Department of Microbial Physiological & Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China; University of Chinese Academy of Sciences, 100049, Beijing, China.
Department of Microbial Physiological & Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China.
Biosens Bioelectron. 2025 Jan 15;268:116904. doi: 10.1016/j.bios.2024.116904. Epub 2024 Nov 1.
This study was undertaken to develop a high-throughput screening strategy using a whole-cell biosensor to enhance methyl-group transfer, a rate-limiting step influenced by intracellular methyl donor availability and methyltransferase efficiency. An l-homocysteine biosensor was designed based on regulatory protein MetR from Escherichia coli, which rapidly reported intracellular l-homocysteine accumulation resulted from S-adenosyl-l-homocysteine (SAH) formation after methyl-group transfer. Using S-adenosyl-l-methionine (SAM) as a methyl donor, this biosensor was applied to caffeic acid 3-O-methyltransferase derived from Arabidopsis thaliana (AtComT). After several rounds of directed evolution, the modified enzyme achieved a 13.8-fold improvement when converting caffeic acid to ferulic acid. The best mutant exhibited a 5.4-fold improvement in catalytic efficiency. Characterization of beneficial mutants showed that improved O-methyltransferase dimerization greatly contributed to enzyme activity. This finding was verified when we switched and compared the N-termini involved in dimerization across different sources. Finally, with tyrosine as a substrate, the evolved AtComT mutant greatly improved ferulic acid biosynthesis, yielding 3448 mg L with a conversion rate of 88.8%. These results have important implications for high-efficiency O-methyltransferase design, which will greatly benefit the biosynthesis of a wide range of natural products. In addition, the l-homocysteine biosensor has the potential for widespread applications in evaluating the efficiency of SAM-based methyl transfer.
本研究旨在开发一种基于全细胞生物传感器的高通量筛选策略，以增强甲基转移，这是一个限速步骤，受细胞内甲基供体可用性和甲基转移酶效率的影响。基于大肠杆菌的调节蛋白 MetR，设计了 l-同型半胱氨酸生物传感器，该传感器可快速报告甲基转移后 S-腺苷-l-同型半胱氨酸（SAH）形成导致的细胞内 l-同型半胱氨酸积累。使用 S-腺苷-l-甲硫氨酸（SAM）作为甲基供体，该生物传感器应用于来自拟南芥的咖啡酸 3-O-甲基转移酶（AtComT）。经过几轮定向进化，修饰后的酶在将咖啡酸转化为阿魏酸时实现了 13.8 倍的提高。最佳突变体的催化效率提高了 5.4 倍。有益突变体的特性表明，改善的 O-甲基转移酶二聚化大大有助于酶活性。当我们切换并比较不同来源涉及二聚化的 N 末端时，验证了这一发现。最后，用酪氨酸作为底物，进化后的 AtComT 突变体大大提高了阿魏酸的生物合成，产量为 3448mg/L，转化率为 88.8%。这些结果对高效 O-甲基转移酶设计具有重要意义，将极大地促进广泛的天然产物的生物合成。此外，l-同型半胱氨酸生物传感器具有评估基于 SAM 的甲基转移效率的广泛应用潜力。
