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
复制标题
DOI：
10.1002/cbic.201100798
复制
发表时间：
2012-04-16
期刊：
影响因子：
3.2
通讯作者：
Watanabe, Kenji
中科院分区：
文献类型：
--
作者：
Ishiuchi, Kan'ichiro;Nakazawa, Takehito;Watanabe, Kenji
真菌基因组测序揭示了许多编码生物合成酶的基因，包括聚酮酶和非核糖体肽合成酶。然而，表征这些酶和鉴定它们合成的化合物仍然是一个挑战，无论这些基因是在其原始宿主还是在更易处理的异源宿主（如酵母）中表达。在这里，我们开发了一种简化的方法，用于从真菌来源中分离生物合成基因，并在工程酿酒酵母宿主菌株中产生生物活性分子。我们使用重叠延伸PCR和酵母同源重组将所需的真菌聚酮合酶或非核糖体肽合成酶基因（520 kb）快速有效地克隆到酵母表达载体中。这种方法被成功地用于克隆五个聚酮酶和一个非核糖体肽合成酶，从各种真菌物种。随后对所得天然产物进行了详细的化学表征，确定了六种聚酮化合物和两种非核糖体肽产物，其中一种是新化合物。我们的系统应有助于调查未知的真菌生物合成基因，确定新的天然产物，合理的工程生物合成途径，生产具有修饰的生物活性的酶类似物。
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 (520 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.