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Ⅲ型聚酮合酶是以合成聚酮类化合物为主的一类重要生物合成酶。本文利用逆转录聚合酶链反应从中草药千层塔新鲜嫩叶中扩增聚酮合酶基因,得到一个Ⅲ型聚酮合酶全长cDNA。该基因全长1212 bp,编码404个氨基酸。与已知的其他植物来源的聚酮合酶氨基酸序列有约50%～66%的同源性。cDNA经双酶切后克隆至pQE81L,并导入大肠杆菌(E.coli)M15中表达,产生大量带寡聚组氨酸标记的重组酶,重组酶分子质量大小约为46.4 kDa。酶活性鉴定研究表明,该重组酶可催化芳香族底物、脂肪族底物生成系列非天然聚酮产物,尤其是其可催化N-甲基邻氨基苯甲酰CoA和丙二酰CoA生成1,3-二羟基-N-甲基-吖啶酮,吖啶酮生物碱一直被认为只能由吖啶酮合酶合成。该工作为研究千层塔中Ⅲ型聚酮合酶在天然药物石杉碱甲生物合成中的作用奠定基础。
Type Ⅲ polyketide synthase polyketides is a class of important biosynthetic enzymes. In this paper, polyketide synthase gene was amplified from fresh leaves of Chinese herbal medicine Melaleuca with reverse transcriptase-polymerase chain reaction to obtain a full-length polyketide synthase cDNA. The gene is 1212 bp in length and encodes 404 amino acids. There are about 50% ~ 66% homology with known polyketide amino acid sequences from other plants. After double digestion, the cDNA was cloned into pQE81L and introduced into E. coli M15 for production of a large amount of recombinant enzyme labeled with an oligohistidine. The molecular mass of the recombinant enzyme was about 46.4 kDa. Identification of enzyme activity showed that the recombinase can catalyze the formation of a series of non-natural polyketides by aromatic substrates and aliphatic substrates. In particular, it can catalyze the formation of N-methyl anthranilate CoA and malonyl-CoA , 3-dihydroxy-N-methyl-acridone, acridone alkaloids have long been considered only by acridone synthase synthesis. This work lays the foundation for the study of the role of type Ⅲ polyketide synthase in melaleuca in the natural drug huperzine A biosynthesis.