Exploration of Hygromycin B Biosynthesis Utilizing CRISPR-Cas9-Associated Base Editing
Exploration of Hygromycin B Biosynthesis Utilizing CRISPR-Cas9-Associated Base Editing
复制标题
利用 CRISPR-Cas9 相关碱基编辑探索潮霉素 B 生物合成
DOI：
10.1021/acschembio.0c00071
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发表时间：
2020
影响因子：
4
通讯作者：
Sun Yuhui
中科院分区：
文献类型：
--
作者：
Li Sicong;Liu Qian;Zhong Zhiyu;Deng Zixin;Sun Yuhui
潮霉素B是一种氨基糖苷类抗生素，广泛应用于工业和生物研究。然而，由于生产菌株的遗传操作的巨大困难，其大部分生物合成途径尚未完全确定。为了解决这个问题，我们开发了一种有效的系统，该系统结合了成簇规则间隔短回文重复序列（CRISPR）-Cas9相关碱基编辑和位点特异性重组，而不是传统的基于双交叉的同源重组。该策略成功地应用于通过产生终止密码子或突变编码区内的保守残基来体内失活参与潮霉素B生物合成的5个候选基因。结果表明，HygJ、HygL和HygD负责核苷二磷酸（NDP）-庚糖的连续脱氢、转氨和转糖基化。值得注意的是，HygY作为一个不寻常的自由基S-腺苷甲硫氨酸（SAM）依赖性差向异构酶的羟基碳，和HygM作为一个通用的甲基转移酶在多个平行的代谢网络。根据体内外实验结果，提出了潮霉素B的生物合成途径。
Hygromycin B is an aminoglycoside antibiotic widely used in industry and biological research. However, most of its biosynthetic pathway has not been completely identified due to the immense difficulty in genetic manipulation of the producing strain. To address this problem, we developed an efficient system that combines clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-associated base editing and site-specific recombination instead of conventional double-cross-based homologous recombination. This strategy was successfully applied to the in vivo inactivation of five candidate genes involved in the biosynthesis of hygromycin B by generating stop codons or mutating conserved residues within the encoding region. The results revealed that HygJ, HygL and HygD are responsible for successive dehydrogenation, transamination and transglycosylation of nucleoside diphosphate (NDP)-heptose. Notably, HygY acts as an unusual radical S-adenosylmethionine (SAM)-dependent epimerase for hydroxyl carbons, and HygM serves as a versatile methyltransferase in multiple parallel metabolic networks. Based on in vivo and in vitro evidence, the biosynthetic pathway for hygromycin B is proposed.