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Light serves as one of the major environmental signals that stimulates anthocyanin biosynthesis in higher plants. In this study, we demonstrate that AtGLK2, an Arabidopsis nuclear GARP transcription factor, plays a positive role in the regulation of anthocyanin biosynthesis. The disruption of AtGLK2 decreased the anthocyanin contents in Arabidopsis seedlings, whereas the overexpression of AtGLK2 resulted in significant enhancements to the accumulation of anthocyanin. Gene expression analysis demonstrated that the expression of late biosynthetic genes in the anthocyanin biosynthetic pathway was dramatically reduced in glk2 but elevated in AtGLK2-overexpressing seedlings. It was found that the disruption of AtHY5 decreased the expression of AtGLK2 and that AtGLK2 exhibited light-responsive expression patterns in an AtHY5-dependent manner. Further studies demonstrated that AtHY5-mediated light signaling is required for AtGLK2-regulated anthocyanin accumulation and photosynthetic development. Moreover, AtGLK2 and AtMYBL2 antagonistically regulate the expression of anthocyanin biosynthetic genes and the subsequent anthocyanin accumulation process. Taken together, our data reveal that AtGLK2 positively regulates anthocyanin biosynthesis through AtHY5-mediated light signaling in Arabidopsis.

The advent of synthetic biology has yielded fruitful studies on orsellinic acid-derived meroterpenoids, which reportedly possess important biological activities. Genomics and transcriptomics have significantly accelerated the discovery of the biosynthetic genes for orsellinic acid-derived fungal and plant meroterpenoids. Subsequently, a well-developed heterologous host provides a convenient platform to generate a supply of useful natural products. Furthermore, in vitro reconstitution and genome editing tools have been increasingly employed as efficient means to fully understand the enzyme reaction mechanisms. With the knowledge of the biosynthetic machinery, combinatorial and engineered biosyntheses have yielded novel molecules with improved bioactivities. These studies will lay the foundation for the production of meroterpenoids with novel medicinal properties.

The Lrp and MarR families are two groups of transcriptional regulators widely distributed among prokaryotes. However, the hierarchical-regulatory relationship between the Lrp family and the MarR family remains unknown. Our previous study found that an Lrp (SACE_Lrp) from Saccharopolyspora erythraea indirectly repressed the biosynthesis of erythromycin. In this study, we characterized a novel MarR family protein (SACE_6745) from S. erythraea, which is controlled by SACE_Lrp and plays a direct regulatory role in erythromycin biosynthesis and export. SACE_Lrp directly regulated the expression of marR by specifically binding a precise site OM (5'-CTCCGGGAACCATT-3'). Gene disruption of marR increased the production of erythromycin by 45% in S. erythraea A226. We found that MarR has direct DNA-binding activity for the promoter regions of the erythromycin biosynthetic genes, as well as an ABC exporter SACE_2701-2702 which was genetically proved to be responsible for erythromycin efflux. Disruption of SACE_Lrp in industrial S. erythraea WB was an efficient strategy to enhance erythromycin production. Herein, we jointly engineered SACE_Lrp and its target MarR by deleting marR in WBΔSACE_Lrp, resulting in 20% increase in erythromycin yield in mutant WBΔLrpΔmarR compared to WBΔSACE_Lrp, and 39% to WB. Overall, our findings provide new insights into the hierarchical-regulatory relationship of Lrp and MarR proteins and new avenues for coordinating antibiotic biosynthesis and export by joint engineering regulators in actinomycetes. KEY POINTS: • The hierarchical-regulatory relationship between SACE_Lrp and MarR was identified. • MarR directly controlled the expression of erythromycin biosynthesis and export genes. • Joint engineering of SACE_Lrp-MarR regulatory element enhanced erythromycin production.

It has been proposed that biosyntheses of many natural products involve pericyclic reactions, including Diels–Alder (DA) reaction. However, only a small set of enzymes have been proposed to catalyze pericyclic reactions. Most surprisingly, there has been no formal identification of natural enzymes that can be defined to catalyze DA reactions (DAases), despite the wide application of the reaction in chemical syntheses of complex organic compounds. However, recent studies began to accumulate a growing body of evidence that supports the notion that enzymes that formally catalyze DA reactions, in fact exist. In this review, I will begin by describing a short history behind the discovery and characterization of macrophomate synthase, one of the earliest enzymes that was proposed to catalyze an intermolecular DA reaction during the biosynthesis of a substituted benzoic acid in a phytopathogenic fungus Macrophoma commelinae. Then, I will discuss representative enzymes that have been chemically authenticated to catalyze DA reactions, with emphasis on more recent discoveries of DAases involved mainly in fungal secondary metabolite biosynthesis except for one example from a marine streptomycete. The current success in identification of a series of DAases and enzymes that catalyze other pericyclic reactions owes to the combined efforts from both the experimental and theoretical approaches in discovering natural products. Such efforts typically involve identifying the chemical features derived from cycloaddition reactions, isolating the biosynthetic genes that encode enzymes that generate such chemical features and deciphering the reaction mechanisms for the enzyme-catalyzed pericyclic reactions.

Here, we identified sequence variations of CYP72A69 and UGT73F4 genes from the Sojeongja and Haepum cultivars, and revealed that accumulation patterns of soyasaponin Aa and Ab were different in the Sojeongja and Haepum cultivars. Several genes related in soyasaponin biosynthetic pathway showed significant changes in their expression patterns in two soybean cultivars and soyasaponin contents were also altered under several abiotic stress conditions, albeit no significant correlations between soyasaponin contents and biosynthetic gene expression were observed. We also demonstrated that UGT73F4 gene could be responsible for biosynthesis of soyasaponin Aa in the Socheongja by investigating gene expression and soyasaponin accumulations. Our results provide a molecular basis to improve the commercial properties of soyasaponin in Korean domestic soybean cultivars.

BACKGROUND: Anthocyanins, a major flavonoid class, determine the color and quality of wine. Recent research revealed that basal leaf removal can increase the content of these compounds. This study determined the effects of basal leaf removal on the anthocyanin profiles of Cabernet Sauvignon grapes. RESULTS: The effects of basal leaf removal on anthocyanin composition in Cabernet Sauvignon grapes were investigated over two growing seasons. Leaf removal at 5% veraison (VB6) and at 100% veraison (VC6) was compared with a control. Reducing sugar and total anthocyanin contents in the leaf removal group were significantly higher than in the control group at harvest for both vintages. Leaf removal increased the content of individual anthocyanins and significantly improved the malvidin‐3‐O‐glucoside (Mv‐3‐glc), peonidin‐3‐O‐glucoside (Pn‐3‐glc), and malvidin‐3‐O‐(6‐acetyl)‐glucoside (Mv‐3‐acglc) content of the VB6 group. At harvest, VB6 treatment increased the transcript abundance of structural and regulator genes in the anthocyanin pathway, especially VvF3′5′H, VvLDOX, and VvDFR. CONCLUSIONS: Our results suggest that leaf removal at 5% veraison may be useful for improving the anthocyanin content in grapes. © 2020 Society of Chemical Industry

While exploring phytotoxic metabolites from phytopathogenic fungi in the 1970s, we became interested in biosynthetic enzymes that catalyze Diels-Alder reactions involving biosynthesis of several phytotoxins that we isolated. Target enzymes were successfully characterized, and this triggered the identification of various Diels-Alderases in a recent decade. Through our Diels-Alderase project in 1990s, we recognized a highly efficient expression system of various biosynthetic genes with Aspergillus oryzae as a host. With the development of tools such as genomic data and bioinformatics analysis to identify biosynthetic gene clusters for natural products, we developed a highly reliable methodology such as hot spot knock-in to elucidate the biosynthetic pathways of representative fungal metabolites including phytotoxic substances. This methodology allows total biosynthesis of natural products and genome mining using silent biosynthetic gene clusters to obtain novel bioactive metabolites. Further applications of this technology are discussed.

Penicillium verrucosum survives in soil and on cereal debris. It colonises grain during harvesting, drying and storage. There is no information on the relative tolerance of P. verrucosum to solute and matric stress in terms of colonisation, or on the biosynthetic toxin gene clusters or ochratoxin A (OTA) production. The objectives were to examine the effect of ionic and non-ionic solute and matric potential stress on (a) growth, (b) expression of two toxin biosynthetic genes otapksPV and otanrpsPV, and (c) OTA production by a strain of P. verrucosum. Optimum growth and OTA production were at -7.0 MPa (= 0.95 water activity, aw) and -1.4 MPa (= 0.99 aw), respectively, regardless of whether solute (Ψs) or matric (Ψm) stress was imposed. P. verrucosum was more sensitive to ionic solute stress (NaCl) with no growth at -19.6 MPa (=0.86 aw) while growth still occurred in the non-ionic solute (glycerol) and matric stress treatments. Relative gene expression of the biosynthetic genes using PCR (RT-qPCR) showed that the otapksPV gene was expressed over a wide range of ionic/non-ionic solute stress conditions (-1.4 to -14.0 MPa; = 0.99-0.90 aw). The highest expression was in the non-ionic Ψs stress treatments at -7.0 MPa (= 0.95 aw). However, the otanrpsPV gene was significantly up regulated under Ψm stress, especially with freely available water (-1.4 MPa = 0.99 aw). OTA production was significantly decreased as Ψs or Ψm stress were imposed. Limited OTA production occurred in the driest treatments under Ψs and Ψm stress respectively. The impact of these two types of stresses on the growth of P. verrucosum was quite different from that for OTA production. The results are discussed in the context of the life cycle and ecological characteristics of this species in contaminating cereals with OTA in the post-harvest phase of the cereal chain.

Anthocyanins confer the wide range of colors for plants and also play beneficial health roles as potentially protective factors against heart disease and cancer. Brassica juncea is cultivated as an edible oil resource and vegetable crop worldwide, thus elucidating the anthocyanin biosynthetic pathway would be helpful to improve the nutritional quality of Brassica juncea through the breeding and cultivating of high anthocyanin content varieties. Herein, 129 genes in B. juncea were identified as orthologs of 41 anthocyanin biosynthetic genes (ABGs) in Arabidopsis thaliana by comparative genomic analyses. The B. juncea ABGs have expanded by whole genome triplication and subsequent allopolyploidizatoin, but lost mainly during the whole genome triplication between B. rapa/B. nigra and A. thaliana, rather than the allopolyploidization process between B. juncea and B. rapa/B. nigra, leading to different copy numbers retention of A. thaliana homologous genes. Although the overall expansion levels ABGs were similar to the whole genome, more negative regulatory genes were retained in the anthocyanin biosynthesis regulatory system. Transcriptional analysis of B. juncea with different anthocyanin accumulation showed that BjDFR, BjTT19, BjTT8 are significantly up-regulated in plants with purple leaves as compared with green leaves. The overexpression of BjTT8 and these target genes which were involved in late anthocyanin biosynthesis and transport might account for increasing levels of anthocyanin accumulation in purple leaves. Our results could promote the understanding of the genetic mechanism of anthocyanin biosynthesis in B. juncea.

Milbemycins and their semisynthetic derivatives are recognized as effective and eco-friendly pesticides, whereas the high price limits their widespread applications in agriculture. One of the pivotal questions is the accumulation of milbemycin-like by-products, which not only reduces the yield of the target products milbemycin A3/A4, but also brings difficulty to the purification. With other analogous by-products abolished, α9/α10 and β-family milbemycins remain to be eliminated. Herein, we solved these issues by engineering of post-modification steps. First, Cyp41, a CYP268 family cytochrome P450, was identified to participate in α9/α10 biosynthesis. By deleting cyp41, milbemycin α9/α10 was eliminated with an increase of milbemycin A3/A4 titer from 2382.5 ± 55.7 mg/L to 2625.6 ± 64.5 mg/L. Then, MilE, a CYP171 family cytochrome P450, was determined to be responsible for the generation of the furan ring between C6 and C8a of milbemycins. By further overexpression of milE, the production of β-family milbemycins was reduced by 77.2%. Finally, the titer of milbemycin A3/A4 was increased by 53.1% to 3646.9 ± 69.9 mg/L. Interestingly, overexpression of milE resulted in increased transcriptional levels of milbemycin biosynthetic genes and production of total milbemycins, which implied that the insufficient function of MilE was a limiting factor to milbemycin biosynthesis. Our research not only provides an efficient engineering strategy to improve the production of a commercially important product milbemycins, but also offers the clues for future study about transcriptional regulation of milbemycin biosynthesis.

In the course of our studies on the heterologous expression of giant biosynthetic genes, we discovered a novel cryptic biosynthetic gene cluster in Streptomyces rochei IFO12908. During our efforts to express biosynthetic genes using the host SUKA strain derived from Streptomyces avermitilis, a novel polyene macrolactam compound designated as JBIR-156 was produced. We report herein the cloning and heterologous expression of the JBIR-156 biosynthetic gene cluster, and the isolation, structure determination, and cytotoxic activity of this novel compound.

Advances in next-generation DNA sequencing technologies, bioinformatics, and mass spectrometry-based metabolite detection have ushered in a new era of natural product discovery. Microbial secondary metabolomes are complex, especially when otherwise silent biosynthetic genes are activated, and there is therefore a need for data analysis software to explore and map the resulting multidimensional datasets. To that end, we herein report the Metabolomics Explorer (MetEx), a publicly available web application for the analysis of parallel liquid chromatography-coupled mass spectrometry (LC-MS)-based metabolomics data. MetEx is a highly interactive application that facilitates visualization and analysis of complex metabolomics datasets, consisting of retention time, m/z, and MS intensity features, as a function of hundreds of conditions or elicitors. The software enables prioritization of leads from three-dimensional maps, extraction of two-dimensional slices from various higher order plots, organization of datasets by elicitor chemotypes, customizable library-based dereplication, and automatically scored lead selection. We describe the application of MetEx to the first UPLC-MS-guided high-throughput elicitor screen in which Burkholderia gladioli was challenged with 750 elicitors, and the resulting profiles were interrogated by UPLC-Qtof-MS and subsequently analyzed with the app. We demonstrate the utility of MetEx by reporting elicitors for several cryptic metabolite groups and by uncovering new natural products that remain to be characterized. MetEx is available at https://mo.princeton.edu/MetEx/.

Heparin is an essential anticoagulant drug discovered over a century ago. Heparin is the second most highly used natural drug and remains a mainstay of therapy with an expected global market share of more than $14 billion in the next 10 years. However, it is still naturally derived from unsustainable animal sources, such as bovine lungs and porcine intestines, as an unfractionated, heterogeneous complex mixture with unpredictable pharmacokinetic properties. Extensive research has been done in devising bioengineering and chemical approaches to produce structurally specific heparin and heparin-like polymers. Though several challenges remain, one of the main bottlenecks is the rapid, high-yield production of recombinant heparosan, a heparin precursor, which is originally isolated from a pathogenic E. coli K5 strain. Herein, we outline the methods for producing metabolically engineered size-specific heparosan, by transforming the essential heparosan biosynthetic genes into nonpathogenic E.coli strain BL21(DE3), in a highly controlled manner. The methods described herein are promising and can be easily scaled up for large-scale production of heparin-like structures.

Environmental microorganisms continue to serve as a major source of bioactive natural products (NPs) and as an inspiration for many other scaffolds in the toolbox of modern medicine. Nearly all microbial NP-inspired therapies can be traced to field expeditions to collect samples from the environment. Despite the importance of these expeditions in the search for new drugs, few studies have attempted to document the extent to which NPs or their corresponding production genes are distributed within a given environment. To gain insights into this, the geographic occurrence of NP ketosynthase (KS) and adenylation (A) domains was documented across 53 and 58 surface sediment samples, respectively, covering 59,590 square kilometers of Lake Huron. Overall, no discernible NP geographic distribution patterns were observed for 90,528 NP classes of nonribosomal peptides and polyketides detected in the survey. While each sampling location harbored a similar number of A domain operational biosynthetic units (OBUs), a limited overlap of OBU type was observed, suggesting that at the sequencing depth used in this study, no single location served as a NP "hotspot". These data support the hypothesis that there is ample variation in NP occurrence between sampling sites and suggest that extensive sample collection efforts are required to fully capture the functional chemical diversity of sediment microbial communities on a regional scale.

Bacterial natural product biosynthetic genes, canonically clustered, have been increasingly found to rely on cryptic enzymes encoded elsewhere in the genome for completion of biosynthesis. The study and application of lanthipeptides are frequently hindered by cryptic proteases required for final maturation. Here, we establish a global correlation network bridging the gap between lanthipeptide precursors and cryptic proteases. Applying our analysis to 161,954 bacterial genomes, we establish 5,209 correlations between precursors and cryptic proteases, with 91 prioritized. We use network predictions and co-expression analysis to reveal a previously missing protease for the maturation of class I lanthipeptide paenilan. We further discover widely distributed bacterial M16B metallopeptidases of previously unclear biological function as a new family of lanthipeptide proteases. We show the involvement of a pair of bifunctional M16B proteases in the production of novel class III lanthipeptides with high substrate specificity. Together, these results demonstrate the strength of our correlational networking approach to the discovery of cryptic lanthipeptide proteases and potentially other missing enzymes for natural products biosynthesis.Graphical abstract

Glucosinolates (GSLs) are important secondary metabolites in Brassicales related to insect and disease resistance, flavor formation, and human health. Here, we determined the GSL profile with sinigrin as the predominant GSL in Brassica nigra. A total of 184 GSL biosynthetic genes (BniGSLs) were identified in B. nigra by a genome-wide search for orthologs of 82 of the 95 known GSL genes in Arabidopsis thaliana. Transcriptome data demonstrated that at least one BniGSL was highly expressed in stems and leaves at each step of the sinigrin synthesis pathway, which ensured the synthesis of a large amount of sinigrin in B. nigra. Among these key candidates of BniGSLs, the high expression of BniMAM1-2, BniCYP79F1, and BniAOP2-1/2, and the absence of MAM3 and AOP3, may contribute remarkably to the synthesis and accumulation of sinigrin. In addition, the low expression of some key BniGSLs partially explains the low content of indolic and aromatic GSLs in B. nigra. This study provided a genetic explanation for the formation of the unique GSL profile with sinigrin as the main GSL in B. nigra. The results of this study will be valuable for further functional analysis of BniGSLs and genetic improvement of GSLs in B. nigra and other Brassica species.

Aberrant splicing of pre-mRNA is implicated in many human genetic disorders. Small molecules that target the spliceosome are important leads as therapeutics and research tools, and one compound of significant interest is the polyketide natural product pladienolide B. Here, we describe the reactivation of quiescent pladienolide B production in the domesticated lab strain Streptomyces platensis AS6200 by overexpression of the pathway-specific activator PldR. The resulting dysregulation of the biosynthetic genes led to the accumulation and isolation of five additional intermediate or shunt metabolites of pladienolide B biosynthesis, including three previously unreported congeners. These compounds likely comprise the entire pladienolide biosynthetic pathway and demonstrate the link between polyketide tailoring reactions and bioactivity, particularly the importance of the 18,19-epoxide. Each congener demonstrated specific inhibitory activity against mammalian cell lines, with successive modifications leading to increased activity (IC50: 8 mM to 5 μM)., Reactivation of quiescent polyketide production in a domesticated lab strain.

Soybean seeds are consumed worldwide owing to their nutritional value and health benefits. In this study we investigated the metabolic properties of 208 soybean mutant diversity pool (MDP) lines by measuring the isoflavone and fatty acid contents of the seed. The total isoflavone content (TIC) ranged from 0.88 mg/g to 7.12 mg/g and averaged 3.08 mg/g. The proportion of oleic acid among total fatty acids (TFA) ranged from 0.38% to 24.66% and averaged 11.02%. Based on the TIC and TFA among the 208 MDP lines, we selected six lines with altered isoflavone content and six lines with altered oleic acid content compared with those of the corresponding wild-types for measuring gene expression. Each of twelve genes from the isoflavone and fatty acid biosynthesis pathways were analyzed at three different seed developmental stages. Isoflavone biosynthetic genes, including CHI1A, IFS1, and IFS2, showed differences in stages and expression patterns among individuals and wild-types, whereas MaT7 showed consistently higher expression levels in three mutants with increased isoflavone content at stage 1. Expression patterns of the 12 fatty acid biosynthetic genes were classifiable into two groups that reflected the developmental stages of the seeds. The results will be useful for functional analysis of the regulatory genes involved in the isoflavone and fatty acid biosynthetic pathways in soybean.

Natural products have drastically improved our lives by providing an excellent source of molecules to fight cancer, pathogens, and cardiovascular diseases that have revolutionized medicine. Fungi are prolific producers of diverse natural products and several recent advances in synthetic biology, genetics, bioinformatics, and natural product chemistry have greatly enhanced our ability to efficiently mine their genomes for the discovery of novel drugs. In this article, we provide an overview of improved heterologous expression platforms for targeted production of fungal secondary metabolites, of advances in chemical and bioinformatics dereplication, and of novel bioinformatic platforms to discover biosynthetic genes involved in the production of metabolites with specific bioactivities. These advances, coupled with the presence of vast numbers of biosynthetic gene clusters in fungal genomes whose natural products remain unknown, have revitalized efforts to mine the fungal treasure chest and renewed the promise of discovering new drugs.

Cherry tomatoes are highly nutritious, flavory with a pleasant taste and are becoming increasingly popular to the consumers. The cherry tomato cv. ‘Minichal’ produced some dwarf plants along with normal plants. Lignin, a phenolic biopolymer is the key component of cell walls in plants. Here, we analyzed lignin biosynthesis-related genes in leaves, inflorescences and fruits of dwarf and normal cherry tomato plants by reverse-transcription quantitative PCR (RT-qPCR). Among analyzed genes, SlCCOAOMT1, SlCCOAOMT2, SlCCOAOMT3, SlF5H, and SlCOMT showed significantly higher expressions, in leaf and inflorescence of dwarf plants compared with the normal plants, while SlPAL1 showed a significantly higher expression only in the leaves. On the contrary, SlHCT and SlC3H showed significantly lower expression levels in the leaves and inflorescences of dwarf plants compared with normal ones. The results suggest that SlHCT and SlC3H might have an impact on the dwarf cherry tomato plants.

Improving the quality of life and human health has a timely demand for the ample supply of fruits with high quality. Apple is one of the most consumed fruit in daily life. Facilitating the coloration of the apple with a simple and available method is important to their timely supply. We propose for the first time that utilizing photocatalytic nanoparticles facilitates the coloration of apple. The photocatalytic nanoparticles with the size of ∼18 nm can effectively promote the utilization of ultraviolet irradiation of the apple skin. The photocatalytic nanoparticles treatment remarkably upregulates the expression of the anthocyanin biosynthetic genes in apple skin, in which MdDFR is even more than 26 times that of the control fruits. The area and color examination indicates that the coloration efficiency of photocatalytic treated apples is as twice that of the control samples. This work provides an available nanomaterial to accelerate the coloration of fruit skin and proposes a new application of photocata...

In Nigeria and many other developing countries of the world, the incidence of mycotoxin- contamination of foods and food products has attracted attention and stirred a lot of concern for food safety. This work aims at detection of aflatoxigenic and ochratoxigenic synthetic genes from fungal isolates of palm oil as a veritable means for the evaluation of foods for possible mycotoxin contamination. In this study, fungal isolates from palm oil samples collected from the five states of South-east geopolitical zone in Nigeria were screened for aflatoxin and ochratoxin biosynthetic genes using Multiplex Polymerase Chain Reaction (mPCR). The assay relied on three sets of primers that amplify aflatoxgenic Aspergillus, ochratoxigenic Aspergillus and Penicillium species under optimized PCR conditions. Optimum multiplex PCR assay was standardized for simultaneous detection of toxigenic Aspergillus and ochratoxin producing Penicillium species targeting AflR, AflS and pks genes involved in aflatoxin and ochratoxin metabolic pathways respectively. AflR primer pair gave specific amplification for aflatoxigenic A. flavus but did not give amplification for A. niger and P. chrysogenum. While AflS and pks gave amplification for only aflatoxigenic and ochratoxigenic A. niger and P. chrysogenum. In the evaluation and monitoring of mycotoxin-producing fungi during the processing of food and feed commodities, Multiplex PCR approach could be a veritable tool to supplement the conventional analytical techniques.

The objectives of this study were to investigate the impact that interactions between key climate change (CC) related environmental factors of temperature (20, 25, 30°C), water activity (aw; 0.995, 0.98) and CO2 exposure (400, 1000 ppm) may have on (a) growth, (b) gene expression of biosynthetic toxin genes (Tri5, Tri6, Tri16), and (c) phenotypic T-2/HT-2 production by Fusarium langsethiae on oat-based agar medium and in stored oats. Fungal growth was optimum at 25°C and 0.995 aw and reduced significantly at 30°C and intermediate stress (0.98 aw, elevated CO2 (1000 ppm) exposure by approx. 4-fold. Lag phases prior to growth paralleled these results with the longest lag phase in this treatment (24 hrs). On oat-based medium, the relative Tri5 gene expression was increased in elevated CO2 conditions. The expression of both the Tri6 and Tri16 genes was reduced when compared to control (20°C, 0.995 aw, 400 ppm), especially in elevated CO2 conditions. In stored oats, the Tri5 gene expression was reduced in all conditions except at 30°C, 0.98 aw, elevated CO2 where there was a significant (5.3-fold) increase. The expression of the Tri6 was slightly over-expressed in elevated CO2 and the Tri16 gene was upregulated, especially in elevated CO2 conditions. For mycotoxin production, both on oat-based medium and in stored oats the production was higher at 25°C when compared to 30°C. In stored oats, at 0.98 aw, elevated CO2 led to higher T2/HT-2 toxin production at both 25 and 30°C with a significant increase (73-fold higher) at 30°C. In elevated CO2 conditions, Tri16 (Spearman test; 0.68; p-value=0.0019) and Tri5 gene expression (Spearman test; 0.56; p-value=0.0151) were correlated with T-2+HT-2 production. Nine T-2 and HT-2 metabolites were detected by LC-MS/MS including a new dehydro T-2 toxin and the conjugate, HT-2 toxin glucuronide (in plantae). The new dehydro T-2 toxin was the most abundant metabolites and showed correlation (R2=0.8176) with T-2 production. This is the first study to examine the impact of CC factors on growth and mycotoxin production by a strain of F. langsethiae. The influence of such scenarios on relative risk of oats contamination with these toxins in relation to the food security agenda is discussed.

Abscisic acid (ABA) is one of the main participants in the regulation of plant responses to water deficiency. Knowledge of the ABA signal transduction pathways in gymnosperms is rather limited, especially in comparison with those in angiosperms. Seedlings of Scots pine and Norway spruce are known for their contrasting behaviour strategies under water deficit. To characterize the possible role of ABA in these differences, ABA dynamics were investigated under conditions of water deficit in seedlings of these two species. The content of ABA and its catabolites was followed in the roots and needles of seedlings of Pinus sylvestris and Picea abies under conditions of polyethylene glycol (PEG)-induced water deficiency (−0.15 and −0.5 MPa) for 10 days. The expression of the main genes for ABA-biosynthetic enzymes was also analysed. ABA showed more pronounced stress-dependent dynamics in pine roots than in spruce roots, whereas in needles, the response was greater for spruce than pine. The ABA increase during drought was mainly due to de novo synthesis and the shift in the balance between ABA synthesis and catabolism towards synthesis. The ABA-glucosyl ester did not serve as a reserve for the release of free ABA under water deficiency. The expression levels of the main ABA biosynthetic genes showed a weak or no correlation with changes in ABA content under water stress, i.e., the ABA content in the seedlings of both species was not directly linked to the transcript levels of the main ABA biosynthetic genes. Less-pronounced stress-induced changes in ABA in pine needles than in spruce needles may be related to pine seedlings having a less conservative strategy of growth and maintenance of water balance under water deficit.

Fungi from the order Onygenales include human pathogens. Although secondary metabolites are critical for pathogenic interactions, relatively little is known about Onygenales compounds. Here, we use chemical and genetic methods on Aioliomyces pyridodomos, the first representative of a candidate new family within Onygenales. We isolated 14 new bioactive metabolites, nine of which are first disclosed here. Thirty-two specialized metabolite biosynthetic gene clusters (BGCs) were identified. BGCs were correlated to some of the new compounds by heterologous expression of biosynthetic genes. Some of the compounds were found after one year of fermentation. By comparing BGCs from A. pyridodomos with those from 68 previously sequenced Onygenales fungi, we delineate a large biosynthetic potential. Most of these biosynthetic pathways are specific to Onygenales fungi and have not been found elsewhere. Family level specificity and conservation of biosynthetic gene content are evident within Onygenales. Identification of these compounds may be important to understanding pathogenic interactions.

With more than 50,000 members, terpenoids are one of the most important classes of natural products and show an enormous diversity. Due to their unique odors and specific bioactivities they already find wide application in the flavor, fragrance and pharma industries. Since most terpenoids can only be obtained by natural product extraction, the discovery of biosynthetic genes for the generation of terpene diversity becomes increasingly important. This study describes the discovery of three novel sesquiterpene synthases from Streptomyces chartreusis with preference for the formation of germacradiene-11-ol, α-eudesmol and α-amorphene respectively. The α-eudesmol synthase showed formation of 10-epi-δ-eudesmol and elemol as side products. Eudesmol-isomers are known to have repellent activity, which makes this enzyme a potential catalyst for products for the prevention of mosquito-related disease. The determination of the structure of the apo-enzyme of α-eudesmol synthase from S. chartreusis provides the first structural insights into an eudesmol-forming enzyme.

Wild and cultivated rice show a significant difference in anthocyanin biosynthesis in the leaf. The regulation system of anthocyanin biosynthesis in rice leaf and the causal mechanism of the difference in this biosynthesis between wild and cultivated rice remain largely unknown. In this study, a genome-wide association study and transcriptome analysis were performed to identify the determinant factors and dissect the regulatory system for anthocyanin biosynthesis in rice leaves. OsC1, OsRb and OsDFR were identified as the determinants of anthocyanin biosynthesis in rice leaves. Artificial selection of certain null mutations of OsC1 and OsRb was the main causal mechanism underlying the loss of anthocyanin pigmentation in most cultivated rice. OsP1 and the MYB-bHLH-WD40 complexes regulate anthocyanin biosynthetic genes in rice leaves with partial functional overlap. OsP1 specifically activates upstream biosynthetic genes (OsCHS, OsCHI and OsF3'H) for anthocyanin biosynthesis, whereas the ternary MYB-bHLH-WD40 complex activates all anthocyanin biosynthetic genes including OsCHS, OsCHI, OsF3'H, OsF3H, OsDFR and OsANS. OsC1 and OsRb are tissue-specific regulators that do not influence anthocyanin biosynthesis in the pericarp. Our results reveal the determinant factors, regulatory system and domestication of anthocyanin biosynthesis in rice leaves, and show the potential of engineering anthocyanin biosynthesis in rice.

Nuts, such as peanut, almond, and chestnut, are valuable food crops for humans being important sources of fatty acids, vitamins, minerals, and polyphenols. Polyphenols, such as flavonoids, stilbenoids, and hydroxycinnamates, represent a group of plant-specialized (secondary) metabolites which are characterized as health-beneficial antioxidants within the human diet as well as physiological stress protectants within the plant. In food chemistry research, a multitude of polyphenols contained in culinary nuts have been studied leading to the identification of their chemical properties and bioactivities. Although functional elucidation of the biosynthetic genes of polyphenols in nut species is crucially important for crop improvement in the creation of higher-quality nuts and stress-tolerant cultivars, the chemical diversity of nut polyphenols and the key biosynthetic genes responsible for their production are still largely uncharacterized. However, current technical advances in whole-genome sequencing have facilitated that nut plant species became model plants for omics-based approaches. Here, we review the chemical diversity of seed polyphenols in majorly consumed nut species coupled to insights into their biological activities. Furthermore, we present an example of the annotation of key genes involved in polyphenolic biosynthesis in peanut using comparative genomics as a case study outlining how we are approaching omics-based approaches of the nut plant species.

In the backdrop of an abundance of lignin in jute, the main focus of the present study was to conduct a quality assessment of four delignified jute lines (in which four lignin biosynthetic genes were individually downregulated) across advanced generations for industrial applications. To this end, the transgenic lines were advanced to 7th (COMT and C4H lines) and 5th (C3H and F5H lines) transformed generations. The results exhibit approximately 16–25% reduction in acid-insoluble lignin for the whole stem and 13–14% reduction in fiber lignin content for all four transgenic lines compared to the control. The altered lignin composition led to a 3–6% increase in the cellulose content and a small improvement in the enzymatic release of glucose. Lignin reduction led to an exposure of the underlying fibrils in transgenic lines as observed through a scanning electron microscope whereas, it was undiscernible in the control fiber. Furthermore, an analysis of the mechanical properties appears almost similar to that of the control with no morphological deformities. Jute fibers from the transgenic lines offer tremendous cost-effective implications from an economic perspective., Jute, lignocellulose, lignin biosynthetic genes, tensile strength, jute fiber.

Covering: up to September 2020 Mushroom-forming fungi of the division Basidiomycota have traditionally been recognised as prolific producers of structurally diverse and often bioactive secondary metabolites, using the methods of chemistry for research. Over the past decade, -omics technologies were applied on these fungi, and sophisticated heterologous gene expression platforms emerged, which have boosted research into the genetic and biochemical basis of the biosyntheses. This review provides an overview on experimentally confirmed natural product biosyntheses of basidiomycete polyketides, amino acid-derived products, terpenoids, and volatiles. We also present challenges and solutions particular to natural product research with these fungi. 222 references are cited.

Summary We demonstrate the production of cholesterol by Enhygromyxa salina, a heterotrophic marine bacterium. Cholesterol is a highly modified sterol that is preserved as cholestane in the rock record. Cholestane is often indicative of heterotrophic microbial eukaryotes. The production of cholesterol by E. salina is the first reported instance of cholesterol synthesis by a bacterium. Through genomic analyses, we show that E. salina has cholesterol biosynthetic genes that are homologous to the eukaryotic cholesterol pathway. However, we do observe some biosynthetic proteins that are unique to E. salina. Further, extensive lipid analyses demonstrate that cholesterol in this organism is conjugated to either a lipid or protein moiety and does not exist as a free sterol. Conjugated sterols play significant physiological roles in eukaryotes and understanding the nature of conjugated cholesterol in E. salina may provide insight into the physiological roles of these lipids in bacteria. Through these studies, we should be able to better assess what implications, if any, cholesterol production by bacteria has on our interpretation of sterol biomarkers in the rock record.

Terrestrial animals, especially insects, contain various carotenoids that show structural diversity. These animals accumulated carotenoids derived from plants and other animals and modified them through metabolic reactions. Therefore, most of the carotenoids found in terrestrial animals originated from plants. Conversely, recent investigation revealed that some species of aphids and spider mites synthesized carotenoid themselves by carotenoid biosynthetic genes, which were horizontally transferred from fungi. In this chapter, carotenoids in terrestrial animals are described from the viewpoints of natural product chemistry, metabolism, food chain, and chemosystematics.

In the recent climate change regime, industrial demand for renewable materials to replace petroleum-derived polymers continues to rise. Of particular interest is polyhydroxybutyrate (PHB) as a substitute for polypropylene. Accumulating evidence indicates that PHB is highly produced as a carbon storage material in various microorganisms. The effects of growth conditions on PHB production have been widely studied in chemolithotrophs, particularly in Rhodobacter. However, the results on PHB production in Rhodobacter have been somewhat inconsistent due to different strains and experimental conditions, and it is currently unclear how diverse environmental factors are linked with PHB production. Here, we report optimized growth conditions for PHB production and show that the growth conditions are closely related to reactive oxygen species (ROS) regulation. PHB accumulates in cells up to approximately 50% at the highest level under dark-aerobic conditions as opposed to light aerobic/anaerobic conditions. According to the time-course, PHB contents increased at 48 h and then gradually decreased. When observing the effect of temperature and medium composition on PHB production, 30 °C and a carbon/nitrogen ratio of 9:1 or more were found to be most effective. Among PHB biosynthetic genes, PhaA and PhaB are highly correlated with PHB production, whereas PhaC and PhaZ showed little change in overall expression levels. We found that, while the amount of hydrogen peroxide in cells under dark conditions was relatively low compared to the light conditions, peroxidase activities and expression levels of antioxidant-related genes were high. These observations suggest optimal culture conditions for growth and PHB production and the importance of ROS-scavenging signaling with regard to PHB production.

Global climate change and the industrial revolution have increased the concentration of tropospheric ozone, a photochemical air pollutant that can negatively affect plant growth and crop production. In the present study, we investigated the effects of O3 on the metabolites and transcripts of tartary buckwheat. A total of 36 metabolites were identified by gas chromatography coupled with time-of-flight mass spectrometry, and principal component analysis was performed to verify the metabolic differences between nontreated and O3-treated tartary buckwheat. The content of threonic acid increased after 2 days of the O3 treatment, whereas it decreased after 4 days of exposure, after which it gradually increased until the eighth day of exposure. In addition, the levels of most metabolites decreased significantly after the O3 treatment. On the contrary, the levels of two anthocyanins, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside, increased more than 11.36- and 11.43-fold, respectively, after the O3 treatment. To assess the effect of O3 on the genomic level, we analyzed the expression of anthocyanin biosynthesis pathway genes in O3-treated and nontreated buckwheat using quantitative real-time reverse transcription polymerase chain reaction (PCR). We found that the expression of all anthocyanin pathway genes increased significantly in the O3-treated buckwheat compared to that in the nontreated buckwheat. Altogether, our results suggested that O3 affected the transcripts and metabolites of tartary buckwheat, which would eventually cause phenotypic changes in plants.

Aspergillus section Circumdati includes 27 species, some of which are considered ochratoxin A (OTA) producers. However, there is considerable controversy about their potential OTA synthesis ability. In this work, the complete genomes of 13 species of Aspergillus section Circumdati were analyzed in order to study the cluster of OTA biosynthetic genes and the region was compared to those previously reported in A. steynii and A. westerdijkiae. The results obtained reveal that the genomes of some species in this section, including A. affinis, A. cretensis, A. elegans, A. muricatus, A. pulvericola, A. roseoglobulosus, and A. subramanianii, contain a potentially functional OTA biosynthetic cluster. Therefore, they might be able to synthesize the toxin. On the contrary, A. melleus, A. ochraceus, A. ostianus, A. persii, A. sclerotiorum, A. sesamicola, and A. westlandensis contain a truncated version of the cluster that lacks many of the genes involved in OTA biosynthesis, which might be related to their inability to produce OTA. The gain/loss pattern is different in all species, which suggests that the genetic evolution of this region might be due to independent events.

Colchicine is among the oldest plant natural products (NPs) still used for treating a broad spectrum of human diseases including gout and other articular inflammation disorders. This molecule is synthesized by several herbaceous species related to the Liliaceae family, but in very low quantities in whole plants. As for many pharmaceutical compounds from plants, the production of colchicine still depends on the natural resource from which it is extracted. From the past decade, metabolic engineering has progressively become a credible alternative for the cost-effective large-scale production of several valuable NPs. In the same vein, Nett and colleagues recently reported an unprecedented advance in the field for colchicine. By using a combination of transcriptomics, metabolomics and pathway reconstitution, Sattely's group deciphered a near-complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes. Besides constituting a benchmark for the elucidation of natural product biosynthetic pathways, it opens unprecedented perspectives regarding metabolic engineering of colchicine biosynthesis.

8-Azaguanine (1) is a special 1,2,3-triazole containing natural product that possesses potent antibacterial and antitumor activities. In the present study, the entire 8-azaguanine biosynthetic gene cluster was located from Streptomyces CGMCC4.1633. Targeted gene disruption, heterologous expression analysis, and feeding experiments identified crucial genes for 8-azaguanine production. Moreover, we characterized the structure of two novel metabolites, analyzed NO (or reactive nitrogen species) related genes 8-azgA/B and radical SAM enzyme homologous 8-AzgG, and verified the non-enzymatic ring formation reaction of 8-azaguanine 1,2,3-triazole. All of the data and presumptions provide insight into the timing and mechanism of the enzymatic and non-enzymatic pathway that produce 8-azaguanine-type 1,2,3-triazole., Graphical Abstract

Indolmycin is an antibiotic from Streptomyces griseus ATCC 12648 with activity against Helicobacter pylori, Plasmodium falciparum, and methicillin-resistant Staphylococcus aureus. Here we describe the use of the indolmycin biosynthetic genes in E. coli to make indolmycenic acid, a chiral intermediate in indolmycin biosynthesis, which can then be converted to indolmycin through a three-step synthesis. To expand indolmycin structural diversity, we introduce a promiscuous tryptophanyl-tRNA synthetase gene (trpS) into our E. coli production system and feed halogenated indoles to generate the corresponding indolmycenic acids, ultimately allowing us to access indolmycin derivatives through synthesis. Bioactivity testing against methicillin-resistant Staphylococcus aureus showed modest antibiotic activity for 5-, 6-, and 7-fluoro-indolmycin., A semi-synthetic system for producing indolmycin, an antibiotic, was developed and used to make indole-substituted, halogenated derivatives of indolmycin, some with modest bioactivity against methicillin-resistant Staphylococcus aureus.

Penicillium verrucosum is responsible for ochratoxin A (OTA) contamination of temperate cereals during harvesting and storage. Inoculum comes from soil and crop debris. This study examined the effect of temperature (25 vs 30 °C), CO2 (400 vs 1000 ppm) and matric and solute stress (-2.8 vs -7.0 MPa) on (i) growth, (ii) key OTA biosynthetic genes and (iii) OTA production on a milled wheat substrate. Growth was generally faster under matric than solute stress at 25 °C, regardless of CO2 concentrations. At 30 °C, growth of P. verrucosum was significantly reduced under solute stress in both CO2 treatments, with no growth observed at -2.8 MPa (=0.98 water activity, aw) and 1000 ppm CO2. Overall, the growth patterns under solute stress was slower in elevated CO2 than under matric stress conditions when compared with existing conditions. The otapksPV gene expression was increased under elevated CO2 levels in matric stress treatments. There was fewer effects on the otanrpsPV biosynthetic gene. This pattern was paralleled with the production of OTA under these conditions. This suggest that P. verrucosum is able to actively grow and survive in both soil and on crop debris under three way interacting climate-related abiotic factors. This resilience suggests that they would still be able to pose an OTA contamination risk in temperate cereals post-harvest.

Ganoderic acids (GAs) produced by Ganoderma are a type of lanostane-type triterpenoids with anticancer and antimetastatic activities; however, low production of GAs limits its wide application. In this study, a novel strategy by promoting sporulation of Ganoderma was developed to increase GA production. First, a high-spore producing Ganoderma strain G. 260125 was obtained from dikaryotic strain CGMCC 5.0026, and the sporulation-specific gene of this strain exhibits a higher transcription level than CGMCC 5.0026. Then, the effect of promoting sporulation on GA content was investigated. The maximum ganoderic acid (GA)-T, GA-Mk, and GA-Me contents in G. 260125 in shake flasks were 358.97, 78.32, and 12.75 μg/100 mg dry weight, respectively, which were 3.42, 2.91, and 1.73 times higher than those obtained in CGMCC 5.0026. Moreover, total and individual GA contents in spores were significantly higher than those in liquid static culture. Both concentrations of intermediates and transcription levels of GA biosynthetic genes also improved in G. 260125 during fermentation compared with those in CGMCC 5.0026. For scaling-up experiments, GA-T, GA-Me, and GA-Mk production in G. 260125 improved by 2.2-, 2.6-, and 2.1-fold compared with those in CGMCC 5.0026. In addition, the effectiveness of the developed strategy was also confirmed in three different Ganoderma strains. This work illustrated that promoting sporulation efficiently improves GA production in liquid static cultures of Ganoderma.

Lindera aggregata(L. aggregata) is a wild shrub growing in the forests of Southeast Asia, whose main bioactive constituents are isoquinoline alkaloids. They are widely used in food and pharmaceutical industries. The studies on the metabolites and biosynthesis pathways inL. aggregata remain poorly understood. Nine isoquinoline alkaloid compounds were identified by UPLC Triple TOF-MS/MS in this study. Except for N-methyllaurotetanine, most isoquinoline alkaloid compounds were widely distributed in various parts ofL. aggregata and accumulated preferentially in roots than in leaves. Transcriptome data showed that several isoquinoline alkaloid biosynthetic genes, such as TyrAT, PPO, TDC, and SOMT, were identified to play important roles in generating differential metabolites in roots and leaves ofL. aggregata. Concentration-dependent analgesic effects and toxic effects of them were demonstrated in zebrafish experiments, and the overall ranking was JRAL > TRAL > LAL. The results of this study would provide useful information for the synthesis mechanisms of isoquinoline alkaloids inL. aggregata, and provide valuable information for the application of traditional non-medicinal parts ofL. aggregata in food and pharmaceutical industries.

Marine microorganisms have drawn great attention as novel bioactive natural product sources, particularly in the drug discovery area. Using different strategies, marine microbes have the ability to produce a wide variety of molecules. One of these strategies is the co-culturing of marine microbes; if two or more microorganisms are aseptically cultured together in a solid or liquid medium in a certain environment, their competition or synergetic relationship can activate the silent biosynthetic genes to produce cryptic natural products which do not exist in monocultures of the partner microbes. In recent years, the co-cultivation strategy of marine microbes has made more novel natural products with various biological activities. This review focuses on the significant and excellent examples covering sources, types, structures and bioactivities of secondary metabolites based on co-cultures of marine-derived microorganisms from 2009 to 2019. A detailed discussion on future prospects and current challenges in the field of co-culture is also provided on behalf of the authors’ own views of development tendencies.

The content of pullulan and melanin in 500 mutants of Aureobasidum pullulans obtained by ultraviolet mutagenesis were examined and statistically analyzed, and a strong positive correlation was found between them. The result was further confirmed by culturing wild type strain As3.3984 in different media. Then we constructed melanin-deletion mutant As-Δalb1 and pullulan-deletion mutant As-Δpul. As-Δalb1 was a melanin-free strain with the yield of pullulan decreased by 41.01%. The supplementation of melanin in the culture of As-Δalb1 increased the production of pullulan. As-Δpul synthesized neither pullulan nor melanin and recovered melanin synthesis by adding pullulan to the medium. The results suggested that high concentration- of pullulan induced morphological transformation and synthesis of melanin, and melanin promoted the synthesis of pullulan. The pullulan biosynthetic genes, upt, pgm, ugp, and pul, were down-regulated, while the negative regulatory gene of pullulan synthesis, creA, was up-regulated by melanin deficiency.

A fluorometabolite, 5-fluoro-5-deoxy-D-ribulose (5-FDRul), from the culture broth of the soil bacterium Streptomyces sp. MA37, was identified through a combination of genetic manipulation, chemo-enzymatic synthesis and NMR comparison. Although 5-FDRul has been chemically synthesized before, it was not an intermediate or a shunt product in previous studies of fluorometalism in S. cattleya. Our study of MA37 demonstrates that 5-FDRul is a naturally occurring fluorometabolite, rendering it a new addition to this rare collection of natural products. The genetic inactivation of key biosynthetic genes involved in the fluorometabolisms in MA37 resulted in the increased accumulation of unidentified fluorometabolites as observed from 19F-NMR spectral comparison among the wild type (WT) of MA37 and the mutated variants, providing evidence of the presence of other new biosynthetic enzymes involved in the fluorometabolite pathway in MA37.

The molybdenum cofactor (Moco) represents an ancient metal‑sulfur cofactor, which participates as catalyst in carbon, nitrogen and sulfur cycles, both on individual and global scale. Given the diversity of biological processes dependent on Moco and their evolutionary age, Moco is traced back to the last universal common ancestor (LUCA), while Moco biosynthetic genes underwent significant changes through evolution and acquired additional functions. In this review, focused on eukaryotic Moco biology, we elucidate the benefits of gene fusions on Moco biosynthesis and beyond. While originally the gene fusions were driven by biosynthetic advantages such as coordinated expression of functionally related proteins and product/substrate channeling, they also served as origin for the development of novel functions. Today, Moco biosynthetic genes are involved in a multitude of cellular processes and loss of the according gene products result in severe disorders, both related to Moco biosynthesis and secondary enzyme functions.

The widely application of metallic oxide nanoparticles (NPs) has led to an increase in their accumulation in farmland. Previous studies have found that the metallic oxide NPs have negative effect on plants development and growth. Nonetheless, the underlying mechanism of response to metallic oxide NPs in rice remains elusive. In this study, we show that rice FT-INTERACTING PROTEIN 7 (OsFTIP7) plays an essential role in NPs of CuO and ZnO-mediated physiological and biochemical changes in rice. Loss of function of OsFTIP7 reduced the toxicity of the NPs of CuO and ZnO to the seedlings by accumulating more biomass and chlorophyll contents. Furthermore, after high exposure to metallic oxide NPs, more indole-3-acetic acid (IAA) were determined in Osftip7 with higher expression of auxin biosynthetic genes than the control seedlings. What's more, IAA-treated seedlings displayed the similar phenotype as Osftip7 under high concentrations of NPs of CuO and ZnO. Taken together, the results substantiate that OsFTIP7 is involved in metallic oxide nanoparticle-mediated physiological and biochemical changes by negatively regulating auxin biosynthesis in rice.

Plants count on ecological services provided by pollinators or seed-dispersal animals through attracting them with volatile compounds from reproductive organs. On the other hand, volatiles from vegetative organs, such as leaves, stems, and roots, doesn’t seem to have the same function despite the organs have abilities to form, emit, and in some cases, accumulate volatiles. In most cases, vegetative plant volatiles are to defend against organisms that eat plants. Plants have acquired several sophisticated ways to make their ability to form volatiles effective against attackers. This chapter gives updates of current knowledge of biosynthetic pathways for green leaf volatiles, volatile terpenes, and phenylpropanoid volatiles, in vegetative organs. The production of volatile compounds at the level sufficient for efficient defense requires appropriate transcriptional/posttranscriptional regulation of biosynthetic pathways and/or transport and deposition of final products on the accurate tissues/cells/subcellular compartments. The precise mechanism of regulation has become clear gradually but steadily. An array of findings about the mechanism obtained so far has significantly contributed to our understanding of evolution of the biosynthetic genes, tissue-localized production, and a glycosylated storage form for vegetative volatiles.