The social amoeba Polysphondylium violaceum uses chemoattractants different from those of Dictyoctelium discoideum for cell aggregation. However, the detailed mechanisms in P. violaceum remain unknown. We have previously reported that the polyketide synthase StlA is involved in inducing aggregation in this species. To elucidate the mechanism of StlA-induced aggregation in P. violaceum, we analysed the phenotype of Pv-stlA- mutants in more detail. Unlike our previous results, the mutant cells did not exhibit proper chemotaxis towards glorin. Defective aggregation was not restored by glorin pulses, 8Br-cAMP, or deletion of the homologue of PufA that is a translational repressor of PKA, whereas mutant cells grown in the presence of 4-methyl-5-pentylbenzene-1,3-diol (MPBD), the putative Pv-StlA product, aggregated normally without it after starvation. Furthermore, the early developmental marker gene, dscA, was down-regulated in the mutant cells. Our data thus suggested that StlA is required for the transition from growth to development in P. violaceum.

Modular polyketide synthases (PKSs) are capable of synthesizing diverse natural products with fascinating bioactivities. Canonical enoyl-CoA hydratases (ECHs) are components of the β-branching cassette that modifies the polyketide chain by adding a β-methyl branch. Herein, it is demonstrated that the deletion of an atypical ECHQ domain (featuring a Q280 residue) of Art21, a didomain protein contains an ECHQ domain and a thioesterase (TE) domain, reprograms the polyketide assembly line from synthesizing tetracyclic aurantinins (ARTs) to bicyclic auritriacids (ATAs) with much lower antibacterial activities. Genes encoding the ECHQ-TE didomain proteins distribute in many PKS gene clusters from different bacteria. Significantly, the ART PKS machinery can be directed to make ARTs, ATAs, or both of them by employing appropriate ECHQ-TE proteins, implying a great potential for using this reprogramming strategy in polyketide structure diversification.

The catastrophic loss of aquatic life in the Central European Oder River in 2022, caused by a toxic bloom of the haptophyte microalga Prymnesium parvum (in a wide sense, s.l.), underscores the need to improve our understanding of the genomic basis of the toxin. Previous morphological, phylogenetic, and genomic studies have revealed cryptic diversity within P. parvum s.l. and uncovered three clade-specific (types A, B, and C) prymnesin toxins. Here, we used state-of-the-art long-read sequencing and assembled the first haplotype-resolved diploid genome of a P. parvum type B from the strain responsible for the Oder disaster. Comparative analyses with type A genomes uncovered a genome-size expansion driven by repetitive elements in type B. We also found conserved synteny but divergent evolution in several polyketide synthase (PKS) genes, which are known to underlie toxin production in combination with environmental cues. We identified an approximately 20-kbp deletion in the largest PKS gene of type B that we link to differences in the chemical structure of types A and B prymnesins. Flow cytometry and electron microscopy analyses confirmed diploidy in the Oder River strain and revealed differences to closely related strains in both ploidy and morphology. Our results provide unprecedented resolution of strain diversity in P. parvum s.l. and a better understanding of the genomic basis of toxin variability in haptophytes. The reference-quality genome will enable us to better understand changes in microbial diversity in the face of increasing environmental pressures and provides a basis for strain-level monitoring of invasive Prymnesium in the future.

Polyketides are a major class of natural products, including bioactive medicines such as erythromycin and rapamycin. They are often rich in stereocenters biosynthesized by the ketoreductase (KR) domain within the polyketide synthase (PKS) assembly line. Previous studies have identified conserved motifs in KR sequences that enable the bioinformatic prediction of product stereochemistry. However, the reliability and applicability of these prediction methods have not been thoroughly assessed. In this study, we conducted a comprehensive bioinformatic analysis of 1,762 KR sequences from cis-AT PKSs to reevaluate the residues involved in conferring stereoselectivity. Our findings indicate that the previously identified fingerprint motifs remain valid for KRs in β-modules from actinobacteria, but their reliability diminishes for KRs from other module types or taxonomic origins. Additionally, we have identified several new motifs that exhibit a strong correlation with the stereochemical outcomes of KRs. These updated fingerprint motifs for stereochemical prediction not only enhance our understanding of the enzymatic mechanisms governing stereocontrol but also facilitate accurate stereochemical prediction and genome mining of polyketides derived from modular cis-AT PKSs.

Utilizing bioinformatics tools, this study expands our understanding of secondary metabolism in Botrytis cinerea, identifying novel genes within polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), sesquiterpene cyclase (STC), diterpene cyclase (DTC), and dimethylallyltryptophan synthase (DMATS) families. These findings enrich the genetic framework associated with B. cinerea\'s pathogenicity and ecological adaptation, offering insights into uncharted metabolic pathways. Significantly, the discovery of previously unannotated genes provides new molecular targets for developing targeted antifungal strategies, promising to enhance crop protection and advance our understanding of fungal biochemistry. This research not only broadens the scope of known secondary metabolites but also opens avenues for future exploration into B. cinerea\'s biosynthetic capabilities, potentially leading to novel antifungal compounds. Our work underscores the importance of integrating bioinformatics and genomics for fungal research, paving the way for sustainable agricultural practices by pinpointing precise molecular interventions against B. cinerea. This study sets a foundation for further investigations into the fungus\'s secondary metabolism, with implications for biotechnology and crop disease management.

The Diels-Alder (DA) reaction, specifically referring to the [4 + 2] cycloaddition reaction in pericyclic reactions, is a process that forms two carbon-carbon covalent bonds in a single step via an electron ring transition state. Among the secondary metabolites produced by microorganisms, numerous compounds are biosynthesized through DA reactions, most of which are enzymatic. Our research group has discovered an enzyme named Diels-Alderase (DAase) that catalyzes the DA reaction in filamentous fungi, and we have been investigating its catalytic mechanism. This review describes the reported microbial DAase enzymes, with a particular focus on those involved in the construction of the decalin ring.

BACKGROUND: Cladosporium phlei is a phytopathogenic fungus that produces a pigment called phleichrome. This fungal perylenequinone plays an important role in the production of a photosensitizer that is a necessary component of photodynamic therapy. We applied synthetic biology to produce phleichrome using Saccharomyces cerevisiae.
RESULTS: The gene Cppks1, which encodes a non-reducing polyketide synthase (NR-PKS) responsible for the biosynthesis of phleichrome in C. phlei, was cloned into a yeast episomal vector and used to transform S. cerevisiae. In addition, a gene encoding a phosphopantetheinyl transferase (PPTase) of Aspergillus nidulans was cloned into a yeast integrative vector and also introduced into S. cerevisiae for the enzymatic activation of the protein product of Cppks1. Co-transformed yeasts were screened on a leucine/uracil-deficient selective medium and the presence of both integrative as well as episomal recombinant plasmids in the yeast were confirmed by colony PCR. The episomal vector for Cppks1 expression was so dramatically unstable during cultivation that most cells lost their episomal vector rapidly in nonselective media. This loss was also observed to a less degree in selective media. This data strongly suggests that the presence of the Cppks1 gene exerts a significant detrimental effect on the growth of transformed yeast cells and that selection pressure is required to maintain the Cppks1-expressing vector. The co-transformants on the selective medium showed the distinctive changes in pigmentation after a period of prolonged cultivation at 20 °C and 25 °C, but not at 30 °C. Furthermore, thin layer chromatography (TLC) revealed the presence of a spot corresponding with the purified phleichrome in the extract from the cells of the co-transformants. Liquid chromatography (LC/MS/MS) verified that the newly expressed pigment was indeed phleichrome.
CONCLUSIONS: Our results indicate that metabolic engineering by multiple gene expression is possible and capable of producing fungal pigment phleichrome in S. cerevisiae. This result adds to our understanding of the characteristics of fungal PKS genes, which exhibit complex structures and diverse biological activities.

Histone acetylation modifications in filamentous fungi play a crucial role in epigenetic gene regulation and are closely linked to the transcription of secondary metabolite (SM) biosynthetic gene clusters (BGCs). Histone deacetylases (HDACs) play a pivotal role in determining the extent of histone acetylation modifications and act as triggers for the expression activity of target BGCs. The genus Chaetomium is widely recognized as a rich source of novel and bioactive SMs. Deletion of a class I HDAC gene of Chaetomium olivaceum SD-80A, g7489, induces a substantial pleiotropic effect on the expression of SM BGCs. The C. olivaceum SD-80A ∆g7489 strain exhibited significant changes in morphology, sporulation ability, and secondary metabolic profile, resulting in the emergence of new compound peaks. Notably, three polyketides (A1-A3) and one asterriquinone (A4) were isolated from this mutant strain. Furthermore, our study explored the BGCs of A1-A4, confirming the function of two polyketide synthases (PKSs). Collectively, our findings highlight the promising potential of molecular epigenetic approaches for the elucidation of novel active compounds and their biosynthetic elements in Chaetomium species. This finding holds great significance for the exploration and utilization of Chaetomium resources. KEY POINTS: • Deletion of a class I histone deacetylase activated secondary metabolite gene clusters. • Three polyketides and one asterriquinone were isolated from HDAC deleted strain. • Two different PKSs were reported in C. olivaceum SD-80A.

Three decades of studies on the multifunctional 6-deoxyerythronolide B synthase have laid a foundation for understanding the chemistry and evolution of polyketide antibiotic biosynthesis by a large family of versatile enzymatic assembly lines. Recent progress in applying chemical and structural biology tools to this prototypical assembly-line polyketide synthase (PKS) and related systems has highlighted several features of their catalytic cycles and associated protein dynamics. There is compelling evidence that multiple mechanisms have evolved in this enzyme family to channel growing polyketide chains along uniquely defined sequences of 10-100 active sites, each of which is used only once in the overall catalytic cycle of an assembly-line PKS. Looking forward, one anticipates major advances in our understanding of the mechanisms by which the free energy of a repetitive Claisen-like reaction is harnessed to guide the growing polyketide chain along the assembly line in a manner that is kinetically robust yet evolutionarily adaptable.

2-(2-Phenylethyl) chromone (PEC) and its derivatives are markers of agarwood formation and are also related to agarwood quality. However, the biosynthetic and regulatory mechanisms of PECs still remain mysterious. Several studies suggested that type III polyketide synthases (PKSs) contribute to PEC biosynthesis in Aquilaria sinensis. Furthermore, systematic studies on the evolution of PKSs in A. sinensis have rarely been reported. Herein, we comprehensively analyzed PKS genes from 12 plant genomes and characterized the AsPKSs in detail. A unique branch contained only AsPKS members was identified through evolutionary analysis, including AsPKS01 that was previously indicated to participate in PEC biosynthesis. AsPKS07 and AsPKS08, two tandem-duplicated genes of AsPKS01 and lacking orthologous genes in evolutionary models, were selected for their transient expression in the leaves of Nicotiana benthamiana. Subsequently, PECs were detected in the extracts of N. benthamiana leaves, suggesting that AsPKS07 and AsPKS08 promote PEC biosynthesis. The interaction between the promoters of AsPKS07, AsPKS08 and five basic leucine zippers (bZIPs) from the S subfamily indicated that their transcripts could be regulated by these transcription factors (TFs) and might further contribute to PECs biosynthesis in A. sinensis. Our findings provide valuable insights into the molecular evolution of the PKS gene family in A. sinensis and serve as a foundation for advancing PEC production through the bioengineering of gene clusters. Ultimately, this contribution is expected to shed light on the mechanism underlying agarwood formation.