Large-scale genome-mining analyses have revealed that microbes potentially harbor a huge reservoir of unchar-acterized natural product(NP)biosynthetic gene clusters(BGCs),and this has spurred a renaissance of novel dr...Large-scale genome-mining analyses have revealed that microbes potentially harbor a huge reservoir of unchar-acterized natural product(NP)biosynthetic gene clusters(BGCs),and this has spurred a renaissance of novel drug discovery.However,the majority of these BGCs are often poorly or not at all expressed in their native hosts under laboratory conditions,and thus are regarded as silent/orphan BGCs.Currently,connecting silent BGCs to their corresponding NPs quickly and on a large scale is particularly challenging because of the lack of universal strategies and enabling technologies.Generally,the heterologous host-based genome mining strategy is believed to be a suitable alternative to the native host-based approach for prioritization of the vast and ever-increasing number of uncharacterized BGCs.In the last ten years,a variety of methods have been reported for the direct cloning of BGCs of interest,which is the first and rate-limiting step in the heterologous expression strategy.Es-sentially,each method requires that the following three issues be resolved:1)how to prepare genomic DNA;2)how to digest the bilateral boundaries for release of the target BGC;and 3)how to assemble the BGC and the capture vector.Here,we summarize recent reports regarding how to directly capture a BGC of interest and briefly discuss the advantages and disadvantages of each method,with an emphasis on the notion that direct cloning is very beneficial for accelerating genome mining research and large-scale drug discovery.展开更多
Aromatic polyketides(APKs)are renowned for their structural diversity and potent biological activities,making them valuable resources for drug discovery in antibiotics,anticancer agents,and antiparasitic therapies.Bip...Aromatic polyketides(APKs)are renowned for their structural diversity and potent biological activities,making them valuable resources for drug discovery in antibiotics,anticancer agents,and antiparasitic therapies.Bipentaromycins,a unique class of dimeric APKs with a cyclic head-to-tail linkage,exhibit broad-spectrum antibacterial activities.Acylation modifications in bipentaromycins C–F enhance their pharmacological properties,yet the responsible acyltransferase remains enigmatic.Herein,we present REGAIN,an innovative strategy combining restriction enzyme digestion,Gibson assembly,and in vivo Cre-lox recombinatio n,enabling rapid and efficient cloning of biosynthetic gene clusters(BGCs).Using REGAIN,we successfully cloned a 40 kb bipentaromycin BGC(bpm).By integrating genetic experiments and computational modeling,we speculated BpmP as the acyltransferase responsible for regioselective acylation.This study establishes a robust platform for exploring novel bioactive molecules and advances the understanding of bipentaromycin biosynthesis.展开更多
基金supported by the National Key Research and Development Program of China(2021YFC2100600)the National Natural Science Foundation of China(31870034)the Science and Technology Commission of Shanghai Municipality(20ZR1469100).
文摘Large-scale genome-mining analyses have revealed that microbes potentially harbor a huge reservoir of unchar-acterized natural product(NP)biosynthetic gene clusters(BGCs),and this has spurred a renaissance of novel drug discovery.However,the majority of these BGCs are often poorly or not at all expressed in their native hosts under laboratory conditions,and thus are regarded as silent/orphan BGCs.Currently,connecting silent BGCs to their corresponding NPs quickly and on a large scale is particularly challenging because of the lack of universal strategies and enabling technologies.Generally,the heterologous host-based genome mining strategy is believed to be a suitable alternative to the native host-based approach for prioritization of the vast and ever-increasing number of uncharacterized BGCs.In the last ten years,a variety of methods have been reported for the direct cloning of BGCs of interest,which is the first and rate-limiting step in the heterologous expression strategy.Es-sentially,each method requires that the following three issues be resolved:1)how to prepare genomic DNA;2)how to digest the bilateral boundaries for release of the target BGC;and 3)how to assemble the BGC and the capture vector.Here,we summarize recent reports regarding how to directly capture a BGC of interest and briefly discuss the advantages and disadvantages of each method,with an emphasis on the notion that direct cloning is very beneficial for accelerating genome mining research and large-scale drug discovery.
基金supported by the Key R&D Program of Shandong Province,China(2024CXPT029)the National Natural Science Foundation of China(22407132)+3 种基金the 100 Talents Program of the Chinese Academy of Sciences,the Shanghai Pujiang Program(23PJ1415100)the Shandong Provincial Excellent Youth Science Fund Project(Overseas)(2025HWYQ-O69)the TaiShan Scholars Program(tsqn202408316)the Shandong Provincial Natural Science Foundation(ZR2024QC204).
文摘Aromatic polyketides(APKs)are renowned for their structural diversity and potent biological activities,making them valuable resources for drug discovery in antibiotics,anticancer agents,and antiparasitic therapies.Bipentaromycins,a unique class of dimeric APKs with a cyclic head-to-tail linkage,exhibit broad-spectrum antibacterial activities.Acylation modifications in bipentaromycins C–F enhance their pharmacological properties,yet the responsible acyltransferase remains enigmatic.Herein,we present REGAIN,an innovative strategy combining restriction enzyme digestion,Gibson assembly,and in vivo Cre-lox recombinatio n,enabling rapid and efficient cloning of biosynthetic gene clusters(BGCs).Using REGAIN,we successfully cloned a 40 kb bipentaromycin BGC(bpm).By integrating genetic experiments and computational modeling,we speculated BpmP as the acyltransferase responsible for regioselective acylation.This study establishes a robust platform for exploring novel bioactive molecules and advances the understanding of bipentaromycin biosynthesis.
