Exploring strategies for accessing bioactive secondary metabolites from Streptomyces bacteria

It is impossible to imagine modern medicine without antibiotics. Unfortunately, rapid global rise of antimicrobial resistance (AMR) threatens to push humanity back to the pre-antibiotic era - a bleak reality when a simple, previously easily treatable infection could quickly turn deadly, and surgical interventions now considered routine would be too dangerous to perform. Similarly, cancer treatment relies on chemotherapeutic drugs - and a vast majority of fatal chemotherapy failures is attributed to drug resistance. It is therefore essential to prioritise research into drug discovery and development, and one of the most abundant sources of both antimicrobial and anticancer compounds are bacteria from the genus Streptomyces.

Streptomyces, the largest genus of the diverse bacterial phylum Actinomycetota, are the source of many useful secondary metabolites and two-thirds of all known antibiotics. Historical approaches to bacterial drug discovery focused mostly on phenotypic screening of random environmental isolates, especially soil-dwelling streptomycetes following Selman Waksman's breakthrough discovery of the first effective anti-TB drug streptomycin. Although most Streptomyces species carry dozens of secondary metabolite biosynthetic gene clusters (BGC) in their genomes, they often remain silent under laboratory conditions and were missed in early research prior to the development of molecular genetics. Next generation sequencing of genomic and environmental DNA can generate large quantities of data for in silico genome mining, offering a rational starting point for wet biochemistry. Modern dereplication methods that combine high-throughput screening with artificial elicitation can be used to screen vast existing libraries of natural compounds and producing organisms, and synthetic biology techniques such as heterologous gene expression and metabolic engineering, combined with bioinformatics, traditional microbiology, chemical purification and instrumental analysis create a powerful and adaptable discovery pipeline. Moreover, a deep understanding of the genetics, biology, ecology and evolutionary history of Streptomyces, as well as examining any existing scientific biases, will lead to a more targeted and successful bioprospecting.

The methods described above were utilised to explore the biosynthetic potential of several Streptomyces species. First, the arcyriaflavin biosynthetic gene cluster acf from S. venezuelae was analysed bioinformatically together with a previously uncharacterised hydroxylase AcfX from the same cluster. Two variants of the cluster (acfODCP/acfXODCP) were cloned and successfully expressed in their native producer, leading to overproduction of arcyriaflavins A and F, respectively, and a direct conversion of arcyriaflavin A to F in vitro was also demonstrated with purified recombinant AcfX. S. venezuelae acfXODCP and acfODCP exconjugants were then used for the biosynthesis of new-to-nature arcyriaflavins by substituted precursor feeding with synthetic tryptophan analogues, and the presence of novel arcyriaflavins was confirmed with LC-MS analysis of culture extracts. Finally, a previously uncharacterised cluster duo related to duocarmycin A was identified in the genome of S. corchorusii and analysed bioinformatically. Following phenotypic screening against indicator strains and attempts to isolate the bioactive fraction, the duo BGC was reassembled and heterologously expressed in S. venezuelae and S.albus. Extract from one S.venezuelae exconjugant produced an immediate cytotoxic effect against B. subtilis recA-, and the HPLC-resolved spectrum of the culture extract was shown to display a unique peak bearing characteristics of two compounds from the duocarmycin family.