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ABSTRACT: Redox enzymes play a central role in generating structural complexity during natural product biosynthesis. In the post-assembly tailoring steps, redox cascades can transform nascent chemical scaffolds into structurally complex final products. Chaetoglobosin A (1) is biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase. It belongs to the chaetoglobosin family of natural products comprised of many analogs having different degrees of oxidation introduced during their biosynthesis. We report here the determination of the complete biosynthetic steps leading to the formation of 1 from prochaetoglobosin I (2). Each oxidation step was elucidated using Chaetomium globosum strains carrying various combinations of deletion of the three redox enzymes, one FAD-dependent monooxygenase and two cytochrome P450 oxygenases, and in vivo biotransformation of intermediates by heterologous expression of the three genes in Saccharomyces cerevisiae. Five analogs were identified in this study as intermediates formed during oxidization of 2 to 1 by those redox enzymes. Furthermore, stereochemical course of each oxidation step was clearly revealed with the absolute configurations of five intermediates determined from X-ray crystal structure. This ap-proach allowed us to quickly determine the biosynthetic intermediates and the enzymes responsible for their formation. Moreover, by addressing the redox enzymes we were able to discover that promiscuity of the redox enzymes allowed the formation of a net-work of pathways that results in a combinatorial formation of multiple intermediate compounds during the formation of 1 from 2. Our approach should expedite elucidation of pathways for other natural products biosynthesized by many uncharacterized enzymes of this fungus.
Journal of the American Chemical Society 04/2013; · 9.91 Impact Factor
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The Journal of Antibiotics 05/2012; 65(7):377-80. · 1.65 Impact Factor
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Kan'ichiro Ishiuchi, Takehito Nakazawa,
Takashi Ookuma,
Satoru Sugimoto,
Michio Sato,
Yuta Tsunematsu,
Noriyasu Ishikawa,
Hiroshi Noguchi,
Kinya Hotta,
Hisao Moriya,
Kenji Watanabe
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ABSTRACT: Fungal genome sequencing has revealed many genes coding for biosynthetic enzymes, including polyketide synthases and nonribosomal peptide synthetases. However, characterizing these enzymes and identifying the compounds they synthesize remains a challenge, whether the genes are expressed in their original hosts or in more tractable heterologous hosts, such as yeast. Here, we developed a streamlined method for isolating biosynthetic genes from fungal sources and producing bioactive molecules in an engineered Saccharomyces cerevisiae host strain. We used overlap extension PCR and yeast homologous recombination to clone desired fungal polyketide synthase or a nonribosomal peptide synthetase genes (5-20 kb) into a yeast expression vector quickly and efficiently. This approach was used successfully to clone five polyketide synthases and one nonribosomal peptide synthetase, from various fungal species. Subsequent detailed chemical characterizations of the resulting natural products identified six polyketide and two nonribosomal peptide products, one of which was a new compound. Our system should facilitate investigating uncharacterized fungal biosynthetic genes, identifying novel natural products, and rationally engineering biosynthetic pathways for the production of enzyme analogues possessing modified bioactivity.
ChemBioChem 03/2012; 13(6):846-54. · 3.94 Impact Factor
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ABSTRACT: Fungal genomes carry many gene clusters seemingly capable of natural product biosynthesis, yet most clusters remain silent. This places a major constraint on the conventional approach of cloning these genes in more amenable heterologous host for the natural product biosynthesis. One way to overcome this difficulty is to activate the silent gene clusters within the context of the target fungus. Here, we successfully activated a silent polyketide biosynthetic gene cluster in Aspergillus oryzae by overexpressing a transcriptional regulator found within the cluster from a plasmid. This strategy allowed us to isolate a new polyketide product and to efficiently decipher its biosynthetic pathway. Through this exercise, we also discovered unexpected activities of the biosynthetic enzymes found in the cluster. These results indicate that our approach would be valuable for isolating novel natural products and engineering analogues of comparable, if not more potent, bioactivity.
ChemBioChem 03/2012; 13(6):855-61. · 3.94 Impact Factor
