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Discovery of parallel pathways of kanamycin biosynthesis allows antibiotic manipulation

Abstract

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.

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Figure 1: The previously proposed biosynthetic pathway of kanamycin.
Figure 2: HPLC-ESI-MS/MS analysis of kanamycin pseudodisaccharide intermediates obtained from the in vivo expression of candidate kanamycin gene sets in recombinant S. venezuelae hosts and in vitro reactions using cell-free extracts of recombinants.
Figure 3: HPLC-ESI-MS/MS analysis of kanamycin pseudotrisaccharides obtained from the in vivo expression of candidate kanamycin gene sets in recombinant S. venezuelae hosts and in vitro reactions using cell-free extracts of recombinants.
Figure 4: Biosynthetic pathway of kanamycin complex.
Figure 5: HPLC-ESI-MS/MS analysis of kanamycin biosynthetic intermediates from recombinant S. venezuelae hosts in which the first glycosyltransferase-encoding gene has been swapped.
Figure 6: HPLC-ESI-MS/MS analysis of 1-N-AHBA-kanamycins and 3′-deoxykanamycins produced by recombinant S. venezuelae hosts.

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Acknowledgements

We thank E. Cundliffe for discussions and for critically reading this manuscript. This work was supported by the National Research Laboratory (NRL) program (R0A-2008-000-20030-0) and Global Frontier Program for Intelligent Synthetic Biology through the National Research Foundation of Korea (NRF), NRF grants (2010-0001487 and 2010-0028193) funded by the Ministry of Education, Science & Technology and the Marine and Extreme Genome Research Center Program of the Ministry of Land, Transportation and Maritime Affairs, Republic of Korea. J.W.P. gratefully acknowledges a grant (20100623) from the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture and Fisheries, Republic of Korea.

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J.W.P., S.R.P., J.K.S. and Y.J.Y. designed research and wrote the paper; J.W.P., S.R.P., K.K.N., A.R.H., Y.H.B., Y.J.Y., E.J.K., E.M.K. and D.K. performed research; and J.W.P., S.R.P. and Y.J.Y. analyzed data.

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Correspondence to Jae Kyung Sohng or Yeo Joon Yoon.

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Park, J., Park, S., Nepal, K. et al. Discovery of parallel pathways of kanamycin biosynthesis allows antibiotic manipulation. Nat Chem Biol 7, 843–852 (2011). https://doi.org/10.1038/nchembio.671

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