Shi Shuobo, Ji Haichuan, Siewers Verena, Nielsen Jens
Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden.
Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden Department of Cell and Molecular Biology, University of Gothenburg, Medicinaregatan 9C, SE-405 30 Gothenburg, Sweden.
FEMS Yeast Res. 2016 Feb;16(1):fov108. doi: 10.1093/femsyr/fov108. Epub 2015 Dec 10.
Biological production of fatty acid (FA)-derived products has gained increasing attention to replace petroleum-based fuels and chemicals. FA biosynthesis is highly regulated, and usually it is challenging to design rational engineering strategies. In addition, the conventional 'one sample at a time' method for lipid determination is time consuming and laborious, and it is difficult to screen large numbers of samples. Here, a method for detecting free FAs in viable cells using Nile red staining was developed for use in large-scale screening. Following optimization of the method, it was used for screening a cDNA library from the oleaginous yeast Yarrowia lipolytica for identification of genes/enzymes that were able to enhance free FA accumulation in Saccharomyces cerevisiae. Several novel enzymes resulting in increasing FA accumulation were discovered. These targets include a GPI anchor protein, malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, FA hydroxylase, farnesyltransferase, anoctamin, dihydrolipoamide dehydrogenase and phosphatidylethanolamine-binding protein. The best enzyme resulted in a 2.5-fold improvement in production of free FAs. Our findings not only provide a novel method for high-throughput evaluation of the content of free FAs, but also give new insight into how enzymes from Y. lipolytica may increase the production of fatty acids in S. cerevisiae.
脂肪酸（FA）衍生产品的生物生产已越来越受到关注，以替代石油基燃料和化学品。FA生物合成受到高度调控，通常设计合理的工程策略具有挑战性。此外，传统的“一次一个样品”脂质测定方法既耗时又费力，且难以筛选大量样品。在此，开发了一种使用尼罗红染色检测活细胞中游离FA的方法用于大规模筛选。在对该方法进行优化后，将其用于筛选产油酵母解脂耶氏酵母的cDNA文库，以鉴定能够增强酿酒酵母中游离FA积累的基因/酶。发现了几种导致FA积累增加的新型酶。这些靶点包括一种糖基磷脂酰肌醇（GPI）锚定蛋白、苹果酸脱氢酶、甘油醛-3-磷酸脱氢酶、FA羟化酶、法尼基转移酶、八聚体通道蛋白、二氢硫辛酰胺脱氢酶和磷脂酰乙醇胺结合蛋白。最佳的酶使游离FA的产量提高了2.5倍。我们的研究结果不仅提供了一种高通量评估游离FA含量的新方法，还为解脂耶氏酵母的酶如何提高酿酒酵母中脂肪酸的产量提供了新的见解。
