Biosynthesis Of Specialized Metabolites Research Articles

The acetyltransferase SCO0988 controls positively specialized metabolism and morphological differentiation in the model strains Streptomyces coelicolor and Streptomyces lividans.

Streptomycetes are well-known antibiotic producers possessing in their genomes numerous silent biosynthetic pathways that might direct the biosynthesis of novel bio-active specialized metabolites. It is thus of great interest to find ways to enhance the expression of these pathways to discover most needed novel antibiotics. In this study, we demonstrated that the over-expression of acetyltransferase SCO0988 up-regulated the production of specialized metabolites and accelerated sporulation of the weak antibiotic producer, Streptomyces lividans and that the deletion of this gene had opposite effects in the strong antibiotic producer, Streptomyces coelicolor. The comparative analysis of the acetylome of a S. lividans strain over-expressing sco0988 with that of the original strain revealed that SCO0988 acetylates a broad range of proteins of various pathways including BldKB/SCO5113, the extracellular solute-binding protein of an ABC-transporter involved in the up-take of a signal oligopeptide of the quorum sensing pathway. The up-take of this oligopeptide triggers the "bald cascade" that regulates positively specialized metabolism, aerial mycelium formation and sporulation in S. coelicolor. Interestingly, BldKB/SCO5113 was over-acetylated on four Lysine residues, including Lys425, upon SCO0988 over-expression. The bald phenotype of a bldKB mutant could be complemented by native bldKB but not by variant of bldKB in which the Lys425 was replaced by arginine, an amino acid that could not be acetylated or by glutamine, an amino acid that is expected to mimic acetylated lysine. Our study demonstrated that Lys425 was a critical residue for BldKB function but was inconclusive concerning the impact of acetylation of Lys425 on BldKB function.

Multi-omic analyses reveal key sectors of jasmonate-mediated defense responses in rice.

The phytohormone jasmonate (JA) plays a central role in plant defenses against biotic stressors. However, our knowledge of the JA signaling pathway in rice (Oryza sativa) remains incomplete. Here, we integrated multi-omic data from three tissues to characterize the functional modules involved in organizing JA-responsive genes. In the core regulatory sector, MYC2 transcription factor transcriptional cascades are conserved. in different species but with distinct regulators (e.g. bHLH6 in rice)., in which genes are early expressed across all tissues. In the feedback sector, MYC2 also regulates the expression of JA repressor and catabolic genes, providing negative feedback that truncates the duration of JA responses. For example, the MYC2-regulated NAC (NAM, ATAF1/2 and CUC2) transcription factor genes NAC1, NAC3, and NAC4 encode proteins that repress JA signaling and herbivore resistance. In the tissue-specific sector, many late-expressed genes are associated with the biosynthesis of specialized metabolites that mediate particular defensive functions. For example, the terpene synthase gene TPS35 is specifically induced in the leaf sheath and TPS35 functions in defense against oviposition by brown planthoppers and the attraction of this herbivore's natural enemies. Thus, by characterizing core, tissue-specific, and feedback sectors of JA-elicited defense responses, this work provides a valuable resource for future discoveries of key JA components in this important crop.

Machine learning assists prediction of genes responsible for plant specialized metabolite biosynthesis by integrating multi-omics data

BackgroundPlant specialized (or secondary) metabolites (PSM), also known as phytochemicals, natural products, or plant constituents, play essential roles in interactions between plants and environment. Although many research efforts have focused on discovering novel metabolites and their biosynthetic genes, the resolution of metabolic pathways and identified biosynthetic genes was limited by rudimentary analysis approaches and enormous number of candidate genes.ResultsHere we integrated state-of-the-art automated machine learning (ML) frame AutoGluon-Tabular and multi-omics data from Arabidopsis to predict genes encoding enzymes involved in biosynthesis of plant specialized metabolite (PSM), focusing on the three main PSM categories: terpenoids, alkaloids, and phenolics. We found that the related features of genomics and proteomics were the top two crucial categories of features contributing to the model performance. Using only these key features, we built a new model in Arabidopsis, which performed better than models built with more features including those related with transcriptomics and epigenomics. Finally, the built models were validated in maize and tomato, and models tested for maize and trained with data from two other species exhibited either equivalent or superior performance to intraspecies predictions.ConclusionsOur external validation results in grape and poppy on the one hand implied the applicability of our model to the other species, and on the other hand showed enormous potential to improve the prediction of enzymes synthesizing PSM with the inclusion of valid data from a wider range of species.

CwJAZ4/9 negatively regulates jasmonate-mediated biosynthesis of terpenoids through interacting with CwMYC2 and confers salt tolerance in Curcuma wenyujin.

Plant JASMONATE ZIM-DOMAIN (JAZ) genes play crucial roles in regulating the biosynthesis of specialized metabolites and stressful responses. However, understanding of JAZs controlling these biological processes lags due to numerous JAZ copies. Here, we found that two leaf-specific CwJAZ4/9 genes from Curcuma wenyujin are strongly induced by methyl-jasmonate (MeJA) and negatively correlated with terpenoid biosynthesis. Yeast two-hybrid, luciferase complementation imaging and in vitro pull-down assays confirmed that CwJAZ4/9 proteins interact with CwMYC2 to form the CwJAZ4/9-CwMYC2 regulatory cascade. Furthermore, transgenic hairy roots showed that CwJAZ4/9 acts as repressors of MeJA-induced terpenoid biosynthesis by inhibiting the terpenoid pathway and jasmonate response, thus reducing terpenoid accumulation. In addition, we revealed that CwJAZ4/9 decreases salt sensitivity and sustains the growth of hairy roots under salt stress by suppressing the salt-mediated jasmonate responses. Transcriptome analysis for MeJA-mediated transgenic hairy root lines further confirmed that CwJAZ4/9 negatively regulates the terpenoid pathway genes and massively alters the expression of genes related to salt stress signaling and responses, and crosstalks of multiple phytohormones. Altogether, our results establish a genetic framework to understand how CwJAZ4/9 inhibits terpenoid biosynthesis and confers salt tolerance, which provides a potential strategy for producing high-value pharmaceutical terpenoids and improving resistantC. wenyujin varieties by a genetic approach.

A bird's-eye view: exploration of the flavin-containing monooxygenase superfamily in common wheat.

The Flavin Monooxygenase (FMO) gene superfamily in plants is involved in various processes most widely documented for its involvement in auxin biosynthesis, specialized metabolite biosynthesis, and plant microbial defense signaling. The roles of FMOs in defense signaling and disease resistance have recently come into focus as they may present opportunities to increase immune responses in plants including leading to systemic acquired resistance, but are not well characterized. We present a comprehensive catalogue of FMOs found in genomes across vascular plants and explore, in depth, 170 wheat TaFMO genes for sequence architecture, cis-acting regulatory elements, and changes due to Transposable Element insertions. A molecular phylogeny separates TaFMOs into three clades (A, B, and C) for which we further report gene duplication patterns, and differential rates of homoeologue expansion and retention among TaFMO subclades. We discuss Clade B TaFMOs where gene expansion is similarly seen in other cereal genomes. Transcriptome data from various studies point towards involvement of subclade B2 TaFMOs in disease responses against both biotrophic and necrotrophic pathogens, substantiated by promoter element analysis. We hypothesize that certain TaFMOs are responsive to both abiotic and biotic stresses, providing potential targets for enhancing disease resistance, plant yield and other important agronomic traits. Altogether, FMOs in wheat and other crop plants present an untapped resource to be exploited for improving the quality of crops.

The Madagascar palm genome provides new insights on the evolution of Apocynaceae specialized metabolism

Specialized metabolites possess diverse interesting biological activities and some cardenolides- and monoterpene indole alkaloids- (MIAs) derived pharmaceuticals are currently used to treat human diseases such as cancers or hypertension. While these two families of biocompounds are produced by specific subfamilies of Apocynaceae, one member of this medicinal plant family, the succulent tree Pachypodium lamerei Drake (also known as Madagascar palm), does not produce such specialized metabolites. To explore the evolutionary paths that have led to the emergence and loss of cardenolide and MIA biosynthesis in Apocynaceae, we sequenced and assembled the P. lamerei genome by combining Oxford Nanopore Technologies long-reads and Illumina short-reads. Phylogenomics revealed that, among the Apocynaceae whose genomes have been sequenced, the Madagascar palm is so far the species closest to the common ancestor between MIA producers/non-MIA producers. Transposable elements, constituting 72.48% of the genome, emerge as potential key players in shaping genomic architecture and influencing specialized metabolic pathways. The absence of crucial MIA biosynthetic genes such as strictosidine synthase in P. lamerei and non-Rauvolfioideae species hints at a transposon-mediated mechanism behind gene loss. Phylogenetic analysis not only showcases the evolutionary divergence of specialized metabolite biosynthesis within Apocynaceae but also underscores the role of transposable elements in this intricate process. Moreover, we shed light on the low conservation of enzymes involved in the final stages of MIA biosynthesis in the distinct MIA-producing plant families, inferring independent gains of these specialized enzymes along the evolution of these medicinal plant clades. Overall, this study marks a leap forward in understanding the genomic dynamics underpinning the evolution of specialized metabolites biosynthesis in the Apocynaceae family, with transposons emerging as potential architects of genomics restructuring and gene loss.

Targeting of Specialized Metabolites Biosynthetic Enzymes to Membranes and Vesicles by Posttranslational Palmitoylation: A Mechanism of Non-Conventional Traffic and Secretion of Fungal Metabolites.

In nature, the formation of specialized (secondary) metabolites is associated with the late stages of fungal development. Enzymes involved in the biosynthesis of secondary metabolites in fungi are located in distinct subcellular compartments including the cytosol, peroxisomes, endosomes, endoplasmic reticulum, different types of vesicles, the plasma membrane and the cell wall space. The enzymes traffic between these subcellular compartments and the secretion through the plasma membrane are still unclear in the biosynthetic processes of most of these metabolites. Recent reports indicate that some of these enzymes initially located in the cytosol are later modified by posttranslational acylation and these modifications may target them to membrane vesicle systems. Many posttranslational modifications play key roles in the enzymatic function of different proteins in the cell. These modifications are very important in the modulation of regulatory proteins, in targeting of proteins, intracellular traffic and metabolites secretion. Particularly interesting are the protein modifications by palmitoylation, prenylation and miristoylation. Palmitoylation is a thiol group-acylation (S-acylation) of proteins by palmitic acid (C16) that is attached to the SH group of a conserved cysteine in proteins. Palmitoylation serves to target acylated proteins to the cytosolic surface of cell membranes, e.g., to the smooth endoplasmic reticulum, whereas the so-called toxisomes are formed in trichothecene biosynthesis. Palmitoylation of the initial enzymes involved in the biosynthesis of melanin serves to target them to endosomes and later to the conidia, whereas other non-palmitoylated laccases are secreted directly by the conventional secretory pathway to the cell wall space where they perform the last step(s) of melanin biosynthesis. Six other enzymes involved in the biosynthesis of endocrosin, gliotoxin and fumitremorgin believed to be cytosolic are also targeted to vesicles, although it is unclear if they are palmitoylated. Bioinformatic analysis suggests that palmitoylation may be frequent in the modification and targeting of polyketide synthetases and non-ribosomal peptide synthetases. The endosomes may integrate other small vesicles with different cargo proteins, forming multivesicular bodies that finally fuse with the plasma membrane during secretion. Another important effect of palmitoylation is that it regulates calcium metabolism by posttranslational modification of the phosphatase calcineurin. Mutants defective in the Akr1 palmitoyl transferase in several fungi are affected in calcium transport and homeostasis, thus impacting on the biosynthesis of calcium-regulated specialized metabolites. The palmitoylation of secondary metabolites biosynthetic enzymes and their temporal distribution respond to the conidiation signaling mechanism. In summary, this posttranslational modification drives the spatial traffic of the biosynthetic enzymes between the subcellular organelles and the plasma membrane. This article reviews the molecular mechanism of palmitoylation and the known fungal palmitoyl transferases. This novel information opens new ways to improve the biosynthesis of the bioactive metabolites and to increase its secretion in fungi.

Enhanced protein secretion in reduced genome strains of Streptomyces lividans

BackgroundS. lividans TK24 is a popular host for the production of small molecules and the secretion of heterologous protein. Within its large genome, twenty-nine non-essential clusters direct the biosynthesis of secondary metabolites. We had previously constructed ten chassis strains, carrying deletions in various combinations of specialized metabolites biosynthetic clusters, such as those of the blue actinorhodin (act), the calcium-dependent antibiotic (cda), the undecylprodigiosin (red), the coelimycin A (cpk) and the melanin (mel) clusters, as well as the genes hrdD, encoding a non-essential sigma factor, and matAB, a locus affecting mycelial aggregation. Genome reduction was aimed at reducing carbon flow toward specialized metabolite biosynthesis to optimize the production of secreted heterologous protein.ResultsTwo of these S. lividans TK24 derived chassis strains showed ~ 15% reduction in biomass yield, 2-fold increase of their total native secretome mass yield and enhanced abundance of several secreted proteins compared to the parental strain. RNAseq and proteomic analysis of the secretome suggested that genome reduction led to cell wall and oxidative stresses and was accompanied by the up-regulation of secretory chaperones and of secDF, a Sec-pathway component. Interestingly, the amount of the secreted heterologous proteins mRFP and mTNFα, by one of these strains, was 12 and 70% higher, respectively, than that secreted by the parental strain.ConclusionThe current study described a strategy to construct chassis strains with enhanced secretory abilities and proposed a model linking the deletion of specialized metabolite biosynthetic clusters to improved production of secreted heterologous proteins.

Engineered and total biosynthesis of fungal specialized metabolites.

Filamentous fungi produce a very wide range of complex and often bioactive metabolites, demonstrating their inherent ability as hosts of complex biosynthetic pathways. Recent advances in molecular sciences related to fungi have afforded the development of new tools that allow the rational total biosynthesis of highly complex specialized metabolites in a single process. Increasingly, these pathways can also be engineered to produce new metabolites. Engineering can be at the level of gene deletion, gene addition, formation of mixed pathways, engineering of scaffold synthases and engineering of tailoring enzymes. Combination of these approaches with hosts that can metabolize low-value waste streams opens the prospect of one-step syntheses from garbage.

Gene Co-expression Network Analysis to Mine Genes Involved in Plant Secondary Metabolite Biosynthesis and Regulation.

Plants produce diverse specialized metabolites (SMs) that do not participate in plant growth and development but help them adapt to various environmental conditions. In addition to aiding in plant adaptation, different SMs serve as active ingredients for pharmaceutical and cosmetics products. However, despite their significant role in plant adaptation and industrial importance, the genes involved in the biosynthesis and regulation of many SMs remain largely unknown. This hinders deciphering the specific role of SMs in plant adaptation and limits their industrial utilization. Since many SMs pathway genes are expected to act in tight association with each other within a coexpression network, the network biology approach, such as weighted gene coexpression network analysis, could be used to identify the unknown genes. This chapter describes a workflow for constructing a gene coexpression network to identify genes that could be associated with the biosynthesis and regulation of SMs.

Nanoparticle elicitation: A promising strategy to modulate the production of bioactive compounds in hairy roots

Hairy root culture is one of the promising biotechnological tools to obtain the stable and sustainable production of specialized metabolites from plants under controlled environment conditions. Various strategies have been adopted to enhance the accumulation of bioactive compounds in hairy roots yet their utilization at the commercial scale is restricted to only a few products. Recently, nanotechnology has been emerged as an active technique that has revolutionized the many sectors in an advantageous way. Elicitation using nanoparticles has been recognized as an effective strategy for enhancing the bioactive compounds of interest in plants. Nanoparticles elicit the activity of defense-related compounds through activation of the specific transcription factors involved in specialized metabolites production. This review discusses the recent progress in using nanoparticles to enhance specialized metabolite biosynthesis using hairy root culture system and the significant achievements in this area of research. Biotic and abiotic elicitors to improve the production of bioactive compounds in hairy roots, different types of nanoparticles as eliciting agents, their properties as dependent on shape, most widely used nanoparticles in plant hairy root systems are described in detail. Further challenges involved in application of nanoparticles, their toxicity in plant cells and risks associated to human health are also envisaged. No doubt, nanoparticle elicitation is a remarkable approach to obtain phytochemicals from hairy roots to be utilized in various sectors including food, medicines, cosmetics or agriculture but it is quite essential to understand the inter-relationships between the nanoparticles and the plant systems in terms of specifics such as type, dosage and time of exposure as well as other important parameters.

An atypical two-component system, AtcR/AtcK, simultaneously regulates the biosynthesis of multiple secondary metabolites in Streptomyces bingchenggensis.

Streptomyces bingchenggensis is an industrial producer of milbemycins, which are important anthelmintic and insecticidal agents. Two-component systems (TCSs), which are typically situated in the same operon and are composed of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Here, an atypical TCS, AtcR/AtcK, in which the encoding genes (sbi_06838/sbi_06839) are organized in a head-to-head pair, was demonstrated to be indispensable for the biosynthesis of multiple secondary metabolites in S. bingchenggensis. With the null TCS mutants, the production of milbemycin and yellow compound was abolished but nanchangmycin was overproduced. Transcriptional analysis and electrophoretic mobility shift assays showed that AtcR regulated the biosynthesis of these three secondary metabolites by a MilR3-mediated cascade. First, AtcR was activated by phosphorylation from signal-triggered AtcK. Second, the activated AtcR promoted the transcription of milR3. Third, MilR3 specifically activated the transcription of downstream genes from milbemycin and yellow compound biosynthetic gene clusters (BGCs) and nanR4 from the nanchangmycin BGC. Finally, because NanR4 is a specific repressor in the nanchangmycin BGC, activation of MilR3 downstream genes led to the production of yellow compound and milbemycin but inhibited nanchangmycin production. By rewiring the regulatory cascade, two strains were obtained, the yield of nanchangmycin was improved by 45-fold to 6.08 g/L and the production of milbemycin was increased twofold to 1.34 g/L. This work has broadened our knowledge on atypical TCSs and provided practical strategies to engineer strains for the production of secondary metabolites in Streptomyces.IMPORTANCEStreptomyces bingchenggensis is an important industrial strain that produces milbemycins. Two-component systems (TCSs), which consist of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Coupled encoding genes of TCSs are typically situated in the same operon. Here, TCSs with encoding genes situated in separate head-to-head neighbor operons were labeled atypical TCSs. It was found that the atypical TCS AtcR/AtcK played an indispensable role in the biosynthesis of milbemycin, yellow compound, and nanchangmycin in S. bingchenggensis. This atypical TCS regulated the biosynthesis of specialized metabolites in a cascade mediated via a cluster-situated regulator, MilR3. Through rewiring the regulatory pathways, strains were successfully engineered to overproduce milbemycin and nanchangmycin. To the best of our knowledge, this is the first report on atypical TCS, in which the encoding genes of RR and HK were situated in separate head-to-head neighbor operons, involved in secondary metabolism. In addition, data mining showed that atypical TCSs were widely distributed in actinobacteria.

Rhythmic histone acetylation acts in concert with day-night oscillation of the floral volatile metabolic network.

The biosynthesis of specialized metabolites is strictly regulated by environmental inputs such as the day-night cycle, but the underlying mechanisms remain elusive. In Petunia hybrida cv. Mitchell flowers, the biosynthesis and emission of volatile compounds display a diurnal pattern with a peak in the evening to attract nocturnal pollinators. Using petunia flowers as a model system, we found that chromatin level regulation, especially histone acetylation, plays an essential role in mediating the day-night oscillation of the biosynthetic gene network of specialized metabolites. By performing time-course chromatin immunoprecipitation assays for histone modifications, we uncovered that a specific group of genes involved in the regulation, biosynthesis, and emission of floral volatile compounds, which displays the greatest magnitude in day-night oscillating gene expression, is associated with highly dynamic histone acetylation marks H3K9ac and H3K27ac. Specifically, the strongest oscillating genes featured a drastic removal of histone acetylation marks at night, potentially to shut down the biosynthesis of floral volatile compounds during the morning when they are not needed. Inhibiting daytime histone acetylation led to a compromised evening induction of these genes. Overall, our study suggested an active role of chromatin modification in the diurnal oscillation of specialized metabolic network.

Heterologous expression of PtAAS1 reveals the metabolic potential of the common plant metabolite phenylacetaldehyde for auxin synthesis in planta.

Aromatic aldehydes and amines are common plant metabolites involved in several specialized metabolite biosynthesis pathways. Recently, we showed that the aromatic aldehyde synthase PtAAS1 and the aromatic amino acid decarboxylase PtAADC1 contribute to the herbivory-induced formation of volatile 2-phenylethanol and its glucoside 2-phenylethyl-β-D-glucopyranoside in Populus trichocarpa. To unravel alternative metabolic fates of phenylacetaldehyde and 2-phenylethylamine beyond alcohol and alcohol glucoside formation, we heterologously expressed PtAAS1 and PtAADC1 in Nicotiana benthamiana and analyzed plant extracts using untargeted LC-qTOF-MS and targeted LC-MS/MS analysis. While the metabolomes of PtAADC1-expressing plants did not significantly differ from those of control plants, expression of PtAAS1 resulted in the accumulation of phenylacetic acid (PAA) and PAA-amino acid conjugates, identified as PAA-aspartate and PAA-glutamate. Herbivory-damaged poplar leaves revealed significantly induced accumulation of PAA-Asp, while levels of PAA remained unaltered upon herbivory. Transcriptome analysis showed that members of auxin-amido synthetase GH3 genes involved in the conjugation of auxins with amino acids were significantly upregulated upon herbivory in P. trichocarpa leaves. Overall, our data indicates that phenylacetaldehyde generated by poplar PtAAS1 serves as a hub metabolite linking the biosynthesis of volatile, non-volatile herbivory-induced specialized metabolites, and phytohormones, suggesting that plant growth and defense can be balanced on a metabolic level.

Blueberry and cranberry pangenomes as a resource for future genetic studies and breeding efforts.

Domestication of cranberry and blueberry began in the United States in the early 1800s and 1900s, respectively, and in part owing to their flavors and health-promoting benefits are now cultivated and consumed worldwide. The industry continues to face a wide variety of production challenges (e.g. disease pressures), as well as a demand for higher-yielding cultivars with improved fruit quality characteristics. Unfortunately, molecular tools to help guide breeding efforts for these species have been relatively limited compared with those for other high-value crops. Here, we describe the construction and analysis of the first pangenome for both blueberry and cranberry. Our analysis of these pangenomes revealed both crops exhibit great genetic diversity, including the presence-absence variation of 48.4% genes in highbush blueberry and 47.0% genes in cranberry. Auxiliary genes, those not shared by all cultivars, are significantly enriched with molecular functions associated with disease resistance and the biosynthesis of specialized metabolites, including compounds previously associated with improving fruit quality traits. The discovery of thousands of genes, not present in the previous reference genomes for blueberry and cranberry, will serve as the basis of future research and as potential targets for future breeding efforts. The pangenome, as a multiple-sequence alignment, as well as individual annotated genomes, are publicly available for analysis on the Genome Database for Vaccinium-a curated and integrated web-based relational database. Lastly, the core-gene predictions from the pangenomes will serve useful to develop a community genotyping platform to guide future molecular breeding efforts across the family.

Reductases Produce Nitric Oxide in an Alternative Pathway to Form the Diazeniumdiolate Group of l-Alanosine.

l-Alanosine is a diazeniumdiolate (N-nitrosohydroxylamine) antibiotic that inhibits MTAP-deficient tumor cells by blocking de novo adenine biosynthesis. Previous work revealed the early steps in the biosynthesis of l-alanosine. In the present study, we used genome mining to discover two new l-alanosine-producing strains that lack the aspartate-nitrosuccinate pathway genes found in the original l-alanosine producer. Instead, nitrate is reduced with a unique set of nitrate-nitrite reductases. These enzymes are typically used as part of the nitrogen cycle for denitrification or assimilation, and our report here shows how enzymes from the nitrogen cycle can be repurposed for the biosynthesis of specialized metabolites. The widespread distribution of nitric-oxide-producing reductases also indicates a potential for the discovery of new nitric-oxide-derived natural products.

CRISPR/Cas9-mediated mutagenesis of the mediator complex subunits MED5a and MED5b genes impaired secondary metabolite accumulation in hop (Humulus lupulus)

Hop (Humulus lupulus L.) is an important commercial crop known for the biosynthesis of valuable specialized secondary metabolites in glandular trichomes (lupulin glands), which are used for the brewing industry. To achieve burgeoning market demands is the essentiality of comprehensive understanding of the mechanisms of biosynthesis of secondary metabolites in hop. Over the past year, several studies using structural biology and functional genomics approaches have shown that Mediator (MED) serves as an integrative hub for RNAP II-mediated transcriptional regulation of various physiological and cellular processes, including involvement of MED5a and MED5b in hyperaccumulation of phenylpropanoid in A. thaliana. In the present work, an unprecedented attempt was made to generate Hlmed5a/med5b double loci mutant lines in hop using a CRISPR/Cas9-based genome editing system. The Hlmed5a/med5b double loci mutant lines showed reduced expression of structural genes of the flavonoid, humulone, and terpenoid biosynthetic pathways, which was more pronounced in the lupulin gland compared to leaf tissue and was consistent with their reduced accumulation. Phenotypic and anatomical observations revealed that Hlmed5a/med5b double loci mutant line exhibited robust growth, earlier flowering, earlier cone maturity, reduced cone size, variations in floral structure patterns, and distorted lupulin glands without any remarkable changes in leaf morphology, intensity of leaf color, and chlorophyll content. Comparative transcriptome analysis of leaf and lupulin gland tissues indicates that the expression of enzymatic genes related to secondary metabolite biosynthesis, phytohormone biosynthesis, floral organs, flowering time, and trichome development, including other genes related to starch and sucrose metabolism and defense mechanisms, were differentially modulated in the Hlmed5a/med5b lines. The combined results from functional and transcriptomic analyses illuminates the pivotal function of HlMED5a and HlMED5b in homeostasis of secondary meatbolites accumulation in hop.

Polyploidy as a strategy to increase taxane production in yew cell cultures: Obtaining and characterizing a Taxus baccata tetraploid cell line

Novel approaches to optimize the production of plant specialized metabolites are crucial to reach maximum productivity of plant biofactories. Plant polyploidization frequently enhances protein synthesis and thereby increases the biosynthesis of specialized metabolites. Paclitaxel is a valuable anticancer agent scarcely produced in nature. Therefore, plant biofactories represent a sustainable alternative source of this compound and related taxanes. With the aim of improving the productivity of Taxus spp. cell cultures, we induced polyploidy in vitro by treating immature embryos of Taxus baccata with colchicine. To obtain the polyploid cell lines, calli were induced from T. baccata plantlets previously treated with colchicine and ploidy levels were accurately identified using flow cytometry. In terms of cell morphology, tetraploid cells were about 3-fold bigger than the diploid cells. The expression of taxane pathway genes was higher in the tetraploid cell line compared to the diploid cells. Moreover, taxane production was 6.2-fold higher and the production peak was achieved 8 days earlier than in the diploid cell line, indicating a higher productivity. The obtained tetraploid cell line proved to be highly productive, constituting a step forward towards the development of a bio-sustainable production system for this chemotherapeutic drug.

The transcription factor NtERF13a enhances abiotic stress tolerance and phenylpropanoid compounds biosynthesis in tobacco

The AP2/ERF (APETALA2/ETHYLENE RESPONSE FACTOR) transcription factors play multiple roles in modulating the biosynthesis of diverse specialized metabolites in response to various environmental stresses. ERF13 has been shown to participate in plant resistance to biotic stress as well as in repressing the synthesis of fatty acid. However, its full roles in regulating plant metabolism and stress resistance still remains to be further studied. In this study, we identified two NtERF genes from N. tabacum genome that belong to Ⅸa subgroup of ERF family. Over-expression and knock-out of NtERF13a showed that NtERF13a could enhance plant resistance to salt and drought stresses, as well as promoted the biosynthesis of chlorogenic acid (CGA), flavonoids, and lignin in tobacco. Transcriptome analysis between WT and NtERF13a-OE plants revealed 6 differentially expressed genes (DEGs) that encode enzymes catalyzing the key steps of phenylpropanoid pathway. Chromatin immunoprecipitation, Y1H, and Dual-Luc assays further clarified that NtERF13a could directly bind to the fragments containing GCC box or DRE element in the promoters of NtHCT, NtF3'H, and NtANS genes to induce the transcription of these genes. Knock-out of NtHCT, NtF3'H, or NtANS in the NtERF13a-OE background significantly repressed the increase of phenylpropanoid compound contents caused by over-expression of NtERF13a, indicating that the promotion of NtERF13a on the phenylpropanoid compound contents depends on the activity of NtHCT, NtF3'H, and NtANS. Our study demonstrated new roles of NtERF13a in promoting plant resistance to abiotic stresses, and provided a promising target for modulating the biosynthesis of phenylpropanoid compounds in tobacco.

The putative role of the epipeptide EpeX in Bacillus subtilis intra-species competition.

Bacteria engage in competitive interactions with neighbours that can either be of the same or different species. Multiple mechanisms are deployed to ensure the desired outcome and one tactic commonly implemented is the production of specialised metabolites. The Gram-positive bacterium Bacillus subtilis uses specialized metabolites as part of its intra-species competition determinants to differentiate between kin and non-kin isolates. It is, however, unknown if the collection of specialized metabolites defines competitive fitness when the two isolates start as a close, interwoven community that grows into a densely packed colony biofilm. Moreover, the identity of specialized metabolites that have an active role in defining the outcome of an intra-species interaction has not been revealed. Here, we determine the competition outcomes that manifest when 21 environmental isolates of B. subtilis are individually co-incubated with the model isolate NCIB 3610 in a colony biofilm. We correlated these data with the suite of specialized metabolite biosynthesis clusters encoded by each isolate. We found that the epeXEPAB gene cluster was primarily present in isolates with a strong competitive phenotype. This cluster is responsible for producing the epipeptide EpeX. We demonstrated that EpeX is a competition determinant of B. subtilis in an otherwise isogenic context for NCBI 3610. However, when we competed the NCIB 3610 EpeX-deficient strain against our suite of environmental isolates we found that the impact of EpeX in competition is isolate-specific, as only one of the 21 isolates showed increased survival when EpeX was lacking. Taken together, we have shown that EpeX is a competition determinant used by B. subtilis that impacts intra-species interactions but only in an isolate-specific manner.