School of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece.
FEMS Yeast Res. 2010 Sep;10(6):699-707. doi: 10.1111/j.1567-1364.2010.00645.x. Epub 2010 Jun 17.
The constructed strains AM63, having an extra copy of the HMG2 gene with a K6R stabilizing mutation in Hmg2p expressed under the control of the inducible galactose promoter and stably integrated into the chromosomal HO locus, and AM64, a derivative of AM63 with an additional deletion of the ERG6 gene, were used as tools to test the squalene accumulation capacity of Saccharomyces cerevisiae. Kinetic data indicated high squalene levels in the early stages of semi-anaerobic cultivation of these strains. The stable Hmg2p induced a strong increase in the squalene pool and a smaller increase in the lanosterol pool. In AM63, the squalene content was approximately 20-fold higher than in the wild-type EGY48 parental strain. In AM64, the combined Hmg2p stabilization and ERG6 deletion did not further enhance squalene accumulation, as lack of ergosterol feedback inhibition led to an elevated transfer of surplus squalene into C27 sterols. The obtained maximum capacity of the selected strains to accumulate squalene and our observations provide a further understanding of the regulation of ergosterol pathway and may also be used as a reference value for its production using food-grade strains of S. cerevisiae.
构建的菌株 AM63 和 AM64 被用作工具，用于测试酿酒酵母的角鲨烯积累能力。菌株 AM63 具有在 Hmg2p 中表达的受诱导半乳糖启动子控制的额外 HMG2 基因拷贝和 K6R 稳定突变，并且稳定整合到染色体 HO 位点，而菌株 AM64 是 AM63 的衍生物，其 ERG6 基因额外缺失。动力学数据表明，这些菌株在半厌氧培养的早期阶段具有高角鲨烯水平。稳定的 Hmg2p 诱导角鲨烯库强烈增加，而羊毛甾醇库略有增加。在 AM63 中，角鲨烯含量比野生型 EGY48 亲本菌株高约 20 倍。在 AM64 中，Hmg2p 的稳定化和 ERG6 的缺失联合作用并没有进一步增强角鲨烯的积累，因为缺乏麦角固醇反馈抑制导致过剩的角鲨烯被转移到 C27 甾醇中。所选菌株积累角鲨烯的最大能力和我们的观察结果进一步了解了麦角固醇途径的调节，也可以作为使用食品级酿酒酵母菌株生产角鲨烯的参考值。
