Geerlings A, Redondo F J, Contin A, Memelink J, van der Heijden R, Verpoorte R
Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands.
Appl Microbiol Biotechnol. 2001 Aug;56(3-4):420-4. doi: 10.1007/s002530100663.
A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for strictosidine synthase (STR) and strictosidine beta-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.
构建了一种转基因酿酒酵母，其包含来自药用植物长春花的编码异胡豆苷合酶（STR）和异胡豆苷β-葡萄糖苷酶（SGD）的cDNA。这两种酶都参与萜类吲哚生物碱的生物合成。发现酵母培养物中这两种酶均高水平表达。STR活性在细胞内（13.2纳 katal/克鲜重）和培养基中（高达25纳 katal/升培养基）均有发现，而SGD活性仅存在于酵母细胞内（2.5微 katal/克鲜重）。在加入色胺和番木鳖苷后，这种转基因酵母培养物在培养基中产生了高水平的异胡豆苷；测得的水平高达2克/升。在酵母细胞内也检测到了异胡豆苷，尽管含量要低得多（0.2毫克/克细胞）。这是由于细胞对底物番木鳖苷和色胺的低渗透性所致。然而，在使酵母细胞通透后，培养基中存在的异胡豆苷被完全水解为番木鳖胺。此外，转基因酿酒酵母能够在白花越橘浆果提取物上生长，该提取物可作为番木鳖苷和碳水化合物的来源。在这些条件下，添加色胺足以使转基因酵母培养物产生吲哚生物碱。我们的结果表明，转基因酵母培养物是生产植物生物碱的一种有趣的替代方法。
