PDF(Pubmed)
Abstract：
Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, their development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs within the promoter regions of highly expressed genes across six different algal species, we identified cis-regulatory elements (CREs) associated with high transcriptional activity. Combining the algorithms POWRS, STREME, and PhyloGibbs, we predicted 1511 CREs and inserted them into a minimal synthetic promoter sequence in 1, 2, or 3 copies, resulting in 4533 distinct synthetic promoters. These promoters were evaluated in vivo for their capacity to drive the expression of a transgene in a high-throughput manner through next-generation sequencing post antibiotic selection and fluorescence-activated cell sorting. To validate our approach, we sequenced hundreds of transgenic lines showing high levels of GFP expression. Further, we individually tested 14 identified promoters, revealing substantial increases in GFP expression─up to nine times higher than the baseline synthetic promoter, with five matching or even surpassing the performance of the native AR1 promoter. As a result of this study, we identified a catalog of CREs that can now be used to build superior synthetic algal promoters. More importantly, here we present a validated pipeline to generate building blocks for innovative synthetic genetic tools applicable to any algal species with a sequenced genome and transcriptome data set.
摘要：
藻类生物技术通过各种生物产品的可持续和可扩展生产来彻底改变生物经济。然而,缺乏先进的遗传工具阻碍了它们的发展。这项研究引入了一种合成生物学方法来开发这种工具，专注于合成启动子的构建和测试。通过分析6种不同藻类高度表达基因的启动子区域内的保守DNA基序，我们确定了与高转录活性相关的顺式调节元件(CREs)。结合算法POWRS，STREME,和PhyloGibbs,我们预测了1511个CRE，并将它们插入到1、2或3个拷贝的最小合成启动子序列中，产生4533个不同的合成启动子。通过抗生素选择和荧光激活细胞分选后的下一代测序,在体内评估这些启动子以高通量方式驱动转基因表达的能力。为了验证我们的方法，我们测序了数百个转基因品系，显示出高水平的GFP表达。Further,我们分别测试了14个鉴定的启动子，显示GFP表达的大幅增加-比基线合成启动子高9倍，具有五个匹配甚至超越天然AR1启动子的性能。作为这项研究的结果，我们确定了可用于构建高级合成藻类启动子的CRE目录。更重要的是,在这里，我们提供了一个经过验证的管道，以生成适用于具有测序基因组和转录组数据集的任何藻类物种的创新合成遗传工具的构建模块。
