Hydroperoxide Lyase Research Articles

Saccharomyces cerevisiae whole cell biotransformation for the production of aldehyde flavors and fragrances

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

Intricate Evolution of Multifunctional Lipoxygenase in Red Algae.

Lipoxygenases (LOXs) from lower organisms have substrate flexibility and function versatility in fatty acid oxidation, but it is not clear how these LOXs acquired the ability to execute multiple functions within only one catalytic domain. This work studied a multifunctional LOX from red alga Pyropia haitanensis (PhLOX) which combined hydroperoxidelyase (HPL) and allene oxide synthase (AOS) activity in its active pocket. Molecular docking and site-directed mutagenesis revealed that Phe642 and Phe826 jointly regulated the double peroxidation of fatty acid, Gln777 and Asn575 were essential to the AOS function, and the HPL activity was improved when Asn575, Gln777, or Phe826 was replaced by leucine. Phylogenetic analysis indicated that Asn575 and Phe826 were unique amino acid sites in the separated clades clustered with PhLOX, whereas Phe642 and Gln777 were conserved in plant or animal LOXs. The N-terminal START/RHO_alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) domain of PhLOX was another key variable, as the absence of this domain disrupted the versatility of PhLOX. Moreover, the functions of two homologous LOXs from marine bacterium Shewanella violacea and red alga Chondrus crispus were examined. The HPL activity of PhLOX appeared to be inherited from a common ancestor, and the AOS function was likely acquired through mutations in some key residues in the active pocket. Taken together, our results suggested that some LOXs from red algae attained their versatility by amalgamating functional domains of ancestral origin and unique amino acid mutations.

Organizing the Chaos: Novel Insights into the Regulation of Z-3-Hexenal Production in Damaged Maize Leaves

Green leaf volatiles (GLVs) are important signaling compounds that help to regulate plant defenses against pests and pathogens. Made through the hydroperoxide lyase (HPL) pathway, they are rapidly produced upon damage and can signal to other parts of the same plant or even plants nearby, where they can induce rapid defense responses directly or prime them against impending danger. In this primed state, plants can respond faster and/or stronger should pests or pathogens attack. However, while all proteins and genes involved in the biosynthesis of GLVs have been identified, little is still known about how the first two steps in the pathway, e.g., oxygenation by a lipoxygenase (LOX) and subsequent cleavage by HPL, are facilitated within the damaged tissue, resulting in the production of Z-3-hexenal (Z3al) as the first committed product of the pathway. Here, we provide evidence that several factors might be involved in the production of Z3al, including pH, Ca2+, and an environment that is highly hydrophobic. We present a model in which the extraordinary circumstances that are present at the site of Z3al production are considered, and shine new light on potential regulatory mechanisms.

Low temperature conditioning and nitric oxide treatment reduced chilling injury and altered fatty acid metabolism of flat peaches

Low-temperature conditioning is a recognized method for alleviating chilling injuries in flat peaches. However, long-term low-temperature storage can mitigate volatile organic compounds, thereby limiting fruit availability. To improve the supply of flat peaches, we investigated the combined effects of low-temperature conditioning and nitric oxide treatment on various parameters including physiological quality, volatile organic compounds, fatty acids, associated enzymes, and genes involved in the fatty acid pathway with the transcriptome sequencing analysis. The results indicated that low-temperature conditioning coordinated with 10 µL L−1 nitric oxide treatment possessed higher firmness and vitamin C content and restricted the content of malonaldehyde and relative electrical conductivity, thus maintaining the overall quality of flat peaches. In the study, 10 µL L−1 nitric oxide was employed for subsequent experiments. Moreover, higher contents of volatile organic compounds were supported by increased contents of palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, alcohol dehydrogenase, hydroperoxide lyase, and up-regulated genes of LOX2, LOX3, LOX6, ADH2, and ADH3. In summary, nitric oxide treatment effectively facilitated the recovery of volatile organic compounds in flat peaches during cold storage. Therefore, low-temperature conditioning and nitric oxide treatment can be a promising strategy to prevent the loss of volatile organic compounds by modulating the fatty acid pathway and associated genes.

CYP74B34 Enzyme from Carrot (Daucus carota) with a Double Hydroperoxide Lyase/Epoxyalcohol Synthase Activity: Identification and Biochemical Properties.

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 carotaL.) and described the biochemical properties of the corresponding recombinant enzyme. TheCYP74B34 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. Atthe 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.

Calcium lactate treatment modulated fatty acid metabolism to attenuate senescence in ‘Jinfeng’ pear fruit during storage

Fatty acid metabolism not only affects cell membrane functionality but also correlates with the fruit quality and postharvest senescence. This study aims to assess the influence of calcium lactate immersion on lipid metabolism and senescence in ‘Jinfeng’ pear fruit during room temperature storage. The results demonstrated that calcium lactate effectively suppressed malondialdehyde content, respiratory rate, mass loss, and ethylene release, as well as phospholipase D (PLD), hydroperoxide lyase (HPL), lipoxygenase (LOX), lipase (LPS), alcohol acyltransferase (AAT), and alcohol dehydrogenase (ADH) activities in ‘Jinfeng’ pears. Additionally, calcium lactate resulted in an up-regulation of the gene expression levels associated with fatty acid biosynthesis, including PcACC, PcFabG, PcFabI, PcFabZ, PcKASII, PcACP, and PcFAD. Furthermore, there was an increase in the expression of genes involved in fatty acid elongation, such as PcKCS4, PcKCS6, PcKCS7, PcKCS10, PcLACS2, PcLACS4, PcLACS8, and PcCER. Conversely, it caused a down-regulation of the gene expression levels associated with fatty acid degradation, including PcLOX, PcAAT, PcADH, PcPLD, PcACD, PcECH, and PcACOX3. Simultaneously, calcium lactate treatment diminished the content of palmitic acid, while elevating the contents of linolenic acid, linoleic acid, and oleic acid. In summary, postharvest calcium lactate-immersed can efficaciously retard senescence of ‘Jinfeng’ pear fruit through regulating fatty acid lipid metabolism.

Whole-cell one-pot biosynthesis of dodecanedioic acid from renewable linoleic acid

BackgroundDodecanedioic acid (DDA), a typical medium-chain dicarboxylic fatty acid with widespread applications, has a great synthetic value and a huge industrial market demand. Currently, a sustainable, eco-friendly and efficient process is desired for dodecanedioic acid production.ResultsHerein, a multi-enzymatic cascade was designed and constructed for the production of DDA from linoleic acid based on the lipoxygenase pathway in plants. The cascade is composed of lipoxygenase, hydroperoxide lyase, aldehyde dehydrogenase, and unidentified double-bond reductase in E. coli for the main cascade reactions, as well as NADH oxidase for cofactor recycling. The four component enzymes involved in the cascade were co-expressed in E. coli, together with the endogenous double-bond reductase of E. coli. After optimizing the reaction conditions of the rate-limiting step, 43.8 g L− 1 d− 1 of DDA was obtained by a whole-cell one-pot process starting from renewable linoleic acid.ConclusionsThrough engineering of the reaction system and co-expressing the component enzymes, a sustainable and eco-friendly DDA biosynthesis route was set up in E. coli, which afforded the highest space time yield for DDA production among the current artificial multi-enzymatic routes derived from the LOX-pathway, and the productivity achieved here ranks the second highest among the current research progress in DDA biosynthesis.Graphical

Oxylipin biosynthesis via an unprecedented 16-hydroperoxide lyase pathway in green tissues of cucumber (Cucumis sativus L.) plants

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 (9Z,12Z)-15-oxo-9,12-pentadecadienoic acid, undergoing a spontaneous isomerization into (9Z,13E)-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 C15 fragment, 15-oxo-9,12-pentadecadienoic acid, and a complementary volatile C3 fragment, propionic aldehyde. The 16-LOX/16-HPL route of oxylipin biosynthesis presents a novel facet of the plant LOX pathway.

Formation of lipid-derived volatile products through lipoxygenase (LOX)- and hydroperoxide lyase (HPL)- mediated pathway in oat, barley and soy bean

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.

Impact of abscisic acid treatment on postharvest storage quality and volatile flavor substances in blueberries

AbstractTo investigate the impact of abscisic acid (ABA) on the postharvest flavor and quality of blueberries, this study employed different concentrations of ABA (400 μM ABA, 600 μM ABA, and 800 μM ABA) to treat the blueberries. The treated blueberries were then stored at 4°C (~80% relative humidity) for 35 day. Subsequently, genes associated with the metabolism of volatile aromatic substances and fatty acids in postharvest blueberries were assessed using real‐time PCR. Our results revealed that the treatment of ABA could maintain a high level of nutrient and aromatic substances in blueberries. Particularly, 600 μM ABA caused most significant changes in fatty acid metabolism‐related enzyme activities, such as lipoxygenase, hydroperoxide lyase, alcohol dehydrogenase, as well as related gene expressions, like VcLOX2S, VcHPL, VcADH1‐1, VcADH1‐2, VcADH1‐3, and VcADH1‐4. In addition, Furthermore, the blueberries treated with 600 μM ABA exhibited a superior quality when compared to the control group. This superior quality included higher levels of total acid, ascorbic acid, total phenol, anthocyanin, soluble sugar contents, and so forth. In conclusion, postharvest ABA treatment decelerated the decrease in volatile aroma substances and effectively maintained the flavor of blueberries.

Epoxyalcohol Synthase Branch of Lipoxygenase Cascade.

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.

The beneficial effects of the tobacco hydroperoxide lyase pathway in whitefly host adaptation

Plants respond to herbivore attack by emitting complex mixtures of volatile compounds to repel herbivores or attract predators and parasitoids. In this study, however, we revealed that tobacco green leaf volatiles (GLVs) were beneficial compounds for whitefly adaptation to host plants. Our study indicates that GLVs are closely associated with plant susceptibility to whiteflies. Whitefly infestation elevated the transcript of the hydroperoxide lyase (HPL) gene, one gene responsible for catalyzing the synthesis of C6 or C9 aldehydes from fatty acid hydroperoxides. Overexpression of HPL increased the emission of GLVs (hexanal, 1- hexanal, trans-2- hexanal and cis-3- hexanal) and resulted in improved performance for whiteflies. Exogenous application of GLVs promoted whitefly survival and fecundity and increased the plant's attraction to whiteflies. Thus, our study provides new insights into the role of the HPL pathway and GLVs in influencing herbivore adaptation to host plants.

Analysis of Anti-Inflammatory Properties of Plant Oxylipins Produced in the Hydroperoxide Lyase Branch

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.

Analysis of Anti-Inflammatory Properties of Plant Oxylipins Produced in the Hydroperoxide Lyase Branch

Analysis of Anti-Inflammatory Properties of Plant Oxylipins Produced in the Hydroperoxide Lyase Branch

Melatonin enhances the synthesis of volatile esters and lactones in apricot during low temperature storage

Low temperature storage often leads to chilling injury in apricots, causing a loss of aroma and a decrease in its commodity value. In this study, melatonin (MT, 0.1, 0.2, and 0.4 mmol L−1) dipping treatment was used to improve the aroma quality of apricot (‘Jintaiyang’ and ‘Pingdingxiang’) at low temperature (4 °C). Results demonstrated that the application of 0.2 mmol L−1 MT treatment effectively maintained firmness and soluble solids content (SSC), and inhibit the ethylene production and respiration rate of apricots. Additional observations revealed that MT was capable of inhibiting the activities of LOX (lipoxygenase), HPL (hydroperoxidelyase), and ADH (alcohol dehydrogenase) enzymes, thereby reducing the levels of alcohols and aldehydes in apricots. Conversely, MT upregulated the activities of LOX, HPL, ADH, ACX (acyl-CoA oxidase) and AAT (alcohol acyltransfera) enzymes, promoting the biosynthesis of lactones and esters in apricots. Thus, melatonin might be an effective method for low temperature storage of apricot by promoting the recovery of aroma substances.

Influence of Benzothiadiazole on the Accumulation and Metabolism of C6 Compounds in Cabernet Gernischt Grapes (Vitis vinifera L.).

Pre-harvest application of elicitors improves grape quality, specifically the phenolic compounds and color characteristics. Limited research has been conducted on the impact of elicitors on the C6 compounds found in grapes. This is due to lack of comprehensive studies examining the combined effects of bound aroma compounds, enzyme activity, and substrate availability. This study aimed to assess the impact of benzothiadiazole (BTH) on the physicochemical properties and C6 compounds of Cabernet Gernischt grapes during ripening. Compared with the control group (CK), BTH treatment significantly increased the 100-berry weight, skin/berry ratio, pH, total phenolic content, and total flavonoid content in ripe grapes. Additionally, BTH treatment led to significant reductions in reducing sugar, total soluble solids, titratable acidity, linoleic acid, linolenic acid, and free C6 aldehydes. Furthermore, BTH treatment significantly decreased the contents of free C6 alcohols and increased the levels of free and bound C6 esters. BTH treatment also increased the activities of lipoxygenase, alcohol dehydrogenase, and alcohol acetyltransferase enzymes, while it decreased the activity of hydroperoxide lyase enzyme. The application of BTH resulted in changes to the physicochemical properties and levels of C6 compounds in Cabernet Gernischt grapes by up-regulating enzyme activity and down-regulating precursors.

Comparative Analysis of Bio- and Chemo-Catalysts for the Synthesis of Flavour Compound Hexanal from Linoleic Acid

Hexanal, hexenal, nonenal and their corresponding alcohols are used as green notes in the fragrance and flavour industry. The production of bio-based hexanal starts from linoleic acid, which can be obtained from sunflower or safflower oil. The biocatalytic process utilizes C13-specific lipoxygenase (LOX) for hydroperoxidation and consecutive splitting with hydroperoxide lyase (HPL). In this study, we investigated the chemical splitting of the LOX product 13-HPODE in comparison to HPL catalysis. In addition, 13-HPODE was synthesized using enriched linoleic acid from safflower oil. Varying amounts of soybean flour suspension as a source of LOX yielded up to 60% HPODE with a regioselectivity of 92% towards 13-HPODE. Using low-toxicity Lewis acids like AlCl3 and ZrCl4, cleavage of the produced 13-HPODE was possible. A maximum hexanal yield of 22.9% was reached with AlCl3 under mild reaction conditions, though product degradation was an interfering process. Comparative trials with N-terminal truncated HPL from papaya revealed hexanal recovery within a comparable range. Additionally, we successfully demonstrated the viability of Hock rearrangement of 13-HPODE through heterogeneous catalysts. Notably, Beta zeolite and Montmorillonite K10 exhibited a turnover frequency (TOF) on par with common heterogeneous catalysts employed in industrial processes.

Optimizing the First Step of the Biocatalytic Process for Green Leaf Volatiles Production: Lipase-Catalyzed Hydrolysis of Three Vegetable Oils.

Green leaf volatiles (GLVs), including short chain volatile aldehydes, are widely used in the flavor and food industries because of their fresh aroma. To meet the growing demand for natural GLVs with high added value, the use of biocatalytic processes appears as a relevant application. In such processes, vegetable oils are bioconverted into GLVs. First, the triacylglycerols of the oils are hydrolyzed by a lipase. Then, the free polyunsaturated fatty acids are converted by a lipoxygenase. Finally, volatile C6 or C9 aldehydes and 9- or 12-oxoacids are produced with a hydroperoxide lyase. Optimization of each biocatalytic step must be achieved to consider a scale-up. In this study, three oils (sunflower, hempseed, and linseed oils) and three lipases (Candida rugosa, Pseudomonas fluorescens, and Rhizomucor miehei lipases) have been tested to optimize the first step of the process. The experimental design and response surface methodology (RSM) were used to determine the optimal hydrolysis conditions for each oil. Five factors were considered, i.e., pH, temperature, reaction duration, enzyme load, and oil/aqueous ratio of the reaction mixture. Candida rugosa lipase was selected as the most efficient enzyme to achieve conversion of 96 ± 1.7%, 97.2 ± 3.8%, and 91.8 ± 3.2%, respectively, for sunflower, hempseed, and linseed oils under the defined optimized reaction conditions.

Volatilom und Transkriptom des Speisepilzes Agrocybe aegerita

Mushrooms are part of the human diet since time immemorial, appreciated for their nutritional value and especially for their delicious flavors. Hundreds of volatile organic compounds (VOCs) have been identified in fungi contributing to the unique aroma of each species. Generally, studies on mushroom VOCs are carried out with chopped fruiting bodies of more or less one developmental stage. For determine fungal aromas for assessment of the food quality this procedure might be adequate. Nonetheless, for analysis of the biological role of fungal VOCs in context of inter alia VOC biosynthesis or fungal communication this approach can suffer from drawbacks. First of all, damaging fruiting bodies can lead to VOC artefacts due to cell disruption and the occurrence of unnatural enzymatic reactions. Furthermore, fungal VOC profiles are dynamic, changing with ongoing development. For better understanding of the biological function of fungal VOCs it is therefore helpful to know which volatile patterns are characteristic for a certain developmental stage.Against this background, an approach was developed enabling on one hand the cultivation of fungi during different developmental stages, including the growth of fruiting bodies, and on the other hand the non‐invasive analysis of VOCs in the headspace (HS) of fungal cultures. These requirements were complied with modified crystallizing dishes for culture purposes and a HS‐ SPME‐GC‐MS approach to analyze the VOCs. This method was applied to analyze the volatilomes of the dikaryotic strain C. aegerita AAE‐3 and four monokaryotic offspring siblings with different fruiting phenotypes throughout ten life stages. At early stages, in the HS of all tested strains alcohols and ketones, such as oct‐1‐en‐3‐ol, 2‐methylbutan‐1‐ol and cyclopentanone, were the most prominent VOCs. Particularly counting for the dikaryon, the volatilome altered with continued fruiting body development exhibiting remarkable changes during sporulation. Here, sesquiterpenes, especially Δ6‐protoilludene, α‐cubebene and δ‐cadinene, were the most abundant VOCs in the HS of C. aegerita AAE‐3. After sporulation, the amount of sesquiterpenes decreased along with the appearance of other VOCs including octan‐3‐one. In contrast, less VOCs were present in the HS of the monokaryotic strains of which all were as well detectable in the HS of the dikaryon. The changes of the volatilome were the fundament for a subsequent transcriptome analysis aiming to identify enzymes involved in fungal VOC biosynthesis, especially regarding C8 VOC formation, which is, despite the fact that these substances are ubiquitous found in fungi, still barely understood. The transcriptomic study was carried out with seven developmental stages of C. aegerita AAE‐3, which during the volatilome study exhibited interesting volatile patterns. Additionally, fruiting bodies (five stages) and mycelia (seven stages) samples were harvested separately to get further insights about the putative origin of the VOCs observed in the HS of C. aegerita. Combining transcriptome and volatilome data, enzymes putatively involved in the biosynthesis of C8 oxylipins in C. aegerita including lipoxygenases (LOXs), dioxygenases (DOXs), hydroperoxide lyases (HPLs), alcohol dehydrogenases (ADHs) and ene‐ reductases could be identified. Especially the putative DOX AAE3_13098, the putative HPLs AAE3_05330 and AAE3_09203, the putative ADHs AAE3_00054 and AAE3_06559 as well as the putative ene‐reductase AAE3_15349 exhibit remarkable transcriptomic patterns making these enzymes highly interesting for future characterization studies. Furthermore, the study showed that the mycelium is probably the main source for sesquiterpenes observed during sporulation in the HS of C. aegerita AAE‐3 cultures whereas changes in the Cs profile detected in late stages of development are probably due to the activity of enzymes located in the fruiting bodies.

Oxylipin biosynthetic gene families of Cannabis sativa.

Cannabis sativa is a global multi-billion-dollar cash crop with numerous industrial uses, including in medicine and recreation where its value is largely owed to the production of pharmacological and psychoactive metabolites known as cannabinoids. Often underappreciated in this role, the lipoxygenase (LOX)-derived green leaf volatiles (GLVs), also known as the scent of cut grass, are the hypothetical origin of hexanoic acid, the initial substrate for cannabinoid biosynthesis. The LOX pathway is best known as the primary source of plant oxylipins, molecules analogous to the eicosanoids from mammalian systems. These molecules are a group of chemically and functionally diverse fatty acid-derived signals that govern nearly all biological processes including plant defense and development. The interaction between oxylipin and cannabinoid biosynthetic pathways remains to be explored. Despite their unique importance in this crop, there has not been a comprehensive investigation focusing on the genes responsible for oxylipin biosynthesis in any Cannabis species. This study documents the first genome-wide catalogue of the Cannabis sativa oxylipin biosynthetic genes and identified 21 LOX, five allene oxide synthases (AOS), three allene oxide cyclases (AOC), one hydroperoxide lyase (HPL), and five 12-oxo-phytodienoic acid reductases (OPR). Gene collinearity analysis found chromosomal regions containing several isoforms maintained across Cannabis, Arabidopsis, and tomato. Promoter, expression, weighted co-expression genetic network, and functional enrichment analysis provide evidence of tissue- and cultivar-specific transcription and roles for distinct isoforms in oxylipin and cannabinoid biosynthesis. This knowledge facilitates future targeted approaches towards Cannabis crop improvement and for the manipulation of cannabinoid metabolism.