Discovery and biosynthesis of novel natural products from Streptomyces venezuelae

Streptomyces bacteria are prolific producers of secondary metabolites. These natural products find uses ranging from antibiotics and antifungals to immunomodulatory agents and pesticides. Biosynthesis of secondary metabolites is often tightly controlled; therefore novel strategies for activation of cryptic secondary metabolites are required. The metabolites of the Streptomyces venezuelae wild type and a mutant strain lacking the ArpA-like transcriptional repressor GbnR have been compared, leading to the identification of the gaburedins – novel, ureido-linked dipeptides – in the gbnR mutant. A combination of in vivo precursor-directed studies has led to a proposed biosynthetic route to gaburedins. Metabolic profiling of a range of other mutant strains has identified gbnB as an essential gene in the gbnABC gene cluster that is responsible for gaburedin biosynthesis. Synthesis of an authentic standard of gaburedin A combined with chiral HPLC analysis of culture extracts has allowed the proposed structure of gaburedins to be confirmed. Bioinformatic analyses of the gbnABC cluster responsible for gaburedin biosynthesis have revealed orthologous systems in a range of other bacterial genera and efforts to reconstitute the S. venezuelae gbnABC pathway in E. coli have begun. The regulatory mechanism controlling gaburedin biosynthesis in S. venezuelae has also been investigated, leading to the discovery of new AHFCA signalling molecules which have been shown to induce gaburedin biosynthesis in a mutant strain unable to produce AHFCAs. This work demonstrates for the first time that AHFCA signalling is involved in regulation of other natural products as well as the methylenomycin cluster from which AHFCAs were first identified. Gaburedins represent the first example by which deletion of an arpA-like regulatory gene has been used as a strategy for de-repression of a biosynthetic pathway for cryptic natural product biosynthesis. The current project demonstrates that rational deletion of proposed regulatory genes is a powerful approach to natural product discovery

Overproduction and identification of butyrolactones SCB1-8 in the antibiotic production superhost Streptomyces M1152

Gamma-butyrolactones (GBLs) are signalling molecules that control antibiotic production in Streptomyces bacteria. The genetically engineered strain S. coelicolor M1152 was found to overproduce GBLs SCB1-3 as well as five novel GBLs named SCB4-8. Incorporation experiments using isotopically-labelled precursors confirmed the chemical structures of SCB1-3 and established those of SCB4-8

Diversity of secoiridoid glycosides in leaves of UK and Danish ash provide new insight for ash dieback management

Secoiridoid glycosides are anti-feeding deterrents of the Oleaceae family recently highlighted as potential biomarkers in Danish ash trees to differentiate between those tolerant and susceptible to the fungal disease ash dieback. With the knowledge that emerald ash borer has recently entered Europe from Russia, and that extensive selection trials are ongoing in Europe for ash dieback tolerant European ash (Fraxinus excelsior), we undertook comprehensive screening of secoiridoid glycosides in leaf extracts of trees tolerant and susceptible to ash dieback sampled from sites in the UK and Denmark. Here we report an unexpected diversity of secoiridoid glycosides in UK trees and higher levels of secoiridoid glycosides in the UK sample group. While it is unlikely that secoiridoid glycosides generally can serve as reliable markers for ash dieback susceptibility, there are differences between tolerant and susceptible groups for specific secoiridoids. We predict that the high levels—and structural diversity—of secoiridoids present in the UK group may provide a robust reservoir of anti-feeding deterrents to mitigate future herbivore threats such as the Emerald ash borer

In vitro elucidation of the crucial but complex oxidative tailoring steps in rufomycin biosynthesis enables one pot conversion of rufomycin B to rufomycin C

The antimycobacterial peptides, rufomycins, have their antibiotic activity conferred by oxidative tailoring of the cyclic peptide. Here we elucidate the roles of cytochrome P450s RufS and RufM in regioselective epoxidation and alkyl oxidation respectively and demonstrate how RufM and RufS create a complex product profile dependent on redox partner availability. Finally, we report the in vitro one pot conversion of rufomycin B to rufomycin C

Plant and prokaryotic TIR domains generate distinct cyclic ADPR NADase products

Toll/interleukin-1 receptor (TIR) domain proteins function in cell death and immunity. In plants and bacteria, TIR domains are often enzymes that produce isomers of cyclic adenosine 5′-diphosphate–ribose (cADPR) as putative immune signaling molecules. The identity and functional conservation of cADPR isomer signals is unclear. A previous report found that a plant TIR could cross-activate the prokaryotic Thoeris TIR–immune system, suggesting the conservation of plant and prokaryotic TIR-immune signals. Here, we generate autoactive Thoeris TIRs and test the converse hypothesis: Do prokaryotic Thoeris TIRs also cross-activate plant TIR immunity? Using in planta and in vitro assays, we find that Thoeris and plant TIRs generate overlapping sets of cADPR isomers and further clarify how plant and Thoeris TIRs activate the Thoeris system via producing 3′cADPR. This study demonstrates that the TIR signaling requirements for plant and prokaryotic immune systems are distinct and that TIRs across kingdoms generate a diversity of small-molecule products

Gamma-butyrolactone and furan signaling systems in streptomyces

Streptomyces bacteria produce different classes of diffusible signaling molecules that trigger secondary metabolite production and/or morphological development within the cell population. The biosynthesis of gamma-butyrolactones (GBLs) and 2-alkyl-4-hydroxymethylfuran-3-carboxylic acids (AHFCAs) signaling molecules is related and involves an essential AfsA-like butenolide synthase. This chapter first describes the catalytic role of AfsA-like enzyme then provides details about methods for the discovery and characterization of potentially novel signaling molecules. In section 4, one approach for establishing the biological role of these signaling molecules is presented