Exploiting a global regulator for small molecule discovery in Photorhabdus luminescens

Abstract

Bacterially produced small molecules demonstrate a remarkable range of structural and functional diversity and include some of our most useful biological probes and therapeutic agents. Annotations of bacterial genomes reveal a large gap between the number of known small molecules and the number of biosynthetic genes/loci that could produce such small molecules, a gap that most likely originates from tight regulatory control by the producing organism. This study coupled a global transcriptional regulator, HexA, to secondary metabolite production in Photorhabdus luminescens, a member of the Gammaproteobacteria that participates in a complex symbiosis with nematode worms and insect larvae. HexA is a LysR-type transcriptional repressor, and knocking it out to create a P. luminescens DeltahexA mutant led to dramatic upregulation of biosynthesized small molecules. Use of this mutant expanded a family of stilbene-derived small molecules, which were known to play important roles in the symbiosis, from three members to at least nine members.

Figure 1

Representative HPLC trace overlay (210 nm) of organic extracts of WT (black) and ΔhexA (blue) P. luminescens cultures, both grown in 5 mM L-proline. Starred peaks denote stilbene derivatives, upregulated in the ΔhexA strain. Numbers above peaks refer to compounds in Figure 2.

Figure 2

Structures of new and known stilbene derivatives isolated from P. luminescens. 1 – 3 have been previously isolated from Photorhabdus. 4 – 9 are new natural products. All four stereoisomers of 5 were isolated in a single fraction. 9 was previously described as a side product in a synthetic scheme (30).

Figure 3

Fold change in production of stilbene metabolites by WT (black •) and ΔhexA (blue ) P. luminescens with increasing concentrations of supplementary L-proline. Numbers above curves refer to structures in Figure 2. Compounds 6 – 8 are overlapped on the HPLC trace and were therefore integrated together.

Scheme 1

Proposed biosynthesis of reduced stilbene 4, based on the established biosynthesis of 1. The pathways to these compounds diverge at prephenate, and oxidation of any intermediate along the route to 4 leads to the convergent production of 1.