Inverted repeat of a large segment unveiled on the right arm of <i>Saccharomyces cerevisiae</i> chromosome II

Ohnishi, Katsunori; Ono, Bun-ichiro

doi:10.1002/yea.1213

Cited by 4 publications

(1 citation statement)

References 33 publications

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“…Transformation and cultivation for compound production: For de novo production of target compounds, an engineered yeast host, SCKW5, was used as an expression host. SCKW5 was derived from S. cerevisiae BY4741 (genotype: MAT α his3‐ Δ 1 leu2‐ Δ 0 met15‐ Δ 0 ura3‐ Δ 0 ),32 which was obtained from Open Biosystems Inc. SCKW5 was obtained by engineering the chromosomal DNA of BY4741 to integrate three genes, matB , npgA and G418 resistance marker, into FCY1 , which is responsible for 5‐fluorocytosine toxicity in yeast. Each expression vector was transformed into SCKW5 by using a standard transformation procedure 33.…”

Section: Methodsmentioning

confidence: 99%

Establishing a New Methodology for Genome Mining and Biosynthesis of Polyketides and Peptides through Yeast Molecular Genetics

et al. 2012

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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.

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Section: Methodsmentioning

confidence: 99%

Establishing a New Methodology for Genome Mining and Biosynthesis of Polyketides and Peptides through Yeast Molecular Genetics

et al. 2012

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Current awareness on yeast

2005

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New natural products isolated from Metarhizium robertsii ARSEF 23 by chemical screening and identification of the gene cluster through engineered biosynthesis in Aspergillus nidulans A1145

et al. 2016

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To rapidly identify novel natural products and their associated biosynthetic genes from underutilized and genetically difficult-to-manipulate microbes, we developed a method that uses (1) chemical screening to isolate novel microbial secondary metabolites, (2) bioinformatic analyses to identify a potential biosynthetic gene cluster and (3) heterologous expression of the genes in a convenient host to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. The chemical screen was achieved by searching known natural product databases with data from liquid chromatographic and high-resolution mass spectrometric analyses collected on the extract from a target microbe culture. Using this method, we were able to isolate two new meroterpenes, subglutinols C (1) and D (2), from an entomopathogenic filamentous fungus Metarhizium robertsii ARSEF 23. Bioinformatics analysis of the genome allowed us to identify a gene cluster likely to be responsible for the formation of subglutinols. Heterologous expression of three genes from the gene cluster encoding a polyketide synthase, a prenyltransferase and a geranylgeranyl pyrophosphate synthase in Aspergillus nidulans A1145 afforded an α-pyrone-fused uncyclized diterpene, the expected intermediate of the subglutinol biosynthesis, thereby confirming the gene cluster to be responsible for the subglutinol biosynthesis. These results indicate the usefulness of our methodology in isolating new natural products and identifying their associated biosynthetic gene cluster from microbes that are not amenable to genetic manipulation. Our method should facilitate the natural product discovery efforts by expediting the identification of new secondary metabolites and their associated biosynthetic genes from a wider source of microbes.

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Inverted repeat of a large segment unveiled on the right arm of Saccharomyces cerevisiae chromosome II

Cited by 4 publications

References 33 publications

Establishing a New Methodology for Genome Mining and Biosynthesis of Polyketides and Peptides through Yeast Molecular Genetics

Establishing a New Methodology for Genome Mining and Biosynthesis of Polyketides and Peptides through Yeast Molecular Genetics

Current awareness on yeast

New natural products isolated from Metarhizium robertsii ARSEF 23 by chemical screening and identification of the gene cluster through engineered biosynthesis in Aspergillus nidulans A1145

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