Expression Of Biosynthetic Genes Research Articles

Comparative physiological and transcriptome analysis in cultivated and wild sugarcane species in response to hydrogen peroxide-induced oxidative stress

BackgroundSugarcane is an important energy crop grown worldwide,supplementing various renewable energy sources. Cultivated and wild sugarcane species respond differently to biotic and abiotic stresses. Generally, wild species are tolerant to various abiotic stresses. In the present study, the physiological and molecular responses of cultivated and wild sugarcane species to oxidative stress at the transcriptional levels were compared. Transcriptional responses were determined using RNAseq. The representative RNA-seq transcript values were validated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and confirmed through physiological responses.ResultsOxidative stress causes leaf-rolling and -tip drying in cultivated sugarcane, but the wild species are tolerant. Higher chlorophyll fluorescence was observed in the wild species than that in the cultivated varieties under stress. Wild species can maintain a higher chlorophyll stability index than the cultivated species, which was confirmed by the lower transcripts of the chlorophyllase gene in the wild species than that in the cultivated variety. Transcription factor genes (NAC, MYB, and WRKY) were markedly expressed in response to oxidative stress, revealing their involvement in stress tolerance. The analysis revealed synchronized expression of acetyl-transferase, histone2A, cellulose synthase, and secondary cell wall biosynthetic genes in the wild species. The validation of selected genes and 15 NAC transcription factors using RT-qPCR revealed that their expression profiles were strongly correlated with RNA-seq. To the best of our knowledge, this is the first report on the oxidative stress response in cultivated and wild sugarcane species.ConclusionPhysiological and biochemical changes in response to oxidative stress markedly differ between cultivated and wild sugarcane species. The differentially expressed stress-responsive genes are grouped intothe response to oxidative stress, heme-binding, peroxidase activity, and metal ion binding categories. Chlorophyll maintenance is a stress tolerance response enhanced by the differential regulation of the chlorophyllase gene.There is a considerable difference in the chlorophyll stability index between wild and cultivated varieties. We observed a substantial regulation of secondary wall biosynthesis genes in the wild species compared with that in the cultivated variety, suggesting differences in stress tolerance mechanisms.

Effects of branched-chain amino acids on Shiraia perylenequinone production in mycelium cultures

BackgroundPerylenequinones from Shiraia fruiting bodies are excellent photosensitizers and widely used for anti-cancer photodynamic therapy (PDT). The lower yield of Shiraia perylenequinones becomes a significant bottleneck for their medical application. Branched-chain amino acids (BCAAs) not only serve as important precursors for protein synthesis, but also are involved in signaling pathway in cell growth and development. However, there are few reports concerning their regulation of fungal secondary metabolism. In present study, the eliciting effects of BCAAs including l-isoleucine (l-Ile), l-leucine (l-Leu) and l-valine (l-Val) on Shiraia perylenequinone production were investigated.ResultsBased on the analysis of the transcriptome and amino acid contents of Shiraia in the production medium, we revealed the involvement of BCAAs in perylenequinone biosynthesis. The fungal conidiation was promoted by l-Val treatment at 1.5 g/L, but inhibited by l-Leu. The spore germination was promoted by both. The production of fungal perylenequinones including hypocrellins A (HA), HC and elsinochromes A-C (EA–EC) was stimulated significantly by l-Val at 1.5 g/L, but sharply suppressed by l-Leu. After l-Val treatment (1.5 g/L) in Shiraia mycelium cultures, HA, one of the main bioactive perylenequinones reached highest production 237.92 mg/L, about 2.12-fold than that of the control. Simultaneously, we found that the expression levels of key genes involved in the central carbon metabolism and in the late steps for perylenequinone biosynthesis were up-regulated significantly by l-Val, but most of them were down-regulated by l-Leu.ConclusionsOur transcriptome analysis demonstrated that BCAA metabolism was involved in Shiraia perylenequinone biosynthesis. Exogenous BCAAs exhibit contrasting effects on Shiraia growth and perylenequinones production. l-Val could promote perylenequinone biosynthesis via not only enhancing the central carbon metabolism for more precursors, but also eliciting perylenequinone biosynthetic gene expressions. This is the first report on the regulation of BCAAs on fungal perylenequinone production. These findings provided a basis for understanding physiological roles of BCAAs and a new avenue for increasing perylenequinone production in Shiraia mycelium cultures.Graphical

AflQ1-Q2 represses lincomycin biosynthesis via multiple cascades in Streptomyces lincolnensis.

Lincomycin is a broad-spectrum antibiotic and particularly effective against Gram-positive pathogens. Albeit familiar with the biosynthetic mechanism of lincomycin, we know less about its regulation, limiting the rational design for strain improvement. We therefore analyzed two-component systems (TCSs) in Streptomyces lincolnensis, and selected eight TCS gene(s) to construct their deletion mutants utilizing CRISPR/Cas9 system. Among them, lincomycin yield increased in two strains (Δ3900-3901 and Δ5290-5291) while decreased in other four strains (Δ3415-3416, Δ4153-4154, Δ4985, and Δ7949). Considering the conspicuous effect, SLINC_5291-5290 (AflQ1-Q2) was subsequently studied in detail. Its repression on lincomycin biosynthesis was further proved by gene complementation and overexpression. By binding to a 16-bp palindromic motif, the response regulator AflQ1 inhibits the transcription of its encoding gene and the expression of eight operons inside the lincomycin synthetic cluster (headed by lmbA, lmbJ, lmbK, lmbV, lmbW, lmbU, lmrA, and lmrC), as demonstrated by quantitative RT-PCR and electrophoretic mobility shift assays. Besides, the regulatory genes including bldD, glnR, lcbR1, and ramR are also regulated by the TCS. According to the screening towards nitrogen sources, aspartate affects the regulatory behavior of histidine kinase AflQ2. And in return, AflQ1 accelerates aspartate metabolism via ask-asd, asd2, and thrA. In summary, we acquired six novel regulators related to lincomycin biosynthesis, and elucidated the regulatory mechanism of AflQ1-Q2. This highly conserved TCS is a promising target for the construction of antibiotic high-yield strains. KEY POINTS: • AflQ1-Q2 is a repressor for lincomycin production. • AflQ1 modulates the expression of lincomycin biosynthetic and regulatory genes. • Aspartate affects the behavior of AflQ2, and its metabolism is promoted by AflQ1.

Postharvest light-induced flavonoids accumulation in mango (Mangifera indica L.) peel is associated with the up-regulation of flavonoids-related and light signal pathway genes.

Flavonoids are important secondary metabolites in plants and light is a crucial environmental factor regulating flavonoids biosynthesis. However, effect of light on the different flavonoids compositions accumulation in mango and the relevant molecular mechanism still need to be clarified. In this study, green-mature fruits of red mango cultivar 'Zill' were subjected to postharvest light treatment, and fruit peel color, total soluble solids content, total organic acid, and firmness of flesh were measured. The flavonoids metabolites profile, and the expression of flavonoids-related genes and light signal pathway genes were also analyzed. Results showed that light treatment promoted the red coloration of fruit peel and increased the total soluble solids content and firmness of flesh. The concentration of flavonols, proanthocyanidins and anthocyanins, and expression of key flavonoids biosynthetic genes including MiF3H, MiFLS, MiLAR, MiANS, MiUFGT1, and MiUFGT3 were significantly induced by light. The MYBs regulating flavonols and proanthocyanidins, i.e. MiMYB22 and MiMYB12, as well as the key light signal pathway transcription factors (TFs) MiHY5 and MiHYH, were identified in mango. The transcription of MiMYB1, MiMYB12, MiMYB22, MiHY5 and MiHYH was up-regulated by light. Our results provide a postharvest technology to improve mango fruit appearance quality, and are helpful to reveal the molecular mechanism of light-induced flavonoids biosynthesis in mango.

Enhancement of herbicolin A production by integrated fermentation optimization and strain engineering in Pantoea agglomerans ZJU23

BackgroundThe lipopeptide herbicolin A (HA) secreted by the biocontrol agent Pantoea agglomerans ZJU23 is a promising antifungal drug to combat fungal pathogens by targeting lipid rafts, both in agricultural and clinical settings. Improvement of HA production would be of great significance in promoting its commercialization. This study aims to enhance the HA production in ZJU23 by combining fermentation optimization and strain engineering.ResultsBased on the results in the single-factor experiments, corn steep liquor, temperature and initial pH were identified as the significant affecting factors by the Plackett–Burman design. The fermentation medium and conditions were further optimized using the Box-Behnken response surface method, and the HA production of the wild type strain ZJU23 was improved from ~ 87 mg/mL in King’s B medium to ~ 211 mg/mL in HA induction (HAI) medium. A transposon library was constructed in ZJU23 to screen for mutants with higher HA production, and two transcriptional repressors for HA biosynthesis, LrhA and PurR, were identified. Disruption of the LrhA gene led to increased mRNA expression of HA biosynthetic genes, and subsequently improved about twofold HA production. Finally, the HA production reached ~ 471 mg/mL in the ΔLrhA mutant under optimized fermentation conditions, which is about 5.4 times higher than before (~ 87 mg/mL). The bacterial suspension of the ΔLrhA mutant fermented in HAI medium significantly enhanced its biocontrol efficacy against gray mold disease and Fusarium crown rot of wheat, showing equivalent control efficacies as the chemical fungicides used in this study. Furthermore, HA was effective against fungicide resistant Botrytis cinerea. Increased HA production substantially improved the control efficacy against gray mold disease caused by a pyrimethanil resistant strain.ConclusionsThis study reveals that the transcriptional repressor LrhA negatively regulates HA biosynthesis and the defined HAI medium is suitable for HA production. These findings provide an extended basis for large-scale production of HA and promote biofungicide development based on ZJU23 and HA in the future.Graphical

Mrhst4 gene, coding for NAD+-dependent deacetylase is involved in citrinin production of Monascus ruber.

In this study, Mrhst4, encoding a member of NAD+-dependent histone deacetylase (HDAC), was deleted to evaluate its regulation on the production of Monascus azaphilone pigments (MonAzPs) and mycotoxin, as well as the developmental process in Monascusruber. Agrobacterium tumefaciens-mediated transformation was applied in this study to generate the Mrhst4 null strain. Mrhst4-deleted strain did not display obvious differences in the sexual and asexual reproduction, colonial morphology, and micro-morphology. UV-Vis scan and UPLC detection showed that disruption of Mrhst4 significantly increased the MonAzPs yields, and citrinin content was dramatically enhanced during the tested period. RT-qPCR results showed that the absence of Mrhst4 significantly increased the relative expression of citrinin biosynthetic pathway genes including pksCT, mrl1, mrl2, mrl4, mrl6, and mrl7. The Western blot assay suggested that deletion of Mrhst4 could significantly elevate the acetylation levels of H3K4, H3K9, H3K18, H3K56, and H4K12, but attenuated the lysine acetylation modification of H4Pan, H4K8, and H4K16. MrHst4 is an important regulator involved in secondary metabolism in Monascus ruber. In particular, MrHst4 plays a pivotal role in regulation of citrinin production.

Histone deacetylase 9 regulates disease resistance through fine-tuning histone deacetylation of melatonin biosynthetic genes and melatonin accumulation in cassava.

Melatonin participates in plant growth and developmentand bioticand abiotic stress responses. Histone acetylation regulates many plant biological processes via transcriptional reprogramming. However, the direct relationship between melatonin and histone acetylation in plant disease resistance remains unclear. In this study, we identified cassava bacterial blight (CBB) responsive histone deacetylase 9 (HDA9), which negatively regulated disease resistance to CBB by reducing melatonin content. In addition, exogenous melatonin alleviated disease sensitivity of MeHDA9 overexpressed plants to CBB. Importantly, MeHDA9 inhibited the expression of melatonin biosynthetic genes through decreasing lysine 5 of histone 4 (H4K5) acetylation at the promoter regions of melatonin biosynthetic genes, thereby modulating melatonin accumulation in cassava. Furthermore, protein phosphatase 2C 12 (MePP2C12) interacted with MeHDA9 in vivo and in vitro, and it was involved in MeHDA9-mediated disease resistance via melatonin biosynthetic pathway. In summary, this study highlights the direct interaction between histone deacetylation and melatonin biosynthetic genes in cassava disease resistance via histone deacetylation, providing new insights into the genetic improvement of disease resistance via epigenetic regulation of melatonin level in tropical crops.

Suppression of the target of rapamycin kinase accelerates tomato fruit ripening through reprogramming the transcription profile and promoting ethylene biosynthesis.

Tomato fruit ripening is a unique process of nutritional and energy metabolism. Target of rapamycin (TOR), a conserved serine/threonine protein kinase in eukaryotes, controls cell growth and metabolism by integrating nutrient, energy, and hormone signals. However, it remains unclear whether TOR participates in the modulation of tomato fruit ripening. Here, we showed that the manipulation of SlTOR by chemical or genetic methods greatly alters the process of tomato fruit maturation. Expression pattern analysis revealed that the transcripts of SlTOR declined as fruit ripening progressed. Moreover, suppression of SlTOR by TOR inhibitor AZD8055 or knock down of its transcripts by inducible RNA interference, accelerated fruit ripening, and led to overall effects on fruit maturity, including changes in colour and metabolism, fruit softening, and expression of ripening-related genes. Genome-wide transcription analysis indicated that silencing SlTOR reprogrammed the transcript profile associated with ripening, including cell wall and phytohormone pathways, elevated the expression of ethylene biosynthetic genes, and further promoted ethylene production. In contrast, the ethylene action inhibitor 1-MCP efficiently blocked fruit maturation, even following SlTOR inhibition. These results suggest that accelerated fruit ripening caused by SlTOR inhibition depends on ethylene, and that SlTOR may function as a regulator in ethylene metabolism.

Biosynthetic Gene Expression and Tissue Distribution of Diosgenin in Dioscorea japonica.

Diosgenin is an aglycone of dioscin, a major bioactive steroidal saponin found in plants, including Himalayan Paris (Paris polyphylla), fenugreek (Trigonella foenum-graecum), and yam (Dioscorea spp.). We have previously demonstrated that a species of natural yam, Dioscorea japonica, contains a promising bioactive compound diosgenin, which induces anti-carcinogenic and anti-hypertriacylglycerolemic activities. Here, we found for the first time that Japanese yam (D. japonica) bulbils are richer in diosgenin than the edible tubers (rhizomes) and leaves. LC-MS and imaging-MS analyses revealed that diosgenin accumulated in the peripheral region of D. japonica bulbils. Additionally, we performed RNA-seq analysis of D. japonica, and multiple sequence alignment identified D. japonica CYP90 (DjCYP90), the orthologous gene of CYP90G4 in P. polyphylla, CYP90B50 in T. foenum-graecum, CYP90G6 in Dioscorea zingiberensis, and CYP90G in Dioscorea villosa, which encodes a diosgenin biosynthetic rate-limiting enzyme. The expression levels of DjCYP90 were significantly upregulated in D. japonica bulbils than in its rhizomes and leaves. Since diosgenin is one of the most promising functional food factors executing several favorable bioactivities, D. japonica bulbils rich in diosgenin would be a beneficial natural resource.

A Zn(II)(2)Cys(6) transcription factor MPsGeI suppresses pigment biosynthesis in Monascus

High-quality natural edible pigments known as monascus pigments (MPs) are widely used in food, medicine, and chemical industries as active functional ingredients. At the transcriptional level, the expression of MPs genes are tightly controlled, limiting their productivity and color value. Hitherto our understanding of the regulation of expression of MPs genes has been rather limited. Here, we describe a pathway-specific Zn(II)(2)Cys(6) transcription factor involved in the MPs biosynthetic cluster named MPsGeI, which encodes a 813-amino-acid protein with six introns. Expression of all MPs biosynthetic genes and accumulation of MPs were remarkably increased in ΔMPsGeI strain, and MPs production was significantly reduced in MPsGeI over-expressing strain. Results clearly demonstrated that MPsGeI negatively regulates MPs accumulation via transcriptional regulation of MPs biosynthetic genes, and plays a central repressive role in MPs' biosynthesis. Transcriptomic analyses revealed that MPsGeI disruptant regulated higher concentrations of precursors flowing to pigment and resulted in accumulation of a large amount of red MPs in hyphae. This work offers an efficient method for increasing MPs's productivity and color value and provides novel insights into the regulatory mechanisms of fungal cellular processes, which will assist to enhance MPs production and application.

Diclofenac and Meloxicam Exhibited Anti-Virulence Activities Targeting Staphyloxanthin Production in Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a worldwide leading versatile pathogen that causes a wide range of serious infections. The emergence of antimicrobial resistance against S. aureus resulted in an urgent need to develop new antimicrobials in the new era. The methicillin-resistant S. aureus (MRSA) prevalence in hospital and community settings necessitates the discovery of novel anti-pathogenic agents. Staphyloxanthin (STX) is a key virulence factor for the survival of MRSA against host innate immunity. The current work aimed to demonstrate the anti-virulence properties of meloxicam (MXM) as compared to diclofenac (DC), which was previously reported to mitigate the virulence of multidrug-resistant Staphylococcus aureus and test their activities in STX production. A total of 80 S. aureus clinical isolates were included, wherein a qualitative and quantitative assessment of STX inhibition by diclofenac and meloxicam was performed. The quantitative gene expression of STX biosynthetic genes (crtM, crtN and sigB) and hla (coded for α-hemolysin) as a virulence gene with and without DC and MXM was conducted, followed by molecular docking analysis for further confirmation. DC and MXM potently inhibited the synthesis of STX at 47 and 59 µg/mL to reach 79.3-98% and 80.6-96.7% inhibition, respectively. Treated cells also revealed a significant downregulation of virulence genes responsible for STX synthesis, such as crtM, crtN and global transcriptional regulator sigB along with the hla gene. Furthermore, computational studies unveiled strong interactions between the CrtM binding site and DC/MXM. In conclusion, this study highlights the potential role and repurposing of DC and MXM as adjuvants to conventional antimicrobials and as an anti-virulent to combat MRSA infections.

Improving spinosad production by tuning expressions of the forosamine methyltransferase and the forosaminyl transferase to reduce undesired less active byproducts in the heterologous host Streptomyces albus J1074

BackgroundSpinosad is a macrolide insecticide with the tetracyclic lactone backbone to which forosamine and tri-o-methylrhamnose are attached. Both the sugar moieties are essential for its insecticidal activity. In biosynthesis of spinosad, the amino group of forosamine is dimethylated by SpnS and then transferred onto the lactone backbone by SpnP. Because the spinosad native producer is difficult to genetically manipulate, we previously changed promoters, ribosome binding sites and start codons of 23 spinosad biosynthetic genes to construct an artificial gene cluster which resulted in a 328-fold yield improvement in the heterologous host Streptomyces albus J1074 compared with the native gene cluster. However, in fermentation of J1074 with the artificial gene cluster, the N-monodesmethyl spinosad with lower insecticidal activity was always produced with the same titer as spinosad.ResultsBy tuning expression of SpnS with an inducible promotor, we found that the undesired less active byproduct N-monodesmethyl spinosad was produced when SpnS was expressed at low level. Although N-monodesmethyl spinosad can be almost fully eliminated with high SpnS expression level, the titer of desired product spinosad was only increased by less than 38%. When the forosaminyl transferase SpnP was further overexpressed together with SpnS, the titer of spinosad was improved by 5.3 folds and the content of N-desmethyl derivatives was decreased by ~ 90%.ConclusionN-monodesmethyl spinosad was produced due to unbalanced expression of spnS and upstream biosynthetic genes in the refactored artificial gene cluster. The accumulated N-desmethyl forosamine was transferred onto the lactone backbone by SpnP. This study suggested that balanced expression of biosynthetic genes should be considered in the refactoring strategy to avoid accumulation of undesired intermediates or analogues which may affect optimal production of desired compounds.

Jasmonic acid regulates the biosynthesis of medicinal metabolites via the JAZ9-MYB76 complex in Salvia miltiorrhiza.

Jasmonic acid (JA) signaling pathway plays an important role in tanshinone and phenolic acid biosynthesis in Salvia miltiorrhiza. However, the specific regulatory mechanism remains largely unclear. Previous work showed that a JASMONATE ZIM-domain (JAZ) protein, SmJAZ9, acted as a repressor of tanshinone production in S. miltiorrhiza. In this study, we revealed that SmJAZ9 reduced both phenolic acid accumulation and related biosynthetic gene expression, confirming that SmJAZ9 also negatively affected phenolic acid biosynthesis. Then, we identified a novel MYB transcription factor, SmMYB76, which interacted with SmJAZ9. SmMYB76 repressed phenolic acid biosynthesis by directly downregulating SmPAL1, Sm4CL2, and SmRAS1. Further investigation demonstrated that JA mediated phenolic acids biosynthesis via SmJAZ9-SmMYB76 complex. Taken together, these findings state the molecular mechanism that SmJAZ9-SmMYB76 regulated phenolic acid biosynthesis at the transcriptional and protein levels, which provided new insights into JA signaling pathway regulating plant metabolism.

β-Carotene and lutein accumulation, and carotenoid biosynthetic gene expression during fruit development and fruit ripening of A genome banana

• β -carotene and lutein content, affecting to orange-yellow pulp color in KK2. • Expression of MaPSY2a gene in KK2 affected to carotenoids accumulation. • Carotenoid biosynthetic genes correlated with β -carotene and lutein level. • Difference of A genome composition in banana affects carotenoid accumulation. Bananas ( Musa spp.) are an important source of pro-vitamin A, derived from carotenoids. Some cultivated bananas originated from intra-specific hybridization resulting in genotypes which are diploid (AA) and triploid (AAA) genomes that are the most important bananas for desserts. The accumulation of carotenoids and transcripts of genes related to carotenoid biosynthesis during fruit development and ripening in cultivars carrying the banana A genome (AA and AAA) were investigated. The accumulation of total carotenoids was greatest in ‘Khai Kasetsart2’ (KK2; AA) and ‘Sa’ (SA; AA), and comparatively lower in other cultivars. The predominant carotenoids in this fruit were β- carotene and lutein, with a small amount of α- carotene, which correlate with the orange-yellow pulp color (in particular KK2). In contrast, Leb Mu Nang had the lowest total carotenoids during fruit development while lutein and β -carotene in Hom Thong increased with fruit ripening. MaPSY2a gene expression in KK2 and SA cultivars occurred throughout fruit development but declined during fruit ripening. The expression of MaLCYB, MaLCYE, MaCHYB , and MaCHYE genes increased throughout fruit development and during fruit ripening resulting in β- carotene and lutein high content. In cultivars with low carotenoid levels, MaLCYB gene expression was reduced, while the MaLCYE, MaCHYE, and MaCHYB genes were up-regulated during fruit ripening. Besides, the MaCCD4 gene (a gene associated with carotenoid turnover) had lower expression throughout fruit development and ripening in KK2 compared to the other cultivars. These results suggested that the different carotenoid accumulation patterns in the cultivars could be attributed to differential gene expression and A genome composition.

Detoxication of Citrinin with Kojic Acid by the Formation of the Citrinin-Kojic Acid Adduct, and the Enhancement of Kojic Acid Production by Citrinin via Oxidative Stress in Aspergillus parasiticus

Our previous work showed that citrinin (CTN) produced bay Penicillium citrinum inhibited the production of aflatoxin by Aspergillus parasiticus. We also reported that CTN was non-enzymatically converted to a novel CTN-KA adduct with kojic acid (KA) in aqueous condition. We herein observed that unlike CTN, the CTN-KA adduct does not show antimicrobial activity against Escherichia coli or Bacillus subtilis or any cytotoxic effect on HeLa cells, suggesting that CTN was detoxified by KA by the formation of the CTN-KA adduct. To examine the function of KA production by fungi, we isolated A. parasiticus mutants with impaired KA production. When the mutants were incubated in either liquid or agar medium supplemented with CTN, they were more susceptible to CTN than the wild KA-producing strain. The same results were obtained when we used the A. oryzae KA-producing strain RIB40 and KA-non-producing strains. When KA was added to the CTN-containing agar medium, the inhibition of growth by CTN was remarkably mitigated, suggesting that the production of KA protected the fungal growth from CTN's toxicity. We also observed that CTN enhanced the production of KA by A. parasiticus as well as A. oryzae strains. Reverse transcription-PCR showed that CTN enhanced the expression of KA biosynthetic genes (kojA, kojR, and kojT) of A. parasiticus. However, the enhancement of KA production with CTN was repressed by the addition of α-tocopherol or butylated hydroxy anisole, suggesting that KA production is enhanced by oxidative stress via the formation of reactive oxygen species caused by CTN. In contrast, α-tocopherol did not affect inhibition of AF production as well as fungal growth by CTN, suggesting that the regulation of these inhibitions with CTN might be different from that of KA production. We propose a regulation scheme of CTN for each of KA production, AF production, and fungal growth in A. parasiticus.

Transcriptome analysis reveals that jasmonic acid biosynthesis and signaling is associated with the biosynthesis of asperosaponin VI in Dipsacus asperoides.

Dipsacus asperoides is a perennial herb, the roots of which are abundant in asperosaponin VI, which has important medicinal value. However, the molecular mechanism underlying the biosynthesis of asperosaponin VI in D. asperoides remains unclear. In present study, a comprehensive investigation of asperosaponin VI biosynthesis was conducted at the levels of metabolite and transcript during root development. The content of asperosaponin VI was significantly accumulated in two-leaf stage roots, and the spatial distribution of asperosaponin VI was localized in the xylem. The concentration of asperosaponin VI gradually increased in the root with the development process. Transcriptome analysis revealed 3916 unique differentially expressed genes (DEGs) including 146 transcription factors (TFs) during root development in D. asperoides. In addition, α-linolenic acid metabolism, jasmonic acid (JA) biosynthesis, JA signal transduction, sesquiterpenoid and triterpenoid biosynthesis, and terpenoid backbone biosynthesis were prominently enriched. Furthermore, the concentration of JA gradually increased, and genes involved in α-linolenic acid metabolism, JA biosynthesis, and triterpenoid biosynthesis were up-regulated during root development. Moreover, the concentration of asperosaponin VI was increased following methyl jasmonate (MeJA) treatment by activating the expression of genes in the triterpenoid biosynthesis pathway, including acetyl-CoA acetyltransferase (DaAACT), 3-hydroxy-3-methylglutaryl coenzyme A synthase (DaHMGCS), 3-hydroxy-3-methylglutaryl coenzyme-A reductase (DaHMGCR). We speculate that JA biosynthesis and signaling regulates the expression of triterpenoid biosynthetic genes and facilitate the biosynthesis of asperosaponin VI. The results suggest a regulatory network wherein triterpenoids, JA, and TFs co-modulate the biosynthesis of asperosaponin VI in D. asperoides.

The modular pYT vector series employed for chromosomal gene integration and expression to produce carbazoles and glycolipids in P. putida.

The expression of biosynthetic genes in bacterial hosts can enable access to high-value compounds, for which appropriate molecular genetic tools are essential. Therefore, we developed a toolbox of modular vectors, which facilitate chromosomal gene integration and expression in Pseudomonas putida KT2440. To this end, we designed an integrative sequence, allowing customisation regarding the modes of integration (random, at attTn7, or into the 16S rRNA gene), promoters, antibiotic resistance markers as well as fluorescent proteins and enzymes as transcription reporters. We thus established a toolbox of vectors carrying integrative sequences, designated as pYT series, of which we present 27 ready-to-use variants along with a set of strains equipped with unique 'landing pads' for directing a pYT interposon into one specific copy of the 16S rRNA gene. We used genes of the well-described violacein biosynthesis as reporter to showcase random Tn5-based chromosomal integration leading to constitutive expression and production of violacein and deoxyviolacein. Deoxyviolacein was likewise produced after gene integration into the 16S rRNA gene of rrn operons. Integration in the attTn7 site was used to characterise the suitability of different inducible promoters and successive strain development for the metabolically challenging production of mono-rhamnolipids. Finally, to establish arcyriaflavin A production in P. putida for the first time, we compared different integration and expression modes, revealing integration at attTn7 and expression with NagR/PnagAa to be most suitable. In summary, the new toolbox can be utilised for the rapid generation of various types of P. putida expression and production strains.

An ABCG-Type Transporter Facilitates ABA Influx and Regulates Camptothecin Biosynthesis in Camptotheca acuminata

Camptothecin (CPT) and its derivatives from Camptotheca acuminata have antitumor effects as a DNA topoisomerase I inhibitor. Previous studies have shown that application of exogenous abscisic acid (ABA) significantly promoted the accumulation level of CPT and induced the expression of CPT biosynthetic genes, which revealed that ABA signaling is effectively involved in regulating CPT biosynthesis in C. acuminata. In this study, an ABA transporter, CaABAT, which encodes a plasma membrane protein belonging to the ABCG subfamily, was identified in C. acuminata, and its ABA import activity was confirmed by transport assay in yeast cells. Real-time PCR analysis showed that CaABAT was predominately expressed in C. acuminata leaves and its expression could be significantly upregulated by exogenous ABA treatment. Silencing of CaABAT down-regulated the expression of ABA response genes, which indicated that translocation of ABA by CaABAT should initiate changes in plant physiological status in response to ABA signaling, thus leading to decreased expression of CPT biosynthesis pathway genes and low accumulation levels of CPT in C. acuminata.

Aspirin Inhibition of Prostaglandin Synthesis Impairs Mosquito Egg Development.

Several endocrine signals mediate mosquito egg development, including 20-hydroxyecdysone (20E). This study reports on prostaglandin E2 (PGE2) as an additional, but core, mediator of oogenesis in a human disease-vectoring mosquito, Aedes albopictus. Injection of aspirin (an inhibitor of cyclooxygenase (COX)) after blood-feeding (BF) inhibited oogenesis by preventing nurse cell dumping into a growing oocyte. The inhibitory effect was rescued by PGE2 addition. PGE2 was found to be rich in nurse cells and follicular epithelium after BF. RNA interference (RNAi) treatments of PG biosynthetic genes, including PLA2 and two COX-like peroxidases, prevented egg development. Interestingly, 20E treatment significantly increased the expressions of PG biosynthetic genes, while the RNAi of Shade (which is a 20E biosynthetic gene) expression prevented inducible expressions after BF. Furthermore, RNAi treatments of PGE2 receptor genes suppressed egg production, even under PGE2. These results suggest that a signaling pathway of BF-20E-PGE2 is required for early vitellogenesis in the mosquito.

ZmEREB92 interacts with ZmMYC2 to activate maize terpenoid phytoalexin biosynthesis upon Fusarium graminearum infection through jasmonic acid/ethylene signaling.

Maize (Zea mays) terpenoid phytoalexins (MTPs) induced by multiple fungi display extensive antimicrobial activities, yet how maize precisely regulates MTP accumulation upon pathogen infection remains elusive. In this study, pretreatment with jasmonic acid (JA)/ethylene (ET)-related inhibitors significantly reduced Fusarium graminearum-induced MTP accumulation and resulted in enhanced susceptibility to F. graminearum, indicating the involvement of JA/ET in MTP regulatory network. ZmEREB92 positively regulated MTP biosynthetic gene (MBG) expression by correlation analysis. Knockout of ZmEREB92 significantly compromised maize resistance to F. graminearum with delayed induction of MBGs and attenuated MTP accumulation. The activation of ZmEREB92 on MBGs is dependent on the interaction with ZmMYC2, which directly binds to MBG promoters. ZmJAZ14 interacts both with ZmEREB92 and with ZmMYC2 in a competitive manner to negatively regulate MBG expression. Altogether, our findings illustrate the regulatory mechanism for JA/ET-mediated MTP accumulation upon F. graminearum infection with the involvement of ZmEREB92, ZmMYC2, and ZmJAZ14, which provides new insights into maize disease responses.