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Cited by 1melanin and skin pigmentationMicrobial Metabolism and ApplicationsMicrobial Natural Products and Biosynthesis

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Multiple mutations in polyketide synthase led to disruption of Psittacofulvin production across diverse parrot species

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Ghosh Roy, Shatadru, Brejcha, Jindřich, Maršík, Petr, Bakhrat, Anna, Abdu, Moty, Arbore, Roberto, Araújo, Pedro Miguel, Afonso, Sandra, Carneiro, Miguel, Grossman-Haham, Iris, Abdu, Uri·2025·Communications Biology
DOI 10.1038/s42003-025-07537-7PMC PMC11739604

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Abstract

Abstract Polyketide synthases (PKSs) are crucial multidomain enzymes in diverse natural product biosynthesis. Parrots use a type I PKS to produce a unique pigment called psittacofulvin in their feathers. In domesticated budgerigars and lovebirds, the same amino acid substitution (R644W) within malonyl/acetyltransferase (MAT) domain of this enzyme has been shown to cause the blue phenotype with no psittacofulvin pigmentation, proposing a strong evolutionary constraint mechanism. Here, we identified six new causal variants in PKS associated with defective psittacofulvin production in four diverse species, including three nonsense mutations. Remaining three nonsynonymous substitutions reside within the ketoacyl synthase (KS) domain. Heterologous expression of these PKS variants in yeast confirmed complete or reduced loss of psittacofulvin production, additionally suggesting presence of an auxiliary polymorphism at the MAT domain. Our findings establish PKS as a functionally conserved key-enzyme determining psittacofulvin-based hues among diverse parrots, highlighting multiple conserved domains essential for the PKS function.

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Figures

Fig. 1 Phenotypic characterization of diverse parrots. a Color morphs analyzed from family psittaculidae b Color morphs analyzed from family cacatuidae (wild-type birds are at the left and blue phenotypes are at right) (c) Phylogenetic relation between previously analyzed species (marked in blue) and species analyzed in the current study (d) Key changes in psittacofulvin-based hues in different species. [Pictures were taken by Shatadru Ghosh Roy or available in the public domain].
Fig. 1 Phenotypic characterization of diverse parrots. a Color morphs analyzed from family psittaculidae b Color morphs analyzed from family cacatuidae (wild-type birds are at the left and blue phenotypes are at right) (c) Phylogenetic relation between previously analyzed species (marked in blue) and species analyzed in the current study (d) Key changes in psittacofulvin-based hues in different species. [Pictures were taken by Shatadru Ghosh Roy or available in the public domain].CC BY-NC-ND · PubMed Central
Fig. 2 Histological sections of remex barb from P. krameri under a light microscope. a Green barbs and yellow to black barbules of contour feathers from wild type P. krameri b Light microscope images of longitudinal section of the contour barb from wild type P. krameri. c blue barbs and white to black barbules of contour feathers from P. krameri (blue) d Light microscope images of longitudinal section of the contour barb from P. krameri (blue). [Cr- Cortex; Md- Medulla. Scale bars for images are 200 µm.].
Fig. 2 Histological sections of remex barb from P. krameri under a light microscope. a Green barbs and yellow to black barbules of contour feathers from wild type P. krameri b Light microscope images of longitudinal section of the contour barb from wild type P. krameri. c blue barbs and white to black barbules of contour feathers from P. krameri (blue) d Light microscope images of longitudinal section of the contour barb from P. krameri (blue). [Cr- Cortex; Md- Medulla. Scale bars for images are 200 µm.].CC BY-NC-ND · PubMed Central
Fig. 3 Non-synonymous substitutions found in Sanger sequencing. a Position of affected residues on the domain structure of a PKS, homologous to mammalian fatty-acid synthase. b Conservation of affected amino acid residues found in different PKS homologs. The shades of blue in the alignment represent the percentage identity of the amino acid residues. Affected residue is marked with red box. [Mu: M. undulatus; Pk: P. krameri; Pe: P. eupatria; Ecr: E. roratus; Eor: E. roseicapilla; Gg: G. gallus; Ac: A. carolinensis; Dr: D. rerio; Mm: M. musculus; Hs: H. sapiens; Dm: D. melanogaster].
Fig. 3 Non-synonymous substitutions found in Sanger sequencing. a Position of affected residues on the domain structure of a PKS, homologous to mammalian fatty-acid synthase. b Conservation of affected amino acid residues found in different PKS homologs. The shades of blue in the alignment represent the percentage identity of the amino acid residues. Affected residue is marked with red box. [Mu: M. undulatus; Pk: P. krameri; Pe: P. eupatria; Ecr: E. roratus; Eor: E. roseicapilla; Gg: G. gallus; Ac: A. carolinensis; Dr: D. rerio; Mm: M. musculus; Hs: H. sapiens; Dm: D. melanogaster].CC BY-NC-ND · PubMed Central
Fig. 4 Biochemical validation of candidate missense variants. a Graphical representation of methanol extraction from yeast strain BJ5464-NpgA expressing HA-tagged GgPKS Wild-type or with the substitutions found in blue phenotypes. b The extracts, illuminated with white light or UV A and corresponding GgPKS expression in total soluble protein extracts from the same yeast cultures by western blot with α-HA antibody. c the results of the analysis using UHPLC-HRAM QTOF showed that the compounds produced in the feathers of the parrot P. krameri, which elute from the Phenyl-Hexyl column at 6.3, 6.7, and 7.1 min and have characteristic exact molecular masses of 217.1229 m/z, 243.1385 m/z, and 269.1542 m/z—corresponding to carboxy-psittacofulvins with chain lengths of 14, 16, and 18 carbons, respectively—are also produced in yeast cultures expressing wild-type GgPKS. These compounds are not found in extracts from yeast lacking GgPKS (empty vector) or with GgPKS containing G112R or R243K substitutions. A partial production of these compounds is seen in GgPKS with N428K, T792A substitutions and their combinations. Wild-type GgPKS sample shows the highest amount of psittacofulvins, followed by lower peaks in the T792A sample, with the N428K sample yielding the least. When N428K and T792A mutations are combined, the production of psittacofulvins is even lower than when these two mutations are expressed individually.
Fig. 4 Biochemical validation of candidate missense variants. a Graphical representation of methanol extraction from yeast strain BJ5464-NpgA expressing HA-tagged GgPKS Wild-type or with the substitutions found in blue phenotypes. b The extracts, illuminated with white light or UV A and corresponding GgPKS expression in total soluble protein extracts from the same yeast cultures by western blot with α-HA antibody. c the results of the analysis using UHPLC-HRAM QTOF showed that the compounds produced in the feathers of the parrot P. krameri, which elute from the Phenyl-Hexyl column at 6.3, 6.7, and 7.1 min and have characteristic exact molecular masses of 217.1229 m/z, 243.1385 m/z, and 269.1542 m/z—corresponding to carboxy-psittacofulvins with chain lengths of 14, 16, and 18 carbons, respectively—are also produced in yeast cultures expressing wild-type GgPKS. These compounds are not found in extracts from yeast lacking GgPKS (empty vector) or with GgPKS containing G112R or R243K substitutions. A partial production of these compounds is seen in GgPKS with N428K, T792A substitutions and their combinations. Wild-type GgPKS sample shows the highest amount of psittacofulvins, followed by lower peaks in the T792A sample, with the N428K sample yielding the least. When N428K and T792A mutations are combined, the production of psittacofulvins is even lower than when these two mutations are expressed individually.CC BY-NC-ND · PubMed Central
Fig. 5 Structural analysis of causal SNPs. a Predicted structure of the KS, linker, and MAT domains of the P. krameri (blue) PKS generated using AlphaFold (Jumper et al., 21) and colored according to per-residue confidence (pLDDT) scale (see legend). Note the high confidence in prediction (indicated in blue). b Left - the prediction shown in (a) colored by domain, showing the positions of the four residues identified as SNPs (indicated in red). Right - the crystal structure of porcine fatty-acid synthase (PDB: 2VZ8) (Maier et al. 22) with each domain differently colored is shown for context. Note that the structure is a homodimer containing two polypeptide chains. The boxed region includes the relevant domains for which the P. krameri structure was predicted. KS - β-ketoacyl synthase domain, MAT - malonyl/acetyltransferase domain, DH – dehydrase domain, ME - pseudo-methyltransferase domain, KR - β-ketoacyl reductase domain, and ER - enoyl reductase domain. c Closer view of G112 (in red), showing it is positioned in a loop, at the interface of a linker domain (gray) and the KS domain (blue). d Closer view of R243 (in red), showing it is positioned near the catalytic residues (colored green) and the residues that form the entrance to the substrate binding tunnel (colored purple). e Closer view of N428, showing its proximity to the residues that form the entrance of the substrate binding tunnel (colored purple) and that it is buried under an α helix. f Closer view of T792, showing a predicted hydrogen bond (indicated with a dashed line) between the sidechain OH group and the main chain of the adjacent loop.
Fig. 5 Structural analysis of causal SNPs. a Predicted structure of the KS, linker, and MAT domains of the P. krameri (blue) PKS generated using AlphaFold (Jumper et al., 21) and colored according to per-residue confidence (pLDDT) scale (see legend). Note the high confidence in prediction (indicated in blue). b Left - the prediction shown in (a) colored by domain, showing the positions of the four residues identified as SNPs (indicated in red). Right - the crystal structure of porcine fatty-acid synthase (PDB: 2VZ8) (Maier et al. 22) with each domain differently colored is shown for context. Note that the structure is a homodimer containing two polypeptide chains. The boxed region includes the relevant domains for which the P. krameri structure was predicted. KS - β-ketoacyl synthase domain, MAT - malonyl/acetyltransferase domain, DH – dehydrase domain, ME - pseudo-methyltransferase domain, KR - β-ketoacyl reductase domain, and ER - enoyl reductase domain. c Closer view of G112 (in red), showing it is positioned in a loop, at the interface of a linker domain (gray) and the KS domain (blue). d Closer view of R243 (in red), showing it is positioned near the catalytic residues (colored green) and the residues that form the entrance to the substrate binding tunnel (colored purple). e Closer view of N428, showing its proximity to the residues that form the entrance of the substrate binding tunnel (colored purple) and that it is buried under an α helix. f Closer view of T792, showing a predicted hydrogen bond (indicated with a dashed line) between the sidechain OH group and the main chain of the adjacent loop.CC BY-NC-ND · PubMed Central

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Citations

Cites

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Metadata

Authors
Ghosh Roy, S., Brejcha, J., Maršík, P., Bakhrat, A., Abdu, M., Arbore, R., Araújo, P. M., Afonso, S., Carneiro, M., Grossman-Haham, I., & Abdu, U.
Affiliations
  • Charles University (CZ)
  • Czech University of Life Sciences Prague (CZ)
  • Ben-Gurion University of the Negev (IL)
  • Universidade do Porto (PT)
Corresponding author
Abdu, Uri
ORCID
Venue
Communications Biology
Publisher
Springer Science and Business Media LLC
Published
2025-01-17
Volume
8
Issue
1
Pages
69-69
ISSN
2399-3642 (ISSN-L)
DOI
10.1038/s42003-025-07537-7
PMC
PMC11739604
OpenAlex
W4406446488
Cited by
1
References
2
Citations by year
2026: 1
FWCI
0.50
Type
journal article
Language
en
Open access
Licence
Locations
  • Communications Biology (publishedVersion, cc-by-nc-nd)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Israel Science Foundation (941/21)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › melanin and skin pigmentation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Microbial Metabolism and Applications
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
Keywords
polyketide synthase, parrots, feather pigmentation, budgerigars, lovebirds, nonsense mutation, nonsynonymous substitutions, heterologous expression in yeast, natural product biosynthesis, evolutionary constraints
MeSH terms
Animals, Feathers, Mutation, Parrots, Pigmentation, Pigments, Biological, Polyketide Synthases

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