Interfacing Polyketide Synthase and Non-Ribosomal Peptide Synthetase Pathways Through Exploitation of Hairpin Docking Domain Pairs

多酮合成酶(PKs)和非核糖体肽合成酶(NRPS)在许多具有生物活性的天然产物的生物合成中起着至关重要的作用。作为分子装配线，这些模块化的大合成酶(ET)从简单的前体逐步构建代谢产物。为了保持生物合成的保真度，这些酶机器的亚基之间的相互作用是由对接结构域(DDS)介导的；对接结构域是附加在亚基的C-末端和N-末端的短多肽。这个项目专注于短线状基序(SLIM)-发夹对接结构域(HDD)对的作用，这是已知的为数不多的在混合NRPS-PKS系统中介导亚单位相互作用的对接结构域类型之一。这些对接结构域连接到PKS载体蛋白的C末端和它们相互作用的NRPS结构域的N末端。Challis小组最近的工作表明，这些DDs参与了HDAC抑制剂的生物合成，假设它们是为了调节装配线上PKS和NRPS组件之间的相互作用。将采用一系列计算、化学和生化方法来探索超薄-HDD对在混合PKS-NRPS中的流行情况，以及如何利用它们的相互作用来产生新的生物合成途径。在大肠杆菌中过量生产相关的蛋白结构域将形成生化分析的基础，以评估重组PKS和包含对接结构域的NRPS组件之间的相互作用。将进行生物合成工程，从不同的途径融合PKS和NRPS组分，使新的HDAC抑制剂能够从头开始生产。

Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) play a critical role in the biosynthesis of numerous biologically-active natural products. Acting as molecular assembly lines, these modular megasynth(et)ases construct metabolic products in a stepwise fashion from simple precursors. To maintain biosynthetic fidelity, the interactions between subunits of these enzymatic machines are mediated by docking domains (DDs); short polypeptides appended to the C- and N-termini of the subunits. This project focusses on the action of Short Linear Motif (SLiM) -hairpin docking domain (HDD) pairs, one of a handful of docking domain types known to mediate subunit interactions in hybrid NRPS-PKS systems. These docking domains are attached to the C-terminus of PKS carrier proteins and the N-terminus of their interacting NRPS domain. Recent work in the Challis group has shown these DDs are involved in the biosynthesis of HDAC inhibitors, where they are hypothesised to mediate the interaction between PKS and NRPS components of the assembly line. A range of computational, chemical and biochemical approaches will be taken to explore the prevalence of SLiM-HDD pairs in hybrid PKS-NRPSs, and how their interactions can be exploited to generate novel biosynthetic pathways. Overproduction of relevant protein domains in E.coli will form the basis for biochemical assays to assess the interaction between recombinant PKS and NRPS components containing the docking domains. Biosynthetic engineering will be undertaken to fuse the PKS and NRPS components from separate pathways, enabling the de novo production of novel HDAC inhibitors.