Molecular Mechanism and Engineering of Sequential Reactions in Natural Products Biosynthesis in Plants

非蛋白质氨基酸是植物次生代谢物中的主要类群之一。这些化合物是通过特定的酶催化的顺序反应合成的。通过在缺失CSase基因的Cys-E.Coli中进行遗传互补，从西瓜幼苗的表达cDNA文库中分离到编码b-PA/DSase的基因。该克隆表达的蛋白具有b-PA合成活性，与编码b-PA/CSase的克隆一致。利用基于T7启动子的载体，建立了b-PA/CSase在大肠杆菌中的高效表达系统。重组西瓜b-PA/CSase能在体外合成含羞草素、奎宁酸和b-PA。以吡唑和丝氨酸为生物合成前体，通过体内实验形成B-PA。从西瓜中分离得到SATase基因克隆，并在大肠杆菌中高效表达。用纯化的重组蛋白对重组蛋白进行了生化鉴定。

The non-protein amino acids represent one of the major groups in secondary metabolites in plants. These compounds were synthesized via sequential reactions catalyzed by specific enzymes. The cDNA encoding b-PA/DSase was isolated from an expression cDNA library of watermelon seedlings by genetic conmplementation in Cys-E.coli lacking CSase loci. The expressed protein by the clone exhibited the activity of b-PA synthesis, indicating the identity of the clone encoding b-PA/CSase. Over-expression system of b-PA/CSase in E.coli was established by using a T7 promoter-based vector. The recombinant b-PA/CSase from watermelon was able to synthesize mimosine and quisqualic acid as well as b-PA in vitro. b-PA was also formed by in vivo experiments fed with pyrazole and serine as the biosynthetic precursors. The cDNA clone of SATase was isolated from watermelon and over-expressed in E.coli. Biochemical characterization has been performed by using the purified recombinant protein.