Helmholtz International Lab for Anti-Infectives, Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.
Suzhou Research Institute, Shandong University, Suzhou, Jiangsu, 215123, China.
Nat Commun. 2021 Jan 12;12(1):296. doi: 10.1038/s41467-020-20548-8.
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.
非核糖体肽合成酶包含起始缩合结构域，指导非核糖体脂肽的生物合成，这些脂肽通常表现出广泛的生物活性。酰基链对生物活性和毒性有很大的影响，但由于对起始缩合结构域介导的脂肽起始的深入了解不足，限制了非核糖体肽合成酶的生物工程来获得具有所需酰基链的新型衍生物。在这里，我们通过工程化起始缩合结构域来修饰脂肽雷佐霉素、霍罗霉素和粘菌素的酰基链，表明这是一种可行的改变酰基链的方法。基于 rhizomide 生物合成酶 RzmA 与辛酰辅酶 A 结合的突变起始缩合结构域的结构和相关点突变实验，我们确定了一组负责底物酰基链选择性的残基，并将酰基链从乙酰基扩展到棕榈酰基。此外，我们在脂肽起始过程的反应循环中说明了起始缩合结构域的三种可能的构象状态。我们的研究为脂肽起始的机制和非核糖体肽合成酶的工程提供了进一步的见解。
