Liu Qi, Li Yun-Hao, Tao Liu-Fei, Yang Jia-Yi, Zhang Yi-Lun, Cai Meng-Hao
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Shanghai Collaborative Innovation Center for Biomanufacturing, Shanghai, China.
Appl Environ Microbiol. 2025 Jan 31;91(1):e0219124. doi: 10.1128/aem.02191-24. Epub 2024 Dec 19.
The C1 and C2 alcohols hold great promise as substrates for biomanufacturing due to their low cost and rich resources. is considered a preferred host for methanol and ethanol bioconversion due to its natural utilization of methanol and ethanol. However, the scarcity of strong and tightly regulated alcohol-inducible promoters limits its extended use. This study aimed to develop enhanced methanol- and ethanol-inducible promoters capable of improving gene expression in . Rational design strategies were employed to rewire the upstream regulatory sequence of the methanol-inducible P, generating several high-strength methanol-inducible promoters with a stringent regulatory pattern. Eleven strong promoters were identified from 36 endogenous ethanol-inducible candidates recognized from transcriptome analysis. Core promoter regions, the crucial element influencing transcriptional strength, were also characterized. Five high-activity core promoters were then combined with four upstream regulatory sequences of high-strength promoters, resulting in four groups of synthetic promoters. Ultimately, the highly active methanol-inducible P and ethanol-inducible P and P were selected for the expression of an α-amylase and yielded enzyme activity 1.6, 2.6, and 4.5 times higher as compared to that of P. This work expands the genetic toolkit available for , providing more precise and efficient options for regulating gene expression. It benefits the use of as an efficient platform for the C1 and C2 alcohol-based biotransformation in industrial biotechnology.IMPORTANCE represents a preferred microbial host for the bio-utilization of C1 and C2 alcohols that are regarded as renewable carbon sources based on clean energy. However, lack of efficient and regulated expression tools highly limits the C1 and C2 alcohols based bioproduction. By exploring high-strength and strictly regulated alcohol-inducible promoters, this study expands the expression toolkit for on C1 and C2 alcohols. The newly developed methanol-inducible P and ethanol-inducible P demonstrate significantly higher expression levels than the commercial P system. The endogenous and synthetic promoter series established in this study provides new construction references and alternative tools for expression control in for C1 and C2 alcohols based biomanufacturing.
C1和C2醇因其低成本和丰富资源，在生物制造领域作为底物具有巨大潜力。由于其对甲醇和乙醇的天然利用能力，[具体生物名称]被认为是甲醇和乙醇生物转化的优选宿主。然而，强大且调控严格的醇诱导型启动子的稀缺限制了其广泛应用。本研究旨在开发能够增强[具体生物名称]中基因表达的甲醇和乙醇诱导型启动子。采用合理设计策略对甲醇诱导型P的上游调控序列进行重新设计，产生了几个具有严格调控模式的高强度甲醇诱导型启动子。从转录组分析识别出的36个内源性乙醇诱导型候选启动子中鉴定出11个强启动子。还对影响转录强度的关键元件核心启动子区域进行了表征。然后将5个高活性核心启动子与4个高强度启动子的上游调控序列组合，产生了4组合成启动子。最终，选择高活性的甲醇诱导型P以及乙醇诱导型P和P用于α-淀粉酶的表达，其产生的酶活性分别比P高1.6倍、2.6倍和4.5倍。这项工作扩展了[具体生物名称]可用的遗传工具集，为调控基因表达提供了更精确和高效的选择。它有利于将[具体生物名称]用作工业生物技术中基于C1和C2醇的生物转化的高效平台。重要性[具体生物名称]是将C1和C2醇作为基于清洁能源的可再生碳源进行生物利用的优选微生物宿主。然而，缺乏高效且调控的表达工具极大地限制了基于C1和C2醇的生物生产。通过探索高强度且调控严格的醇诱导型启动子，本研究扩展了[具体生物名称]在C1和C2醇上的表达工具集。新开发的甲醇诱导型P和乙醇诱导型P的表达水平明显高于商业P系统。本研究中建立的内源性和合成启动子系列为基于C1和C2醇的生物制造中[具体生物名称]的表达控制提供了新的构建参考和替代工具。
