Since the first use of streptomycin as an effective antibiotic drug in the treatment of tuberculosis, aminoglycoside antibiotics have been widely used against a variety of bacterial infections for over six decades. However, the pathways for aminoglycoside biosynthesis still remain unclear, mainly because of difficulty in genetic manipulation of actinomycetes producing this class of antibiotics. Gentamicin belongs to the group of 4,6-disubstituted aminoglycosides containing a characteristic core aminocyclitol moiety, 2-deoxystreptamine (2-DOS), and the recent discovery of its biosynthetic gene cluster in Micromonospora echinospora has enabled us to decipher its biosynthetic pathway. To determine the minimal set of genes and their functions for the generation of gentamicin A(2), the first pseudotrisaccharide intermediate in the biosynthetic pathway for the gentamicin complex, various sets of candidate genes from M. echinospora and other related aminoglycoside-producing strains were introduced into a nonaminoglycoside producing strain of Streptomyces venezuelae. Heterologous expression of different combinations of putative 2-DOS biosynthetic genes revealed that a subset, gtmB-gtmA-gacH, is responsible for the biosynthesis of this core aminocyclitol moiety of gentamicin. Expression of gtmG together with gtmB-gtmA-gacH led to production of 2'-N-acetylparomamine, demonstrating that GtmG acts as a glycosyltransferase that adds N-acetyl-d-glucosamine (GLcNA) to 2-DOS. Expression of gtmM in a 2'-N-acetylparomamine-producing recombinant S. venezuelae strain generated paromamine. Expression of gtmE in an engineered paromamine-producing strain of S. venezuelae successfully generated gentamicin A(2), indicating that GtmE is another glycosyltransferase that attaches d-xylose to paromamine. These results represent in vivo evidence elucidating the complete biosynthetic pathway of the pseudotrisaccharide aminoglycoside
aminoglycoside biosynthesis, Micromonospora echinospora, Streptomyces venezuelae, pathway engineering
NCBI PubMed ID: 18550838Publication DOI: 10.1073/pnas.0803164105Journal NLM ID: 7505876Publisher: National Academy of Sciences
Correspondence: joonyoon@ewha.ac.kr
Institutions: Division of Nano Sciences, Ewha Womans University, Seoul, South Korea, Interdisciplinary Program of Biochemical Engineering and Biotechnology, Seoul National University, Seoul, South Korea, Genotech Corporations, Daejon, Korea, Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, Chungnam, Korea
Methods: 13C NMR, 1H NMR, NMR-2D, PCR, DNA techniques, ESI-MS/MS, extraction, HPLC-MS
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