Translocation of the thioesterase domain for the redesign of plipastatin synthetase

Abstract

Non-ribosomal peptide synthetases (NRPSs) are large enzymatic complexes that catalyse the synthesis of biologically active peptides in microorganisms. Genetic engineering has recently been applied to reprogram NRPSs to produce lipopeptides with a new sequence. The carboxyl-terminal thioesterase (TE) domains from NRPSs catalyse cleavage products by hydrolysis or complex macrocyclization. In this study, we modified plipastatin synthetase by moving the intrinsic TE region to the end of the internal thiolation (T) domains, thus generating Bacillus subtilis strains that could produce new truncated cyclic or linear peptides of the predicted sequence, which further provided an important insight into the regioselectivity of plipastatin TE. The TE was capable of recognizing and catalysing the lactone formation between _L-Try₃ with the last few residues _L-Pro7 and _L-Gln8 at the C-terminus. Additionally, the unmatched linkers connecting the TE region and T domain resulted in nonproduction strains, suggesting that the native T-TE linker is necessary and sufficient for the TE domain to release the products from the hybrid enzymes. This is the first report to demonstrate truncated cyclic lipopeptides production and module skipping by simply moving the TE domain forward in an NRPS system.

Figure 1. Structure of plipastatin (PubChem CID:102466606) and schematic diagram of the plipastatin biosynthesis operon (pps).

Five NRPSs are encoded by the genes ppsABCDE. Based on their function, the five distinct synthetases, PpsA–E, can be divided into modules and domains. Each module is comprised of condensation (C), adenylation (A), thiolation (T), and epimerization (E) domains that are responsible for the activation, attachment, and modification of one constitutive amino acid residue. At module 10, a terminal thioesterase (TE) domain catalyses linear peptide cyclization and releases the final product, plipastatin.

Figure 2. The linkers connecting the thioesterase (TE) domain with various thiolation (T) domains in new hybrid non-ribosomal peptide synthetases.

The 7ProT-C, 8GlnT-C, and 9TyrT-E linkers were connected to the TE domain, respectively (A–C). TE_Long, containing the native 10IleT-TE linker, was connecting to different T domains of the PPSD subunit (D–F). 7ProT-C: linker region between the T domain of module 7 and the condensation (C) domain of module 8. 8GlnT-C: linker region between the T domain of module 8 and the C domain of module 9. 9TyrT-E: linker region between the T and epimerization (E) domains in module 9.

Figure 3

Liquid chromatography-electrospray ionization-tandem mass spectra of [M + H]⁺ ions of the linear heptapeptide at 1119.65 m/z (A) and the cyclic heptapeptide at 1101.65 m/z (B).

Figure 4

Liquid chromatography-electrospray ionization-tandem mass spectra of [M + H]⁺ ions of the linear octapeptide at 1247.72 m/z (A) and the cyclic octapeptide at 1229.71 m/z (B).

Figure 5. Liquid chromatography-electrospray ionization-tandem mass spectra of [M + H]⁺ ions of the linear nonapeptide at 1410.78 m/z.