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.

The Burkholderia genus possesses ecological and metabolic diversities. A large number of silent biosynthetic gene clusters (BGCs) in the Burkholderia genome remain uncharacterized and represent a promising resource for new natural product discovery. However, exploitation of the metabolomic potential of Burkholderia is limited by the absence of efficient genetic manipulation tools. Here, we screened a bacteriophage recombinase system Redγ-BAS, which was functional for genome modification in the plant pathogen Burkholderia gladioli ATCC 10248. By using this recombineering tool, the constitutive promoters were precisely inserted in the genome, leading to activation of two silent nonribosomal peptide synthetase gene clusters (bgdd and hgdd) and production of corresponding new classes of lipopeptides, burriogladiodins A–H (1–8) and haereogladiodins A–B (9–10). Structure elucidation revealed an unnatural amino acid Z- dehydrobutyrine (Dhb) in 1–8 and an E-Dhb in 9–10. Notably, compounds 2–4 and 9 feature an unusual threonine tag that is longer than the predicted collinearity assembly lines. The structural diversity of burriogladiodins was derived from the relaxed substrate specificity of the fifth adenylation domain as well as chain termination conducted by water or threonine. The recombinase-mediating genome editing system is not only applicable in B. gladioli, but also possesses great potential for mining meaningful silent gene clusters from other Burkholderia species.

No abstract

Nonribosomal peptide synthetases (NRPSs) can incorporate nonproteinogenic amino acids into peptidyl backbones to increase structural diversity. Genome mining of Schlegelella brevitalea led to the identification of a class of linear lipoheptapeptides, glidomides, featuring two unusual residues: threo‐β‐OH‐L‐His and threo‐β‐OH‐D‐Asp. The β‐hydroxylation of Asp and His is catalyzed by the nonheme FeII/α‐ketoglutarate‐dependent β‐hydroxylases GlmD and GlmF, respectively. GlmD independently catalyzes the hydroxylation of L‐Asp to primarily produce threo‐β‐OH‐L‐Asp on the thiolation domain, and then undergoes epimerization to form threo‐β‐OH‐D‐Asp in the final products. However, β‐hydroxylation of His requires the concerted action of GlmF and the interface (I) domain, a novel condensation domain family clade. The key sites of I domain for interaction with GlmF were identified, suggesting that the mechanism for hydroxylation of His depends on the collaboration between hydroxylase and NRPS.

Biosynthesis. The biosynthesis of glidomides is reported by Youming Zhang, Xiaoying Bian et al. in their Research Article (e202203591). Different mechanisms for the formation of β‐OH amino acid building blocks were elucidated.