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.

Streptomyces venezuelae TX‐TL – a next generation cell‐free synthetic biology tool

“…Recently, Streptomyces venezuelae, the chloramphenicol producer, has been adopted by synthetic biology for its use in metabolic engineering [2], since it is relatively well characterized, has strong promoter tools and genome engineering plasmids for integration [3][4][5]. Whilst it is not as characterized as Streptomyces coelicolor A3(2), in contrast, S. venezuelae provides significant advantages such as a fast growth (≈T D = 40 min) and no aggregation during liquid culture [3].…”

Concentrated biosynthesis of tobramycin by genetically engineered Streptomyces tenebrarius

“…GenBank accession number AJ629123). In this gene cluster, aprD3 and aprD4, which are responsible for C3 deoxygenation of pseudodisaccharides, have been functionally characterized through gene disruption in vivo (Ni et al, 2011) and recombinant protein expression in vitro (Park et al, 2011). These advances greatly improved our understanding of the antibiotic biosynthesis in S. tenebrarius and eventually allowed us to manipulate the biosynthetic pathway for direct fermentative production of tobramycin.…”

“…GenBank accession number AJ810851). In tob-cluster, tobS1 coding for L-glutamine: 2-deoxy-scyllo-inosose aminotransferase (Kharel et al, 2005), tobQ coding for 6 -dehydrogenase (Yu et al, 2008), tobM2 coding for 6-glucosyltransferase (Park et al, 2011) and tobZ (also termed tacA) coding for O-carbamoyltransferase (Parthier et al, 2012) have been characterized. The apramycin biosynthetic gene cluster (apr-cluster) was also isolated from S. tenebrarius (Piepersberg.…”

Aminoglycoside Antibiotics

“…The biochemistry of the biosynthetic pathways leading to 4,6-disubstituted-2-deoxystreptamine family is still a developing field (Wehmeier and Piepersberg 2009). However, the recent discovery of the complete kanamycin pathway and the engineered biosynthesis of a novel kanamycin analog that is active against some kanamycin and amikacinresistant Gram-negative bacteria demonstrate the potential of biological approaches for production of more robust aminoglycosides (Park et al 2011). Recent reviews cover the enzymology of aminoglycoside biosynthesis and the potential of biological approaches for the heterologous production of the existing natural and semi-synthetic aminoglycosides and the creation of new ones by chemo-enzymatic synthesis (Kudo and Eguchi 2009;Park et al 2013;Wehmeier and Piepersberg 2009).…”