title: Assembly of a novel biosynthetic pathway for gentamicin B production in Micromonospora echinospora.
abstract: BACKGROUND
  Isepamicin is a weakly toxic but highly active aminoglycoside antibiotic derivative of gentamicin B. Gentamicin B is a naturally occurring minor component isolated from Micromonospora echinospora. 2'-NH2-containing gentamicin C complex is a dominant compound produced by wild-type M. echinospora; by contrast, 2'-OH-containing gentamicin B is produced as a minor component. However, the biosynthetic pathway of gentamicin B remains unclear. Considering that gentamicin B shares a unique C2' hydroxyl group with kanamycin A, we aimed to design a new biosynthetic pathway of gentamicin B by combining twelve steps of gentamicin biosynthesis and two steps of kanamycin biosynthesis.
  
  
  RESULTS
  We blocked the biosynthetic pathway of byproducts and generated a strain overproducing JI-20A, which was used as a precursor of gentamicin B biosynthesis, by disrupting genK and genP. The amount of JI-20A produced in M. echinospora ∆K∆P reached 911 μg/ml, which was 14-fold higher than that of M. echinospora ∆P. The enzymes KanJ and KanK necessary to convert 2'-NH2 into 2'-OH from the kanamycin biosynthetic pathway were heterologously expressed in M. echinospora ΔKΔP to transform JI-20A into gentamicin B. The strain with kanJK under PermE* could produce 80 μg/ml of gentamicin B, which was tenfold higher than that of the wild-type strain. To enhance gentamicin B production, we employed different promoters and gene integration combinations. When a PhrdB promoter was used and kanJ and kanK were integrated in the genome through gene replacement, gentamicin B was generated as the major product with a maximum yield of 880 μg/ml.
  
  
  CONCLUSIONS
  We constructed a new biosynthetic pathway of high-level gentamicin B production; in this pathway, most byproducts were removed. This method also provided novel insights into the biosynthesis of secondary metabolites via the combinatorial biosynthesis.
authors.affiliation: School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No.103 Wenhua Road, Shenyang, Liaoning, China. nixianpu126@126.com.
authors.forename: Xianpu
authors.initials: X
authors.lastname: Ni
authors.affiliation: School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No.103 Wenhua Road, Shenyang, Liaoning, China. 1026625249@qq.com.
authors.forename: Zhenpeng
authors.initials: Z
authors.lastname: Sun
authors.affiliation: School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No.103 Wenhua Road, Shenyang, Liaoning, China. gyw0636@163.com.
authors.forename: Yawen
authors.initials: Y
authors.lastname: Gu
authors.affiliation: School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No.103 Wenhua Road, Shenyang, Liaoning, China. cuihao102@163.com.
authors.forename: Hao
authors.initials: H
authors.lastname: Cui
authors.affiliation: School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No.103 Wenhua Road, Shenyang, Liaoning, China. xiahz612@sina.com.
authors.forename: Huanzhang
authors.initials: H
authors.lastname: Xia
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D005839:Gentamicins / Q000378:metabolism*; D008847:Micromonospora / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D005839:Gentamicins; C023804:gentamicin B