Je Won Park^1,2,5, Sung Ryeol Park^1,5, Keshav Kumar Nepal², Ah Reum Han³, Yeon Hee Ban¹, Young Ji Yoo¹, Eun Ji Kim¹, Eui Min Kim², Dooil Kim⁴, Jae Kyung Sohng^2* & Yeo Joon Yoon^1*

¹Department of Chemistry and Nanoscience, Ewha Womans University, Seoul, Republic of Korea. ²Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, Chungnam, Republic of Korea. ³Interdisciplinary Program of Biochemical Engineering and Biotechnology, Seoul National University, Seoul, Republic of Korea. ⁴Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea. ⁵These authors contributed equally to this work.

Kanamycin is one of the most widely used antibiotics, yet its biosynthetic pathway remains unclear. Current proposals suggest that the kanamycin biosynthetic products are linearly related via single enzymatic transformations. To explore this system, we have reconstructed the entire biosynthetic pathway through the heterologous expression of combinations of putative biosynthetic genes from Streptomyces kanamyceticus in the non.aminoglycoside-producing Streptomyces venezuelae. Unexpectedly, we discovered that the biosynthetic pathway contains an early branch point, governed by the substrate promiscuity of a glycosyltransferase, that leads to the formation of two parallel pathways in which early intermediates are further modified. Glycosyltransferase exchange can alter flux through these two parallel pathways, and the addition of other biosynthetic enzymes can be used to synthesize known and new highly active antibiotics. These results complete our understanding of kanamycin biosynthesis and demonstrate the potential of pathway engineering for direct in vivo production of clinically useful antibiotics and more robust aminoglycosides.