Site-directed mutagenesis enhances the activity of benzylidene acetone synthase of polyketide synthase from <i>Polygonum cuspidatum</i>.

Zhimin HE; Wenrui MA; Liping YU; Heshu LÜ; Mingfeng Yang

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

Author: Zhimin HE ¹ ; Wenrui MA ¹ ; Liping YU ¹ ; Heshu LÜ ¹ ; Mingfeng YANG ¹
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1. Key Laboratory for Northern Urban Agriculture of Ministry of Agriculture and Rural Affairs, Beijing University of Agriculture, Beijing 102206, China.
Publication Type:Journal Article
Keywords: benzylidene acetone synthase; chalcone synthase; high performance liquid chromatography; site-directed mutagenesis
MeSH: Amino Acid Sequence; Fallopia japonica/metabolism*; Polyketide Synthases/chemistry*; Acetone; Mutagenesis, Site-Directed; Flavonoids/metabolism*; Acyltransferases/metabolism*
From: Chinese Journal of Biotechnology 2023;39(7):2806-2817
CountryChina
Language:Chinese
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.