定点突变提高虎杖聚酮合酶的苯亚甲基丙酮合酶活性

Site-directed mutagenesis enhances the activity of benzylidene acetone synthase of polyketide synthase from Polygonum cuspidatum

摘要:

虎杖（Polygonum cuspidatum）聚酮合酶（polyketide synthase 1，PcPKS1）同时具有查尔酮合酶（chalcone synthase，CHS）及苯亚甲基丙酮合酶（benzylidene acetone synthase，BAS）催化活性，能够催化生成聚酮类化合物柚皮素查尔酮和苯亚甲基丙酮，进而催化合成黄酮类或覆盆子酮等具有多种生物学活性的化合物。本研究通过分析虎杖PcPKS1与掌叶大黄（Rheum palmatum） BAS、拟南芥（Arabidopsis thaliana） CHS等家族成员的序列以及酶催化位点的构象，确定可能影响酶功能的3个氨基酸位点：Thr133、Ser134、Ser339。采用定点突变对PcPKS1进行分子修饰，成功获得2个突变体并进行相关体外酶促反应，高效液相色谱（high performance liquid chromatography，HPLC）产物分析结果表明，在pH 7.0和pH 9.0的体外酶促条件下，突变体T133LS134A和S339V维持BAS和CHS双功能活性，且BAS活性显著高于原PcPKS1。本研究为利用PcPKS1进行基因工程调节黄酮类和覆盆子酮化合物的生物合成提供理论依据。

Abstract:

Polygonum cuspidatum polyketide synthase 1 (PcPKS1) has the catalytic activity of chalcone synthase (CHS) and benzylidene acetone synthase (BAS), which can catalyze the production of polyketides naringenin chalcone and benzylidene acetone, and then catalyze the synthesis of flavonoids or benzylidene acetone. In this study, three amino acid sites (Thr133, Ser134, Ser33) that may affect the function of PcPKS1 were identified by analyzing the sequences of PcPKS1, the BAS from Rheum palmatum and the CHS from Arabidopsis thaliana, as well as the conformation of the catalytic site of the enzyme. Molecular modification of PcPKS1 was carried out by site-directed mutagenesis, and two mutants were successfully obtained. The in vitro enzymatic reactions were carried out, and the differences in activity were detected by high performance liquid chromatography (HPLC). Finally, mutants T133LS134A and S339V with bifunctional activity were obtained. In addition to bifunctional activities of BAS and CHS, the modified PcPKS1 had much higher BAS activity than that of the wild type PcPKS1 under the conditions of pH 7.0 and pH 9.0, respectively. It provides a theoretical basis for future use of PcPKS1 in genetic engineering to regulate the biosynthesis of flavonoids and raspberry ketones.