Biosynthesis of 3″-demethyl-gentamicin C components by gen N disruption strain of Micromonospora echinospora and test their antimicrobial activities in vitro

Abstract

Gentamicin consists primarily of four components, which have different patterns of methylation at C-6' position. The methyl groups have a significant impact on gentamicin antimicrobial activity. Sequence analysis predicted that GenN was a methyltransferase in the gentamicin biosynthetic pathway. To study the function of genN, it was disrupted in Micromonospora echinospora. The genN disruption strains produced 3″-N-demethyl-gentamicin C complex instead of the gentamicin C complex. In this study, 3″-N-demethyl gentamicin C1a was purified from the broth of disruption strain, and its structure was elucidated using MS and NMR. Besides 3″-N-demethyl products corresponding to gentamicin C1a, C2, and C2a, two 3″-N-demethyl products corresponding to gentamicin C1 were detected, which were concluded as C-6' epimers originating from decreased substrate specificity of 6'-N methyltransferase. To explore the effects of 3″-N-methyl on gentamicin antimicrobial activity, antimicrobial activity of these demethyl gentamicin analogues were tested in vitro. 3″-N-Demethyl gentamicin components have identical activity with corresponding components of gentamicin. The results of bioassays showed that the 3″-N-methyl group has little impact on gentamicin activity. However, these highly bioactive compounds afforded a unique opportunity for creating new and high potent aminoglycoside antibiotics.

Methyltransferases of gentamicin biosynthesis

“…For 6 of these, the structure could be determined by MS and MS n methods, and structures could also be proposed for 13 others ( 27 ). In previous work it has been shown that levels of certain metabolites with altered methylation patterns are enhanced upon deletion of either genN ( 19 , 28 ) or genK ( 20 ). We therefore set out systematically to alter the complement of known methyltransferase genes and to study the metabolites produced.…”

“…In total, 19 candidate genes (GenBank accession nos. MF036118–MF036131 and MF036134–MF036138) were identified using the BLASTP program ( 28 ), with percentage sequence identities to GenN ranging from 26.67 to 40.35% ( SI Appendix , Table S4 ). Each candidate gene was cloned into expression vector pET28a(+) in Escherichia coli , and cell extracts were assayed for their ability to catalyze SAM-dependent conversion of C2 into C1, monitored by LC-ESI-HRMS ( SI Appendix , Fig.…”

“…In previous work, three consecutive methylation steps in the later stages of gentamicin biosynthesis have been firmly established as being catalyzed, respectively, by the 3′′- N -methyltransferase GenN, the 4″- C -methyltransferase GenD1, and the 6′- C -methyltransferase GenK ( 19 , 20 , 28 ). However, here we have shown that the three methylations may take place in an alternative order, giving rise to a form of network.…”

“…However, here we have shown that the three methylations may take place in an alternative order, giving rise to a form of network. A commercial batch of gentamicin C has been shown to contain as trace contaminants gentamicins lacking one or more of these methyl groups ( 27 ), and specific deletion of genN has been shown, for example, to increase the yield of several 3′′- N -desmethylgentamicins ( 19 , 28 ). To explore this further, we therefore constructed the complete combinatorial set of seven mutants in which one, two, or all three of methyltransferase genes had been independently deleted in-frame.…”

Mechanistic Insights into Dideoxygenation in Gentamicin Biosynthesis

“…Since the biosynthetic gene cluster of gentamicin was identified more than a decade ago, we and others have elucidated most of the gentamicin biosynthetic pathway in Micromonospora echinospora ATCC15835 [8][9][10][11][12][13][14][15][16][17][18][19] and revealed crystal structures of several of the enzymes involved in the biosynthesis 14,20,21,22 . GenD2, GenN, GenS2 and GenD1 have been demonstrated to be involved in the formation of gentamicin X2 16,21,22 .…”