Toehold Switch-Based Acid-Tolerance Modules

Enhancing acid tolerance of industrial microorganisms is critical for improving fermentation efficiency and sustainability. This study presents a synthetic biology approach that employs toehold switch-based acid-tolerance modules to engineer acid-tolerant strains. This toehold switch-based approach enables the construction of modules consisting of a trigger block and a switch block, generating a synthetic module library of ~105 constructs that integrate four acid-responsive promoters and 18 acid-resistance genes. Through stepwise evaluation, we identified two best synthetic modules, RE-6 and RE-38, which enabled an industrial lysine-producing strain to maintain lysine titers and yields at pH 5.5 comparable to those observed in the parent strain at pH 6.8. Transcriptional analyses revealed that upregulation of key acid-resistance genes involved in protein quality control, reactive oxygen species scavenging, and redox homeostasis contributed to the enhanced acid tolerance of the engineered strains. Our study offers a powerful toehold switch-based approach for constructing synthetic modules of interest, particularly for enhancing the robustness and productivity of industrial strains.

提升工业微生物的酸耐受性，对于优化发酵效率与可持续性至关重要。本研究提出一种合成生物学策略，采用基于toehold开关（toehold switch）的酸耐受性模块，以工程化构建耐酸菌株。该基于toehold开关的策略可构建由触发单元与开关单元组成的模块，进而生成包含约105个重组体的合成模块文库，该文库整合了4个酸响应启动子与18个耐酸基因。通过逐步筛选评估，本研究鉴定出两个最优合成模块RE-6与RE-38；这两个模块可使工业赖氨酸生产菌株在pH 5.5的条件下，维持与亲本菌株在pH 6.8条件下相当的赖氨酸效价与得率。转录分析显示，参与蛋白质质量控制、活性氧清除以及氧化还原稳态的关键耐酸基因的上调表达，是工程菌株酸耐受性提升的重要原因。本研究提供了一种基于toehold开关的高效策略，可用于构建目标合成模块，尤其适用于提升工业菌株的鲁棒性与生产性能。