Abstract
BackgroundIsepamicin 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-NH2-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 C2ʹ 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.ResultsWe 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-NH2 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.ConclusionWe 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 materialThe online version of this article (doi:10.1186/s12934-015-0402-6) contains supplementary material, which is available to authorized users.
Highlights
Isepamicin is a weakly toxic but highly active aminoglycoside antibiotic derivative of gentamicin B
The structural difference between JI-20A and gentamicin B is similar to the difference between kanamycin A and B
Considering that kanamycin B is converted into kanamycin A by KanJ and KanK, we determined whether KanJ and KanK can transform JI-20A into gentamicin B
Summary
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-NH2-containing gentamicin C complex is a dominant compound produced by wild-type M. echinospora; by contrast, 2ʹ-OHcontaining gentamicin B is produced as a minor component. The biosynthetic pathway of gentamicin B remains unclear. Considering that gentamicin B shares a unique C2ʹ 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. Other aminoglycoside antibiotics include 2-deoxystreptamine (2-DOS) containing gentamicin, kanamycin, neomycin, and butirosin. Isepamicin is manufactured from gentamicin B, which is co-produced in Micromonospora echinospora. Gentamicin C complex is produced by M. echinospora as its major product.
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