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

Plasmids used in this study.

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

Bacterial strains used in this study.

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

Kanamycin biosynthetic gene cluster and proposed kanamycin biosynthetic pathways.

<p><b>(a)</b> Kanamycin biosynthetic gene cluster. <b>(b)</b> Proposed parallel and linear kanamycin biosynthetic pathways.</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

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

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

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

Carbon Dots with Red Emission for Sensing of Pt²⁺, Au³⁺, and Pd²⁺ and Their Bioapplications in Vitro and in Vivo

Red emissive carbon dots (CDs) have drawn more and more attention due to their good organ penetration depth and slight biological tissue photodamage. Herein, the fluorescent CDs with red emission were synthesized by the facile one-pot hydrothermal treatment of citric acid and neutral red and they show red fluorescence both in aqueous solution and solid state. The solution of CDs exhibits the quantum yield of 12.1%, good stability against photobleaching, and low cytotoxicity. As-prepared CDs can be used as a fluorescent probe for peculiar detection of Pt²⁺, Au³⁺, and Pd²⁺. Furthermore, the CDs show excellent biocompatibility, which were successfully used as hopeful bioimaging and biosensing of noble metal ions in PC12 cells and zebrafish