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 inMicromonospora echinosporahas 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 fromM. echinosporaand other related aminoglycoside-producing strains were introduced into a nonaminoglycoside producing strain ofStreptomyces 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 ofgtmGtogether withgtmB-gtmA-gacHled to production of 2′-N-acetylparomamine, demonstrating that GtmG acts as a glycosyltransferase that addsN-acetyl-d-glucosamine (GLcNA) to 2-DOS. Expression ofgtmMin a 2′-N-acetylparomamine-producing recombinantS. venezuelaestrain generated paromamine. Expression ofgtmEin an engineered paromamine-producing strain ofS. venezuelaesuccessfully generated gentamicin A₂, indicating that GtmE is another glycosyltransferase that attachesd-xylose to paromamine. These results representin vivoevidence elucidating the complete biosynthetic pathway of the pseudotrisaccharide aminoglycoside.

Covering: 2007 to September 2012 The 2-deoxystreptamine-containing aminoglycosides, such as neomycin, kanamycin and gentamicin, are an important class of antibiotics. A detailed understanding of the complete biosynthetic pathway of aminoglycosides and their biosynthetic enzymes will allow us to not only generate more robust antibiotic agents or drugs with other altered biological activities, but also to produce clinically important semi-synthetic antibiotics by direct fermentation. This Highlight focuses on recent advances in the characterization of their biosynthetic enzymes and pathway as well as some chemo-enzymatic and metabolic engineering approaches for the biological production of natural, semi-synthetic, and novel aminoglycosides.