虎杖中一种新的2-吡喃酮合酶基因的克隆和功能研究（英文）

Isolation and Characterization of a Novel 2-Pyrone-Producing Type Ⅲ Polyketide Synthase From Polygonum cuspidatum

4-羟基-6-甲基-2-吡喃酮(2-吡喃酮)及其衍生物是一类重要的植物次生代谢产物,具有抗虫、抗真菌等功能,在工业上可用于生产可再生化学平台间苯三酚和1,3,5-三氨基-2,4,6-三硝基苯. 2-吡喃酮合酶(2PS),一种Ⅲ型聚酮合酶(PKSs),是合成2-吡喃酮的关键酶.本研究以中药材虎杖(Polygonum cuspidatum Sieb. et Zucc)为材料,从中分离鉴定了一种新的2-吡喃酮合酶(Pc2PS). Pc2PS与已知的几种2PSs的氨基酸序列相似性为54%~56%.通过体外酶促反应鉴定功能发现,Pc2PS可以催化1分子乙酰-CoA与2分子丙二酰-CoA,缩合生成4-羟基-6-甲基-2-吡喃酮;也可以只利用3分子丙二酰-CoA,以相同的效率缩合生成2-吡喃酮.由此可以看出,乙酰-CoA存在与否并不影响该酶的催化效率.随后,我们测定了Pc2PS以丙二酰-CoA为单一底物时的酶动力学参数.虽然之前报道的2PSs也可以只利用丙二酰-CoA生成2-吡喃酮,但与Pc2PS不同的是,乙酰-CoA的缺失会大大降低催化效率.另外,对Pc2PS基因的组织表达特异性检测结果表明,该基因主要在虎杖根中表达,在叶中的表达量很低.本研究丰富了2PS的种类,并为2-吡喃酮的生物合成提供了基因资源.

Derivatives of 4-hydroxy-6-methyl-2-pyrone(2-pyrone)are actively involved in plant resistance to insects and pathogens,and are considered as potential renewable chemical platforms for phloroglucinol and 1,3,5-triamino-2,4,6-trinitrobenzene.2-Pyrone synthase(2 PS),one of the typeⅢpolyketide synthases(PKSs),is the key enzyme to produce 2-pyrone.In this study,a novel Pc2PS was isolated from Polygonum cuspidatum Sieb.et Zucc.,a traditional Chinese herb used for the treatment of cough,suppurative conditions,and hypertension.The Pc2PS shared 54%–56%amino acid sequence identity with 2 PSs of other species.Enzymatic activity analysis showed that Pc2PS could catalyze the condensation of one molecule of acetyl-CoA with two molecules of malonyl-CoA at almost the same efficiency as that of three molecules of malonyl-CoA alone for production of 4-hydroxy-6-methyl-2-pyrone.Thus,the presence or absence of acetyl-CoA in the reaction mixture did not affect catalytic efficiency.This catalytic property of Pc2PS differs from that of previously reported Gh2 PSs,which catalyzed malonyl-CoA condensation to form the same products as Pc2PS but at a lower rate in the absence of acetyl-CoA.In addition,we determined the kinetics of Pc2PS with malonyl-CoA alone as the substrate for the first time.Pc2PS showed tissue-specific expression in P.cuspidatum;Pc2PS was predominantly expressed in the root,whereas few transcripts were detected in the leaf.This study enriches the known diversity of2 PS proteins,and provides a novel genetic resource for 2-pyrone biosynthesis.