Gan Qinhua, Yu Xinjun, Jiang Xiang, Huang Xingwei, Xin Yi, Lu Yandu
State Key Laboratory of Marine Resource Utilization in South China Sea, School of Marine Biology and Fisheries, Hainan University, Haikou, 570228, China.
School of Tropical Crops, Hainan University, Haikou, 570228, China.
Microb Cell Fact. 2025 May 29;24(1):127. doi: 10.1186/s12934-025-02700-5.
The rapidly expanding catalog of sequenced genomes has revolutionized the pace and scale of microalgal cellular metabolism delineation. However, knowledge of the gene regulation in these genomes is lacking. This is true even for , the laboratory model species where transcriptional regulation is best understood, although systematic knowledge of regulatory elements (e.g., promoters) remains elusive. This leads to limitations in microalgae for engineering system designs, which currently rely mainly on characterizing the molecular mechanisms of individual regulatory sequences via low-throughput methods. Here, we take a first step toward multi-promoter dissection and demonstrate systematic dissection of multiple microalgal promoters through quantitative genome-wide comparisons and sequence-based structural annotations. We demonstrate this approach on both well-studied and previously uncharacterized promoters in the oleaginous microalga IMET1. Using in silico design, representative regulatory elements were synthesized and assembled as building blocks for transgene circuits. Assessment of the in vivo activity revealed the maximum transformation efficiency (414 ± 102 transformants/µg DNA) in the plasmid pHN5-2 containing promoter and terminator. Therefore, the present synthetic approach for establishing engineering systems is superior to the conventional empirical methods. Applying these transforming circuits to the creation and characterization of a gene-indexed loss-of-function mutagenesis library verifies the applicability of this strategy to the discovery and standardization of regulatory parts for microalgae. Moreover, the generality of the approach presented here provides the possibility of quantitative promoter dissection and rational transform system design in and a wide range of other microalgae.
The online version contains supplementary material available at 10.1186/s12934-025-02700-5.
快速扩展的已测序基因组目录彻底改变了微藻细胞代谢描绘的速度和规模。然而，这些基因组中的基因调控知识却很匮乏。即使对于实验室模式物种 也是如此，尽管对调控元件（如启动子）的系统知识仍然难以捉摸，但在该物种中对转录调控的理解最为深入。这导致微藻工程系统设计存在局限性，目前主要依靠低通量方法来表征单个调控序列的分子机制。在这里，我们朝着多启动子剖析迈出了第一步，并通过全基因组定量比较和基于序列的结构注释展示了对多个微藻启动子的系统剖析。我们在产油微藻IMET1中对已充分研究和先前未表征的启动子都验证了这种方法。通过计算机设计，合成了代表性调控元件并将其组装成转基因电路的构建模块。体内活性评估显示，在含有 启动子和 终止子的质粒pHN5-2中，最大转化效率为（414±102个转化体/μg DNA）。因此，目前用于建立工程系统的合成方法优于传统的经验方法。将这些转化电路应用于创建和表征基因索引的功能丧失诱变文库，验证了该策略在发现和标准化微藻调控元件方面的适用性。此外，这里提出的方法的通用性为在 和其他多种微藻中进行定量启动子剖析和合理转化系统设计提供了可能性。
在线版本包含可在10.1186/s12934-025-02700-5获取的补充材料。
