Elucidating and exploiting halogenase recruitment beta hairpin docking domains in nonribosomal peptide biosynthesis

非核糖体肽和聚酮是一类天然产物，由多种与生物相关的化合物组成。它们分别由非核糖体肽合成酶(NRPS)和聚酮合成酶(PKS)合成，它们是以流水线方式发挥作用的模块化巨合酶。PKS和NRPS蛋白亚基之间的相互作用对其生物合成的保真度至关重要，在许多情况下，C端和N端的对接结构域有助于PKS和NRPS蛋白的相互作用。在杂交NRPS-PKS系统中，亚基之间的一种相互作用涉及连接到一个亚基N-末端催化结构域的发夹状对接结构域(HDD)与另一个亚基C-末端载体蛋白上的短线性基序(SLIMs)的对接。最近Challis小组的计算工作表明，这种对接结构域系统在聚酮和非核糖体多肽代谢物的生物合成机制中可能比最初认识的要普遍得多。该项目将集中于HDD/SLIM相互作用的特征，预测将发生在蓝藻非核糖体多肽的生物合成途径中。该项目的目的是通过在大肠杆菌中过度生产相关蛋白质并使用合成底物类似物来检测它们的酶活性，来实验表征HDD/SLIM的相互作用。我们还将尝试使用X射线结晶学和核磁共振光谱来获得蛋白质的高分辨率结构信息。一旦确定了特征，该系统将被用来了解HDD/SLIM系统作为生物合成途径工程工具的潜力，包括使用来自不同代谢物生物合成的组件生产混合装配线。

Nonribosomal peptides and polyketides are classes of natural products, comprising a large variety of biologically-relevant compounds. They are synthesised by nonribosomal peptide synthases (NRPSs) and polyketide synthases (PKSs) respectively, which are modular megasynthases that act in an assembly line-like manner. The interaction between subunits of PKS and NRPS proteins is critical to the fidelity of their biosyntheses, and are known in many cases to be facilitated by C-terminal and N-terminal docking domains. One type of interaction between subunits in hybrid NRPS-PKS systems involves the docking of -hairpin docking domains (HDDs) attached to catalytic domains at the N-terminus of one subunit with a short linear motifs (SLiMs) attached to carrier proteins at the C-terminus of another subunit. Recent computational work in the Challis group has suggested that this system of docking domains may be far more prevalent in the biosynthetic machinery of polyketide and nonribosomal peptide metabolites than first appreciated. This project will focus upon the characterisation of HDD/SLiM interactions predicted to occur in the biosynthetic pathway of nonribosomal peptides from cyanobacteria. The aims of the project are to experimentally characterise the HDD/SLiM interactions by overproducing the relevant proteins in E. coli and using synthetic substrate analogues to examine their enzymatic activity. We will also attempt to obtain high resolution structural information for the proteins using X-ray crystallography and NMR spectroscopy. Once characterised, this system will be exploited to understand the potential of the HDD/SLiM systems as a tool for engineering of biosynthetic pathways, including the production of hybrid assembly lines using components from the biosyntheses of different metabolites.