Abstract
There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1–alb6. Bioinformatic analysis of the proteins encoded by alb1–6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2–Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism.
Highlights
As the reported number of antibiotic resistant organisms continues to increase, the number of new antibiotics coming into use has dramatically declined over the past 20 years [1]
In this work we report the unexpected observation that a strain of S. marcescens, an enteric bacterium and an opportunistic pathogen, produces the broad-spectrum antibiotic althiomycin
Biosynthesis of althiomycin by S. marcescens Db10 is directed by a six-gene operon encoding a hybrid non-ribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line, essential tailoring enzymes and a cognate self-resistance protein
Summary
As the reported number of antibiotic resistant organisms continues to increase, the number of new antibiotics coming into use has dramatically declined over the past 20 years [1]. The majority of antibiotics that are in use within the clinic and in agriculture are derivatives of natural products and microorganisms remain an essential source of potential drugs [2]. This has led to a surge in the mining of genomes and analysis of unique environmental niches in the search for novel antimicrobial compounds [3,4]. Many clinically relevant secondary metabolites (including antibiotics) are biosynthesized by non-ribosomal peptide synthetase (NRPS) enzymes, polyketide synthase (PKS) enzymes, or hybrids thereof. The soil-dwelling bacteria Streptomyces and Myxococcus are well known and prolific producers of such compounds [5,6]
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