends in changes of enzymatic activity in the bagged fruits of different genotypes. Overall, this study assesses the information on changing characteristics of fruit volatile composition and nutritional quality among different cucumber genotypes after bagging treatment. Results of this study would contribute to providing reference for mechanism study and cultivation conditions to improve cucumber fruit flavor to a considerable degree.

Oxylipins are one of the most important classes of bioregulators, biosynthesized through the oxidative metabolism of unsaturated fatty acids in various aerobic organisms. Oxylipins are bioregulators that maintain homeostasis at the cellular and organismal levels. The most important oxylipins are mammalian eicosanoids and plant octadecanoids. In plants, the main source of oxylipins is the lipoxygenase cascade, the key enzymes of which are nonclassical cytochromes P450 of the CYP74 family, namely allene oxide synthases (AOSs), hydroperoxide lyases (HPLs), and divinyl ether synthases (DESs). The most well-studied plant oxylipins are jasmonates (AOS products) and traumatin and green leaf volatiles (HPL products), whereas other oxylipins remain outside of the focus of researchers' attention. Among them, there is a large group of epoxy hydroxy fatty acids (epoxyalcohols), whose biosynthesis has remained unclear for a long time. In 2008, the first epoxyalcohol synthase of lancelet Branchiostoma floridae , BfEAS (CYP440A1), was discovered. The present review collects data on EASs discovered after BfEAS and enzymes exhibiting EAS activity along with other catalytic activities. This review also presents the results of a study on the evolutionary processes possibly occurring within the P450 superfamily as a whole.

Okara (soybean residue), a by-product from soymilk and tofu production, has a green, grassy off odour as it contains a large amount of aldehydes. This work investigated the rate-limiting enzyme(s) in the formation of aldehydes in okara and the pathways leading to their bioconversion into fruity, pleasant-smelling esters by the yeast Lindnera saturnus. Lipase and hydroperoxide lyase were shown to be rate-limiting enzymes while endogenous soy lipoxygenase was also crucial for the production of aldehydes in okara. Subsequent fermentation of okara by L. saturnus increased the amount of esters by about 70 times to 165-277 μg/g dried okara. The generation of C7 esters followed our hypothesised pathway, while that for C6 esters was mainly affected by L. saturnus. This study presents a simple and inexpensive one-pot setup for the natural bio-production of esters from okara. [ABSTRACT FROM AUTHOR]

Abstract Hydroperoxide lyases (HPLs) of the CYP74 family (P450 superfamily) are widely distributed enzymes in higher plants and are responsible for the stress-initiated accumulation of short-chain aldehydes. Fatty acid hydroperoxides serve as substrates for HPLs; however, details of the HPL-promoted conversion are still incompletely understood. In the present work, we report first time the micropreparative isolation and the NMR structural studies of fatty acid hemiacetal (TMS/TMS), the short-lived HPL product. With this aim, linoleic acid 9( S )‑hydroperoxide (9( S )‑HPOD) was incubated with recombinant melon hydroperoxide lyase (CmHPL, CYP74C2) in a biphasic system of water/hexane for 60 s at 0 °C, pH 4.0. The hexane layer was immediately decanted and vortexed with a trimethylsilylating mixture. Analysis by GC–MS revealed a major product, i.e. the bis-TMS derivative of a hemiacetal which was conclusively identified as 9‑hydroxy‑9‑[(1′ E ,3′ Z )‑nonadienyloxy]‑nonanoic acid by NMR-spectroscopy. Further support for the hemiacetal structure was provided by detailed NMR-spectroscopic analysis of the bis-TMS hemiacetal generated from [13C 18 ]9( S )‑HPOD in the presence of CmHPL. The results obtained provide incontrovertible evidence that the true products of the HPL group of enzymes are hemiacetals, and that the short-chain aldehydes are produced by their rapid secondary chain breakdown. Therefore, we suggest replacing the name “hydroperoxide lyase”, which does not reflect the factual isomerase (intramolecular oxidoreductase) activity, with “hemiacetal synthase” (HAS). Highlights • Recombinant CmHPL (CYP74C2) was incubated with [13C 18 ]linoleate 9‑hydroperoxide. • The predominant product was a hemiacetal obtained in form of its TMS derivative. • Its structure was found to be 9‑hydroxy‑9‑[(1′ E ,3′ Z )‑nonadienyloxy]‑nonanoic acid. • The data showed that the hemiacetal is the true product of the enzyme. • Renaming the enzyme from “hydroperoxide lyase” to “hemiacetal synthase” is suggested. [ABSTRACT FROM AUTHOR]

The present study was aimed at understanding the effect of micro-perforated polyethylene film packing (MPFP) on the metabolism of fatty acid-derived volatiles and physicochemical attributes in “Red Globe” table grapes under storage at 0 °C for 50 days. Physicochemical properties evaluated in grapes were total soluble solids (TSS), texture profile, decay incidence, fatty acid content, and aroma-related enzyme activities including lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase (ADH), and volatiles in grape peel and flesh. Aroma-related genes (VvLOX, VvHPL, and VvADH) were also evaluated. Grapes treated with MPFP was found to retain the texture profile and decay incidence was reduced (17.4% of reduction compared with control) significantly (P ≤ 0.05). Linoleic acid (LA), the main substrate of LOX/HPL pathway, was the highest in table grapes packed in MPFP, indicating the use of MPFP was able to preserve LA compound. Micro-perforated film packing upregulated VvLOX and VvHPL, downregulated VvADH, increased LOX and HPL activities, and inhibited ADH activity, enhancing C6/C9 compounds. The results indicated that MPFP system was the best choice to improve aroma quality of “Red Globe” table grapes in comparison to polyethylene film packing (PFP) and control (no packing). [ABSTRACT FROM AUTHOR]

The approach of this research was to describe the biochemical transformations of minor components of virgin olive oil with high impact on its sensory quality, specifically “volatile compounds” during its extraction process. For this purpose `Picual´, `Arbequina´ and `Hojiblanca´ cultivars were processed at three different harvesting times. Their volatile profiles and levels of enzymatic activity of “hydroperoxide lyase”, responsible for the biosynthesis of C6 aldehydes related to “green and fruity notes”, were monitored and identified in mesocarp, paste before kneading and paste after kneading based on the catalytic action of crude enzyme extracts. Both of them, volatiles and HPL, were analyzed by incubating with their corresponding substrates and reaction products formed were analyzed by Solid Phase Microextraction-Gas Chromatography and Mass Spectrometry. The results obtained in the present research can be useful in order to develop new markers biochemical whereby improved aroma quality in olive breeding programs or in the design of new protocol of VOO extraction. [ABSTRACT FROM AUTHOR]

As an effective ethylene action inhibitor, 1-Methylcyclopropene (1-MCP) can delay fruit ripening and senescence. However, little is known about the effect of 1-MCP on peach fruit aroma-related volatiles and their biosynthesis-related gene expression during cold storage. Peaches (cv. Xiahui 6) were treated with 1-MCP (10 μL L −1 for 12 h) to investigate the influence of 1-MCP on aromatic volatile biosynthesis and involved genes expression during cold storage. 1-MCP treatment significantly decreased ethylene production and delayed the ripening and senescence of peach at cold storage. A lower production of esters was found in 1-MCP treated fruit, while there were higher alcohol acyltransferase (AAT) activity and PpaAAT1/2 expression in treated fruit during the early storage. The performance patterns of both aldehydes and alcohols were different between 1-MCP and control fruit accompanied by lower lipoxygenase (LOX), alcohol dehydrogenase (ADH), hydroperoxide lyase (HPL) activities in accordance with the expression of PpaLOX3 , PpaADH1 and PpaHPL1 in 1-MCP treated fruit compared with the control in the late storage. All in all, the role of 1-MCP on the volatiles and its related genes expression in peach fruits was discussed, and 1-MCP affected the formation of aroma volatile by functioning in the expression of pivotal enzyme genes including LOX, HPL, ADH and AAT. [ABSTRACT FROM AUTHOR]

Fatty acid hydroperoxides can generate short-chained volatile aldehydes that may participate in plant defence. A grapevine hydroperoxide lyase (VvHPL1) clustering to the CYP74B class was functionally characterized with respect to a role in defence. In grapevine leaves, transcripts of this gene accumulated rapidly to high abundance in response to wounding. Cellular functions of VvHPL1 were investigated upon heterologous expression in tobacco BY-2 cells. A C-terminal green fluorescent protein (GFP) fusion of VvHPL1 was located in plastids. The overexpression lines were found to respond to salinity stress or the bacterial elicitor harpin by increasing cell death. This signal-dependent mortality response was mitigated either by addition of exogenous jasmonic acid or by treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases. By feeding different substrates to recombinantly expressed enzyme, VvHPL1 could also be functionally classified as true 13-HPL. The cognate products generated by this 13-HPL were cis-3-hexenal and trans-2-hexenal. Using a GFP-tagged actin marker line, one of these isomeric products, cis-3-hexenal, was found specifically to elicit a rapid disintegration of actin filaments. This response was not only observed in the heterologous system (tobacco BY-2), but also in a grapevine cell strain expressing this marker, as well as in leaf discs from an actin marker grape used as a homologous system. These results are discussed in the context of a role for VvHPL1 in a lipoxygenase-dependent signalling pathway triggering cell death-related defence that bifurcates from jasmonate-dependent basal immunity. [ABSTRACT FROM AUTHOR]

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT ( Medicago truncatula ) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9( S )-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13( S )-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST ( Solanum tuberosum ), CYP74C2 ( Cucumis melo ), CYP74C1_CS and CYP74C31 (both of Cucumis sativus ), and CYP74C13_GM ( Glycine max ). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9 S, 10 S, 11 S, 12 Z )-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the “hydroperoxide-binding domain” (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs. [ABSTRACT FROM AUTHOR]

The spr2 mutation in tomato (Solanum lycopersicum), which disrupts function of FATTY ACID DESATURASE 7 (FAD7), confers resistance to the potato aphid (Macrosiphum euphorbiae) and modifies the plant's C6 volatile profiles. To investigate whether C6 volatiles play a role in resistance, HYDROPEROXIDE LYASE (HPL), which encodes a critical enzyme in C6 volatile synthesis, was silenced in wild-type tomato plants and spr2 mutants. Silencing HPL in wild-type tomato increased potato aphid host preference and reproduction on 5-week old plants but had no influence on 3-week old plants. The spr2 mutation, in contrast, conferred strong aphid resistance at both 3 and 5 weeks, and silencing HPL in spr2 did not compromise this aphid resistance. Moreover, a mutation in the FAD7 gene in Arabidopsis thaliana also conferred resistance to the green peach aphid (Myzus persicae) in a genetic background that carries a null mutation in HPL. These results indicate that HPL contributes to certain forms of aphid resistance in tomato, but that the effects of FAD7 on aphids in tomato and Arabidopsis are distinct from and independent of HPL. [ABSTRACT FROM AUTHOR]

C6 volatiles are synthesized through lipoxygenase-hydroperoxide lyase (LOX-HPL) pathway and these volatiles play important roles in the aromatic quality of grape berries. This study investigated the evolution of both C6 volatiles and the key genes in the LOX-HPL pathway in different table grape cultivars during the berry development period, and further assessed the correlation between the accumulation of C6 volatiles and the expression of these genes in these cultivars. Results showed that hexanal, (E)-2-hexenal, (E)-2-hexen-1-ol and (Z)-3-hexen-1-ol were found to be the dominant C6 volatiles in these ripened grape cultivars under two consecutive vintages, and their flavor notes were incorporated in the overall aroma of these cultivars. The cultivar "Xiangfei" showed the most abundant level of C6 aldehydes and C6 acid, whereas the cultivar "Tamina" and "Moldova" possessed the highest C6 alcohol content. The "Muscat of Alexandria" cultivar was found to contain the highest level of C6 esters. C6 volatiles were grouped into three evolutionary patterns in these cultivars during berry development, and their evolution was consistent with the evolution of the LOX-HPL pathway genes' expression. Pearson's correlation analysis indicated that the LOX-HPL-pathway-related genes were correlated to the accumulation of C6 volatiles in these cultivars, and VvLOXA appeared to be an important gene that regulated the synthesis of all C6 volatiles. [ABSTRACT FROM AUTHOR]

The stabilization of olive recombinant hydroperoxide lyases (rHPLs) was investigated using selected chemical additives. Two rHPLs were studied: HPL full-length and HPL with its chloroplast transit peptide deleted (matured HPL). Both olive rHPLs are relatively stable at 4 °C, and enzyme activity can be preserved (about 100% of the rHPL activities are maintained) during 5 weeks of storage at −20 or at −80 °C in the presence of glycerol (10%, v/ v). Among the additives used in this study, glycine (2.5% w/ v), NaCl (0.5 M), and NaSO (0.25 M) provided the highest activation of HPL full-length activity, while the best matured HPL activity was obtained with NaSO (0.25 M) and NaCl (1 M). Although the inactivation rate constants ( k) showed that these additives inactivate both rHPLs, their use is still relevant as they strongly increase HPL activity. Results of C6-aldehyde production assays also showed that glycine, NaCl, and NaSO are appropriate additives and that NaCl appears to be the best additive, at least for hexanal production. [ABSTRACT FROM AUTHOR]

Main conclusion : This study describes a new role for hydroperoxide lyase branch of oxylipin biosynthesis pathway in protecting photosynthetic apparatus under high light conditions. Lipid-derived signaling molecules, oxylipins, produced by a multi-branch pathway are central in regulation of a wide range of functions. The two most known branches, allene oxide synthase (AOS) and 13-hydroperoxide lyase (HPL) pathways, are best recognized as producers of defense compounds against biotic challenges. In the present work, we examine the role of these two oxylipin branches in plant tolerance to the abiotic stress, namely excessive light. Towards this goal, we have analyzed variable chlorophyll fluorescence parameters of intact leaves of Arabidopsis thaliana genotypes with altered oxylipin profile, followed by examining the impact of exogenous application of selected oxylipins on functional activity of photosynthetic apparatus in intact leaves and isolated thylakoid membranes. Our findings unequivocally bridge the function of oxylipins to photosynthetic processes. Specifically, HPL overexpressing lines display enhanced adaptability in response to high light treatment as evidenced by lower rate constant of photosystem 2 (PS2) photoinhibition and higher rate constant of PS2 recovery after photoinhibition. In addition, exogenous application of linolenic acid, 13-hydroperoxy linolenic acid, 12-oxophytodienoic acid, and methyl jasmonate individually, suppresses photochemical activity of PS2 in intact plants and isolated thylakoid membranes, while application of HPL-branch metabolites-does not. Collectively these data implicate function of HPL branch of oxylipin biosynthesis pathway in guarding PS2 under high light conditions, potentially exerted through tight regulation of free linolenic acid and 13-hydroperoxy linolenic acid levels, as well as competition with production of metabolites by AOS-branch of the oxylipin pathway. [ABSTRACT FROM AUTHOR]

Lipoxygenase enzymatic pathway is a widely studied mechanism in the plant kingdom. Combined actions of three enzymes: lipase, lipoxygenase (LOX) and hydroperoxide lyase (HPL) convert lipidic substrates such as C18:2 and C18:3 fatty acids into short chain volatiles. These reactions, triggered by cell membrane disruptions, produce compounds known as Green Leaf Volatiles (GLVs) which are C6 or C9-aldehydes and alcohols. These GLVs are commonly used as flavors to confer a fresh green odor of vegetable to food products. Therefore, competitive biocatalytic productions have been developed to meet the high demand in these natural flavors. Vegetable oils, chosen for their lipidic acid profile, are converted by soybean LOX and plant HPL into natural GLVs. However this second step of the bioconversion presents low yield due to the HPL instability and the inhibition by its substrate. This paper will shortly describe the different enzymes involved in this bioconversion with regards to their chemical and enzymatic properties. Biotechnological techniques to enhance their production potentialities will be discussed along with their implication in a complete bioprocess, from the lipid substrate to the corresponding aldehydic or alcoholic flavors.

The presence of selected dehydrogenases, including alcohol dehydrogenase (ADH-YL) and aldehyde dehydrogenase (ALDH-YL), inYarrowia lipolyticaJMY 861, and their potential role in flavor synthesis were investigated. The experimental findings showed that using reduced form of nicotinamide adenine dinucleotide (NADH) as cofactor, the ADH-YL activityin vitrowas 6-fold higher than that with reduced form of nicotinamide adenine dinucleotide phosphate (NADPH); however, under the experimental conditions used in this study, an ALDH-YL activity was not detected. Thein situhexanal reduction reaction was found to be instantaneous; however, when the yeast cells suspension was diluted 150 times, the initial relative hexanal concentration was increased by 84.1%. The chromatographic analyses indicated the conversion,in situ, of linoleic acid hydroperoxides (HPODs) into volatile C6-compounds after 60 min of HPODs addition to the yeast cells suspension. An endogenous alcohol dehydrogenase inYarrowia lipolyticaJMY 861 along with the genetically cloned hydroperoxide lyase from green bell pepper. [ABSTRACT FROM PUBLISHER]

A novel synthesis of sulfonated dihydroisoquinolinones via cascade radical addition and cyclization was developed in the presence of tert-butyl hydroperoxide (TBHP). The reactions generated the desired sulfonated dihydroisoquinolinones in good yields from readily available arylsulfinic acids and N-allylbenzamides under metal-free conditions. [ABSTRACT FROM AUTHOR]

Rice allene oxide synthase-1 mutants carrying F92L, P430A or F92L/P430A amino acid substitution mutations were constructed, recombinant mutant and wild type proteins were purified and their substrate preference, UV–vis spectra and heme iron spin state were characterized. The results show that the hydroperoxide lyase activities of F92L and F92L/P430A mutants prefer 13-hydroperoxy substrate to other hydroperoxydienoic acids or hydroperoxytrienoic acids. The Soret maximum was completely red-shifted in P430A and F92L/P430A mutants, but it was partially shifted in the F92L mutant. ESR spectral data showed that wild type, F92L and P430A mutants occupied high and low spin states, while the F92L/P430A mutant occupied only low spin state. The extent of the red shift of the Soret maximum increased as the population of low spin heme iron increased, suggesting that the spectral shift reflects the high to low transition of heme iron spin state in rice allene oxide synthase-1. Relative to wild type allene oxide synthase-1, the hydroperoxide lyase activities of F92L and F92L/P430A are less sensitive to inhibition by imidazole with (13 S or 9 S )-hydroperoxydienoic acid as substrate and more sensitive than wild type with (13 S )-hydroperoxytrienoic acid as substrate. Our results suggest that hydroperoxydienoic acid is the preferred substrate for the hydroperoxide lyase activity and (13 S )-hydroperoxytrienoic acid is the preferred substrate for allene oxide synthase activity of allene oxide synthase-1. [ABSTRACT FROM AUTHOR]

Arabidopsis produces galactolipids containing esters of 12-oxo-phytodienoic acid (OPDA) and dinor-12-oxo-phytodienoic acid (dnOPDA). These lipids are referred to as arabidopsides and accumulate in response to abiotic and biotic stress. We explored the natural genetic variation found in 14 different Arabidopsis accessions to identify genes involved in the formation of arabidopsides. The accession C24 was identified as a poor accumulator of arabidopsides whereas the commonly used accession Col-0 was found to accumulate comparably large amounts of arabidopsides in response to tissue damage. A quantitative trait loci analysis of an F2 population created from a cross between C24 and Col-0 located a region on chromosome four strongly linked to the capacity to form arabidopsides. Expression analysis of HYDROPEROXIDE LYASE 1 (HPL1) showed large differences in transcript abundance between accessions. Transformation of Col-0 plants with the C24 HPL1 allele under transcriptional regulation of the 35S promoter revealed a strong negative correlation between HPL1 expression and arabidopside accumulation after tissue damage, thereby strengthening the view that HPL1 competes with ALLENE OXIDE SYNTHASE (AOS) for lipid-bound hydroperoxide fatty acids. We further show that the last step in the synthesis of galactolipid-bound OPDA and dnOPDA from unstable allene oxides is exclusively enzyme-catalyzed and not the result of spontaneous cyclization. Thus, the results presented here together with previous studies suggest that all steps in arabidopside biosynthesis are enzyme-dependent and apparently all reactions can take place with substrates being esterified to galactolipids. [ABSTRACT FROM AUTHOR]

Volatile C6-aldehydes are the main contributors to the characteristic odor of plants known as 'green note' and are widely used by the flavor industry. Biotechnological processes were developed to fulfill the high demand in C6-aldehydes in natural flavorants and odorants. Recombinant hydroperoxide lyases (HPLs) constitute an interesting alternative to overcome drawbacks arising from the use of HPL from plant extracts. Thus, olive recombinant 13-HPL was assayed as biocatalysts to produce C6-aldehydes. Firstly, a cDNA encoding for olive HPL of Leccino variety was isolated and cloned in pQE-30 expression vector. In order to improve the enzyme solubility, its chloroplast transit peptide was deleted. Both enzymes (HPL wild type and HPL deleted) were expressed into Escherichia coli strain M15, purified, characterized, and then used for bioconversion of 13-hydroperoxides of linoleic and linolenic acids. Aldehydes produced were extracted, then identified and quantified using gas chromatography and mass spectrometry. Recombinant HPL wild type (HPL wt) allowed producing 5.61 mM of hexanal and 4.39 mM of 3 Z-hexenal, corresponding to high conversion yields of 93.5 and 73 %, respectively. Using HPL deleted (HPL del) instead of HPL wt failed to obtain greater quantities of hexanal or 3 Z-hexenal. No undesirable products were formed, and no isomerization of 3 Z-hexenal in 2 E-hexenal occurred. The olive recombinant HPL wt appears to be a promising efficient biocatalyst for the production of C6-aldehydes. [ABSTRACT FROM AUTHOR]

Green leaf volatiles (GLVs) are C6-aliphatic aldehydes/alcohols/acetates, and biosynthesized from the central precursor fatty acid 13-hydroperoxides by 13-hydroperoxide lyases (HPLs) in various plant species. While GLVs have been implicated as defense compounds in plants, GLVs give characteristic grassy note to a bouquet of aroma in green tea, which is manufactured from young leaves of Camellia sinensis . Here we identify three HPL- related genes from C. sinensis via RNA-Sequencing (RNA-Seq) in silico , and functionally characterized a candidate gene, CYP74B24 , as a gene encoding tea HPL. Recombinant CYP74B24 protein heterologously expressed in Escherichia coli specifically produced ( Z )-3-hexenal from 13-HPOT with the optimal pH 6.0 in vitro . CYP74B24 gene was expressed throughout the aerial organs in a rather constitutive manner and further induced by mechanical wounding. Constitutive expression of CYP74B24 gene in intact tea leaves might account for low but substantial and constitutive formation of a subset of GLVs, some of which are stored as glycosides. Our results not only provide novel insights into the biological roles that GLVs play in tea plants, but also serve as basis for the improvement of aroma quality in tea manufacturing processes. [ABSTRACT FROM AUTHOR]