Search Results (274)

Fungal iterative non-reducing polyketide synthases (NR-PKS) contain a unique product template (PT) domain for aromatic cyclization. Among them, some NR-PKSs, such as the sorbicillin NR-PKS (SorB), have an apparently inactive PT. It is unknown what role such PT plays in NR-PKS programming. In this study, the PT domain of SorB was first dissected and engineered. Removal of the PT domain from SorB did not change the product profile, but the yield decreased. Meanwhile, a significantly lower transcriptional level of the ketoacyl synthase (KS) domain was observed in the knockout mutant (UvSorB∆PT). Phylogenetic tree analysis and multiple sequence alignments revealed this PT belongs to group I (C2–C7, monocyclic ring), and mutations were found at catalytic dyad sites when compared with functional fungal PTs. However, mutating these residues back to the conserved ones did not give rise to products corresponding to a functional PT, but rendered the NR-PKS unproductive. Likewise, removal of the C-methyltransferase (CMT) domain from SorB destroyed the polyketide production. Furthermore, in an attempt to alter the methylation pattern, mutations of the key substrate-binding sites of the CMT domain were made. Site-directed mutations of the C-MT led to cessation of the polyketide production. This reveals CMT is vulnerable to engineering in a collaborating NR-PKS (SorB). These results provide additional insights for catalytic reprogramming in fungal NR-PKS. Full article

Background: Doramectin (CHC-B1) is an excellent antiparasitic drug produced by feeding cyclohexanecarboxylic acid (CHC) to Streptomycesavermitilis bkd⁻ mutants. AveC, a bifunctional enzyme encoded by aveC (sav_0940), catalyzes the stereospecific spiroketalization and selective dehydration of dihydroxy ketone polyketide intermediates and modulates both the yield and the proportion of avermectin/doramectin in Streptomyces avermitilis. In our previous work, we constructed a strain harboring a synthetic aveC* gene encoding ten amino acid mutations, which produced nearly pure doramectin. However, the doramectin yield achieved only approximately 60% of the total doramectin and CHC-B2 output observed in the parental strain. Methods: To investigate the roles of Ser138 and Ala139 of AveC in the biosynthesis of doramectin and avermectin, site-directed mutagenesis was performed at both sites. The production and proportion of avermectin and doramectin were determined using high-performance liquid chromatography (HPLC). AlphaFold2-based molecular docking simulations were used to interpret the results. Results: Among the tested mutants, S138G, S138T, and A139H exhibited the highest doramectin production, achieving 143.87%, 151.22%, and 153.36% of the control level, respectively. Unfortunately, almost none of the tested mutants showed a positive effect on avermectin production. Molecular docking simulations revealed distinct affinities of these mutants for the dihydroxy ketone polyketide intermediate, both with and without a cyclohexyl group. Notably, all three mutants displayed larger substrate-binding cavity volumes compared with the wild-type enzyme, which likely facilitates doramectin synthesis by effectively accommodating the cyclohexyl moiety. Docking results further indicated that Ser138 and Ala139 are positioned within the binding cavity but probably do not directly participate in the dehydration activity. Conclusions: These findings suggest that optimizing cavity size through residue substitutions can enhance substrate specificity for doramectin production while preserving catalytic functionality. Full article

Diet plays a critical role in shaping the composition of gut microbiota in insects. Samia ricini, an economically important Lepidoptera insect, is a polyphagous herbivore that offers a useful model for studying dietary effects on the animal gut microbiome. Here, we fed S. ricini larvae with different food plants, Ricinus communis, Ailanthus altissima, and Manihot esculenta leaves to investigate how host plant species influence growth performance, digestive enzyme activities, and the gut microbial community. Our results showed that the Ricinus group exhibited better growth performance. Regarding digestive enzymes, the midgut lipase activity was significantly higher in the Ricinus group than in the Ailanthus group, while no significant differences were observed in α-amylase, cellulase, or trypsin activities among the three groups. Compared to the Manihot group, the Ricinus group showed increased bacterial richness, while the Ailanthus group showed increased bacterial diversity. β-diversity analysis further revealed distinct microbial community structures among all three dietary groups. Specifically, Acinetobacter, Mammaliicoccus, Roseateles, Methylobacterium, Agrobacterium, Faecalibacterium, and Segatella were the dominant bacterial genera. Functional prediction revealed that gut microbes enriched in the Ricinus group were associated with terpenoid/polyketide metabolism, xenobiotics biodegradation, and glycan biosynthesis, whereas those involved in carbohydrate metabolism and biosynthesis of other secondary metabolites were higher in the Manihot group. Spearman correlation analysis indicated that Methylobacterium, Methylorubrum, and Agrobacterium were significantly positively correlated with larval weight, while Staphylococcus and Cyanothece_PCC-7424 exhibited negative correlations. Collectively, these findings suggest a potential association between different plant-derived diets, gut microbiota composition, and host growth performance, highlighting the pivotal role of diet in shaping insect gut microbial communities. Full article

Streptomyces species are major producers of bioactive molecules via biosynthetic gene clusters (BGCs). However, many BGCs are silent or poorly expressed in their native hosts, making heterologous expression hosts a key strategy for discovering novel natural products and efficiently producing known compounds. In this study, Streptomyces albus ZD11, an industrial salinomycin producer capable of efficiently utilizing soybean oil to supply abundant polyketide precursors, was selected as a candidate host for the expression of polyketide BGCs. A genome-reduced derivative, designated ZD12, was constructed by deleting four endogenous polyketide BGCs from ZD11, aiming to reduce precursor competition and alleviate metabolic burden. To evaluate the polyketide biosynthesis capacity of ZD12, an engineered spectinabilin BGC was heterologously expressed in both ZD12 and a commonly used heterologous host S. albus J1074. The resulting ZD12-derived strain DHM produced 412 mg/L spectinabilin, while the J1074-derived strain J-DHM produced 114 mg/L, both of which were significantly higher than the native production level in S. spectabilis. Notably, the titer in DHM exceeded the highest previously reported heterologous titer by more than threefold. Furthermore, under identical integration conditions, DHM achieved a 2.6-fold higher spectinabilin titer than J-DHM, demonstrating the superior polyketide biosynthesis capacity of ZD12. Full article

Plant pathogens pose a severe threat to global agricultural production, and their pathogenicity is closely linked to the biosynthesis of secondary metabolites. Basidiomycete within the family Ustilaginaceae represent significant plant pathogens, among which Ustilago maydis, as a model species, has been extensively studied for its secondary metabolites. However, the biosynthetic potential of other species within this family remains poorly understood. In this study, we conducted whole-genome bioinformatic analyses of 16 Ustilaginaceae species, including U. maydis, to systematically identify the distribution of biosynthetic gene clusters (BGCs), core gene domain compositions, and interspecies similarities. A total of 181 predicted BGCs were identified, averaging approximately 11 per species. BGCs for mannosylerythritol lipids (MELs), siderophores, and itaconic acid, as well as the melanin-associated genes pks1 and pks2, were widely distributed across most species. Conversely, an additional melanin biosynthetic gene cluster was found exclusively in U. maydis strain 521, indicating species-specific occurrence. Furthermore, this study identified a novel class of polyketide synthase (PKS) gene clusters with uncharacterized functions across 15 species, exhibiting high sequence and structural conservation between species. These findings reveal the rich metabolic diversity and species-specific biosynthetic potential of Ustilaginaceae, and by using U. maydis as a reference model, we highlight several BGCs (e.g., for MELs, siderophores, itaconic acid, and melanin) that are known to contribute to virulence or pathogenicity in plant hosts. This provides new insights into their pathogenic mechanisms. Full article

Abscisic acid (ABA) regulates diverse aspects of plant growth and secondary metabolism, yet its concentration-dependent effects on rhizomatous spice crops remain poorly understood at the systems level. Here, we investigated the phenotypic, physiological, hormonal, and multi-omics responses of ginger (Zingiber officinale) to foliar-applied ABA across a concentration gradient. Exogenous ABA modulated ginger growth in a distinctly non-linear manner. Low-to-moderate concentrations (5–15 mg/L) significantly enhanced aboveground branching and belowground rhizome yield, whereas high concentration (35 mg/L) inhibited branching while promoting structural carbohydrate accumulation, revealing a concentration-dependent trade-off between growth and secondary wall deposition. Hormone profiling uncovered global reprogramming of the endogenous hormonal network, with optimal ABA (15 mg/L) coordinately elevating growth-promoting hormones and defense-related signals, while high concentrations suppressed multiple hormone pathways and triggered negative feedback inhibition of endogenous ABA biosynthesis. Integrated metabolomic and transcriptomic analyses identified convergent enrichment on phenylpropanoid biosynthesis, gingerol biosynthesis, and plant hormone signal transduction. Co-expression network analysis revealed a highly interconnected module of 583 genes linking hormone signaling to secondary metabolism, with coordinated up-regulation of key enzymes from phenylalanine ammonia-lyase (PAL) to polyketide synthase under 15 mg/L ABA explaining the 64% increase in 6-gingerol content. This study establishes a mechanistic chain from ABA perception to improved ginger yield and quality, mediated by hormonal crosstalk and transcriptional activation of the phenylpropanoid-gingerol network. We propose an “ABA optimization window” of 5–15 mg/L for precision cultivation of high-quality ginger, providing a systems-level framework for understanding hormone-mediated regulation of secondary metabolism in medicinal and spice crops. Full article

Castanopsis hystrix (C. hystrix) is one of the most dominant and ecologically important species in subtropical evergreen broad-leaved forests of China. Interactions between its root and rhizosphere microorganisms play a pivotal role in nutrient acquisition and in mediating plant response s to environmental stresses. In this study, high-throughput 16S ribosomal RNA (16S rRNA) sequencing combined with untargeted metabolomics was employed to systematically characterize the rhizosphere microbial community and root exudates in C. hystrix. The results showed that, compared with non-rhizosphere soil, bacterial diversity in the rhizosphere of C. hystrix was significantly reduced, while several specialized and potentially efficient taxa were selectively enriched, particularly Candidatus_Solibacter, Candidatus_Xiphinematobacter, and Candidatus_Koribacter, thereby reshaping a distinct rhizosphere-specific community structure. Metabolomic analyses further revealed that 129 metabolites were significantly enriched in the rhizosphere, including four major classes of compounds associated with plant stress resistance: lipids and lipid-like molecules, organoheterocyclic compounds, organic acids and derivatives, and phenylpropanoids and polyketides. The enrichment of these metabolites likely contributes substantially to stress tolerance in C. hystrix. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified six defense-related metabolic pathways, including pyrimidine metabolism, steroid biosynthesis, nucleotide metabolism, plant hormone signal transduction, ATP-binding cassette transporter (ABC transporters), and the biosynthesis of various plant secondary metabolites. Further correlation analysis and co-occurrence network analysis suggested that C. hystrix may potentially influence the enrichment of beneficial microorganisms through rhizosphere metabolites selectively, which could reduce the reliance on external nutrient acquisition and enhance the stress resilience of C. hystrix. Our study provides a comprehensive perspective for elucidating rhizosphere interaction networks and their ecological functions in C. hystrix, thereby enhancing our understanding of the environmental adaptability of dominant tree species in subtropical forests. Full article

Milbemycin D is a promising 16-membered macrolide insecticide with reported superior efficacy, but its commercial development has been hindered by extremely low natural yields. This study aimed to construct a high-yielding microbial platform for milbemycin D production using combinatorial biosynthesis and advanced genome editing. An optimized CRISPR/Cas9-AcrIIA4 system was employed to seamlessly replace the aveA3 polyketide synthase (PKS) gene in the ivermectin B1b-producing strain Streptomyces avermitilis HU501 with the heterologous milA3 PKS from S. bingchenggensis. The engineered strain was validated genetically and metabolically, followed by high-throughput screening and fermentation optimization in various media. The biosynthesized compound was structurally confirmed by spectroscopy. Bioactivity was evaluated against Bursaphelenchus xylophilus, Hyphantria cunea, and Plutella xylostella. The engineered strain S. avermitilis HU501-M successfully shifted its major product to milbemycin D, reaching a final titer of 679.03 mg/L. Bioassays revealed that milbemycin D exhibited significantly enhanced potency, with LC₅₀ values 8–24% lower than those of milbemycin A3/A4. This work demonstrates an efficient CRISPR/Cas9-mediated PKS replacement strategy to achieve the high-yield production of milbemycin D, offering a promising microbial source and a generalizable framework for engineering complex polyketide pathways. This proof-of-concept establishes a foundation for future process development toward potential commercial application. Full article

Acyl-CoA carboxylase (YCC) complexes generate essential starter and extender units for fatty acid and polyketide biosynthesis in Actinobacteria. In Streptomyces coelicolor, two tri-subunit YCC complexes, acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC), have been characterized. However, comparative genomic analyses indicate that β/ε subunits are more diversified than currently appreciated. Here, we identify a previously unrecognized β/ε pair, AccB2 and AccE2, and demonstrate that they assemble with the canonical α subunit to form a functional YCC complex. Both genes are transcribed in vivo, and co-immunoprecipitation (Co-IP) reveals association with AccA1 and AccA2, with AccE2 showing stronger relative association with AccA1-containing pull-downs. In vitro reconstitution confirms carboxylation activity toward acetyl-CoA, propionyl-CoA, and butyryl-CoA, which is strongly dependent on AccE2. These findings expand the YCC repertoire in S. coelicolor and support a modular assembly model in which alternative β/ε combinations contribute to functional diversification of YCC complexes. Full article

Camellia oleifera Abel, recognized as a woody oil-producing tree species, possesses considerable economic significance. To improve the breeding efficiency of C. oleifera, it is crucial to elucidate the genetic foundation underlying the mechanisms regulating fruit traits. In this study, a total of 6,252,197 high-quality single-nucleotide polymorphisms (SNPs) were identified from 109 germplasm accessions. Through genetic structure analysis, these accessions were categorized into two distinct populations. The average fixation index (Fst) was found to be 0.0153, indicating weak population differentiation. The genome-wide association analysis (GWAS) identified 157 significant loci. From these loci, 110 candidate genes were selected, which were associated with disease resistance, reproduction, development, and RNA biosynthesis. Twenty-three genes were involved in metabolic pathways, including genetic information-processing protein families, metabolic protein families, terpenoids, and polyketides. The identification of gene loci closely related to fruit traits not only provides genetic data for studying the molecular mechanisms of fruit traits but also offers new research avenues for molecular breeding of C. oleifera. Full article

Natural products such as polyketides are a fertile target for drug discovery. Methodologies relating to discovery, metabolism, synthesis and biosynthesis of polyketides have evolved considerably since they were first studied in the early 20th century. The antibiotic erythromycin, produced by the Streptomyces erythreus bacteria, was the first of the macrolide natural products to be discovered in 1952. The biosynthesis of erythromycin is catalysed by a large multifunctional enzyme, which constructs the polyketide intermediate that is acted upon by tailoring enzymes to produce the final construct. It is during this process that molecular diversity is generated, and commercial samples of erythromycin tend to be mixtures of co-metabolites. To fully identify these compounds, a full fragmentation scheme of the main component (erythromycin A) is required, which is absent from the literature. In this study, accurate-mass sequential mass spectrometry is used to propose a fragmentation scheme which is then used to assign structures to eight co-metabolites including the identification of a previously unpublished form of erythromycin. This clearly demonstrates the successful application of the methodology. Full article

(1) Background: The objective of this study was to investigate the modulatory effects of thiamine on BHBA metabolism, milk yield, and the rumen microbial ecosystem. (2) Methods: A total of 24 SCK dairy cows with similar body conditions were selected and randomly allocated to SCK (SCK) or SCK with thiamine supplement (SCKT) treatment. Twelve healthy dairy cows served as the control (CON) treatment. Milk yield, milk quality, ruminal fermentability parameters, rumen and fecal microbial communities were further measured. (3) Results: Thiamine significantly decreased BHBA content, milk CFUs, and somatic cells, while significantly increasing milk yield, milk fat, acetate, and the A/P ratio (p < 0.05). Thiamine-treated cows exhibited significantly increased ruminal and fecal Proteobacteria but significantly decreased ruminal Firmicutes (p < 0.05) as well as fecal Spirochaetes and Cyanobacteria (p < 0.05), compared with SCK cows. Functional analysis showed that differential rumen bacteria exhibited high energy metabolism, nucleotide metabolism, and glycan biosynthesis and metabolism, while the metabolism of terpenoids and polyketides were the primary functional pathways of differential fecal microbiota. (4) Conclusions: Thiamine supplementation in SCK cows effectively alleviated subclinical ketosis by reducing BHBA content, enhancing ruminal fermentability, and proliferating rumen microbial communities, leading to improved milk yield in the early-lactation period. Full article

As agriculture faces increasing pressure to reduce pesticide residues and heavy metal accumulation in soils, marine microalgae are emerging as sustainable sources of biopesticides. Among them, Amphidinium carterae produces amphidinols (AMs), polyketide metabolites with strong antifungal activity against crop pathogens. Currently, large-scale AM production remains constrained by a limited understanding of AM biosynthesis across different A. carterae growth phases and by the lack of high-performing industrial strains. In this study, AM production dynamics were investigated in one wild-type (WT) and five mutagenized A. carterae strains. The production of bioactive AM18 and its sulfated inactive form AM19 was monitored through exponential, linear, and early stationary growth phases. The maximum AM productivity occurred between the linear and early stationary phase, with the average values of 5.58 ± 0.4 and 3.58 ± 0.2 µg/mL/day for AM18 and AM19, respectively. The AM18/AM19 ratio consistently decreased with the culture age, indicating that earlier harvesting favors higher proportions of bioactive AMs. UV mutagenesis increased the AM18 cell content by more than twofold and the growth rate by up to 20% in certain mutagenized strains compared to the WT strain, but did not enhance the volumetric AM productivity. Overall, these results identify optimal AM harvesting windows and clarify the potential benefits of mutagenesis strain improvement for industrial AM production improvement. Full article

Villosiclava virens (anamorph: Ustilaginoidea virens) is a fungal pathogen that causes rice false smut, one of the most devastating diseases of rice. The Uvpks1 gene encoding polyketide synthase is responsible for the biosynthesis of ustilaginoidins, a major group of mycotoxins in V. virens. In this study, three strains, including the Uvpks1 deletion mutant ΔUvpks1, the complementation strain ΔUvpks1-C1, and the wild-type isogenic strain P1 of V. virens, were employed to investigate the role of Uvpks1 in shaping the metabolic profile and in the development, stress responses, and pathogenicity of V. virens. The deletion of Uvpks1 led to both the elimination of ustilaginoidin biosynthesis and the induction of many other secondary metabolite biosynthetic pathways. It decreased mycelial growth and sporulation, fungal tolerance to Congo red-induced cell wall damage stress, and susceptibility to the fungicides epoxiconazole and prochloraz. Meanwhile, it increased hyphal hydrophobicity, resistance to H₂O₂-induced oxidative stress and metal cation stress, and susceptibility to the fungicide azoxystrobin. Furthermore, the deletion of Uvpks1 resulted in reduced fungal pathogenicity toward rice plants. The findings reveal the functions of Uvpks1 in shaping the metabolic profile, development, stress responses, and pathogenicity of V. virens, which will be beneficial for developing new strategies to control rice false smut and ustilaginoidins. Full article

Tocosh, a traditional Peruvian fermented potato product, is known for its health-promoting properties, including its antioxidant, anti-inflammatory, probiotic, and antibiotic effects, which have popularized its consumption, particularly in rural areas. To gain a better understanding of its antimicrobial properties, this study aimed to perform a comprehensive whole-genome analysis and functional assessment of the Bacillus velezensis TCSH0001 strain isolated from tocosh. The isolate was identified through whole-genome sequencing using the MinION nanopore platform. AntiSMASH analysis revealed nine biosynthetic gene clusters (BGCs) potentially responsible for producing secondary metabolites with antibiotic potential. Notably, seven BGCs showed a 100% similarity to known clusters involved in the biosynthesis of polyketide synthases (PKSs) and non-ribosomal peptides (NRPSs), including difficidin, bacillibactin, bacilysin, macrolactin H, bacillaene, fengycin, and bacillomycin D. In vitro analysis revealed antimicrobial activity against S. aureus strains. In addition, RT-qPCR indicated that the expression of the baeJ (bacillaene), bmyA (bacillomycin D), and pks2A (macrolactin H) occurs predominantly during the exponential growth phase. Our results suggest that this B. velezensis strain has the capacity to produce a diverse array of bioactive compounds, supporting the traditional use of tocosh as a natural antimicrobial agent, and revealing the potential of the strain as a high NRPS producer. Full article