Genetic dissection of the biosynthetic route to gentamicin A2 by heterologous expression of its minimal gene set

Je Won Park, Jay Sung Joong Hong, Niranjan Parajuli, Seok Jung Won, Ryeol Park Sung, Si Kyu Lim, Kyung Sohng Jae, Joon Yoon Yeo

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

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 A2, 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 A2, 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.

Original languageEnglish
Pages (from-to)8399-8404
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number24
DOIs
Publication statusPublished - 2008 Jun 17
Externally publishedYes

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Keywords

  • Aminoglycoside biosynthesis
  • Micromonospora echinospora
  • Pathway engineering
  • Streptomyces venezuelae

ASJC Scopus subject areas

  • General

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