聚酮是由短链羧酸连续脱羧聚合而形成的一大类化学结构复杂、生物活性多样的次级代谢产物，其中许多已经被用作临床药物。生物合成研究发现，聚酮合酶（PKS）起始模块中的酰基转移酶（AT）结构域决定聚酮合成起始单元的选择，是PKS设计改造的理想靶点之一。在前期的工作中我们解析了一个起始AT的晶体结构，并结合体外功能分析鉴定了一些参与底物结合的关键氨基酸，但详细的分子机制仍不清楚。本研究中我们将对起始AT底物特异性的分子机制进行系统研究：首先，解析起始AT与底物复合物的晶体结构，确定催化中心参与底物结合的关键位点；其次利用定点突变和体外功能分析验证这些关键位点氨基酸组成对底物特异性的影响；再次，解析突变体的晶体结构，并与野生型AT进行比较，分析催化中心的结构变化；最后，利用所得到的结果指导起始AT底物特异性的设计改造，将预期的起始单元引入到聚酮的生物合成，在微生物体内合成具有预期化学结构的聚酮产物。

Polyketides are a large group of secondary metabolites with complicated structures and diverse bioactivities generated by the repetitive decarboxylative condensation of short-chain carboxylic acids. Many of them have been clinically used as drugs. During the biosynthesis of polyketides, the acyltransferase (AT) domains of loading modules of polyketide synthases (PKSs) control the choice of starter units incorporated into polyketides and are therefore attractive targets for the engineering of PKSs. Although several key residues that affect the substrate specificity of loading AT domains have been identified by structural analysis in our previous research, the detailed mechanism is still unclear. In this proposal, we will use multiple methods to elucidate the molecular basis of the substrate specificity of loading AT domains. Special emphasis will be placed on 1) solving the crystal structures of loading ATs complexed with the substrates by X-ray crystallography to identify residues involved in substrate binding; 2) elucidating the effects of these residues on substrate binding by site-directed mutagenesis and in vitro functional assay; 3) solving the crystal structures of loading AT mutants to reveal the conformation changes of active sites; and 4) generating expected polyketides in vivo using suitable microbial hosts by engineering loading AT domains of PKSs with the resulting information.