基于酰基转移酶结构的聚酮理性设计

聚酮是小分子药物的重要来源，合成前体的多样性是产生其复杂结构的重要原因。生物合成研究揭示模块化聚酮合酶（mPKS）的酰基转移酶（AT）结构域在聚酮延伸中负责选择合成前体。前期工作中，我们解析了多个AT的晶体结构，通过改造酰基结合口袋切换了AT对丙二酰和甲基、乙基丙二酰辅酶A（CoA）的特异性，并发现突变参与CoA结合的保守精氨酸降低对CoA前体的活性，但不影响对喂养实验中常用的乙酰半胱胺（SNAC）前体的活性。本研究将以上述体外结构和酶学分析的结果为基础理性设计mPKS，工程菌株发酵合成新聚酮产物：1）突变酰基口袋切换AT对酰基的特异性，改变聚酮的甲基、乙基取代模式；2）突变AT中结合CoA的保守精氨酸，抑制前体喂养中内源CoA前体的竞争，提高外源添加SNAC前体的利用效率。此外进一步解析AT与更复杂前体的复合物结构，鉴定参与识别的位点，指导AT理性设计，在更大范围内实现底物特异性的切换。

Polyketides are important source of small molecular drugs. The diversity of biosynthetic precursors results in the structural complexity of polyketides. During the elongation of polyketides, acyltransferase (AT) domains of modular polyketide synthases (mPKSs) control the choice of precursors. Previously, we solved crystal structures of several AT domains, switched substrate specificity of an AT for malonyl-, methylmalonyl- and ethylmalonyl-coenzyme A (CoA) by engineering the acyl binding pocket. It is also revealed that mutating the conserved arginine involved in CoA binding decreases activity toward CoA derived precursors, but does not affect activity toward N-acetylcysteamine (SNAC) derived precursors that are widely used in feeding experiments. In this proposal, we'll design mPKSs rationally based on the aforementioned in vitro structural and enzymatic results to generate novel polyketide products by fermentation of engineered strains. Our research will focus on 1) switching specificity of AT for acyl groups by mutating acyl binding pockets to alter the profiles of methyl- and ethyl-substituents of polyketides; 2) increasing the utilization efficiency of exogenously added SNAC derived precursors in feed experiments by inhibiting competition of endogenous CoA precursors in microbial cells through mutating the conserved arginine binding CoA. In addition, we will solve complex structures of AT with more complicated precursors to identify residues involved in substrate recognition and switch specificity of ATs for more substrates by rational design.