Safety assessment of genetically engineered yeast and breeding of non-toxigenic industrially used yeasts

甲基乙二醛（methylglyoxal， MG）是一种由二羟基丙酮磷酸经甲基乙二醛合酶（methylglyoxal synthase， MGS）作用合成的典型毒性酮醛，通过测定其细胞水平变化来评价转基因酵母细胞的安全性。得到了以下结果：(1)用糖酵解酶基因（磷酸葡萄糖异构酶、磷酸果糖激酶和三磷酸异构酶基因）转化的酿酒酵母细胞在大量葡萄糖的发酵条件下培养时，MG在细胞中以致突变水平积累。(2)酶促研究表明，MG的积累完全由MGS引起。(3) MGS缺乏显著降低了细胞MG水平。从这些结果来看，检测基因工程酵母细胞中的MG波动对于在实际发酵过程中安全利用所创造的酵母是必要的。研究了MGS对酵母产孢的生理作用。该酶的活性与清酒酵母的产孢能力密切相关。这一发现被应用于没有产孢能力的清酒酵母细胞。MG的去除使清酵母产孢效率显著提高，为分子杂交育种提供了手段。1995年被日本发酵与生物工程学会授予“Eda奖”。将安全性评价推广到大豆甘氨酸基因转基因马铃薯。甘氨酸的表达量约占马铃薯总蛋白的1%。脂质、碳水化合物、灰分、纤维、维生素和脂肪酸的水平与对照（带载体转化的马铃薯或未转化的天然马铃薯）几乎相同。然而，用含有吉霉素基因的载体或载体转化的马铃薯中，类糖核素（茄素和脉轮素）的含量增加，达到对照的1.5-2倍。结果表明，载体与待转化植物的合适组合对于创建转基因植物和/或转基因食品具有重要意义。少

The safety of genetically engineered yeast cells was assessed by determining the change in cellular level of methylglyoxal (MG), a typical toxic ketoaldehyde biosynthesized from dihydroxyacetonephosphate by an action of methylglyoxal synthase (MGS). The following results were obtained. (1) when the cells of Saccharomyces cerevisiae transformed with glycolytic enzyme genes (phosphoglucose isomerase, phosphofructokinase and triosephosphate isomerase genes) were incubated in fermentation conditions with large amount of glucose, MG was accumulated in cells at a mutagenic level. (2) The enzymatic study indicated that MGS was entirely responsiblefor the observed accumulation of MG.(3) The deficiency of MGS dramatically lowered the cellular MG level. From these results, the inspection of MG fluctuation in genetically engineered yeast cells is necessary for the safety utilization of the created yeast in practical fermenta-tion processes.The physiological function of MGS was also studied in rel … More ation to the yeast sporulation. The activity of the enzyme was closely related to the sporulation capability of Sake yeasts. The finding was applied to the Sake yeast cells that show no sporulation capability. The removal of MG resulted in the marked increase in sporulation efficiency of Sake yeast and provided means for molecular breeding by hybridization. We were awarded a "Eda prize" from Japan Society of Fermentation and Bioengineering in 1995.The safety assessment was extended to the genetically transformed potato with soybean glycinin gene. The expression level of glycinin was about 1% of the total potato protein. The levels of lipid, carbohydrate, ash, fiber, vitamin, and fatty acids were almost the same as those of control (transformed potato with a vector or non-transformed natural potato). However, transformed potato with a vector or the vector habouring giycinin gene showed increased level of glycoalkaroids (solanin and chakonin) and the level reached 1.5-2 fold of the control. The results indicated that the suitable combination of vector and plant to be transformed is important in creation transgenic plant and/or genetically engineered foods. Less