Liang Y-L, Zhao S-J, Xu L-X, Zhang X-Y
Department of Bioengineering, College of Biological and Agricultural Engineering, Jilin University, ChangChun, China.
Lett Appl Microbiol. 2012 Nov;55(5):323-9. doi: 10.1111/j.1472-765X.2012.03295.x. Epub 2012 Sep 10.
Dammarenediol production by an engineered yeast Saccharomyces cerevisiae was investigated.
A dammarenediol-producing engineered yeast was constructed by heterologous expression of the dammarenediol synthase gene from Panax ginseng hairy roots through RT-PCR. Fermentation was carried out in a 5-L GRJY-bioreactor with an inoculum size of 1% v/v at 30°C. Dammarenediol detection was performed with silica gel chromatography and HPLC. Determination of dammarenediol synthase activity subcellular distribution was carried out by surveying the enzyme activity in microsomes, lipid particles and total yeast homogenate. When cultured under aerobic conditions, the engineered yeast could produce dammarenediol up to 250μgl(-1). However, when an anaerobic shift strategy was employed, dammarenediol accumulated at a level as twice as that under aerobic condition. The dammarenediol synthase and dammarenediol were mainly localized in lipid particles.
Dammarenediol could be heterologously produced in engineered yeast. The heterologously expressed dammarenediol synthase is mainly localized in lipid particles. Anaerobic shift strategy could enhance the dammarenediol level in the engineered yeast.
This study showed that the high-value plant product dammarenediol could be produced by heterologous expression of the according gene in yeast. Furthermore, the anaerobic shift strategy could be potentially applied in oxidosqualene-derived compounds production in yeast. Here, the information about subcellular distribution of heterologously expressed dammarenediol synthase in the engineered yeast was also provided.
研究工程化酿酒酵母生产达玛烯二醇的情况。
通过逆转录聚合酶链反应（RT-PCR），异源表达人参毛状根中的达玛烯二醇合酶基因，构建了产达玛烯二醇的工程化酵母。在5升GRJY生物反应器中，接种量为1%（v/v），于30℃进行发酵。采用硅胶柱色谱法和高效液相色谱法检测达玛烯二醇。通过检测微粒体、脂质颗粒和酵母全细胞匀浆中的酶活性，确定达玛烯二醇合酶活性的亚细胞分布。在有氧条件下培养时，工程化酵母能够产生高达250μg l⁻¹的达玛烯二醇。然而，当采用厌氧转换策略时，达玛烯二醇的积累量是有氧条件下的两倍。达玛烯二醇合酶和达玛烯二醇主要定位于脂质颗粒中。
工程化酵母能够异源生产达玛烯二醇。异源表达的达玛烯二醇合酶主要定位于脂质颗粒中。厌氧转换策略可提高工程化酵母中的达玛烯二醇水平。
本研究表明，通过在酵母中异源表达相应基因，可以生产高价值植物产物达玛烯二醇。此外，厌氧转换策略可能应用于酵母中氧化鲨烯衍生化合物的生产。这里还提供了工程化酵母中异源表达的达玛烯二醇合酶亚细胞分布的信息。
