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Strategy for efficient cloning of biosynthetic gene clusters from fungi 被引量:2

Strategy for efficient cloning of biosynthetic gene clusters from fungi
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摘要 Filamentous fungi are excellent sources for the production of a group of bioactive small molecules which are often called secondary metabolites(SMs). The advanced genome sequencing technology combined with bioinformatics analysis reveals a large number of unexplored biosynthetic gene clusters(BGCs) in the fungal genomes. To unlock this fungal SM treasure, many approaches including heterologous expression are being developed and efficient cloning of the BGCs is a crucial step to do this.Here, we present an efficient strategy for the direct cloning of fungal BGCs. This strategy consisted of Splicing by Overlapping Extension(SOE)-PCR and yeast assembly in vivo. By testing 14 BGCs DNA fragments ranging from 7 kb to 52 kb, the average positive rate was over 80%. The maximal insertion size for fungal BGC assembly was 52 kb. Those constructs could be used conveniently for the heterologous expression leading to the discovery of novel natural products. Thus, our results provide an efficient and quick method for the low cost direct cloning of fungal BGCs. Filamentous fungi are excellent sources for the production of a group of bioactive small molecules which are often called secondary metabolites(SMs). The advanced genome sequencing technology combined with bioinformatics analysis reveals a large number of unexplored biosynthetic gene clusters(BGCs) in the fungal genomes. To unlock this fungal SM treasure, many approaches including heterologous expression are being developed and efficient cloning of the BGCs is a crucial step to do this.Here, we present an efficient strategy for the direct cloning of fungal BGCs. This strategy consisted of Splicing by Overlapping Extension(SOE)-PCR and yeast assembly in vivo. By testing 14 BGCs DNA fragments ranging from 7 kb to 52 kb, the average positive rate was over 80%. The maximal insertion size for fungal BGC assembly was 52 kb. Those constructs could be used conveniently for the heterologous expression leading to the discovery of novel natural products. Thus, our results provide an efficient and quick method for the low cost direct cloning of fungal BGCs.
出处 《Science China(Life Sciences)》 SCIE CAS CSCD 2019年第8期1087-1095,共9页 中国科学(生命科学英文版)
关键词 biosynthetic gene CLUSTERS SACCHAROMYCES CEREVISIAE HOMOLOGOUS recombination DNA assembly biosynthetic gene clusters Saccharomyces cerevisiae homologous recombination DNA assembly
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