Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering

Abstract

Both kanamycin A and kanamycin B, antibiotic components produced by Streptomyces kanamyceticus, have medical value. Two different pathways for kanamycin biosynthesis have been reported by two research groups. In this study, to obtain an optimal kanamycin A-producing strain and a kanamycin B-high-yield strain, we first examined the native kanamycin biosynthetic pathway in vivo. Based on the proposed parallel biosynthetic pathway, kanN disruption should lead to kanamycin A accumulation; however, the kanN-disruption strain produced neither kanamycin A nor kanamycin B. We then tested the function of kanJ and kanK. The main metabolite of the kanJ-disruption strain was identified as kanamycin B. These results clarified that kanamycin biosynthesis does not proceed through the parallel pathway and that synthesis of kanamycin A from kanamycin B is catalyzed by KanJ and KanK in S. kanamyceticus. As expected, the kanamycin B yield of the kanJ-disruption strain was 3268±255 μg/mL, 12-fold higher than that of the original strain. To improve the purity of kanamycin A and reduce the yield of kanamycin B in the fermentation broth, four different kanJ- and kanK-overexpressing strains were constructed through either homologous recombination or site-specific integration. The overexpressing strain containing three copies of kanJ and kanK in its genome exhibited the lowest kanamycin B yield (128±20 μg/mL), which was 54% lower than that of the original strain. Our experimental results demonstrate that kanamycin A is derived from KanJ-and-KanK-catalyzed conversion of kanamycin B in S. kanamyceticus. Moreover, based on the clarified biosynthetic pathway, we obtained a kanamycin B-high-yield strain and an optimized kanamycin A-producing strain with minimal byproduct.

Fig 1. Kanamycin biosynthetic gene cluster and proposed kanamycin biosynthetic pathways.

(a) Kanamycin biosynthetic gene cluster. (b) Proposed parallel and linear kanamycin biosynthetic pathways.

Fig 2. Metabolite analysis of S. kanamyceticus CG305 and S. kanamyceticus ΔkanN.

(a) HPLC analysis of metabolites. (b) HPLC-MS analysis of the products of S. kanamyceticus ΔkanN.

Fig 3. Metabolite analysis of S. kanamyceticus CG305 and S. kanamyceticus ΔkanJ.

(a) HPLC analysis of metabolites. (b) Time-course of kanamycin B production. (c) HPLC-MS analysis of the main product (1) of S. kanamyceticus ΔkanJ. (d) HPLC-MS analysis of the byproduct (2) of S. kanamyceticus ΔkanJ. The blue letters indicate the number of carbon rings. (e) HPLC-MS/MS analysis of the byproduct (2) of S. kanamyceticus ΔkanJ.

Fig 4. Genotypes of Streptomyces kanamyceticus CG305 and its recombinant overexpressing strains.

Fig 5. Metabolites of S. kanamyceticus CG305 and its recombinant overexpressing strains.

(a) HPLC analysis of metabolites. (b) Analysis of secondary metabolite yields. (c) Stability of kanamycin B yields from S. kanamyceticus JKE1, JKE2, JKE3, and JKE4.

Fig 6. Cell growth, relative enzyme activity and qRT-PCR gene transcription analysis of kanamycin A-producing strains.

(a) Cell growth of kanamycin A-producing strains. (b) The relative activity of KanJ and KanK in mycelia after fermentation for 72 h. (c) qRT-PCR gene transcription analysis of kanJ and kanK in the original strain and in the S. kanamyceticus JKE1, JKE2, JKE3 and JKE4 strains.