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

在本研究中，我试图建立一种利用生物技术大规模制备THCA、CBDA和CBCA三种生物活性大麻素的方法。首先，将纯化的THCA合成酶和CBDA合成酶用氰基溴或蛋白酶消化，然后对所得的多肽片段进行N末端测序。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。从墨西哥大麻品系(用于克隆THCA合成酶)和CBDA大麻品系(用于克隆CBDA合成酶)中提取mRNA，并通过逆转录酶反应合成cDNA。用简并聚合酶链式反应克隆THCA合成酶和CBDA合成酶基因，然后克隆3‘和5’-RACE。它们的基因分别由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合成酶和CBDA合成酶的氨基酸序列具有很高的同源性(84%)，与黄连素桥酶有较高的同源性(40%)。通过聚合酶链式反应扩增出成熟THCA合成酶和CBDA合成酶的编码区，并将其整合到表达载体pET28a中。转化大肠杆菌JM109后，加入异丙基-L-β-D-硫代半乳糖苷进行诱导表达。重组蛋白的表达得到了证实，但没有任何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合成酶在酵母中的表达。目前，针对CBDA合成酶的表达，利用pYE22m进行类似的实验才刚刚开始。

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.