Phylogenomics reveals subfamilies of fungal nonribosomal peptide synthetases and their evolutionary relationships.

Kathryn E Bushley,B Gillian Turgeon

doi:10.1186/1471-2148-10-26

Abstract

BackgroundNonribosomal peptide synthetases (NRPSs) are multimodular enzymes, found in fungi and bacteria, which biosynthesize peptides without the aid of ribosomes. Although their metabolite products have been the subject of intense investigation due to their life-saving roles as medicinals and injurious roles as mycotoxins and virulence factors, little is known of the phylogenetic relationships of the corresponding NRPSs or whether they can be ranked into subgroups of common function. We identified genes (NPS) encoding NRPS and NRPS-like proteins in 38 fungal genomes and undertook phylogenomic analyses in order to identify fungal NRPS subfamilies, assess taxonomic distribution, evaluate levels of conservation across subfamilies, and address mechanisms of evolution of multimodular NRPSs. We also characterized relationships of fungal NRPSs, a representative sampling of bacterial NRPSs, and related adenylating enzymes, including α-aminoadipate reductases (AARs) involved in lysine biosynthesis in fungi.ResultsPhylogenomic analysis identified nine major subfamilies of fungal NRPSs which fell into two main groups: one corresponds to NPS genes encoding primarily mono/bi-modular enzymes which grouped with bacterial NRPSs and the other includes genes encoding primarily multimodular and exclusively fungal NRPSs. AARs shared a closer phylogenetic relationship to NRPSs than to other acyl-adenylating enzymes. Phylogenetic analyses and taxonomic distribution suggest that several mono/bi-modular subfamilies arose either prior to, or early in, the evolution of fungi, while two multimodular groups appear restricted to and expanded in fungi. The older mono/bi-modular subfamilies show conserved domain architectures suggestive of functional conservation, while multimodular NRPSs, particularly those unique to euascomycetes, show a diversity of architectures and of genetic mechanisms generating this diversity.ConclusionsThis work is the first to characterize subfamilies of fungal NRPSs. Our analyses suggest that mono/bi-modular NRPSs have more ancient origins and more conserved domain architectures than most multimodular NRPSs. It also demonstrates that the α-aminoadipate reductases involved in lysine biosynthesis in fungi are closely related to mono/bi-modular NRPSs. Several groups of mono/bi-modular NRPS metabolites are predicted to play more pivotal roles in cellular metabolism than products of multimodular NRPSs. In contrast, multimodular subfamilies of NRPSs are of more recent origin, are restricted to fungi, show less stable domain architectures, and biosynthesize metabolites which perform more niche-specific functions than mono/bi-modular NRPS products. The euascomycete-only NRPS subfamily, in particular, shows evidence for extensive gain and loss of domains suggestive of the contribution of domain duplication and loss in responding to niche-specific pressures.

Highlights

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes, found in fungi and bacteria, which biosynthesize peptides without the aid of ribosomes
The majority of multimodular NRPSs were composed of one or more standard NRPS modules (AT-C) with or without modifying domains (E, M, etc), while most monomodular NRPSs lacked complete A-TC modules and consisted of a single A domain or an AT unit followed by a variety of C-terminal domains, several of which have not previously been identified as core NRPS domains (Additional file 2)
Phylogenomic analysis and subfamily identification All known NRPS/NRPS-like proteins formed a monophyletic group supported by greater than 90% bootstrap support in ML analyses and greater than 50% bootstrap support in the NJ analysis (Fig. 1), separating them from most other known adenylating enzymes selected as potential outgroups, e.g., Acyl AMP ligases (AAL), CPS1 [54], Long Chain Fatty Acid ligases (LCFAL), AcetylCoA synthetases (ACoAS), and Ochratoxin synthetases (OCHRA)(Fig. 1, Additional file 5)

Summary

Introduction

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes, found in fungi and bacteria, which biosynthesize peptides without the aid of ribosomes Their metabolite products have been the subject of intense investigation due to their life-saving roles as medicinals and injurious roles as mycotoxins and virulence factors, little is known of the phylogenetic relationships of the corresponding NRPSs or whether they can be ranked into subgroups of common function. NRPS encoding genes (NPSs) are plentiful in fungi and bacteria but are not known in plants or animals The enzymes they encode biosynthesize a staggering diversity of chemical products because their substrates can include both D and L forms of the 20 amino acids used in ribosomal protein synthesis, as well as non-proteinogenic amino acids such as ornithine, imino acids, and hydroxy acids such as a-aminoadipic and a-butyric acids [1]. An additional mechanism, which has been reported only in biosynthesis of fungal ergot alkaloids, involves nonenzymatic cyclization by formation of a diketopiperazine ring [16,24]

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