Biocombinatorial Synthesis of Novel Lipopeptides by COM Domain-Mediated Reprogramming of the Plipastatin NRPS Complex

Abstract

Both donors and acceptors of communication-mediating (COM) domains are essential for coordinating intermolecular communication within nonribosomal peptides synthetases (NRPSs) complexes. Different sets of COM domains provide selectivity, allowing NRPSs to utilize different natural biosynthetic templates. In this study, novel lipopeptides were synthesized by reprogramming the plipastatin biosynthetic machinery. A Thr-to-Asp point mutation was sufficient to shift the selectivity of the donor COM domain of ppsB toward that of ppsD. Deletion and/or interchangeability established donor and acceptor function. Variations in acceptor COM domain did not result in novel product formation in the presence of its partner donor, whereas plipastatin formation was completely abrogated by altering donor modules. Five novel lipopeptides (cyclic pentapeptide, linear hexapeptide, nonapeptide, heptapeptide, and cyclic octapeptide) were identified and verified by high-resolution LC-ESI-MS/MS. In addition, we demonstrated the potential to generate novel strains with the antimicrobial activity by selecting compatible COM domains, and the novel lipopeptides exhibited antimicrobial activity against five of the fungal species at a contention of 31.25–125 μg/ml.

Module and individual domain deletions of NRPS to produce plipastatin derivatives in Bacillus subtilis

“…It was difficult to acquire sufficient quantities of purified lipopeptides derivatives from the engineering mutant for biological testing by isolation and separation. In previous study [ 18 ], we chemically synthesized the linear plipastatin derivative (hexapeptide: C 16 β-OHFA-Glu-Orn-Tyr-Thr-Glu-Ala/Val) from company of KareBay (Ningbo, China), and tested its antimicrobial activity. The results showed that this linear hexapeptide was inactive against bacteria, but exhibited a MIC of 62.5 μg mL −1 against Fusarium oxysporum , Rhizopus stolonifer, and Aspergillus ochraceus .…”

“…We recently investigated the production of bioengineered plipastatin analogs by thioesterase domain- and inter-module communication domain-mediated reprogramming of the plipastatin NRPS complex [ 18 , 19 ]. In the present study, we attempted to further modify the plipastatin amino acid core by deleting complete modules or individual domains, and subsequently evaluated the effect of these changes on the plipastatin assembly line.…”

Biocatalytic synthesis of peptidic natural products and related analogues

“…For example, mutagenesis of the COM domains of rhabdopeptide/xenortide NRPS modules resulted in the change of protein affinity and the production of different peptide analogues ( Hacker et al., 2018 ). Furthermore, five novel lipopeptides (cyclic pentapeptide, linear hexapeptide, nonapeptide, heptapeptide, and cyclic octapeptide) were produced through the engineering of the COM domains of plipastatin NRPSs ( Liu et al., 2016 ), further demonstrating the importance of COM domains for the proper interactions of adjacent NRPS modules. On the other hand, the COM domains are not the only factor that influences the biosynthetic process of NRPs ( Bloudoff and Schmeing, 2017 ; Miller et al., 2016 ).…”

Bacillus lipopeptides as powerful pest control agents for a more sustainable and healthy agriculture: recent studies and innovations

“…Also, [∆Leu 6 ] surfactin showed a significant antifungal activity against Fusarium moniliforme. Liu et al (2016) were able to create novel LPs by shifting the selectivity of the donor COM domain (communication-mediating domain, essential for coordinating intermolecular communication within NRPSs complexes). Using this technique, and reprogramming the plipastatin biosynthetic machinery, five new LPs were identified.…”