阳春砂龙脑基二磷酸合酶关键氨基酸位点筛选及突变体的构建

目的 对阳春砂萜类合成关键酶龙脑基二磷酸合酶(bornyl diphosphate synthase,BPPS)催化生成龙脑基二磷酸(bornyl diphosphate,BPP)的关键氨基酸位点解析,为阳春砂BPPS的功能优化并且促进其在代谢工程中的应用奠定基础.方法 比较阳春砂、海南砂的BPPS (AvBPPS、A...

Screening of key amino acid sites of AvBPPS and construction of mutants

Objective Bornyl diphosphate synthase (BPPS) is a special monoterpene synthase which produces bornyl diphosphate (BPP). BPP is the key precursor for the medicinal terpenoids biosynthesis of Amomum villosum. The understanding of the BPPS key amino acid sites catalyzing the production of BPP in A. villosum will help to optimize the function of BPPS and improve its application in metabolic engineering. Methods The comparison of BPPSs from A. villosum, A. longiligulare (another plant source for Fructus Amomi) and three other BPPSs which have been reported showed that AvBPPS had higher ratio of BPP production than other four BPPSs. Through the methods of molecular modeling, substrate docking, prediction of conserved motifs, and multiple alignments to explore the key active sites of AvBPPS that determine the production for BPP. The method of fast Site-Directed Mutagenesis was used to obtain the mutants. Result Twenty key sites of AvBPPS were screened out and predicted, and according to these selected sites, 20 AvBPPS mutants and one AlBPPS mutant were designed, 17 mutants and their purified proteins have been successfully obtained. Conclusion Intergrated analysis methods were used to design BPPS mutants rationally in this study, which provides the basis to explore the catalytic mechanism and to optimize the function of AvBPPS.