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Science Advances Mar 2024The fungal bioluminescence pathway can be reconstituted in other organisms allowing luminescence imaging without exogenously supplied substrate. The pathway starts from...
The fungal bioluminescence pathway can be reconstituted in other organisms allowing luminescence imaging without exogenously supplied substrate. The pathway starts from hispidin biosynthesis-a step catalyzed by a large fungal polyketide synthase that requires a posttranslational modification for activity. Here, we report identification of alternative compact hispidin synthases encoded by a phylogenetically diverse group of plants. A hybrid bioluminescence pathway that combines plant and fungal genes is more compact, not dependent on availability of machinery for posttranslational modifications, and confers autonomous bioluminescence in yeast, mammalian, and plant hosts. The compact size of plant hispidin synthases enables additional modes of delivery of autoluminescence, such as delivery with viral vectors.
Topics: Animals; Luminescence; Plants; Mammals
PubMed: 38457503
DOI: 10.1126/sciadv.adk1992 -
BMC Ecology and Evolution Oct 2023Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid...
BACKGROUND
Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid social amoebas acquired diguanylate cyclase (dgcA) by horizontal gene transfer. Dictyostelium discoideum (Ddis) in taxon group 4 uses c-di-GMP as a secreted signal to induce differentiation of stalk cells, the ancestral somatic cell type that supports the propagating spores. We here investigated how this role for c-di-GMP evolved in Dictyostelia by exploring dgcA function in the group 2 species Polysphondylium pallidum (Ppal) and in Polysphondylium violaceum (Pvio), which resides in a small sister clade to group 4.
RESULTS
Similar to Ddis, dgcA is upregulated after aggregation in Ppal and Pvio and predominantly expressed in the anterior region and stalks of emerging fruiting bodies. DgcA null mutants in Ppal and Pvio made fruiting bodies with very long and thin stalks and only few spores and showed delayed aggregation and larger aggregates, respectively. Ddis dgcA- cells cannot form stalks at all, but showed no aggregation defects. The long, thin stalks of Ppal and Pvio dgcA- mutants were also observed in acaA- mutants in these species. AcaA encodes adenylate cyclase A, which mediates the effects of c-di-GMP on stalk induction in Ddis. Other factors that promote stalk formation in Ddis are DIF-1, produced by the polyketide synthase StlB, low ammonia, facilitated by the ammonia transporter AmtC, and high oxygen, detected by the oxygen sensor PhyA (prolyl 4-hydroxylase). We deleted the single stlB, amtC and phyA genes in Pvio wild-type and dgcA- cells. Neither of these interventions affected stalk formation in Pvio wild-type and not or very mildly exacerbated the long thin stalk phenotype of Pvio dgcA- cells.
CONCLUSIONS
The study reveals a novel role for c-di-GMP in aggregation, while the reduced spore number in Pvio and Ppal dgcA- is likely an indirect effect, due to depletion of the cell pool by the extended stalk formation. The results indicate that in addition to c-di-GMP, Dictyostelia ancestrally used an as yet unknown factor for induction of stalk formation. The activation of AcaA by c-di-GMP is likely conserved throughout Dictyostelia.
Topics: Dictyostelium; Ammonia; Phosphorus-Oxygen Lyases; Dictyosteliida; Oxygen
PubMed: 37803310
DOI: 10.1186/s12862-023-02169-z -
Nature Communications Nov 2023The dominant mutational signature in colorectal cancer genomes is C > T deamination (COSMIC Signature 1) and, in a small subgroup, mismatch repair signature (COSMIC...
The dominant mutational signature in colorectal cancer genomes is C > T deamination (COSMIC Signature 1) and, in a small subgroup, mismatch repair signature (COSMIC signatures 6 and 44). Mutations in common colorectal cancer driver genes are often not consistent with those signatures. Here we perform whole-genome sequencing of normal colon crypts from cancer patients, matched to a previous multi-omic tumour dataset. We analyse normal crypts that were distant vs adjacent to the cancer. In contrast to healthy individuals, normal crypts of colon cancer patients have a high incidence of pks + (polyketide synthases) E.coli (Escherichia coli) mutational and indel signatures, and this is confirmed by metagenomics. These signatures are compatible with many clonal driver mutations detected in the corresponding cancer samples, including in chromatin modifier genes, supporting their role in early tumourigenesis. These results provide evidence that pks + E.coli is a potential driver of carcinogenesis in the human gut.
Topics: Humans; Escherichia coli; Colorectal Neoplasms; Mutation; Carcinogenesis; Colonic Neoplasms
PubMed: 38030613
DOI: 10.1038/s41467-023-43329-5 -
Open Biology Aug 2023Assembly line polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for synthesizing many medicinally relevant natural products with...
Assembly line polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for synthesizing many medicinally relevant natural products with remarkable structural variety and biological activity. The decrease in cost of genomic sequencing paired with development of computational tools like antiSMASH presents an opportunity to survey the vast diversity of assembly line PKS. Mining the genomic data in the National Center for Biotechnology Information database, our updated catalogue (https://orphanpkscatalog2022.stanford.edu/catalog) presented in this article revealed 8799 non-redundant assembly line polyketide synthase clusters across 4083 species, representing a threefold increase over the past 4 years. Additionally, 95% of the clusters are 'orphan clusters' for which natural products are neither chemically nor biologically characterized. Our analysis indicates that the diversity of assembly line PKSs remains vastly under-explored and also highlights the promise of a genomics-driven approach to natural product discovery.
Topics: Polyketide Synthases; Base Sequence; Genomics; Biological Products
PubMed: 37528731
DOI: 10.1098/rsob.230096 -
Chemical Science Oct 2023Protein-reactive natural products such as the fungal metabolite cerulenin are recognized for their value as therapeutic candidates, due to their ability to selectively...
Protein-reactive natural products such as the fungal metabolite cerulenin are recognized for their value as therapeutic candidates, due to their ability to selectively react with catalytic residues within a protein active site or a complex of protein domains. Here, we explore the development of fatty-acid and polyketide-synthase probes by synthetically modulating cerulenin's functional moieties. Using a mechanism-based approach, we reveal unique reactivity within cerulenin and adapt it for fluorescent labeling and crosslinking of fatty-acid and iterative type-I polyketide synthases. We also describe two new classes of silylcyanohydrin and silylhemiaminal masked crosslinking probes that serve as new tools for activity and structure studies of these biosynthetic pathways.
PubMed: 37829009
DOI: 10.1039/d3sc02864j -
PNAS Nexus Mar 2024Parrots have remarkable plumage coloration that result in part from a unique ability to produce pigments called psittacofulvins that yield yellow to red feather colors....
Parrots have remarkable plumage coloration that result in part from a unique ability to produce pigments called psittacofulvins that yield yellow to red feather colors. Little is known about the evolution of psittacofulvin-based pigmentation. Widespread color mutations of captive-bred parrots provide perfect opportunities to study the genetic basis of this trait. An earlier study on budgerigars, which do not possess psittacofulvins, reveals the involvement of an uncharacterized polyketide synthase (MuPKS) in yellow psittacofulvin synthesis. The phenotype had repeatedly appeared in different parrot species, similar to independent experimental replications allowing the study of convergent evolution and molecular mechanism of psittacofulvin-based pigmentation. Here, we investigated the genetic basis of the phenotypes in two species of parrots, Fischer's lovebird () and Yellow-collared lovebird (). Using whole-genome data, we identified a single genomic region with size <2 Mb to be strongly associated with the color difference between and wild-type (WT) birds in both species. Surprisingly, we discovered that the mutation associated with the phenotype was identical to the previously described substitution causing the functional change of MuPKS in budgerigars. Together with the evidence of shared -associated haplotypes and signatures of a selective sweep in this genomic region in both species, we demonstrated both mutation and interspecific introgression play a role in the evolution of this trait in different species. The convergent substitution in the same gene in both lovebirds and budgerigars also indicates a strong evolutionary constraint on psittacofulvin-based coloration.
PubMed: 38528953
DOI: 10.1093/pnasnexus/pgae107 -
Angewandte Chemie (International Ed. in... Nov 2023Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite,...
Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA-A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS shows relaxed substrate specificity, suggesting precise spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.
Topics: Mupirocin; Polyketide Synthases; Hydroxylation; Anti-Bacterial Agents
PubMed: 37768840
DOI: 10.1002/anie.202312514 -
Scientific Reports Sep 2023Docosahexaenoic acid (DHA) and selenium (Se) are nutrients that confer several health benefits to both humans and animals. Widespread use of DHA in milk powder and...
Docosahexaenoic acid (DHA) and selenium (Se) are nutrients that confer several health benefits to both humans and animals. Widespread use of DHA in milk powder and health products requires large-scale mass production via Schizochytrium sp., while Se intended for human consumption is produced as organic Se via yeast. However, producing these nutrients on an industrial scale is constrained by various factors. We found that supplementing Schizochytrium sp. with NaSeO (0.5 mg/L) improves its biomass and DHA production and also provides organic Se. De novo assembled transcriptome and biochemical indicators showed that NaSeO promotes forming acetyl coenzyme A and L-cysteine via the glycerol kinase and cysteine synthase pathways, promoting DHA synthesis through the polyketide synthase pathway. However, high doses of NaSeO (5 mg/L) limited the biomass of Schizochytrium sp. and DHA content. This study provided a theoretical basis for the simultaneous production of organic Se and DHA via Schizochytrium sp.
Topics: Animals; Humans; Selenium; Docosahexaenoic Acids; Stramenopiles; Acetyl Coenzyme A; Biomass; Saccharomyces cerevisiae
PubMed: 37731016
DOI: 10.1038/s41598-023-42900-w -
BioRxiv : the Preprint Server For... Dec 2023Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have...
Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like . Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in frequently results in a myriad of unpredictable issues with protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts, such as BGC refactoring, can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as 1) a valuable expression platform for enzymes that are challenging to synthesize , and as 2) a testbed for rapid prototyping that can improve cellular expression. Here, we use a type III polyketide synthase from , RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques We synergistically tune promoter and codon usage to improve flaviolin production from cell-free expressed RppA. We then assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs. By showing the coordinators between CFE versus expression, this work advances CFE as a tool for natural product research.
PubMed: 38077034
DOI: 10.1101/2023.11.30.569483 -
Marine Drugs Jul 2023Dinoflagellates are unicellular organisms that are implicated in harmful algal blooms (HABs) caused by potent toxins that are produced through polyketide synthase (PKS)...
Dinoflagellates are unicellular organisms that are implicated in harmful algal blooms (HABs) caused by potent toxins that are produced through polyketide synthase (PKS) pathways. However, the exact mechanisms of toxin synthesis are unknown due to a lack of genomic segregation of fat, toxins, and other PKS-based pathways. To better understand the underlying mechanisms, the actions and expression of the PKS proteins were investigated using the toxic dinoflagellate as a model. Cerulenin, a known ketosynthase inhibitor, was shown to reduce acetate incorporation into all fat classes with the toxins amphidinol and sulpho-amphidinol. The mass spectrometry analysis of cerulenin-reacted synthetic peptides derived from ketosynthase domains of multimodular PKS transcripts demonstrated a strong covalent bond that could be localized using collision-induced dissociation. One multi-modular PKS sequence present in all dinoflagellates surveyed to date was found to lack an AT domain in toxin-producing species, indicating -acting domains, and was shown by Western blotting to be post-transcriptionally processed. These results demonstrate how toxin synthesis in dinoflagellates can be differentiated from fat synthesis despite common underlying pathway.
Topics: Cerulenin; Polyketide Synthases; Dinoflagellida; Harmful Algal Bloom; Blotting, Western
PubMed: 37623706
DOI: 10.3390/md21080425