Development of the method for large-scale preparation of bioactive cannabinoids

在本研究中，我尝试开发利用生物技术大规模制备三种生物活性大麻素THCA、CBDA和CBCA的方法。首先，用CNBr或蛋白酶对纯化的THCA合成酶和CBDA合成酶进行酶切，并对得到的肽片段进行n端测序。根据这些氨基酸序列以及两种酶的n端氨基酸序列设计了不同的引物。cDNA模板方面，分别从墨西哥大麻菌株（用于克隆THCA合酶）和CBDA大麻菌株（用于克隆CBDA合酶）制备mRNA，通过逆转录酶反应分别合成cDNA。用简并PCR法克隆THCA合酶和CBDA合酶基因，然后用3′-和5′- race法克隆。他们的基因分别由1635和1632个核苷酸组成，分别编码545和544个氨基酸残基的蛋白质。THCA合酶和CBDA合酶的氨基酸序列具有较高的同源性（84%），与小檗碱桥酶的同源性为中等高（40%）。我尝试利用克隆的基因开发THCA合酶和CBDA合酶的表达系统。通过PCR扩增得到成熟THCA合酶和CBDA合酶的编码区，并将其整合到表达载体pET28a中。重组蛋白经大肠杆菌JM109转化后，加入异丙基- 1 -β- d -硫代半乳糖苷诱导表达。重组蛋白的表达得到证实，而没有蛋白具有大麻素合成酶活性。因此，我们选择pYE22m作为表达载体。因此，我证实了具有催化活性的THCA合酶在酵母中的表达。目前，对于CBDA合酶的表达，使用pYE22m的类似实验才刚刚开始。

In this study, I attempted to develop method for large-scale preparation of three bioactive cannabinoids, THCA, CBDA and CBCA using biotechnology.As a first step, purified THCA synthase and CBDA synthase were digested with CNBr or protease, ant the resulting peptide fragments were N-terminally sequenced. The various primers were designed based on these amino acid sequences, together with N-terminal amino acid sequences of both enzymes. Concerning the cDNA templates, mRNA was prepared from the Mexican Cannabis strain (for cloning of THCA synthase) and CBDA Cannabis strain (for cloning of CBDA synthase), and cDNA was synthesized from each mRNA preparation by reverse transcriptase reaction. Cloning of genes encoding THCA synthase and CBDA synthase was achieved by degenerate PCR, followed by 3'- and 5'-RACE. Their genes were found to consist of 1635- and 1632- nucleotide open reading frames encoding proteins of 545 and 544 amino acid residues, respectively. The deduced amino acid sequences of THCA synthase and CBDA synthase showed high identity (84%) to each other and moderately high identity (40%) to berberine bridge enzyme.I attempted development of expression systems for THCA synthase and CBDA synthase using the cloned genes. The regions coding deduced mature THCA synthase and CBDA synthase were amplified by PCR, and the resulting cDNAs were incorporated into the expression vector pET28a. After transformation of E. coli JM109, expression of the recombinant protein was induced by the addition of isopropyl-l-β-D-thiogalactoside. The expression of recombinant proteins was confirmed, whereas no proteins had cannabinoid synthase activity. Hence, pYE22m was used as a expression vector. Consequently, I confirmed the expression of catalytically active THCA synthase in yeast. Now, for the expression of CBDA synthase, similar experimet using pYE22m is just started.