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Phenazine-based small molecules are nitrogen-containing heterocyclic compounds with diverse bioactivities and electron transfer properties that exhibit promising applications in pharmaceutical and electrochemical industries. However, the biosynthetic mechanism of highly substituted natural phenazines remains poorly understood. In this study, we report the direct cloning and heterologous expression of the lomofungin biosynthetic gene cluster (BGC) from Streptomyces lomondensis S015. Reconstruction and overexpression of the BGCs in Streptomyces coelicolor M1152 resulted in eight phenazine derivatives including two novel hybrid phenazine metabolites, and the biosynthetic pathway of lomofungin was proposed. Furthermore, gene deletion suggested that NAD(P)H-dependent oxidoreductase gene lomo14 is a nonessential gene in the biosynthesis of lomofungin. Cytotoxicity evaluation of the isolated phenazines and lomofungin was performed. Specifically, lomofungin shows substantial inhibition against two human cancer cells, HCT116 and 5637. These results provide insights into the biosynthetic mechanism of lomofungin, which will be useful for the directed biosynthesis of natural phenazine derivatives.

Alternaria species (Deuteromycetes, Ascomycota) as ubiquitous fungi and prolific producers of a variety of toxic compounds are a part of microbiomes of plants, humans, and animals, mainly causing disease, allergic reactions, and toxicosis. However, some species have also been reported as endophytic microorganisms with highly bioactive metabolites. Our previous results indicate that potentially endophytic Alternaria species from Cupressaceae produce bioactive metabolites that possibly contribute to plant holobiont's health. Here, a possible mechanism behind this bioactivity is elucidated. As some endophytic fungi are reported to produce cytotoxic taxane diterpenoids, eight potentially endophytic Alternaria isolates from our collection were analyzed for the presence of the key genes of the paclitaxel (Taxol) biosynthetic pathway, i.e., taxadin synthase (ts), 10-deacetylbaccatin III-10-O-acetyltransferase (dbat), and C-13-phenylpropanoid side-chain CoA acyltransferase (bapt). The presence of all genes, i.e., ts, dbat, and bapt, was detected by PCR in six isolates and dbat and bapt in two isolates. Chemical analyses of the fermentation broths by TLC and HPLC chromatography and IR spectroscopy indicated the synthesis of the final product, i.e., paclitaxel. So, we introduce the synthesis of taxane diterpenoids as a possible mechanism by which Alternaria occupies the plant niches and protects the plant holobiont in the presence of competing microorganisms., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nickel (Ni) is an abundant element on Earth and it can be toxic to all forms of life. Unlike our knowledge of other metals, little is known about the biochemical response to Ni overload. Previous studies in mammals have shown that Ni induces various physiological changes including redox stress, hypoxic responses, as well as cancer progression pathways. However, the primary cellular targets of nickel toxicity are unknown. Here, we used the environmental fungus Cryptococcus neoformans as a model organism to elucidate the cellular response to exogenous Ni. We discovered that Ni causes alterations in ergosterol (the fungal equivalent of mammalian cholesterol) and lipid biosynthesis, and that the Sterol Regulatory Element-Binding transcription factor Sre1 is required for Ni tolerance. Interestingly, overexpression of the C-4 methyl sterol oxidase gene ERG25, but not other genes in the ergosterol biosynthesis pathway tested, increases Ni tolerance in both the wild type and the sre1Δ mutant. Overexpression of ERG25 with mutations in the predicted binding pocket to a metal cation cofactor sensitizes Cryptococcus to nickel and abolishes its ability to rescue the Ni-induced growth defect of sre1Δ. As overexpression of a known nickel-binding protein Ure7 or Erg3 with a metal binding pocket similar to Erg25 does not impact on nickel tolerance, Erg25 does not appear to simply act as a nickel sink. Furthermore, nickel induces more profound and specific transcriptome changes in ergosterol biosynthetic genes compared to hypoxia. We conclude that Ni targets the sterol biosynthesis pathway primarily through Erg25 in fungi. Similar to the observation in C. neoformans, Ni exposure reduces sterols in human A549 lung epithelial cells, indicating that nickel toxicity on sterol biosynthesis is conserved., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Matha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bioinformatic analysis revealed that the genomes of ubiquitous Penicillium spp. might carry dozens of biosynthetic gene clusters (BGCs), yet many clusters have remained uncharacterized. In this study, a detailed investigation of co-culture fermentation including the basidiomycete Armillaria mellea CPCC 400891 and the P. brasilianum CGMCC 3.4402 enabled the isolation of five new compounds including two bisabolene-type sesquiterpenes (arpenibisabolanes A and B), two carotane-type sesquiterpenes (arpenicarotanes A and B), and one polyketide (arpenichorismite A) along with seven known compounds. The assignments of their structures were deduced by the extensive analyses of detailed spectroscopic data, electronic circular dichroism spectra, together with delimitation of the biogenesis. Most new compounds were not detected in monocultures under the same fermentation conditions. Arpenibisabolane A represents the first example of a 6/5-fused bicyclic bisabolene. The bioassay of these five new compounds exhibited no cytotoxic activities in vitro against three human cancer cell lines (A549, MCF-7, and HepG2). Moreover, sequence alignments and bioinformatic analysis to other metabolic pathways, two BGCs including Pb-bis and Pb-car, responsible for generating sesquiterpenoids from co-culture were identified, respectively. Furthermore, based on the chemical structures and deduced gene functions of the two clusters, a hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed. These results demonstrated that the co-culture approach would facilitate bioprospecting for new metabolites even from the well-studied microbes. Our findings would provide opportunities for further understanding of the biosynthesis of intriguing sesquiterpenoids via metabolic engineering strategies. KEY POINTS: • Penicillium and Armillaria co-culture facilitates the production of diverse secondary metabolites • Arpenibisabolane A represents the first example of 6/5-fused bicyclic bisabolenes • A hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed., (© 2024. The Author(s).)

Episomal AMA1-based plasmids are increasingly used for expressing biosynthetic pathways and CRISPR/Cas systems in filamentous fungi cell factories due to their high transformation efficiency and multicopy nature. However, the gene expression from AMA1 plasmids has been observed to be highly heterogeneous in growing mycelia. To overcome this limitation, here we developed next-generation AMA1-based plasmids that ensure homogeneous and strong expression. We achieved this by evaluating various degradation tags fused to the auxotrophic marker gene on the AMA1 plasmid, which introduces a more stringent selection pressure throughout multicellular fungal growth. With these improved plasmids, we observed in Aspergillus nidulans a 5-fold increase in the expression of a fluorescent reporter, a doubling in the efficiency of a CRISPRa system for genome mining, and a up to a 10-fold increase in the production of heterologous natural product metabolites. This strategy has the potential to be applied to diverse filamentous fungi., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Previous studies have reported the functional role, biochemical features and synthesis pathway of podophyllotoxin (PTOX) in plants. In this study, we employed combined morphological and molecular techniques to identify an endophytic fungus and extract PTOX derivatives. Based on the analysis of ITS sequences and the phylogenetic tree, the isolate was classified as Penicillium herquei HGN12.1C, with a sequence identity of 98.58%. Morphologically, the HGN12.1C strain exhibits white colonies, short-branched mycelia and densely packed hyphae. Using PacBio sequencing at an average read depth of 195×, we obtained a high-quality genome for the HGN12.1C strain, which is 34.9 Mb in size, containing eight chromosomes, one mitochondrial genome and a GC content of 46.5%. Genome analysis revealed 10 genes potentially involved in PTOX biosynthesis. These genes include VdtD, Pinoresinollariciresinol reductase (PLR), Secoisolariciresinol dehydrogenase (SDH), CYP719A23, CYP71BE54, O-methyltransferase 1 (OMT1), O-methyltransferase 3 (OMT3), 2-ODD, CYP71CU and CYP82D61. Notably, the VdtD gene in fungi shares functional similarities with the DIR gene found in plants. Additionally, we identified peltatin, a PTOX derivative, in the HGN12.1C extract. Docking analysis suggests a potential role for the 2-ODD enzyme in converting yatein to deoxypodophyllotoxin. These findings offer invaluable insights into the synthesis mechanism of PTOX in fungi, shedding light on the relationship between host plants and endophytes., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Actinobacteria are renowned for their prolific production of diverse bioactive secondary metabolites. In recent years, there has been an increasing focus on exploring "rare" genera within this phylum for biodiscovery purposes, notably the Nocardiopsis genus, which will be the subject of the present study. Recognizing the absence of articles describing the research process of finding bioactive molecules from the genus Nocardiopsis in North African environments. We, therefore, present a historical overview of the discoveries of bioactive molecules of the genus Nocardiopsis originating from the region, highlighting their biological activities and associated reported molecules, providing a snapshot of the current state of the field, and offering insights into future opportunities and challenges for drug discovery. Additionally, we present a genome mining analysis of three genomes deposited in public databases that have been reported to be bioactive. A total of 36 biosynthetic gene clusters (BGCs) were identified, including those known to encode bioactive molecules. Notably, a substantial portion of the BGCs showed little to no similarity to those previously described, suggesting the possibility that the analyzed strains could be potential producers of new compounds. Further research on these genomes is essential to fully uncovering their biotechnological potential. Moving forward, we discuss the experimental designs adopted in the reported studies, as well as new avenues to guide the exploration of the Nocardiopsis genus in North Africa., (© 2024 Wiley‐VCH GmbH.)

This study investigates the transition of Rosa canina L. petals from pink to white, driven by genetic and biochemical factors. It characterizes the expression of ten key genes involved in anthocyanin and flavonoid biosynthesis across five developmental stages, correlating gene expression with flavonoid and anthocyanin concentrations and colorimetric changes. Initially, the petals exhibit a rich flavonoid profile, dominated by Rutin and Kaempferol derivatives. The peak anthocyanin concentration, corresponding to the deepest color saturation, occurs in the subsequent stage. Advanced chromatographic analyses identify key flavonoids persisting into the final white petal stage. Notably, the ANS gene shows a dramatic 137.82-fold increase in expression at the final stage, indicating its crucial role in petal color maturation despite the absence of visible pigmentation. The study provides a comprehensive characterization of the genetic and biochemical mechanisms underlying petal pigmentation, suggesting that reduced anthocyanin synthesis and increased flavonol concentration led to white petals. It also highlights the roles of other genes such as PAL, CCD1, FLS, CHI, CHS, UFGT, F3H, DFR, and RhMYB1, indicating that post-translational modifications and other regulatory mechanisms may influence anthocyanin stability and degradation., Competing Interests: Declaration of competing interest All the authors listed have agreed with the submission and declared no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cananga odorata is known as a natural perfume tree of the Annonaceae family in Magnoliales. However, its phylogenetic position and the molecular mechanisms involved in the biosynthesis of the floral volatile organic compounds (VOCs) remain unclear. Here, by combining a variety of sequencing platforms, we present a telomere-to-telomere (T2T) genome of C. odorata with 735.83 Mb, which represents the highest integrity and assembly quality of genome in magnoliid plants reported to date. Phylogenetic analysis based on multiple datasets and approaches showed that C. odorata, as a member of magnoliids, is sister to eudicots, after their divergence from monocots. Metabolomic of VOCs in the essential oil and flowers scent showed that sesquiterpenes, especially β-caryophyllene, were the major compounds. Two CoTPS21 homologues derived from tandem duplication events were highly expressed during flower development and were identified as the key sesquiterpene synthases for the production of β-caryophyllene. In addition, CoSPL3 and CoSPL9 were considered as potential transcription factors for activating the expression of CoTPS21 homologues. Our results shed light on the molecular mechanisms underlying the biosynthesis of the unique floral fragrance in C. odorata and provide new insights into the phylogenetic position of magnoliids., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Background and Research Purpose: Paeoniflorin and albiflorin are monoterpene glycosides that exhibit various medicinal properties in Paeonia species. This study explored the terpene biosynthesis pathway and analyzed the distribution of these compounds in different tissues of two Korean landraces of Paeonia lactiflora to gain insights into the biosynthesis of monoterpene glycosides in P. lactiflora and their potential applications., Materials and Methods: Two Korean landraces, Hongcheon var. and Hwacheon var, of P. lactiflora were used for the analyses. Contents of the paeoniflorin and albiflorin were analyzed using HPLC. RNA was extracted, sequenced, and subjected to transcriptome analysis. Differential gene expression, KEGG, and GO analyses were performed. Paeoniflorin biosynthesis genes were isolated from the transcriptomes using the genes in Euphorbia maculata with the NBLAST program. Phylogenetic analysis of of 1-Deoxy-D-xylulose 5-phosphate synthase (DOXPS), geranyl pyrophosphate synthase (GPPS), and pinene synthase (PS) was carried out with ClustalW and MEGA v5.0., Results and Discussion: Analysis of paeoniflorin and albiflorin content in different tissues of the two P. lactiflora landraces revealed significant variation. Transcriptome analysis yielded 36,602 unigenes, most of which were involved in metabolic processes. The DEG analysis revealed tissue-specific expression patterns with correlations between landraces. The isolation of biosynthetic genes identified 173 candidates. Phylogenetic analysis of the key enzymes in these pathways provides insights into their evolutionary relationships. The sequencing and analysis of DOXPS, GPPS, PS revealed distinct clades and subclades, highlighting their evolutionary divergence and functional conservation. Our findings highlight the roots as the primary sites of paeoniflorin and albiflorin accumulation in P. lactiflora, underscoring the importance of tissue-specific gene expression in their biosynthesis., Conclusion: this study advances our understanding of monoterpene glycoside production and distribution in Paeonia, thereby guiding further plant biochemistry investigations., (© 2024. The Author(s) under exclusive licence to The Genetics Society of Korea.)

The Fusarium solani species complex (FSSC) is comprised of important pathogens of plants and humans. A distinctive feature of FSSC species is perithecial pigmentation. While the dark perithecial pigments of other Fusarium species are derived from fusarubins synthesized by polyketide synthase 3 (PKS3), the perithecial pigments of FSSC are derived from an unknown metabolite synthesized by PKS35. Here, we confirm in FSSC species Fusarium vanettenii that PKS35 (fsnI) is required for perithecial pigment synthesis by deletion analysis and that fsnI is closely related to phnA from Penicillium herquei, as well as duxI from Talaromyces stipentatus, which produce prephenalenone as an early intermediate in herqueinone and duclauxin synthesis respectively. The production of prephenalenone by expression of fsnI in Saccharomyces cerevisiae indicates that it is also an early intermediate in perithecial pigment synthesis. We next identified a conserved cluster of 10 genes flanking fsnI in F. vanettenii that when expressed in F. graminearum led to the production of a novel corymbiferan lactone F as a likely end product of the phenalenone biosynthetic pathway in FSSC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Saccharomyces cerevisiae is commonly used as a microbial cell factory to produce high-value compounds or bulk chemicals due to its genetic operability and suitable intracellular physiological environment. The current biosynthesis pathway for targeted products is primarily rewired in the cytosolic compartment. However, the related precursors, enzymes, and cofactors are frequently distributed in various subcellular compartments, which may limit targeted compounds biosynthesis. To overcome above mentioned limitations, the biosynthesis pathways are localized in different subcellular organelles for product biosynthesis. Subcellular compartmentalization in the production of targeted compounds offers several advantages, mainly relieving competition for precursors from side pathways, improving biosynthesis efficiency in confined spaces, and alleviating the cytotoxicity of certain hydrophobic products. In recent years, subcellular compartmentalization in targeted compound biosynthesis has received extensive attention and has met satisfactory expectations. In this review, we summarize the recent advances in the compartmentalized biosynthesis of the valuable compounds in S. cerevisiae, including terpenoids, sterols, alkaloids, organic acids, and fatty alcohols, etc. Additionally, we describe the characteristics and suitability of different organelles for specific compounds, based on the optimization of pathway reconstruction, cofactor supplementation, and the synthesis of key precursors (metabolites). Finally, we discuss the current challenges and strategies in the field of compartmentalized biosynthesis through subcellular engineering, which will facilitate the production of the complex valuable compounds and offer potential solutions to improve product specificity and productivity in industrial processes., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Artemisia argyi is an herbaceous plant of the genus Artemisia. Its young and mature leaves are used as food and medicine, respectively. Glandular trichomes (GTs) are distributed on the leaf surface in A. argyi and are generally considered the location of flavonoid biosynthesis and accumulation. However, the mechanism of flavonoid biosynthesis and accumulation in A. argyi remains unclear. In this study, the coregulatory genes involved in flavonoid biosynthesis and trichome development in this species were screened and evaluated, and the biosynthetic pathways for key flavonoids in A. argyi were uncovered. AaMYB1 and AaYABBY1 were screened using weighted gene co-expression network analysis, and both genes were then genetically transformed into Nicotiana tabacum L. cv. K326 (tobacco). Simultaneously, AaYABBY1 was also genetically transformed into Arabidopsis thaliana. The total flavonoid and rutin contents were increased in tobacco plants overexpressing AaMYB1 and AaYABBY1, and the expression levels of genes participating in the flavonoid synthesis pathway, such as PAL, FLS, and F3H, were significantly up-regulated in plants overexpressing these genes. These results indicated that AaMYB1 and AaYABBY1 promote flavonoid biosynthesis in tobacco. Furthermore, compared to that in the wild-type, the trichome density was significantly increased in tobacco and A. thaliana plants overexpressing AaYABBY1. These results confirm that AaYABBY1 might be involved in regulating trichome formation in A. argyi. This indicates the potential genes involved in and provides new insights into the development of trichome cellular factories based on the "development-metabolism" interaction network and the cultivation of high-quality A. argyi., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lycoris radiata is the main source of galanthamine, a clinical drug used in Alzheimer's disease; however, the galanthamine content in L. radiata is low. Lycoris aurea is another Lycoris species with high galanthamine content. Fungal endophytes can enhance plant secondary metabolite accumulation; thus, we compared the fungal communities in these two Lycoris species to identify certain fungal taxa in L. aurea capable of enhancing galanthamine accumulation. Several fungal endophytes, which were enriched in, exclusively isolated from L. aurea, or showed significant correlations with galanthamine, were demonstrated to enhance the accumulation of only galanthamine but no other Amaryllidaceae alkaloids (AAs) in L. radiata. These fungal endophytes mainly upregulated the downstream genes in the biosynthesis pathways of AAs in L. radiata, suggesting that they may allocate more precursors for galanthamine biosynthesis. This study demonstrated that fungal endophytes from L. aurea with higher galanthamine content can specifically enhance the accumulation of this medicinal alkaloid in other Lycoris species, thereby increasing the galanthamine source and reducing galanthamine separation and purification costs. This study broadens our understanding of the complex interactions between plant secondary metabolites and fungal endophytes., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The genetic control underlying natural variation in lignin content and composition in trees is not fully understood. We performed a systems genetic analysis to uncover the genetic regulation of lignin biosynthesis in a natural 'SwAsp' population of aspen (Populus tremula) trees. We analyzed gene expression by RNA sequencing (RNA-seq) in differentiating xylem tissues, and lignin content and composition using Pyrolysis-GC-MS in mature wood of 268 trees from 99 genotypes. Abundant variation was observed for lignin content and composition, and genome-wide association study identified proteins in the pentose phosphate pathway and arabinogalactan protein glycosylation among the top-ranked genes that are associated with these traits. Variation in gene expression and the associated genetic polymorphism was revealed through the identification of 312 705 local and 292 003 distant expression quantitative trait loci (eQTL). A co-expression network analysis suggested modularization of lignin biosynthesis and novel functions for the lignin-biosynthetic CINNAMYL ALCOHOL DEHYDROGENASE 2 and CAFFEOYL-CoA O-METHYLTRANSFERASE 3. PHENYLALANINE AMMONIA LYASE 3 was co-expressed with HOMEOBOX PROTEIN 5 (HB5), and the role of HB5 in stimulating lignification was demonstrated in transgenic trees. The systems genetic approach allowed linking natural variation in lignin biosynthesis to trees´ responses to external cues such as mechanical stimulus and nutrient availability., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Achyranthes aspera is renowned for its rich medicinal properties since the Ayurvedic era. This plant is known for the presence of experimentally validated anticancer compounds like oleanolic acid (OA) and ursolic acid (UA). Our study involved sequencing the RNA from the root tissue of A. aspera to elucidate the genes responsible for synthesizing these two critical secondary metabolites. Through RNA-Seq analysis, we assembled approximately 167,698 transcripts, averaging 847 base pairs in length, with an N50 value of 1509 bp. From this data, we mapped 604 sequences involved in the metabolism of terpenoids and polyketide pathways. Among them, 241 transcripts were mapped to the triterpenoid biosynthesis pathway, which included 127 transcripts involved in OA and UA biosynthesis. From these transcripts, we identified 22 full-length genes coding for all the 21 enzymes required for OA and UA biosynthesis. Identifying these full-length genes will lead to a better understanding of the pathway and adopting genetic engineering approaches., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Golden Gate Assembly (GGA) represents a versatile method for assembling multiple DNA fragments into a single molecule, which is widely used in rapid construction of complex expression cassettes for metabolic engineering. Here we describe the GGA method for facile construction and optimization of lycopene biosynthesis pathway by the combinatorial assembly of different transcriptional units (TUs). Furthermore, we report the method for characterizing and improving lycopene production in the synthetic yeast chassis., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

With the rise of antimicrobial resistance, there is high demand for novel antimicrobials to combat multi-drug resistant pathogens. The bacterial genus Pantoea produces a diversity of antimicrobial natural products effective against a wide range of bacterial and fungal targets. These antimicrobials are synthesized by specialized biosynthetic gene clusters that have unique distributions across Pantoea as well as several other genera outside of the Erwiniaceae. Phylogenetic and genomic evidence shows that these clusters can mobilize within and between species and potentially between genera. Pantoea antimicrobials belong to unique structural classes with diverse mechanisms of action, but despite their potential in antagonizing a wide variety of plant, human, and animal pathogens, little is known about many of these metabolites and how they function. This review will explore the known antimicrobials produced by Pantoea: agglomerins, andrimid, D-alanylgriseoluteic acid, dapdiamide, herbicolins, pantocins, and the various Pantoea Natural Products (PNPs). It will include information on the structure of each compound, their genetic basis, biosynthesis, mechanism of action, spectrum of activity, and distribution, highlighting the significance of Pantoea antimicrobials as potential therapeutics and for applications in biocontrol., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Mycobacterium tuberculosis 's ( Mtb ) autarkic lifestyle within the host involves rewiring its transcriptional networks to combat host-induced stresses. With the help of RNA sequencing performed under various stress conditions, we identified that genes belonging to Mtb sulfur metabolism pathways are significantly upregulated during oxidative stress. Using an integrated approach of microbial genetics, transcriptomics, metabolomics, animal experiments, chemical inhibition, and rescue studies, we investigated the biological role of non-canonical L-cysteine synthases, CysM and CysK2. While transcriptome signatures of RvΔcysM and RvΔcysK2 appear similar under regular growth conditions, we observed unique transcriptional signatures when subjected to oxidative stress. We followed pool size and labelling ( 34 S) of key downstream metabolites, viz. mycothiol and ergothioneine, to monitor L-cysteine biosynthesis and utilization. This revealed the significant role of distinct L-cysteine biosynthetic routes on redox stress and homeostasis. CysM and CysK2 independently facilitate Mtb survival by alleviating host-induced redox stress, suggesting they are not fully redundant during infection. With the help of genetic mutants and chemical inhibitors, we show that CysM and CysK2 serve as unique, attractive targets for adjunct therapy to combat mycobacterial infection., Competing Interests: MK, DH, BS, YK, NS, DP, SD, DS, Ld, VN No competing interests declared, (© 2023, Khan et al.)

Bacteria embellish their cell envelopes with a variety of specialized polysaccharides. Biosynthesis pathways for these glycans are complex, and final products vary greatly in their chemical structures, physical properties, and biological activities. This tremendous diversity comes from the ability to arrange complex pools of monosaccharide building blocks into polymers with many possible linkage configurations. Due to the complex chemistry of bacterial glycans, very few biosynthetic pathways have been defined in detail. As part of an initiative to characterize novel polysaccharide biosynthesis enzymes, we isolated a bacterium from Lake Michigan called Sphingomonas sp. LM7 that is proficient in exopolysaccharide (EPS) production. We identified genes that contribute to EPS biosynthesis in LM7 by screening a transposon mutant library for colonies displaying altered colony morphology. A gene cluster was identified that appears to encode a complete wzy/wzx- dependent polysaccharide assembly pathway. Deleting individual genes in this cluster caused a non-mucoid phenotype and a corresponding loss of EPS secretion, confirming the role of this gene cluster in polysaccharide production. We extracted EPS from LM7 cultures and determined that it contains a linear chain of 3- and 4-linked glucose, galactose, and glucuronic acid residues. Finally, we show that the EPS pathway in Sphingomonas sp. LM7 diverges from that of sphingan-family EPSs and adhesive polysaccharides such as the holdfast that are present in other Alphaproteobacteria . Our approach of characterizing complete biosynthetic pathways holds promise for engineering polysaccharides with valuable properties., Importance: Bacteria produce complex polysaccharides that serve a range of biological functions. These polymers often have properties that make them attractive for industrial applications, but they remain woefully underutilized. In this work, we studied a novel polysaccharide called promonan that is produced by Sphingomonas sp. LM7, a bacterium we isolated from Lake Michigan. We extracted promonan from LM7 cultures and identified which sugars are present in the polymer. We also identified the genes responsible for polysaccharide production. Comparing the promonan genes to those of other bacteria showed that promonan is distinct from previously characterized polysaccharides. We conclude by discussing how the promonan pathway could be used to produce new polysaccharides through genetic engineering., Competing Interests: The authors declare no conflict of interest.

So far, a variety of metabolite components of kiwifruit have been elucidated. However, the identification and analysis of flavonoids in different tissues of kiwifruit are rarely carried out. In this study, we performed transcriptome and metabolome analyses of roots (Gkf_R), stems (Gkf_T), leaves (Gkf_L), and fruits (Gkf_F) to provide insights into the differential accumulation and regulation mechanisms of flavonoids in kiwifruit. Results showed that a total of 301 flavonoids were identified, in four tissues with different accumulation trends, and a large proportion of flavonoids had high accumulation in Gkf_L and Gkf_R. A total of 84 genes have been identified involved in the flavonoid biosynthesis pathway, and the expression levels of five LAR, two DFR, and one HCT were significantly correlated with the accumulation of 16 flavonoids and co-localized in the flavonoid biosynthesis pathway. In addition, a total of 2362 transcription factor genes were identified, mainly MYBs, bHLHs, ERFs, bZIPs and WRKYs, among which the expression level of bHLH74, RAP2.3L/4L/10L, MYB1R1, and WRKY33 were significantly correlated with 25, 56, 43, and 24 kinds of flavonoids. Our research will enrich the metabolomic data and provide useful information for the directed genetic improvement and application in the pharmaceutical industry of kiwifruit., (© 2024. The Author(s).)

Nicotinamide mononucleotide (NMN) serves as a precursor for NAD + synthesis and has been shown to have positive effects on the human body. Previous research has predominantly focused on the nicotinamide phosphoribosyltransferase-mediated route (NadV-mediated route) for NMN biosynthesis. In this study, we have explored the de novo NMN biosynthesis route as an alternative pathway to enhance NMN production. Initially, we systematically engineered Escherichia coli to enhance its capacity for NMN synthesis and accumulation, resulting in a remarkable over 100-fold increase in NMN yield. Subsequently, we progressively enhanced the de novo NMN biosynthesis route to further augment NMN production. We screened and identified the crucial role of MazG in catalyzing the enzymatic cleavage of NAD + to NMN. And the de novo NMN biosynthesis route was optimized and integrated with the NadV-mediated NMN biosynthetic pathways, leading to an intracellular concentration of 844.10 ± 17.40 μM NMN. Furthermore, the introduction of two transporters enhanced the uptake of NAM and the excretion of NMN, resulting in NMN production of 1293.73 ± 61.38 μM. Finally, by engineering an E. coli strain with optimized PRPP synthetase, we achieved the highest NMN production, reaching 3067.98 ± 27.25 μM after 24 h of fermentation at the shake flask level. In addition to constructing an efficient E. coli cell factory for NMN production, our findings provide new insights into understanding the NAD + salvage pathway and its role in energy metabolism within E. coli .

Mucormycoses are emerging fungal infections caused by a variety of heterogeneous species within the Mucorales order. Among the Mucor species complex, Mucor circinelloides is the most frequently isolated pathogen in mucormycosis patients and despite its clinical significance, there is an absence of established genome manipulation techniques to conduct molecular pathogenesis studies. In this study, we generated a spontaneous uracil auxotrophic strain and developed a genetic transformation procedure to analyze molecular mechanisms conferring antifungal drug resistance. With this new model, phenotypic analyses of gene deletion mutants were conducted to define Erg3 and Erg6a as key biosynthetic enzymes in the M. circinelloides ergosterol pathway. Erg3 is a C-5 sterol desaturase involved in growth, sporulation, virulence, and azole susceptibility. In other fungal pathogens, erg3 mutations confer azole resistance because Erg3 catalyzes the production of a toxic diol upon azole exposure. Surprisingly, M. circinelloides produces only trace amounts of this toxic diol and yet, it is still susceptible to posaconazole and isavuconazole due to alterations in membrane sterol composition. These alterations are severely aggravated by erg3 Δ mutations, resulting in ergosterol depletion and, consequently, hypersusceptibility to azoles. We also identified Erg6a as the main C-24 sterol methyltransferase, whose activity may be partially rescued by the paralogs Erg6b and Erg6c. Loss of Erg6a function diverts ergosterol synthesis to the production of cholesta-type sterols, resulting in resistance to amphotericin B. Our findings suggest that mutations or epimutations causing loss of Erg6 function may arise during human infections, resulting in antifungal drug resistance to first-line treatments against mucormycosis., Importance: The Mucor species complex comprises a variety of opportunistic pathogens known to cause mucormycosis, a potentially lethal fungal infection with limited therapeutic options. The only effective first-line treatments against mucormycosis consist of liposomal formulations of amphotericin B and the triazoles posaconazole and isavuconazole, all of which target components within the ergosterol biosynthetic pathway. This study uncovered M. circinelloides Erg3 and Erg6a as key enzymes to produce ergosterol, a vital constituent of fungal membranes. Absence of any of those enzymes leads to decreased ergosterol and consequently, resistance to ergosterol-binding polyenes such as amphotericin B. Particularly, losing Erg6a function poses a higher threat as the ergosterol pathway is channeled into alternative sterols similar to cholesterol, which maintain membrane permeability. As a result, erg6a mutants survive within the host and disseminate the infection, indicating that Erg6a deficiency may arise during human infections and confer resistance to the most effective treatment against mucormycoses., Competing Interests: The authors declare no conflict of interest.

Dendrobium species, which are perennial herbs widely distributed in tropical and subtropical regions, are notable for their therapeutic properties attributed to various bioactive compounds, including dendrobine-type sesquiterpenoid alkaloids (DTSAs). The objective of this review article is to provide a comprehensive overview of recent advances in the biosynthesis of DTSAs, including their extraction from Dendrobium species and endophytes, elucidation of associated genes through genomic and transcriptomic sequencing in both Dendrobium spp. and endophytes, exploration of the biosynthetic pathways of DTSAs, and drawing conclusions and outlining future perspectives in this field. Alkaloids, predominantly nitrogen-containing compounds found in medicinal orchids, include over 140 types discovered across more than 50 species. DTSAs, identified in 37 picrotoxane alkaloids, have a distinctive five-membered nitrogen heterocyclic ring. This review highlights endophytic fungi as alternative sources of DTSAs, emphasizing their potential in pharmaceutical applications when plant-derived compounds are scarce or complex. Genomic and transcriptomic sequencing of Dendrobium spp. and their endophytes has identified key genes involved in DTSAs biosynthesis, elucidating pathways such as the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways. Genes encoding enzymes, such as acetyl-CoA C-acetyltransferase and diphosphomevalonate decarboxylase, are positively associated with dendrobine production. Despite significant advancements, the complexity of terpenoid biosynthesis in different subcellular compartments remains a challenge. Future research should focus on leveraging high-quality genomic data and omics technologies to further understand and manipulate the biosynthetic pathways of DTSAs and enhance their medicinal use.

Complex coumarins (CCs) represent characteristic metabolites found in Apiaceae plants, possessing significant medical value. Their essential functional role is likely as protectants against pathogens and regulators responding to environmental stimuli. Utilizing genomes and transcriptomes from 34 Apiaceae plants, including our recently sequenced Peucedanum praeruptorum, we conduct comprehensive phylogenetic analyses to reconstruct the detailed evolutionary process of the CC biosynthetic pathway in Apiaceae. Our results show that three key enzymes - p-coumaroyl CoA 2'-hydroxylase (C2'H), C-prenyltransferase (C-PT), and cyclase - originated successively at different evolutionary nodes within Apiaceae through various means of gene duplications: ectopic and tandem duplications. Neofunctionalization endows these enzymes with novel functions necessary for CC biosynthesis, thus completing the pathway. Candidate genes are cloned for heterologous expression and subjected to in vitro enzymatic assays to test our hypothesis regarding the origins of the key enzymes, and the results precisely validate our evolutionary inferences. Among the three enzymes, C-PTs are likely the primary determinant of the structural diversity of CCs (linear/angular), due to divergent activities evolved to target different positions (C-6 or C-8) of umbelliferone. A key amino acid variation (Ala161/Thr161) is identified and proven to play a crucial role in the alteration of enzymatic activity, possibly resulting in distinct binding forms between enzymes and substrates, thereby leading to different products. In conclusion, this study provides a detailed trajectory for the establishment and evolution of the CC biosynthetic pathway in Apiaceae. It explains why only a portion, not all, of Apiaceae plants can produce CCs and reveals the mechanisms of CC structural diversity among different Apiaceae plants., (© 2024. The Author(s).)

Solanum xanthocarpum fruits are used in the treatment of cough, fever, and heart disorders. It possesses antipyretic, hypotensive, antiasthmatic, aphrodisiac and antianaphylactic properties. In the present study, 24 elicitors (both biotic and abiotic) were used to enhance the production of glycoalkaloids in cell cultures of S. xanthocarpum. Four concentrations of elicitors were added into the MS culture medium. The maximum accumulation (5.56-fold higher than control) of demissidine was induced by sodium nitroprusside at 50 mM concentration whereas the highest growth of cell biomass (4.51-fold higher than control) stimulated by systemin at 30 mM concentration. A total of 17 genes of biosynthetic pathways of glycoalkaloids were characterized from the cells of S. xanthocarpum. The greater accumulation of demissidine was confirmed with the expression analysis of 11 key biosynthetic pathway enzymes e.g., acetoacetic-CoA thiolase, 3- hydroxy 3-methyl glutaryl synthase, β-hydroxy β-methylglutaryl CoA reductase, mevalonate kinase, farnesyl diphosphate synthase, squalene synthase, squalene epoxidase, squalene-2,3- epoxide cyclase, cycloartenol synthase, UDP-glucose: solanidine glucosyltransferase and UDP-rhamnose: solanidine rhamno-galactosyl transferase. The maximum expression levels of UDP-rhamnose: solanidine rhamno-galactosyl transferase gene was recorded in this study., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Naringenin is a plant polyphenol, widely explored due to its interesting biological activities, namely anticancer, antioxidant, and anti-inflammatory. Due to its potential applications and attempt to overcome the industrial demand, there has been an increased interest in its heterologous production. The microbial biosynthetic pathway to produce naringenin is composed of tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Herein, we targeted the efficient de novo production of naringenin in Escherichia coli by performing a step-by-step validation and optimization of the pathway. For that purpose, we first started by expressing two TAL genes from different sources in three different E. coli strains. The highest p-coumaric acid production (2.54 g/L) was obtained in the tyrosine-overproducing M-PAR-121 strain carrying TAL from Flavobacterium johnsoniae (FjTAL). Afterwards, this platform strain was used to express different combinations of 4CL and CHS genes from different sources. The highest naringenin chalcone production (560.2 mg/L) was achieved by expressing FjTAL combined with 4CL from Arabidopsis thaliana (At4CL) and CHS from Cucurbita maxima (CmCHS). Finally, different CHIs were tested and validated, and 765.9 mg/L of naringenin was produced by expressing CHI from Medicago sativa (MsCHI) combined with the other previously chosen genes. To our knowledge, this titer corresponds to the highest de novo production of naringenin reported so far in E. coli. KEY POINTS: • Best enzyme and strain combination were selected for de novo naringenin production. • After genetic and operational optimizations, 765.9 mg/L of naringenin was produced. • This de novo production is the highest reported so far in E. coli., (© 2024. The Author(s).)

Riboflavin (vitamin B 2 ) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo . Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism and physiology, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins, and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for future studies investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria., Importance: The pathway for biosynthesis and utilization of riboflavin, precursor of the essential coenzymes, FMN and FAD, is of particular interest in the flavin-rich pathogen, Mycobacterium tuberculosis (Mtb), for two important reasons: (i) the pathway includes potential tuberculosis (TB) drug targets and (ii) intermediates from the riboflavin biosynthesis pathway provide ligands for mucosal-associated invariant T (MAIT) cells, which have been implicated in TB pathogenesis. However, the riboflavin pathway is poorly understood in mycobacteria, which lack canonical mechanisms to transport this vitamin and to regulate flavin coenzyme homeostasis. By conditionally disrupting each step of the pathway and assessing the impact on mycobacterial viability and on the levels of the pathway proteins as well as riboflavin, our work provides genetic validation of the riboflavin pathway as a target for TB drug discovery and offers a resource for further exploring the association between riboflavin biosynthesis, MAIT cell activation, and TB infection and disease., Competing Interests: The authors declare no conflict of interest.

Lichens have developed numerous adaptations to optimize their survival in various environmental conditions, largely by producing secondary compounds by the fungal partner. They often have antibiotic properties and are involved in protection against intensive UV radiation, pathogens, and herbivores. To contribute to the knowledge of the arsenal of secondary compounds in a crustose lichen species, we sequenced and assembled the genome of Toniniopsis dissimilis , an indicator of old-growth forests, using Oxford Nanopore Technologies (ONT, Oxford, UK) long reads. Our analyses focused on biosynthetic gene clusters (BGCs) and specifically on Type I Polyketide (T1PKS) genes involved in the biosynthesis of polyketides. We used the comparative genomic approach to compare the genome of T. dissimilis with six other members of the family Ramalinaceae and twenty additional lichen genomes from the database. With only six T1PKS genes, a comparatively low number of biosynthetic genes are present in the T. dissimilis genome; from those, two-thirds are putatively involved in melanin biosynthesis. The comparative analyses showed at least three potential pathways of melanin biosynthesis in T. dissimilis , namely via the formation of 1,3,6,8-tetrahydroxynaphthalene, naphthopyrone, or YWA1 putative precursors, which highlights its importance in T. dissimilis . In addition, we report the occurrence of genes encoding ribosomally synthesized and posttranslationally modified peptides (RiPPs) in lichens, with their highest number in T. dissimilis compared to other Ramalinaceae genomes. So far, no function has been assigned to RiPP-like proteins in lichens, which leaves potential for future research on this topic.

It is unknown why roses are terpene-rich, what the terpene biosynthetic pathways in roses are, and why only a few rose species produce the major components of rose essential oil. Here, we assembled two high-quality chromosome-level genomes for Rosa rugosa and Rosa multiflora. We also re-sequenced 132 individuals from the F1 progeny of Rosa chinensis and Rosa wichuraiana and 36 of their related species. Comparative genomics revealed that expansions of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and terpene synthases (TPSs) gene families led to the enrichment of terpenes in rose scent components. We constructed a terpene biosynthesis network and discovered a TPS-independent citronellol biosynthetic pathway in roses through gene functional identification, genome-wide association studies (GWASs), and multi-omic analysis. Heterologous co-expression of rose citronellol biosynthetic genes in Nicotiana benthamiana led to citronellol production. Our genomic and metabolomic analyses suggested that the copy number of NUDX1-1a determines the citronellol content in different rose species. Our findings not only provide additional genome and gene resources and reveal the evolution of the terpene biosynthetic pathways but also present a nearly complete scenario for terpenoid metabolism that will facilitate the breeding of fragrant roses and the production of rose oil., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Marine microorganisms are increasingly recognized as primary producers of marine secondary metabolites, drawing growing research interest. Many of these organisms are unculturable, posing challenges for study. Metagenomic techniques enable research on these unculturable microorganisms, identifying various biosynthetic gene clusters (BGCs) related to marine microbial secondary metabolites, thereby unveiling their secrets. This review comprehensively analyses metagenomic methods used in discovering marine microbial secondary metabolites, highlighting tools commonly employed in BGC identification, and discussing the potential and challenges in this field. It emphasizes the key role of metagenomics in unveiling secondary metabolites, particularly in marine sponges and tunicates. The review also explores current limitations in studying these metabolites through metagenomics, noting how long-read sequencing technologies and the evolution of computational biology tools offer more possibilities for BGC discovery. Furthermore, the development of synthetic biology allows experimental validation of computationally identified BGCs, showcasing the vast potential of metagenomics in mining marine microbial secondary metabolites., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

The sesquiterpene lactone artemisinin is an important anti-malarial component produced by the glandular secretory trichomes of sweet wormwood (Artemisia annua L.). Light was previously shown to promote artemisinin production, but the underlying regulatory mechanism remains elusive. In this study, we demonstrate that ELONGATED HYPOCOTYL 5 (HY5), a central transcription factor in the light signaling pathway, cannot promote artemisinin biosynthesis on its own, as the binding of AaHY5 to the promoters of artemisinin biosynthetic genes failed to activate their transcription. Transcriptome analysis and yeast two-hybrid screening revealed the B-box transcription factor AaBBX21 as a potential interactor with AaHY5. AaBBX21 showed a trichome-specific expression pattern. Additionally, the AaBBX21-AaHY5 complex cooperatively activated transcription from the promoters of the downstream genes AaGSW1, AaMYB108, and AaORA, encoding positive regulators of artemisinin biosynthesis. Moreover, AaHY5 and AaBBX21 physically interacted with the A. annua E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). In the dark, AaCOP1 decreased the accumulation of AaHY5 and AaBBX21 and repressed the activation of genes downstream of the AaHY5-AaBBX21 complex, explaining the enhanced production of artemisinin upon light exposure. Our study provides insights into the central regulatory mechanism by which light governs terpenoid biosynthesis in the plant kingdom., (© 2024 The Author(s). Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

Ginsenoside F1 has high medicinal values, which is a kind of rare triterpene saponin isolated from Panax plants. The extremely low content of ginsenoside F1 in herbs has limited its research and application in medical field. In this work, we constructed a pathway in tobacco for the biosynthesis of ginsenoside F1 by metabolic engineering. Four enzyme genes (PnDDS, CYP716A47, CYP716S1 and UGT71A56) isolated from Panax notoginseng were introduced into tobacco. Thus, a biosynthetic pathway for ginsenoside F1 synthesis was artificially constructed in tobacco cells; moreover, the four exogenous genes could be expressed in the roots, stems and leaves of transgenic plants. Consequently, ginsenoside F1 and its precursors were successfully synthesized in the transgenic tobacco, compared with Panax plants, the content of ginsenoside F1 in transgenic tobacco was doubled. In addition, accumulation of ginsenoside F1 and its precursors in transgenic tobacco shows organ specificity. Based on these results, a new approach was established to produce rare ginsenoside F1; meanwhile, such strategy could also be employed in plant hosts for the heterologous synthesis of other important or rare natural products., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Chassis strains, derived from Streptomyces coelicolor M145, deleted for one or more of its four main specialized metabolites biosynthetic pathways (CPK, CDA, RED and ACT), in various combinations, were constructed for the heterologous expression of specialized metabolites biosynthetic pathways of various types and origins. To determine consequences of these deletions on the metabolism of the deleted strains comparative lipidomic and metabolomic analyses of these strains and of the original strain were carried out. These studies unexpectedly revealed that the deletion of the peptidic clusters, RED and/or CDA, in a strain deleted for the ACT cluster, resulted into a great increase in the triacylglycerol (TAG) content, whereas the deletion of polyketide clusters, ACT and CPK had no impact on TAG content. Low or high TAG content of the deleted strains was correlated with abundance or paucity in amino acids, respectively, reflecting high or low activity of oxidative metabolism. Hypotheses based on what is known on the bio-activity and the nature of the precursors of these specialized metabolites are proposed to explain the unexpected consequences of the deletion of these pathways on the metabolism of the bacteria and on the efficiency of the deleted strains as chassis strains., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Cellobiose lipids are surface-active compounds or biological detergents produced by distinct Basidiomycetes yeasts, of which the most and best-described ones belong to the Ustilaginomycetes class. The molecules display slight variation in congener type, which is linked to the hydroxylation position of the long fatty acid, acetylation profile of the cellobiose unit, and presence or absence of the short fatty acid. In general, this variation is strain specific. Although cellobiose lipid biosynthesis has been described for about 11 yeast species, hitherto only two types of biosynthetic gene clusters are identified, and this for only three species. This work adds six more biosynthetic gene clusters and describes for the first time a novel type of cellobiose lipid biosynthetic cluster with a simplified architecture related to specific cellobiose lipids synthesized by Trichosporonaceae family members., (© 2024 John Wiley & Sons Ltd.)

Key Message: Transcriptomics and phenotypic data analysis identified 24 transcription factors (TFs) that play key roles in regulating the competitive accumulation of lignin and flavonoids. Tilia tuan Szyszyl. (T. tuan) is a timber tree species with important ecological and commercial value. However, its highly lignified pericarp results in a low seed germination rate and a long dormancy period. In addition, it is unknown whether there is an interaction between the biosynthesis of flavonoids and lignin as products of the phenylpropanoid pathway during seed development. To explore the molecular regulatory mechanism of lignin and flavonoid biosynthesis, T. tuan seeds were harvested at five stages (30, 60, 90, 120, and 150 days after pollination) for lignin and flavonoid analyses. The results showed that lignin accumulated rapidly in the early and middle stages (S1, S3, and S4), and rapid accumulation of flavonoids during the early and late stages (S1 and S5). High-throughput RNA sequencing analysis of developing seeds identified 50,553 transcripts, including 223 phenylpropanoid biosynthetic pathway genes involved in lignin accumulation grouped into 3 clusters, and 106 flavonoid biosynthetic pathway genes (FBPGs) grouped into 2 clusters. Subsequent WGCNA and time-ordered gene co-expression network (TO-GCN) analysis revealed that 24 TFs (e.g., TtARF2 and TtWRKY15) were involved in flavonoids and lignin biosynthesis regulation. The transcriptome data were validated by qRT-PCR to analyze the expression profiles of key enzyme-coding genes. This study revealed that there existed a competitive relationship between flavonoid and lignin biosynthesis pathway during the development of T. tuan seeds, that provide a foundation for the further exploration of molecular mechanisms underlying lignin and flavonoid accumulation in T. tuan seeds., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The biosynthetic capability of the long-chain polyunsaturated fatty acids (LC-PUFA) in teleosts are highly diversified due to evolutionary events such as gene loss and subsequent neo- and/or sub-functionalisation of enzymes encoded by existing genes. In the present study, we have comprehensively characterised genes potentially involved in LC-PUFA biosynthesis, namely one front-end desaturase (fads2) and eight fatty acid elongases (elovl1a, elovl1b, elovl4a, elovl4b, elovl5, elovl7, elovl8a and elovl8b) from an amphidromous teleost, Ayu sweetfish, Plecoglossus altivelis. Functional analysis confirmed Fads2 with Δ6, Δ5 and Δ8 desaturase activities towards multiple PUFA substrates and several Elovl enzymes exhibited elongation capacities towards C 18-20 or C 18-22 PUFA substrates. Consequently, P. altivelis possesses a complete enzymatic capability to synthesise physiologically important LC-PUFA including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) from their C 18 precursors. Interestingly, the loss of elovl2 gene in P. altivelis was corroborated by genomic and phylogenetic analyses. However, this constraint would possibly be overcome by the function of alternative Elovl enzymes, such as Elovl1b, which has not hitherto been functionally characterised in teleosts. The present study contributes novel insights into LC-PUFA biosynthesis in the relatively understudied teleost group, Osmeriformes (Stomiati), thereby enhancing our understanding of the complement of LC-PUFA biosynthetic genes within teleosts., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Bo Zhao reports financial support was provided by China Scholarship Council. Juan C. Navarro, Oscar Monroig reports financial support was provided by Spain Ministry of Science and Innovation. Juan C. Navarro, Oscar Monroig reports financial support was provided by Government of Valencia. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Dimethylsulfoniopropionate (DMSP) is an abundant marine organosulfur compound with roles in stress protection, chemotaxis, nutrient and sulfur cycling and climate regulation. Here we report the discovery of a bifunctional DMSP biosynthesis enzyme, DsyGD, in the transamination pathway of the rhizobacterium Gynuella sunshinyii and some filamentous cyanobacteria not previously known to produce DMSP. DsyGD produces DMSP through its N-terminal DsyG methylthiohydroxybutyrate S-methyltransferase and C-terminal DsyD dimethylsulfoniohydroxybutyrate decarboxylase domains. Phylogenetically distinct DsyG-like proteins, termed DSYE, with methylthiohydroxybutyrate S-methyltransferase activity were found in diverse and environmentally abundant algae, comprising a mix of low, high and previously unknown DMSP producers. Algae containing DSYE, particularly bloom-forming Pelagophyceae species, were globally more abundant DMSP producers than those with previously described DMSP synthesis genes. This work greatly increases the number and diversity of predicted DMSP-producing organisms and highlights the importance of Pelagophyceae and other DSYE-containing algae in global DMSP production and sulfur cycling., (© 2024. The Author(s).)

We determined whether there exists a complementary pathway of cordycepin biosynthesis in wild-type Cordyceps militaris, high-cordycepin-producing strain C. militaris GYS60, and low-cordycepin-producing strain C. militaris GYS80. Differentially expressed genes were identified from the transcriptomes of the three strains. Compared with C. militaris, in GYS60 and GYS80, we identified 145 and 470 upregulated and 96 and 594 downregulated genes. Compared with GYS80, in GYS60, we identified 306 upregulated and 207 downregulated genes. Gene Ontology analysis revealed that upregulated genes were mostly involved in detoxification, antioxidant, and molecular transducer in GYS60. By Clusters of Orthologous Groups of Proteins and Kyoto Encyclopedia of Genes and Genomes analyses, eight genes were significantly upregulated: five genes related to purine metabolism, one to ATP production, one to secondary metabolite transport, and one to RNA degradation. In GYS60, cordycepin was significantly increased by upregulation of ATP production, which promoted 3',5'-cyclic AMP production. Cyclic AMP accelerated 3'-AMP accumulation, and cordycepin continued to be synthesized and exported. We verified the novel complementary pathway by adding the precursor adenosine and analyzing the expression of four key genes involved in the main pathway of cordycepin biosynthesis. Adenosine addition increased cordycepin production by 51.2% and 10.1%, respectively, in C. militaris and GYS60. Four genes in the main pathway in GYS60 were not upregulated., (© 2023. The Author(s).)

Actinobacteria produce a plethora of bioactive secondary metabolites that are often regulated by quorum-sensing signaling molecules via specific binding to their cognate TetR-type receptors. Here, we identified monocyclic α-pyrone as a new class of actinobacterial signaling molecules influencing quorum sensing process in Nocardiopsis sp. LDBS0036, primarily evidenced by a significant reduction in the production of phenazines in the pyrone-null mutant compared to the wild-type strain. Exogenous addition of the α-pyrone can partially restore the expression of some pathways to the wild strain level. Moreover, a unique multicomponent system referred to as a conservon, which is widespread in actinobacteria and generally contains four or five functionally conserved proteins, may play an important role in detecting and transmitting α-pyrone signals in LDBS0036. We found the biosynthetic gene clusters of α-pyrone and their associated conservon genes are highly conserved in Nocardiopsis, indicating the widespread prevalence and significant function of this regulate mechanism within Nocardiopsis genus. Furthermore, homologous α-pyrones from different actinobacterial species were also found to mediate interspecies communication. Our results thus provide insights into a novel quorum-sensing signaling system and imply that various modes of bacterial communication remain undiscovered., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Engineering metabolism to efficiently produce chemicals from multi-step pathways requires optimizing multi-gene expression programs to achieve enzyme balance. CRISPR-Cas transcriptional control systems are emerging as important tools for programming multi-gene expression, but poor predictability of guide RNA folding can disrupt expression control. Here, we correlate efficacy of modified guide RNAs (scRNAs) for CRISPR activation (CRISPRa) in E. coli with a computational kinetic parameter describing scRNA folding rate into the active structure (r S  = 0.8). This parameter also enables forward design of scRNAs, allowing us to design a system of three synthetic CRISPRa promoters that can orthogonally activate (>35-fold) expression of chosen outputs. Through combinatorial activation tuning, we profile a three-dimensional design space expressing two different biosynthetic pathways, demonstrating variable production of pteridine and human milk oligosaccharide products. This RNA design approach aids combinatorial optimization of metabolic pathways and may accelerate routine design of effective multi-gene regulation programs in bacterial hosts., (© 2024. The Author(s).)

Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes. KEY POINTS: Construction of a highly efficient Penicillium crustosum heterologous expression host Reduction of secondary metabolite background by genetic dereplication strategy Integration of wA site to provide an alternative host besides Aspergillus nidulans., (© 2024. The Author(s).)

The metabolic intimacy of symbiosis often demands the work of specialists. Natural products and defensive secondary metabolites can drive specificity by ensuring infection and propagation across host generations. But in contrast to bacteria, little is known about the diversity and distribution of natural product biosynthetic pathways among fungi and how they evolve to facilitate symbiosis and adaptation to their host environment. In this study, we define the secondary metabolism of Escovopsis and closely related genera, symbionts in the gardens of fungus-farming ants. We ask how the gain and loss of various biosynthetic pathways correspond to divergent lifestyles. Long-read sequencing allowed us to define the chromosomal features of representative Escovopsis strains, revealing highly reduced genomes composed of seven to eight chromosomes. The genomes are highly syntenic with macrosynteny decreasing with increasing phylogenetic distance, while maintaining a high degree of mesosynteny. An ancestral state reconstruction analysis of biosynthetic pathways revealed that, while many secondary metabolites are shared with non-ant-associated Sordariomycetes , 56 pathways are unique to the symbiotic genera. Reflecting adaptation to diverging ant agricultural systems, we observe that the stepwise acquisition of these pathways mirrors the ecological radiations of attine ants and the dynamic recruitment and replacement of their fungal cultivars. As different clades encode characteristic combinations of biosynthetic gene clusters, these delineating profiles provide important insights into the possible mechanisms underlying specificity between these symbionts and their fungal hosts. Collectively, our findings shed light on the evolutionary dynamic nature of secondary metabolism in Escovopsis and its allies, reflecting adaptation of the symbionts to an ancient agricultural system.IMPORTANCEMicrobial symbionts interact with their hosts and competitors through a remarkable array of secondary metabolites and natural products. Here, we highlight the highly streamlined genomic features of attine-associated fungal symbionts. The genomes of Escovopsis species, as well as species from other symbiont genera, many of which are common with the gardens of fungus-growing ants, are defined by seven chromosomes. Despite a high degree of metabolic conservation, we observe some variation in the symbionts' potential to produce secondary metabolites. As the phylogenetic distribution of the encoding biosynthetic gene clusters coincides with attine transitions in agricultural systems, we highlight the likely role of these metabolites in mediating adaptation by a group of highly specialized symbionts., Competing Interests: The authors declare no conflict of interest.

Coevolutionary antagonism generates relentless selection that can favour genetic exchange, including transfer of antibiotic synthesis and resistance genes among bacteria, and sexual recombination of disease resistance alleles in eukaryotes. We report an unusual link between biological conflict and DNA transfer in bdelloid rotifers, microscopic animals whose genomes show elevated levels of horizontal gene transfer from non-metazoan taxa. When rotifers were challenged with a fungal pathogen, horizontally acquired genes were over twice as likely to be upregulated as other genes - a stronger enrichment than observed for abiotic stressors. Among hundreds of upregulated genes, the most markedly overrepresented were clusters resembling bacterial polyketide and nonribosomal peptide synthetases that produce antibiotics. Upregulation of these clusters in a pathogen-resistant rotifer species was nearly ten times stronger than in a susceptible species. By acquiring, domesticating, and expressing non-metazoan biosynthetic pathways, bdelloids may have evolved to resist natural enemies using antimicrobial mechanisms absent from other animals., (© 2024. The Author(s).)

Background: Microbial genome sequencing and analysis revealed the presence of abundant silent secondary metabolites biosynthetic gene clusters (BGCs) in streptomycetes. Activating these BGCs has great significance for discovering new compounds and novel biosynthetic pathways., Results: In this study, we found that ovmZ and ovmW homologs, a pair of interdependent transcriptional regulators coding genes, are widespread in actinobacteria and closely associated with the biosynthesis of secondary metabolites. Through co-overexpression of native ovmZ and ovmW in Streptomyces neyagawaensis NRRL B-3092, a silent type II polyketide synthase (PKS) gene cluster was activated to produce gephyromycin A, tetrangomycin and fridamycin E with the yields of 22.3 ± 8.0 mg/L, 4.8 ± 0.5 mg/L and 20.3 ± 4.1 mg/L respectively in the recombinant strain of S.ne/pZ n W n . However, expression of either ovmZ or ovmW failed to activate this gene cluster. Interestingly, overexpression of the heterologous ovmZ and ovmW pair from oviedomycin BGC of S. ansochromogenes 7100 also led to awakening of this silent angucyclinone BGC in S. neyagawaensis., Conclusion: A silent angucyclinone BGC was activated by overexpressing both ovmZ and ovmW in S. neyagawaensis. Due to the wide distribution of ovmZ and ovmW in the BGCs of actinobacteria, co-overexpression of ovmZ and ovmW could be a strategy for activating silent BGCs, thus stimulating the biosynthesis of secondary metabolites., (© 2024. The Author(s).)

Covering: up to October 2023Many bioactive natural products are synthesized by microorganisms that are either difficult or impossible to cultivate under laboratory conditions, or that produce only small amounts of the desired compound. By transferring biosynthetic gene clusters (BGCs) into alternative host organisms that are more easily cultured and engineered, larger quantities can be obtained and new analogues with potentially improved biological activity or other desirable properties can be generated. Moreover, expression of cryptic BGCs in a suitable host can facilitate the identification and characterization of novel natural products. Heterologous expression therefore represents a valuable tool for natural product discovery and engineering as it allows the study and manipulation of their biosynthetic pathways in a controlled setting, enabling innovative applications. Bacillus is a genus of Gram-positive bacteria that is widely used in industrial biotechnology as a host for the production of proteins from diverse origins, including enzymes and vaccines. However, despite numerous successful examples, Bacillus species remain underexploited as heterologous hosts for the expression of natural product BGCs. Here, we review important advantages that Bacillus species offer as expression hosts, such as high secretion capacity, natural competence for DNA uptake, and the increasing availability of a wide range of genetic tools for gene expression and strain engineering. We evaluate different strain optimization strategies and other critical factors that have improved the success and efficiency of heterologous natural product biosynthesis in B. subtilis . Finally, future perspectives for using B. subtilis as a heterologous host are discussed, identifying research gaps and promising areas that require further exploration.

Saffron (Crocus sativus L.) is being embraced as the most important medicinal plant and the commercial source of saffron spice. Despite the beneficial economic and medicinal properties of saffron, the regulatory mechanism of the correlation of TFs and genes related to the biosynthesis of the apocarotenoids pathway is less obvious. Realizing these regulatory hierarchies of gene expression networks related to secondary metabolites production events is the main challenge owing to the complex and extensive interactions between the genetic behaviors. Recently, high throughput expression data have been highly feasible for constructing co-regulation networks to reveal the regulated processes and identifying novel candidate hub genes in response to complex processes of the biosynthesis of secondary metabolites. Herein, we performed Weighted Gene Co-expression Network Analysis (WGCNA), a systems biology method, to identify 11 regulated modules and hub TFs related to secondary metabolites. Three specialized modules were found in the apocarotenoids pathway. Several hub TFs were identified in notable modules, including MADS, C2H2, ERF, bZIP, HD-ZIP, and zinc finger protein MYB and HB, which were potentially associated with apocarotenoid biosynthesis. Furthermore, the expression levels of six hub TFs and six co-regulated genes of apocarotenoids were validated with RT-qPCR. The results confirmed that hub TFs specially MADS, C2H2, and ERF had a high correlation (P < 0.05) and a positive effect on genes under their control in apocarotenoid biosynthesis (CCD2, GLT2, and ADH) among different C. sativus ecotypes in which the metabolite contents were assayed. Promoter analysis of the co-expressed genes of the modules involved in apocarotenoids biosynthesis pathway suggested that not only are the genes co-expressed, but also share common regulatory motifs specially related to hub TFs of each module and that they may describe their common regulation. The result can be used to engineer valuable secondary metabolites of C. sativus by manipulating the hub regulatory TFs., (© 2024. The Author(s).)

Vitamin D deficiencies are linked to multiple human diseases. Optimizing its synthesis, physicochemical properties, and delivery systems while minimizing side effects is of clinical relevance and is of great medical and industrial interest. Biotechnological techniques may render new modified forms of vitamin D that may exhibit improved absorption, stability, or targeted physiological effects. Novel modified vitamin D derivatives hold promise for developing future therapeutic approaches and addressing specific health concerns related to vitamin D deficiency or impaired metabolism, such as avoiding hypercalcemic effects. Identifying and engineering key enzymes and biosynthetic pathways involved, as well as developing efficient cultures, are therefore of outmost importance and subject of intense research. Moreover, we elaborate on the critical role that microbial bioconversions might play in the a la carte design, synthesis, and production of novel, more efficient, and safer forms of vitamin D and its analogs. In summary, the novelty of this work resides in the detailed description of the physiological, medical, biochemical, and epidemiological aspects of vitamin D supplementation and the steps towards the enhanced and simplified industrial production of this family of bioactives relying on microbial enzymes. KEY POINTS: • Liver or kidney pathologies may hamper vitamin D biosynthesis • Actinomycetes are able to carry out 1α- or 25-hydroxylation on vitamin D precursors., (© 2024. The Author(s).)

L-cysteine is an important sulfur-containing amino acid being difficult to produce by microbial fermentation. Due to the lack of high-throughput screening methods, existing genetically engineered bacteria have been developed by simply optimizing the expression of L-cysteine-related genes one by one. To overcome this limitation, in this study, a biosensor-based approach for multilevel biosynthetic pathway optimization of L-cysteine from the DecR regulator variant of Escherichia coli was applied. Through protein engineering, we obtained the DecR N29Y/C81E/M90Q/M99E variant-based biosensor with improved specificity and an 8.71-fold increase in dynamic range. Using the developed biosensor, we performed high-throughput screening of the constructed promoter and RBS combination library, and successfully obtained the optimized strain, which resulted in a 6.29-fold increase in L-cysteine production. Molecular dynamics (MD) simulations and electrophoretic mobility shift analysis (EMSA) showed that the N29Y/C81E/M90Q/M99E variant had enhanced induction activity. This enhancement may be due to the increased binding of the variant to DNA in the presence of L-cysteine, which enhances transcriptional activation. Overall, our biosensor-based strategy provides a promising approach for optimizing biosynthetic pathways at multiple levels. The successful implementation of this strategy demonstrates its potential for screening improved recombinant strains., (© 2024 Wiley Periodicals LLC.)

Background: Bacterial antimicrobial resistance poses a severe threat to humanity, necessitating the urgent development of new antibiotics. Recent advances in genome sequencing offer new avenues for antibiotic discovery. Paenibacillus genomes encompass a considerable array of antibiotic biosynthetic gene clusters (BGCs), rendering these species as good candidates for genome-driven novel antibiotic exploration. Nevertheless, BGCs within Paenibacillus genomes have not been extensively studied., Results: We conducted an analysis of 554 Paenibacillus genome sequences, sourced from the National Center for Biotechnology Information database, with a focused investigation involving 89 of these genomes via antiSMASH. Our analysis unearthed a total of 848 BGCs, of which 716 (84.4%) were classified as unknown. From the initial pool of 554 Paenibacillus strains, we selected 26 available in culture collections for an in-depth evaluation. Genomic scrutiny of these selected strains unveiled 255 BGCs, encoding non-ribosomal peptide synthetases, polyketide synthases, and bacteriocins, with 221 (86.7%) classified as unknown. Among these strains, 20 exhibited antimicrobial activity against the gram-positive bacterium Micrococcus luteus, yet only six strains displayed activity against the gram-negative bacterium Escherichia coli. We proceeded to focus on Paenibacillus brasilensis, which featured five new BGCs for further investigation. To facilitate detailed characterization, we constructed a mutant in which a single BGC encoding a novel antibiotic was activated while simultaneously inactivating multiple BGCs using a cytosine base editor (CBE). The novel antibiotic was found to be localized to the cell wall and demonstrated activity against both gram-positive bacteria and fungi. The chemical structure of the new antibiotic was elucidated on the basis of ESIMS, 1D and 2D NMR spectroscopic data. The novel compound, with a molecular weight of 926, was named bracidin., Conclusions: This study outcome highlights the potential of Paenibacillus species as valuable sources for novel antibiotics. In addition, CBE-mediated dereplication of antibiotics proved to be a rapid and efficient method for characterizing novel antibiotics from Paenibacillus species, suggesting that it will greatly accelerate the genome-based development of new antibiotics., (© 2024. The Author(s).)