Exploring, evolving and exploiting thioester synthetases for industrial biotechnology

生物催化的目标是开发绿色的、酶促的化学转化方法来生产合成靶标和高价值的原料。目前将羧酸转化为酰基硫酸酯的化学方法很复杂，而且需要多个步骤。该项目旨在开发一锅版本的这种转化使用工程吡喃甲酰辅酶A合成酶(PCAS)。酰基硫酯是许多重要的天然产物，如脂肪酸，脂类和抗生素的聚酮途径的中间体。枯草杆菌新发现的PCAS是维生素和羧化辅因子生物素的关键酶，该酶的晶体结构、作用机理和活性中心残基已被确定，并被合理地改造以接受新的脂肪酸底物。该项目将充分利用PCAS，并利用定向进化产生具有更广泛底物和应用的酶。将开发一种高通量筛选方法来鉴定具有新活性的克隆。还将探索目前合成方法无法企及的联结。

goal of biocatalysis is the development of green, enzymatic methods for chemical transformations producing synthetic targets and high-value feedstocks. Current chemical methods for conversion of carboxylic acids to acyl thioesters are complex and multistep. This project aims to develop a one-pot version of this transformation using engineered pimeloyl-CoA synthase (PCAS).Acyl thioesters are intermediates in the polyketide pathways to many important natural products such as fatty acids, lipids and antibiotics. A recently discovered PCAS from B. subtilis is the key enzyme of vitamin and carboxylation cofactor biotin.The crystal structure, mechanism and active site residues of this enzyme have been determined and it has been rationally engineered to accept novel fatty acid substrates. This project will fully exploit PCAS and generate enzymes with a broader range of substrates and applications, using directed evolution. A high-throughput screening method will be developed to identify clones with novel activity. Couplings will also be explored which currently lie beyond the reach of synthetic methods.