A selection of Saccharomyces cerevisiae promoters for biotechnological applications aiming at the use of synthetic glycerol medium

Glycerol offers several advantages as a substrate for biotechnological applications. An important step towards using the popular production host Saccharomyces cerevisiae for glycerol-based bioprocesses have been recent studies in which commonly used S. cerevisiae strains were engineered to grow in synthetic medium containing glycerol as the sole carbon source. In order to boost extensive S. cerevisiae metabolic engineering incentives aiming at the use of glycerol, we realized that promoters with predictable expression levels in synthetic glycerol medium were required. In the current study, we used transcriptome analysis and a yECitrine-based fluorescence reporter assay to select and characterize useful 25 promoters for driving expression of target genes in S. cerevisiae under the given conditions. The promoters of the genes ALD4 and ADH2 showed 4.2- and 3-fold higher activities compared to the well-known strong TEF1 promoter. Moreover, the collection contains promoters with graded activities in synthetic glycerol medium and different degrees of glucose repression. To demonstrate the general applicability of the promoter collection, we successfully used a subset of the characterized promoters with graded activities in order to optimize growth on glycerol in an engineered derivative of CEN.PK, in which glycerol catabolism exclusively occurs via a non-native DHA pathway.甘油作为底物在生物技术应用中具备多重优势。将常用生产宿主酿酒酵母（Saccharomyces cerevisiae）应用于甘油基生物工艺的重要进展，源于近期相关研究：研究人员将常用酿酒酵母菌株进行工程化改造，使其可在以甘油为唯一碳源的合成培养基中生长。为进一步推动以甘油为底物的酿酒酵母代谢工程研究，我们亟需可在合成甘油培养基中实现可预测表达水平的启动子。本研究通过转录组分析与基于yECitrine的荧光报告基因检测法，筛选并鉴定了25个可在该培养条件下驱动酿酒酵母靶基因表达的优质启动子。其中，ALD4与ADH2基因的启动子活性分别相较于经典强启动子TEF1高出4.2倍与3倍。此外，该启动子集合包含在合成甘油培养基中具有梯度活性、且对葡萄糖阻遏程度各异的启动子。为验证该启动子集合的通用适用性，我们选取其中一批具有梯度活性的特征化启动子，对CEN.PK工程菌株的甘油生长性能进行了优化；该工程菌株仅通过非天然二羟丙酮（DHA）通路完成甘油分解代谢。