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

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

Metabolites of <i>S</i>. <i>kanamyceticus</i> CG305 and its recombinant overexpressing strains.

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

Genotypes of <i>Streptomyces kanamyceticus</i> CG305 and its recombinant overexpressing strains.

<p>Genotypes of <i>Streptomyces kanamyceticus</i> CG305 and its recombinant overexpressing strains.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

MOESM2 of Assembly of a novel biosynthetic pathway for gentamicin B production in Micromonospora echinospora

Additional file 2: Figure S2. 1H NMR spectrum of the new compound from kanJK expression strains

Modulation of kanamycin B and kanamycin A biosynthesis in <i>Streptomyces kanamyceticus</i> via metabolic engineering

<div><p>Both kanamycin A and kanamycin B, antibiotic components produced by <i>Streptomyces kanamyceticus</i>, 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 <i>in vivo</i>. Based on the proposed parallel biosynthetic pathway, <i>kanN</i> disruption should lead to kanamycin A accumulation; however, the <i>kanN</i>-disruption strain produced neither kanamycin A nor kanamycin B. We then tested the function of <i>kanJ</i> and <i>kanK</i>. The main metabolite of the <i>kanJ</i>-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 <i>S</i>. <i>kanamyceticus</i>. As expected, the kanamycin B yield of the <i>kanJ</i>-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 <i>kanJ</i>- and <i>kanK</i>-overexpressing strains were constructed through either homologous recombination or site-specific integration. The overexpressing strain containing three copies of <i>kanJ</i> and <i>kanK</i> 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 <i>S</i>. <i>kanamyceticus</i>. 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.</p></div

Metabolite analysis of <i>S</i>. <i>kanamyceticus</i> CG305 and <i>S</i>. <i>kanamyceticus</i> Δ<i>kanN</i>.

<p><b>(a)</b> HPLC analysis of metabolites. <b>(b)</b> HPLC-MS analysis of the products of <i>S</i>. <i>kanamyceticus</i> Δ<i>kanN</i>.</p