Lee Seong-Rae, Kang Jun Su, Lee Pyung Cheon
Department of Molecular Science and Technology and Advanced College of Bio-convergence Engineering, Ajou University, Suwon, Republic of Korea.
Front Bioeng Biotechnol. 2025 Apr 28;13:1586218. doi: 10.3389/fbioe.2025.1586218. eCollection 2025.
: a non-conventional yeast with significant industrial potential for tetraacetyl phytosphingosine (TAPS), remains underutilized due to the lack of a comprehensive genetic toolbox. In this study, we developed a modular genetic system tailored for to enable strain engineering and metabolic pathway optimization. This toolkit includes episomal plasmids incorporating multiple selectable markers, replication origins, and fluorescent reporters. Systematic evaluation of four antibiotic resistance markers demonstrated that nourseothricin, geneticin, and zeocin effectively confer resistance, whereas hygromycin B did not support selection in this host. Among three tested replication origins, 2μ and CEN6/ARS4 enabled stable episomal maintenance, whereas panARS failed to replicate. Expression analysis of six fluorescent proteins under the endogenous promoter revealed significant variability in transcript levels, which correlated with codon adaptation index values, emphasizing the importance of codon optimization for heterologous expression. Additionally, characterization of the endogenous and promoters using two highly expressed fluorescent proteins confirmed that promoter strength is largely independent of the downstream coding sequence. To demonstrate the functional application of this toolkit, we overexpressed a phosphorylation-insensitive mutant of acetyl-CoA carboxylase ( ), resulting in a 2.4-fold increase in TAPS production. Collectively, this study establishes a versatile genetic platform for , providing a robust foundation for future synthetic biology and metabolic engineering applications.
一种具有生产四乙酰植物鞘氨醇（TAPS）巨大工业潜力的非常规酵母，由于缺乏全面的遗传工具箱而未得到充分利用。在本研究中，我们开发了一种为其量身定制的模块化遗传系统，以实现菌株工程改造和代谢途径优化。该工具包包括整合了多个选择标记、复制起点和荧光报告基因的附加体质粒。对四种抗生素抗性标记的系统评估表明，制霉菌素、遗传霉素和博来霉素能有效赋予抗性，而潮霉素B在此宿主中不支持选择。在测试的三个复制起点中，2μ和CEN6/ARS4能实现附加体的稳定维持，而泛ARS无法复制。在内源启动子下对六种荧光蛋白的表达分析显示转录水平存在显著差异，这与密码子适应指数值相关，强调了密码子优化对异源表达的重要性。此外，使用两种高表达荧光蛋白对内源和启动子的表征证实，启动子强度在很大程度上独立于下游编码序列。为了证明该工具包的功能应用，我们过表达了乙酰辅酶A羧化酶的磷酸化不敏感突变体（），导致TAPS产量提高了2.4倍。总的来说，本研究为建立了一个通用的遗传平台，为未来的合成生物学和代谢工程应用提供了坚实的基础。
