Assembly of a novel biosynthetic pathway for gentamicin B production in Micromonospora echinospora

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. 2ʹ-NH(2)-containing gentamicin C complex is a dominant compound produced by wild-type M. echinospora; by contrast, 2ʹ-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 C(2ʹ) 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 2ʹ-NH(2) into 2ʹ-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. CONCLUSION: 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0402-6) contains supplementary material, which is available to authorized users.