Methyltransferase Contingencies in the Pathway of Everninomicin D Antibiotics and Analogues

Abstract

Everninomicins are orthoester oligosaccharide antibiotics with potent activity against multidrug-resistant bacterial pathogens. Everninomicins act by disrupting ribosomal assembly in a distinct region in comparison to clinically prescribed drugs. We employed microporous intergeneric conjugation with Escherichia coli to manipulate Micromonospora for targeted genereplacement studies of multiple putative methyltransferases across the octasaccharide scaffold of everninomicin effecting the A 1 , C, F, and H rings. Analyses of gene-replacement and genetic complementation mutants established the mutability of the everninomicin scaffold through the generation of 12 previously unreported analogues and, together with previous results, permitted assignment of the ten methyltransferases required for everninomicin biosynthesis. The in vitro activity of A 1-and H-ring-modifying methyltransferases demonstrated the ability to catalyze late-stage modification of the scaffold on an A 1-ring phenol and H-ring C-4' hydroxy moiety. Together these results establish the potential of the everninomicin scaffold for modification through mutagenesis and in vitro modification of advanced biosynthetic intermediates.

Microbial Oligosaccharides with Biomedical Applications

“…It is generally acknowledged that the rarely observed orthoester linkage is necessary for the antibiotic properties of the orthosomycins. Although the orthosomycin family includes hygromycin B, everninomicin, avilamycin, flambamycin, curamycin, etc., the former three members have been extensively studied, especially in the aspect of their bioactivity and biosynthesis [ 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 ]. Herein, restricted by the space, everninomicin (EVN) and avilamycin (AVI) will be mainly discussed in this review.…”

“…Until now, twenty EVN ( 43 – 62 ) ( Figure 8 ) have been purified from wide- or mutant-type M. carbonacea var. africana [ 59 , 60 , 61 , 62 , 63 , 71 ], while thirty-seven AVI ( 63 – 99 ) ( Figure 9 ) have been isolated from the wide- or mutant-type Streptomyces viridochromogenes Tü57 [ 59 , 64 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 ]. Structurally, EVN and AVI share the same seven-sugar core (rings B to H) and characteristic substituted partner including two orthoester linkages located between rings C and D and rings G and H and a methylenedioxy bridge attached to ring H. The major differences between EVN and AVI are attributed to the presence (or absence) of the A-ring nitrosugar and the orsellinic acid in EVN (or in AVI).…”

“…Due to the multiple-ring structure of Avi A, its biosynthetic pathway is relatively complicated. Up to now, aided with the bioinformatics analysis, some of the key enzymes in the Avi A biosynthesis pathway have been successfully elucidated by analysis of the corresponding Avi derivatives, gavibamycins (Gav, 79 – 99 ), in situ gene knockout and complementation strategy, heterologous expression systems, such as S. lividans TK24, S. coelicolor CH999, Streptomyces TK66, etc., or feeding experiments [ 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 ]. Herein, four main initial precursors, including propionyl-CoA, d -glucose-1-phosphate, GDP- d -mannose, and UDP- d -glucose, are summarized in Figure 10 .…”

“…In addition, two resistances, rRNA-methyltransferase AviRa and AviRb, two regulators, ABC transporters AviABC1 and AviABC2 [ 80 ], and two transcriptional activators, AviC1 and AviC2 [ 81 ] are also reported. Regarding the Avi A contributes to poor water solubility and difficulties in drug development perhaps ascribed to its multiple methyl groups [ 76 , 82 ], fully understanding the Avi A biosynthetic pathway will provide useful information and contribute to produce novel derivatives with improved polarity and pharmacokinetic properties.…”

Biosynthesis and Engineered Overproduction of Everninomicins with Promising Activity against Multidrug-Resistant Bacteria

“…Therefore, EVN D holds promise for further investigations as a potential drug lead for treating bacterial infections. Compared to the chemical synthesis that requires masterful skill, it is more practical to perform the structural diversification and structure–activity relationship (SAR) analysis of EVNs based on microbial fermentation and the natural, artificial, and semisynthetic derivatives arising therefrom. ,,− However, the wild-type Micromonospora strains usually produce EVNs in extremely low yields under laboratory fermentation conditions. ,, In the past, sufficient quantities of EVNs for structure elucidation and bioactivity evaluation were prepared through industrial-scale fermentation that typically involved volumes greater than 1000 litres, ,,, which discouraged academic researchers that could only perform bench-scale fermentation. Thus, the engineered strains producing sufficient EVNs for bioassay and SAR analysis are urgently demanded.…”