The Landscape of Recombination Events That Create Nonribosomal Peptide Diversity

Abstract

Nonribosomal peptides (NRP) are crucial molecular mediators in microbial ecology and provide indispensable drugs. Nevertheless, the evolution of the flexible biosynthetic machineries that correlates with the stunning structural diversity of NRPs is poorly understood. Here, we show that recombination is a key driver in the evolution of bacterial NRP synthetase (NRPS) genes across distant bacterial phyla, which has guided structural diversification in a plethora of NRP families by extensive mixing and matching of biosynthesis genes. The systematic dissection of a large number of individual recombination events did not only unveil a striking plurality in the nature and origin of the exchange units but allowed the deduction of overarching principles that enable the efficient exchange of adenylation (A) domain substrates while keeping the functionality of the dynamic multienzyme complexes. In the majority of cases, recombination events have targeted variable portions of the Acore domains, yet domain interfaces and the flexible Asub domain remained untapped. Our results strongly contradict the widespread assumption that adenylation and condensation (C) domains coevolve and significantly challenge the attributed role of C domains as stringent selectivity filter during NRP synthesis. Moreover, they teach valuable lessons on the choice of natural exchange units in the evolution of NRPS diversity, which may guide future engineering approaches.

Bifurcation drives the evolution of assembly-line biosynthesis

“…A key feature of desotamide evolution that could be exploited for future NRPS engineering efforts is the intragenomic recombination between distinct NRPS encoding BGCs. The recombination event we predicted takes place within the boundaries of A domains, consistent with previous 38,39,63 and more recent work 18,40 . More specifically, the predicted recombination allows for the substitution of the substrate specificity conferring active site and the ATP/phosphate binding catalytic P-loop 64 while functionally maintaining most of the A-T 65 and all of the C-A interdomain contacts [66][67][68] .…”

“…Similar patterns have been observed in other bacterial NRPS clusters 18,[38][39][40] Many studies have highlighted the role recombination plays in the evolution of assemblylines 1,41 . More specifically, given the reduced rate of horizontal gene transfer between distantly related taxa and the high rate of heterogeneity between recombinant sequences has, it has been speculated that intragenomic recombination within ancestral strains can provide opportunities for assembly-line diversification 18,42 . To assess the possible role of intragenomic recombination in the evolution of the wol BGC we generated a nucleotide sequence alignment of all thirty-four NRPS-associated adenylation domains present in the Streptomyces sp.…”

“…Only two recombination events were supported unanimously, including one event, in which wolG1A2 was identified as a recombinant sequence with wolG2A2 as the major parent and an adenylation domain encoding sequence from elsewhere in the genome, encoded by orf6595A, as the minor parent (Fig 4a-c and Supplementary Table 9). The predicted recombinant region lies between the conserved motif A2 (N-terminal) and motifs A5 and A6 (C-terminal) of the A-domain, thus comprising the flavodoxin subdomain 18,38,40 and a large portion of the N-terminal subdomain (Fig. 4d).…”

“…Insights into structural flexibility, inter-domain communication, and the role of proof-reading by catalytic domains pre-empted novel strategies to engineer assembly-line proteins [10][11][12][13][14] . There is also an increasing body of evidence suggesting that we might further improve our ability to engineer these systems if we had a better understanding of their evolution 1,[15][16][17][18] .…”

Biocatalytic synthesis of peptidic natural products and related analogues

“…They can be short linear, cyclic, or branch-cyclic peptides (Gurevich et al, 2018) (Figure 1). Different from regular peptides, PNPs often contain non-standard amino acids, such as non-proteinogenic a-, b-, or g-amino acids and D-amino acids (Baumann et al, 2017;Maini et al, 2016) (Figure 1). The structural diversity of PNPs is further enriched by modifications on peptide backbone and sidechains, resulting in various ring topologies and the formation of heterocycles (e.g., oxazolines and thiazolines) (Figure 1).…”