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-crossover-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.

中文翻译：

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利用CRISPR-Cas9相关碱基编辑探索潮霉素B生物合成。

潮霉素B是一种氨基糖苷类抗生素，广泛用于工业和生物学研究。然而，由于生产菌株的遗传操作非常困难，因此尚未完全鉴定出其大部分生物合成途径。为了解决这个问题，我们开发了一种有效的系统，该系统将聚类的规则间隔的短回文重复序列（CRISPR）-Cas9相关的碱基编辑和位点特异性重组相结合，而不是传统的基于双交叉的同源重组。该策略已成功应用于体内通过产生终止密码子或突变编码区域内的保守残基，使参与潮霉素B生物合成的五个候选基因失活。结果表明，HygJ，HygL和HygD负责核苷酸二磷酸（NDP）庚糖的连续脱氢，转氨作用和转糖基化。值得注意的是，HygY充当羟基碳的不寻常的自由基S-腺苷甲硫氨酸（SAM）依赖差向异构酶，而HygM在多个平行代谢网络中充当通用的甲基转移酶。基于体内和体外证据，提出了潮霉素B的生物合成途径。