Engineering and elucidation of the lipoinitiation process in nonribosomal peptide biosynthesis

Abstract

Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformational states of starter condensation domains during the reaction cycle of the lipoinitiation process. Our studies provide further insights into the mechanism of lipoinitiation and the engineering of nonribosomal peptide synthetases.

Biosynthesis of Fungal Natural Products Involving Two Separate Pathway Crosstalk

“…The hty BGC provides an important biosynthetic precursor L-homotyrosine which was recognized by the fourth A domain of the NRPS EcdA. Understanding the biosynthetic mechanism of echinocandin B will facilitate us to take advantage of synthetic biology techniques to bioengineer NRPSs to generate bioactive compounds [ 49 , 50 , 51 ].…”

Chemical diversity and cellular effects of antifungal cyclic lipopeptides from cyanobacteria

“…A fully saturated fatty acid residue can be found in puwainaphycins, minutissamides, muscotoxins, and laxaphycins Gregson et al, 1992;Neuhof et al, 2005), while a set of polyunsaturated fatty acid is found in anabaenolysins (Jokela et al, 2012). Bacterial cyclic lipopeptides are synthesized through complex secondary metabolic pathways (Chooi & Tang, 2010;Roongsawang et al, 2010;Zhong et al, 2021). Fatty acyl chains are the first monomers incorporated into the peptidyl backbone via a process known as lipoinitiation (Chooi & Tang, 2010;Zhong et al, 2021).…”

“…Bacterial cyclic lipopeptides are synthesized through complex secondary metabolic pathways (Chooi & Tang, 2010; Roongsawang et al, 2010; Zhong et al, 2021). Fatty acyl chains are the first monomers incorporated into the peptidyl backbone via a process known as lipoinitiation (Chooi & Tang, 2010; Zhong et al, 2021). The lipid residue can be incorporated in a number of ways (Figure 2, Chooi & Tang, 2010).…”

“…The first step in the biosynthesis of hassallidins is postulated to be the N ‐acylation of the exocyclic Thr by HasV (Vestola et al, 2014). The N ‐acylation of lipopeptides using starter condensation domains has been reported from other lipopeptide biosynthetic pathways, including surfactin, lichenysin, fengycin, and arthrofactin (Zhong et al, 2021). A putative biosynthetic scheme for the lipidation of hassallidins implicates three proteins (HasG, HasH, and HasL) in the addition of lipids (Figure 2, Vestola et al, 2014).…”

Saccharochelins A–H, Cytotoxic Amphiphilic Siderophores from the Rare Marine Actinomycete Saccharothrix sp. D09

“…Saccharochelins harbor linear or branched fatty acyl chains ranging from C12 to C15, but foroxymithine contains only an acetyl chain (C2), which implied that SacE_C S might possess flexible but comparatively higher specificity toward longer fatty acid groups, resulting in the diverse amphiphilic saccharochelin derivatives (Figure S4). Based on our recent progress in genetic engineering and structural exploration of RzmA_C S , the C S domain of the rhizomide biosynthetic synthetase RzmA, 26 we also investigated the key residues in defining the length of fatty acyl chains in SacE_C S by alignment and structure homology modeling (Figure S5). The W29, Y123, and C133 residues in SacE_C S corresponding to Q36, Y138, and R148, respectively, in RzmA_C S were predicted to control the length of acyl donors (Figure S5).…”