Genetic dissection of the biosynthetic route to gentamicin A2 by heterologous expression of its minimal gene set.
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.
Park
Je Won
JW
Division of Nano Sciences, Ewha Womans University, 11-1 Daehyung-dong, Seodaemun-gu, Seoul 120-750, Republic of Korea.
Hong
Jay Sung Joong
JS
Parajuli
Niranjan
N
Jung
Won Seok
WS
Park
Sung Ryeol
SR
Lim
Si-Kyu
SK
Sohng
Jae Kyung
JK
Yoon
Yeo Joon
YJ
D000617:Aminoglycosides / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000096:biosynthesis / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005839:Gentamicins / Q000096:biosynthesis* / Q000302:isolation & purification; D006595:Hexosamines / Q000096:biosynthesis / Q000235:genetics; D008847:Micromonospora / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D051146:N-Acylsphingosine Galactosyltransferase / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics
C530071:2'-N-acetylparomamine; D000617:Aminoglycosides; D004187:Disaccharides; D005839:Gentamicins; D006595:Hexosamines; C030880:gentamicin A; C005936:paromamine; D051146:N-Acylsphingosine Galactosyltransferase; C007265:2-deoxystreptamine