|Budget Amount *help
¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1999 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1998 : ¥1,900,000 (Direct Cost : ¥1,900,000)
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.