Synthetic Biotechnology to Study and Engineer Ribosomal Bottromycin Biosynthesis

Abstract

Bottromycins represent a promising class of antibiotics binding to the therapeutically unexploited A-site of the bacterial ribosome. By inhibiting translation they are active against clinically important pathogens, such as vancomycin-resistant Enterococci. Structurally, bottromycins are heavily modified peptides exhibiting various unusual biosynthetic features. To set the stage for compound modification and yield optimization, we identified the biosynthetic gene cluster, used synthetic biotechnology approaches to establish and improve heterologous production, and generated analogs by pathway genetic engineering. We unambiguously identified three radical SAM methyltransferase-encoding genes required for various methylations at unactivated carbons yielding tert-butyl valine, methyl-proline, and β-methyl-phenylalanine residues, plus a gene involved in aspartate methyl-ester formation. Evidence for the formation of the exo-thiazole unit and for a macrocyclodehydration mechanism leading to amidine ring formation is provided.

Dissecting Bottromycin Biosynthesis Using Comparative Untargeted Metabolomics

“…Instead, a P450 enzyme, BtmJ, was predicted to catalyze the oxidative decarboxylation of the thiazoline into a thiazole 5, 6, 7. This is an uncommon role for a P450, although it has been reported for thiazole formation in the biosynthesis of the plant alkaloid camalexin32 and could be mechanistically similar to the fatty acid P450 decarboxylase OleT 33.…”

Dissecting Bottromycin Biosynthesis Using Comparative Untargeted Metabolomics

“…[5,7] In S. scabies,b ottromycin production is either severely reduced or entirely abolished when either RSMT gene, btmC or btmG,i sd eleted. BtmG methylates Va l4 and Va l5,and BtmC methylates Phe 6, [5,7] but it is unclear why the pathway stalls when either step is missed.…”

“…[29] Instead, aP 450 enzyme,B tmJ, was predicted to catalyze the oxidative decarboxylation of the thiazoline into at hiazole. [5][6][7] This is an uncommon role for aP 450, although it has been reported for thiazole formation in the biosynthesis of the plant alkaloid camalexin [32] and could be mechanistically similar to the fatty acid P450 decarboxylase OleT. [33] Analysis of DbtmJ revealed two abundant compounds with m/z 841.43 and 855.44 (Figure 4), which were confirmed to be carboxylated O-desmethyl bottromycins B 2 and A 2 ,r espectively (12 and 13)u sing MS 2 (Supporting Information, Figure S11).…”

“…[2][3][4] However,e lucidating the biosynthesis of RiPPs can be hindered by the difficulty of isolating intermediates,asthe biosynthesis takes place on alarger precursor peptide,a nd intermediates may be rapidly proteolyzed. Therefore,i mproved methods for the identification of RiPP intermediates are desirable.Bottromycin A 2 (1,Scheme 1) [5][6][7][8] possesses potent antibacterial activity towards multidrugresistant bacteria, [9] and is structurally unique owing its unprecedented macrocyclic amidine,r are b-methylated amino acids residues,and aterminal thiazole.Nature employs av ariety of strategies for peptide macrocyclization, [10][11][12] but amidine formation has only been observed for bottromycin. Initial studies on bottromycin biosynthesis showed that its amino acids were b-methylated by radical SAM methyltransferases [5,7] (RSMTs), but the rest of the bottromycin pathway represented abiosynthetic black box, where little was known about key steps in the pathway,including the unprecedented macrocyclization.…”

“…Therefore,i mproved methods for the identification of RiPP intermediates are desirable.Bottromycin A 2 (1,Scheme 1) [5][6][7][8] possesses potent antibacterial activity towards multidrugresistant bacteria, [9] and is structurally unique owing its unprecedented macrocyclic amidine,r are b-methylated amino acids residues,and aterminal thiazole.Nature employs av ariety of strategies for peptide macrocyclization, [10][11][12] but amidine formation has only been observed for bottromycin. Initial studies on bottromycin biosynthesis showed that its amino acids were b-methylated by radical SAM methyltransferases [5,7] (RSMTs), but the rest of the bottromycin pathway represented abiosynthetic black box, where little was known about key steps in the pathway,including the unprecedented macrocyclization. In this study,weemploy untargeted metabolomics and mass spectral networking to deduce the biosynthetic route to bottromycins in Streptomyces scabies.T his analysis identifies the enzymes responsible for macrocyclization, thiazole formation, and aspartate epimerization, thereby demonstrating the utility of an untargeted metabolomic approach for elucidating atargeted biosynthetic pathway.…”