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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 endophytic actinobacteria associated with Artemisia herba-alba (synonym: Seriphidium herba-alba) are highly diverse. This study aimed to illustrate the extent of their differences from the free-living actinobacteria in the surrounding environment. A selection of eighteen actinobacteria inhabiting A. herba-alba were compared with twenty and ten actinobatceria isolates from the surrounding desert and groundwater, respectively, representing six genera. Antagonistic and enzymatic activities, plant growth-promoting traits, and the occurrence of biosynthetic genes were compared among the isolates. Data were analyzed statistically using principal component analysis (PCA) and were visualized using heat map. Endophytic strains showed higher antimicrobial activity and production of plant growth promoters compared to desert and groundwater strains. Polyketide synthase and non-ribosomal peptide synthetase gene clusters were detected at higher frequencies in the endophytic strains (8 and 11 strains, respectively) than the desert strains (1 and 2 strains, respectively). In contrast, both gene clusters were not detected in the groundwater strains. The PCA revealed unique metabolic characteristics of the endophytes. The heatmap clustered the endophytic strains apart from the free-living strains, indicating distinctive qualitative and quantitative bioactivities. Analysis of 16S rRNA genes confirmed the chemotaxonomic identity of all but two strains, with > 94.5% similarity. Six endophytes displayed

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.

Natural products of lichen-forming fungi are structurally diverse and have a variety of medicinal properties. Despite this, they have limited implementation in industry mostly because the corresponding genes are unknown for most of their natural products. Here, we implement a long-read sequencing and bioinformatic approach to identify the putative biosynthetic gene cluster of the bioactive natural product gyrophoric acid (GA). Using 15 high-quality genomes representing nine GA-producing species of the lichen-forming fungal genus

Shikonins (SK) and acetyl-shikonins (acetyl-SK) are known to possess great pharmaceutical potentials however, their ability to disrupt infectious bacterial communication system viz. quorum sensing (QS) remains unexplored. Therefore, the rationale behind this research was to investigate the QS-quenching potential of SK and acetyl-SK against C. violaceum through biofilm formation and violacein production assays. The quantitative evaluation was followed by the investigation of transcript abundance of violacein-regulating genes using RT-qPCR. Quantitative analysis indicated marked inhibition of QS-related traits with more obvious inhibition (87.5% biofilm, and 58% violacein inhibition) with 0.4 mM SK-treatment. Our RT-qPCR results displayed a substantially significant decrease in the expression level of CviR in SK-treated cells. Furthermore, SK treatment resulted in reduced transcript abundance of violacein biosynthetic genes such as vioB, vioC, and vioD. It was found that SK-induced QS inhibition in C. violaceum occurred through CviR down-regulation, which might further potentially disrupt the AHL-CviR receptor interaction. Both SK and acetyl-SK treatments were found to be ineffective against pre-mature biofilms, implying that SK-mediated biofilm attenuation is caused by interference with the expression of genes involved in biofilm attachment. In our study, SK strongly interfered with AHL-regulated physiological attributes while acetyl-SK was effective in inhibiting biofilm formation only. Collectively we found SK as anti-QS agents that mediate C. violaceum QS inhibition and bring new hope in combating C. violaceum infections. In addition, our findings can help prevent drug resistance in microbial pathogens in the future.

Tubulysins are a group of secondary metabolites produced by myxobacteria that inhibit the function of the eukayotic cytoskeleton. We developed a pair of PCR primers that specifically amplified tubulysin biosynthetic genes. Using these primers, eight out of the eighty-one strains of myxobacteria belonging to the Cystobacteraceae family that harbored putative tubulysin biosynthetic genes were screened through PCR analysis. The selected strains included two Archangium gephyra, two Stigmatella sp., two Vitiosangium cumulatum, and two unidentified myxobacteria. LC-MS analysis of the culture extracts from the selected strains revealed that A. gephyra KYC4066 produced putative tubulysin A and B.

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.

Current research energies are fixated on the synthesis of environmentally friendly and non-hazardous products, which include finding and recognizing biosurfactants that can substitute synthetic surfactants. Microbial biosurfactants are surface-active compounds synthesized intracellularly or extracellularly. To use biosurfactants in various industries, it is essential to understand scientific engagements that demonstrate its potentials as real advancement in the 21st century. Other than applying a substantial effect on the world economic market, engineered hyper-producing microbial strains in combination with optimized cultivation parameters have made it probable for many industrial companies to receive the profits of 'green' biosurfactant innovation. There needs to be an emphasis on the worldwide state of biosurfactant synthesis, expression of biosurfactant genes in expressive host systems, the recent developments, and prospects in this line of research. Thus, molecular dynamics with respect to genetic engineering of biosynthetic genes are proposed as new biotechnological tools for development, improved synthesis, and applications of biosurfactants. For example, mutant and hyper-producing recombinants have been designed efficaciously to advance the nature, quantity, and quality of biosurfactants. The fastidious and deliberate investigation will prompt a comprehension of the molecular dynamics and phenomena in new microorganisms. Throughout the decade, valuable data on the molecular genetics of biosurfactant have been produced, and this solid foundation would encourage application-oriented yields of the biosurfactant production industry and expand its utilization in diverse fields. Therefore, the conversations among different interdisciplinary experts from various scientific interests such as microbiology, biochemistry, molecular biology, and genetics are indispensable and significant to accomplish these objectives.

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.

The main objective of the study is the evaluation of the diversity of actinobacteria associated with Polytrichum strictum — dominant species of widespread Antarctic Тall moss turf subformation and their characteristics as the producers of biologically active compounds. The actinobacterial isolates were isolated by direct inoculation, phenol pretreatment, and heated treatment. The cultural properties of the isolates were evaluated using diagnostic media. The antimicrobial activity of the isolates was determined by the point inoculations method. The phylogenetic analysis was based on sequence analysis of the 16S rRNA gene. The biosynthetic genes screening was performed using polymerase chain reaction. A total of 23 actinobacterial isolates associated with P. strictum were isolated, the four identified genera being Streptomyces (7 isolates), Micromonospora (14 isolates), Kribbella (1 isolate), and Micrococcus (1 isolate). Eight psychrotrophic strains of all identified genera were identified. The optimal pH values for all isolates were in the range 6–10. Four isolates grew on the medium with 7.5% NaCl. A significant number of the isolates showed a wide range of enzymatic activities. Antagonists of a wide range of pathogenic microorganisms were found, including against multidrug-resistant strain of Candida albicans and Methicillin-resistant Staphylococcus aureus. Some strains were active against phytopathogenic bacteria, namely three strains against Erwinia amylovora, one strain against Agrobacterium tumefaciens, and one strain against Pectobacterium carotovorum. More than half of the isolates showed antifungal activity against Fusarium oxysporum and Aspergillus niger. The biosynthetic genes involved in synthesizing a wide range of bioactive compounds were found in more than 80% of isolates. Antarctic actinobacteria isolated in this study demonstrate potential as the producers of a wide range of biologically active compounds. Further studies of these actinobacteria may lead to the identification of previously unknown biologically active compounds.

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

Human Milk Oligosaccharides (HMOs) are abundant carbohydrates fundamental to infant health and development. Although these oligosaccharides were discovered more than half a century ago, their biosynthesis in the mammary gland remains largely uncharacterized. Here, we used a systems biology framework that integrated glycan and RNA expression data to construct an HMO biosynthetic network and predict glycosyltransferases involved. To accomplish this, we constructed models describing the most likely pathways for the synthesis of the oligosaccharides accounting for >95% of the HMO content in human milk. Through our models, we propose candidate genes for elongation, branching, fucosylation, and sialylation of HMOs. We further explored selected enzyme activities through kinetic assay and their co-regulation through transcription factor analysis. These results provide the molecular basis of HMO biosynthesis necessary to guide progress in HMO research and application with the ultimate goal of understanding and improving infant health and development.Significance statementWith the HMO biosynthesis network resolved, we can begin to connect genotypes with milk types and thereby connect clinical infant, child and even adult outcomes to specific HMOs and HMO modifications. Knowledge of these pathways can simplify the work of synthetic reproduction of these HMOs providing a roadmap for improving infant, child, and overall human health with the specific application of a newly limitless source of nutraceuticals for infants and people of all ages.

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.

The transgene‐directed accumulation of non‐native omega‐3 long chain polyunsaturated fatty acids in the seed oil of Camelina sativa (Camelina) was evaluated in the field, in distinct geographical and regulatory locations. A construct, DHA2015.1, containing an optimal combination of biosynthetic genes, was selected for experimental field release in the UK, USA and Canada, and the accumulation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) determined. The occurrence of these fatty acids in different triacylglycerol species was monitored and found to follow a broad trend irrespective of the agricultural environment. This is a clear demonstration of the stability and robust nature of the transgenic trait for omega‐3 long chain polyunsaturated fatty acids in Camelina. Examination of non‐seed tissues for the unintended accumulation of EPA and DHA failed to identify their presence in leaf, stem, flower, anther or capsule shell material, confirming the seed‐specific accumulation of these novel fatty acids. Collectively, these data confirm the promise of GM plant‐based sources of so‐called omega‐3 fish oils as a sustainable replacement for oceanically derived oils.

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.

Natural products (NPs) represent one of the most important resources for discovering new drugs. Here we asked whether NP origin can be assigned from their molecular structure in a subset of 60,171 NPs in the recently reported Collection of Open Natural Products (COCONUT) database assigned to plants, fungi, or bacteria. Visualizing this subset in an interactive tree-map (TMAP) calculated using MAP4 (MinHashed atom pair fingerprint) clustered NPs according to their assigned origin (https://tm.gdb.tools/map4/coconut_tmap/), and a support vector machine (SVM) trained with MAP4 correctly assigned the origin for 94% of plant, 89% of fungal, and 89% of bacterial NPs in this subset. An online tool based on an SVM trained with the entire subset correctly assigned the origin of further NPs with similar performance (https://np-svm-map4.gdb.tools/). Origin information might be useful when searching for biosynthetic genes of NPs isolated from plants but produced by endophytic microorganisms. Supplementary Information The online version contains supplementary material available at 10.1186/s13321-021-00559-3.

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.

Τα τριτερπένια είναι φυτικοί δευτερογενείς μεταβολίτες που προέρχονται από την κυκλοποίηση του 2,3-οξειδοσκουαλενίου από εξειδικευμένα ένζυμα, τις συνθάσες ή κυκλάσες του οξειδοσκουαλενίου. Ο ρόλος της συνθάσης της λουπεόλης, που κωδικοποιείται από το γονίδιο OSC3, καθώς και το προϊόν του, λουπεόλη, διερευνήθηκε σε αναπτυσσόμενες ρίζες και φυμάτια του μοντέλου-ψυχανθούς Lotus japonicus. Τα πρότυπα έκφρασης του γονιδίου OSC3 σε διάφορα αναπτυξιακά στάδια μη μολυσμένων και μολυσμένων ριζών με το συμβιώτη Mesorhizobium loti καθορίστηκαν. Η ιστοειδική έκφραση του γονιδίου OSC3 καθορίστηκε με in situ υβριδισμό. Πραγματοποιήθηκε λειτουργική ανάλυση σε μετασχηματισμένες ρίζες που παρουσίαζαν αποσιώπηση του γονιδίου OSC3. Στις μετασχηματισμένες ρίζες καθορίστηκε η απουσία παραγωγής λουπεόλης με GC-MS. Τα μετασχηματισμένα φυτά παρουσίασαν το φαινότυπο της πιο ταχείας φυματιογένεσης σε σύγκριση με τα φυτά – μάρτυρες. Επιπλέον, η έκφραση του γονιδίου ENOD40, γονίδιο δείκτης της έναρξης σχηματισμού των καταβολών των φυματίων, αυξήθηκε σημαντικά στα μετασχηματισμένα φυτά. Η εξωγενής παροχή λουπεόλης σε αγρίου τύπου φυτά παρείχε επιπλέον ενδείξεις για τον αρνητικό ρυθμιστικό ρόλο της λουπεόλης στην έκφραση του γονιδίου ENOD40. Η βιοσύνθεση της λουπεόλης μπορεί να αποδωθεί αποκλειστικά στο γονίδιο OSC3. Λαμβάνοντας όλα αυτά υπ’όψιν, προτείνεται στη λουπεόλη ένας ρόλος στο σχηματισμό των φυματίων μέσω της ρύθμισης της έκφρασης του γονιδίου ENOD40. Επιπρόσθετα, τα γονίδια που συμμετέχουν σε μονοπάτια βιοσύνθεσης τριτερπενοειδών οργανώνονται σε γονιδιακές συστοιχίες στα φυτά της βρώμης και του Arabidopsis thaliana. Η παρουσία μιας ανάλογης γονιδιακής συστοιχίας στο φυτό L. japonicus ταυτοποιήθηκε. Στη γονιδιωματική περιοχή που εδράζεται το γονίδιο της συνθάσης της β-αμυρίνης, εντοπίστηκαν δύο γονίδια του κυτοχρώματος Ρ450 καθώς και μία ρεδουκτάση. Ο λειτουργικός χαρακτηρισμός της γονιδιακής συστοιχίας πραγματοποιήθηκε με ετερόλογη έκφραση σε φύλλα Nicotiana benthamiana. Μια καινούρια δομή τριτερπενοειδούς, η διυδρολουπεόλη, παράγεται από την συνθάση της β-αμυρίνης. Επιπλέον, ένα καινούριο ένζυμο του κυτοχρώματος Ρ450, το CYP71D353, χαρακτηρίστηκε, το οποίο καταλύει το σχηματισμό του 20-hydroxybetulinic acid μέσω τριών διαδοχικών βημάτων οξείδωσης της διυδρολουπεόλης. Τα πρότυπα έκφρασης των γονιδίων μελετήθηκαν σε διαφορετικές αναπτυξιακές και περιβαλλοντικές συνθήκες. Τα γονίδια της συστοιχίας συν-εκφράζονται κατά τη διάρκεια της ανάπτυξης των φυματίων και των ριζών, σε φυτά που υποβληθήκαν σε καταπονήσεις και σε φυτά που αναπτύχθηκαν παρουσία ορμονών. Ο φυσιολογικός ρόλος της γονιδιακής συστοιχίας στη φυματιογένεση και στην φυτική ανάπτυξη μελετήθηκε σε μετασχηματισμένα φυτά με αποσιώπηση. Επιπλέον, αποκαλύφθηκε ένας μηχανισμός επιγενετικής τροποποίησης που πιθανά εμπλέκεται στη ρύθμιση της γονιδιακής συστοιχίας. Μια γονιδιακή συστοιχία που αποτελείται από λειτουργικά συσχετιζόμενα γονίδια εμπλέκεται στη βιοσύνθεση τριτερπενοειδών στα ψυχανθή και πιθανά εμπλέκεται στη φυτική ανάπτυξη.

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

Natural products (NPs) represent one of the most important resources for discovering new drugs. Here we asked whether NP origin can be assigned from their molecular structure in a subset of 60,171 NPs in the recently reported Collection of Open Natural Products (COCONUT) database assigned to plants, fungi, or bacteria. Visualizing this subset in an interactive tree-map (TMAP) calculated using MAP4 (MinHashed atom pair fingerprint) clustered NPs according to their assigned origin (https://tm.gdb.tools/map4/coconut_tmap/), and a support vector machine (SVM) trained with MAP4 correctly assigned the origin for 94% of plant, 89% of fungal, and 89% of bacterial NPs in this subset. An online tool based on an SVM trained with the entire subset correctly assigned the origin of further NPs with similar performance (https://np-svm-map4.gdb.tools/). Origin information might be useful when searching for biosynthetic genes of NPs isolated from plants but produced by endophytic microorganisms.

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.

Drought-tolerant rootstocks have been used to improve production efficiency of graft-propagated crops. Kiwifruit is a high-valued fruit crop of the world. Drought stress widely occurs in many of kiwifruit-growing regions including China. The present study used micrografting for evaluation of drought tolerance in four kiwifruit cultivars micrografted on the drought-tolerant rootstock MX1. When stressed by PEG-induced drought, the micrografted kiwifruit cultivars showed significant differences in accumulation of ROS (O2·− and H2O2), physiological metabolism (total soluble sugar, total soluble protein and free proline), activities of antioxidants (SOD and CAT), endogenous ABA levels and expression levels of ABA biosynthetic genes (DREB1 and DREB2). Drought tolerance of the micrografts varied with the four kiwifruit cultivars, with the highest and lowest drought tolerance found in ‘Yuxiang’ and ‘Hayward’, respectively. Micrografting provides an alternative strategy for fast and efficient evaluation of drought tolerance of plant cultivars, thus assisting sustainable development of production of the graft-propagated plant species. In vitro micrografting provided a fast and efficient approach to evaluate drought tolerance of kiwifruit, and has potential applications to other graft-propagated plant species.

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.

3-Deoxyanthocyanidins are a rare type of anthocyanins that are present in mosses, ferns, and some flowering plants. They are water-soluble pigments and impart orange-red and blue-violet color to plants and play a role as phytoalexins against microbial infection and environmental stress. In contrast to anthocyanins, the lack of a hydroxyl group at the C-3 position confers unique chemical and biochemical properties. They are potent natural antioxidants with a number of potential health benefits including cancer prevention. 3-Deoxyanthocyanidin pigments have attracted much attention in the food industry as natural food colorants, mainly due to their higher stability during processing and handling conditions compared with anthocyanins. They are also photochromic compounds capable of causing a change in "perceived" color, when exposed to UV light, which can be used to design novel foods and beverages. Due to their interesting properties and potential industrial applications, great efforts have been made to synthesize these compounds. For biosynthesis, researchers have discovered the 3-deoxyanthocyanidin biosynthetic pathway and their biosynthetic genes. For chemical synthesis, advances have been made to synthesize the compounds in a simpler and more efficient way as well as looking for its novel derivative with enhanced coloration properties. This review summarizes the developments in the research on 3-deoxyanthocyanidin as a colorant, from natural sources to chemical syntheses and from health benefits to applications and future prospects, providing comprehensive insights into this group of interesting compounds.

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.

Aflatoxins residues in different poultry products were determined using HPLC technique and the expression of biosynthetic nor-1 gene of these aflatoxins by PCR assay. 90 different chicken products were analyzed for presence of aflatoxins (AFB1, AFB2, AFG1 and AFG2) using highly accurate, robust, selective, sensitive and precise HPLC-FLD assay. Incidence of aflatoxins in examined samples showed that the level of AFB1, AFB2 and AFG2 were 10 % for each and of AFG1 was 16.7%. The highest aflatoxins levels of analyzed samples were found in the liver. Expression of nor-1 gene of aflatoxins in polluted poultry products by PCR assay. A Real time PCR (RT-PCR) technique was applied to detect the gene expression of structural nor-1, which catalyzed the first step in the biosynthetic pathway of aflatoxins. The obtained results showed a very strong relationship between the presences of aflatoxins biosynthetic genes as analyzed by multiplex PCR and aflatoxins determination by HPLC/FLD.

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.

Lanthipeptides are ribosomally synthesized and posttranslationally modified peptides, with modifications that are incorporated during biosynthesis by dedicated enzymes. Various modifications of the peptides are possible, resulting in a highly diverse group of bioactive peptides that offer a potential reservoir for use in the fight against a plethora of diseases. Their activities range from the antimicrobial properties of lantibiotics, especially against antibiotic-resistant strains, to antiviral activity, immunomodulatory properties, antiallodynic effects, and the potential to alleviate cystic fibrosis symptoms. Lanthipeptide biosynthetic genes are widespread within bacterial genomes, providing a substantial repository for novel bioactive peptides. Using genome mining tools, novel bioactive lanthipeptides can be identified, and coupled with rapid screening and heterologous expression technologies, the lanthipeptide drug discovery pipeline can be significantly sped up. Lanthipeptides represent a group of bioactive peptides that hold great potential as biotherapeutics, especially at a time when novel and more effective therapies are required. With this review, we provide insight into the latest developments made toward the therapeutic applications and production of lanthipeptides, specifically looking at heterologous expression systems.

Secondary metabolites (SMs) are a vast group of compounds with different structures and properties. Humankind uses SMs as drugs, food additives, dyes, and as monomers for novel plastics. In many cases, the biosynthesis of SMs is catalysed by enzymes whose corresponding genes are co-localized in the genome in biosynthetic gene clusters (BGCs). Notably, BGCs may contain so-called gap genes, that are not involved in the biosynthesis of the SM. Current genome mining tools can identify BGCs but they have problems with distinguishing essential genes from gap genes and defining the borders of a BGC. This can and must be done by expensive, laborious, and time-consuming comparative genomic approaches or co-expression analyses. In this study, we developed a novel tool that allows automated identification of essential genes in a BGC based solely on genomic data. The Functional Order (FunOrder) tool – Identification of essential biosynthetic genes through computational molecular co-evolution – searches for co-evolutionary linked genes in the BGCs. In light of the growing number of genomic data available, this will contribute to the studies of BGCs in native hosts and facilitate heterologous expression in other organisms with the aim of the discovery of novel SMs, including antibiotics and other pharmaceuticals.

Camptothecin (CPT), a well-known monoterpenoid indole alkaloid with broad-spectrum anti-cancer activity, is produced from plants and endophytes. In view of the limitations of plants as sources of camptothecin in productivity and efficiency, endophytes serve as the fast growth, high cost-effectiveness, good reproducibility, and feasible genetic manipulation, so they have the potential to meet the huge market demand of the pharmaceutical industry. In this review, we summarized the isolation, identification and fermentation of CPT-producing endophytes, as well as the biosynthesis, extraction and detection of camptothecin from endophytes. Among them, we put emphasis on increasing the production of camptothecin in endophytes through different strategies such as changing the proportion of carbon, nitrogen and phosphate source, adding the precursors, elicitors or adsorbent resin, utilizing co-culture fermentation or fermenter culture. However, cell subculture and metabolic reprogramming affect the expression of camptothecin biosynthetic genes in CPT-producing endophytes, which poses a challenge to the industrial production of camptothecin. Therefore, it will be useful to gain insights through the review of these researches and provide alternative approaches to develop economical, eco-friendly and reliable natural products.

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.

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.