Study On Hygromicin B Biosynthesis Based On Base Editing

Hygromycin B is an aminoglycoside antibiotic generated by Streptomyces hygroscopicus subsp.hygroscopicus DSM40578.Its structure is characterized by the parent nucleus of 2-deoxystreptamine(2-DOS)which is common in aminoglycosides,as well as an orthoester linkage.As an antibiotic,Hygromycin B inhibits bacteria cells by blocking protein synthesis mainly through targeting 30 S ribosomal subunit and it is aslo useful in biological experiments as a screening marker for eukaryotic cells.It has also been used in animal husbandry because of its good anti-parasitic activity.Although hygromycin B has been widely applicated in variety areas,its biosynthetic pathway is still not fully charaterized.One of the biggest obstacles is that the genetic manipulation system of hygromycin B producing bacteria is difficult to be established.Since traditional methods based on homologous recombination is time-consuming and is also inefficient in hygromycin B producing bacteria,base-editing technology based on CRISPR/Cas9 was applied in this study.As results,candidate genes postulated to be involved in hygromycin B biosynthesis were successfully inactivated in vivo through introducing nonsense mutation or changing its conserved sequence by base editor and further confirmed by biochemical assay in vitro.The results showed that hyg J,hyg L and hyg D were the main functional genes involved in the dehydrogenation,transamination and glycation,respectively.So far,among the glycosyltransferases that use NDP-heptylose as substrate to form aminoglycoside compounds,the glycosyltransferase Hyg D involved in this study is the first case,which will provide an important reference for similar studies of this class of antibiotics.Of note,Hyg Y was a S-adenosylmethionine(SAM)-dependent hydroxyl isomerase,which was rarely seen in the biosynthesis of other aminoglycosides.Hyg M,as a methyltransferase,could recognize a variety of substrates and function in multiple parallel metabolic networks.