Evolution-guided engineering of nonribosomal peptide synthetase adenylation domains

Abstract

Hormaomycin is a structurally unusual morphogenic and antibiotic peptide biosynthesized by a bacterial nonribosomal peptide synthetase (NRPS). Bioinformatic analysis suggested that parts of the NRPS adenylation (A) domains had recombined during evolution, resulting in a major switch of substrate specificity. This feature inspired us to create A domains with altered substrates based on the putative recombination points. Following characterization of all native hormaomycin A domains, engineered versions were constructed and characterized. Three of the enzymes displayed an almost identical specificity profile to that of native domains recognizing the same substrates. The data support the evolutionary hypothesis regarding the emergence of the hormaomycin pathway and suggest new strategies in NRPS engineering.

An environmental bacterial taxon with a large and distinct metabolic repertoire

“…Overexpression of soluble CthA2 was greatly improved by transforming the expression plasmids into E. coli Bl21-A1 and growing the clones as above except a final concentration of 0.2% arabinose was added at when the D 600 nm reached 0.4. The protein-purification and mass-exchange-based adenylation assays were performed as reported previously 26,62,63 . UPLC high-resolution HESI-MS analysis of sponge and bacterial fractions.…”

Identification and Characterization of the Sulfazecin Monobactam Biosynthetic Gene Cluster

“…Interestingly, the SulI A1T1 didomain in addition showed approximately 47% activity with D-Gln, suggesting that D-Gln could be an alternative substrate. To date, none of the known monobactam producers incorporate more than one side chain (Parker et al, 1986), but the incorporation of D-Gln in vitro suggests the biosynthetic pathway might be engineered to produce sulfazecin analogs in the future (Crusemann et al, 2013; Kries et al, 2015). …”

Engineering polyketide synthases and nonribosomal peptide synthetases

“…As might be expected for a selection strategy that relied only on binding affinity, and one that omitted the carrier protein, improvements to K m were largely responsible for large specificity shifts of selected variants. New A-domain substrate specificities have also been created using the more invasive mutagenic approach of chimeragenesis, using A-domains from the biosynthesis of hormaomycin [46]. Inspired by bioinformatic analysis that suggested A-domains acquired new substrate specificities by recombination of A-domain fragments during evolution, chimeric A-domains were created by replacing the core active site regions of a [β-Me]Phe activating “scaffold” A-domain (HrmO3 A ) with core regions derived from three A-domains which each activated (3-Ncp)Ala, threonine, and valine, respectively.…”