非天然氨基酸拓展生物酶功能及定向进化数据集

基因密码子的扩展是蛋白质工程和合成生物学中的一个关键研究领域。非天然氨基酸的引入极大地拓宽了酶催化的功能，使研究人员能够设计出新颖的催化剂。这种扩展不仅丰富了蛋白质的化学多样性，还赋予酶新的生物学特性，从而提升其在药物开发、环境催化和生物材料设计等领域的应用潜力。因此，开发识别和插入非天然氨基酸的氨酰tRNA合成酶，成为这一进程的核心技术之一。
针对3-甲基组氨酸，我们构建了基于PylRS的定点饱和突变库，经过三轮正向筛选和两轮负向筛选，成功筛选出特异性识别3-甲基组氨酸的突变体。平板验证实验确认了这些突变体在氯霉素选择压力下的强依赖性。通过SDS-PAGE分析，我们验证了非天然氨基酸MeH成功插入到目标蛋白中，并利用ESI-MS质谱分析证实了MeH的插入。
针对硒代苯丙氨酸，我们通过一系列反应合成了该化合物，并使用1H-NMR和13C-NMR对中间产物和终产物进行了验证。在构建基于MjYRS的定点饱和突变文库后，经过筛选识别出特异性识别SeF的酪氨酰tRNA合成酶（SeFRS）。通过SDS-PAGE分析，确认了SeF成功插入到目标蛋白中，并通过胶内酶解质谱进一步验证了其成功插入。
近几年，光酶催化的还原反应研究受到广泛关注，尤其是基于非天然氨基酸辅因子的人工光酶展现出巨大潜力。这些光酶能够实现二氧化碳的还原及卤代芳烃的脱卤，并通过非天然氨基酸的结合显著提升催化效率与选择性。与筛选非天然氨基酸突变体的研究相辅相成，人工光酶的发展需有效的筛选和进化方法。为应对这一挑战，我们采用了“细菌表面展示+光催化邻近标记+磁珠分选/流式分析”的方法，对光敏元件进行优化，从而发挥非天然氨基酸辅因子在人工光酶催化中的核心作用。
在光敏元件和还原元件的优化开发方面，我们进行了DNA测序，并对构建的光敏酶突变体库进行了验证。通过荧光检测、Western blot等方法，我们确认了插入非天然氨基酸的光敏酶在细菌表面展示和光催化邻近标记的效果。流式分析则用于比较不同突变体的反应活性，同时利用ESI-MS质谱进一步分析了光敏酶催化的光敏反应。

Expansion of the genetic codon is a critical research area in protein engineering and synthetic biology. The introduction of non-canonical amino acids (ncAAs) greatly broadens the catalytic functions of enzymes, enabling researchers to design novel catalysts. This expansion not only enriches the chemical diversity of proteins but also endows enzymes with new biological properties, thereby enhancing their application potential in fields such as drug development, environmental catalysis, and biomaterial design. Therefore, developing aminoacyl-tRNA synthetases (aaRSs) that recognize and incorporate non-canonical amino acids has become one of the core technologies in this process.

Targeting 3-methylhistidine, we constructed a site-directed saturation mutagenesis library based on PylRS. After three rounds of positive selection and two rounds of negative selection, we successfully screened out mutants that specifically recognize 3-methylhistidine. Plate-based validation experiments confirmed the strong dependence of these mutants under chloramphenicol selection pressure. Through SDS-PAGE analysis, we verified that the non-canonical amino acid MeH was successfully incorporated into the target protein, and ESI-MS mass spectrometry analysis confirmed the incorporation of MeH.

Targeting selenophenylalanine, we synthesized this compound through a series of reactions, and verified the intermediate and final products using 1H-NMR and 13C-NMR. After constructing a site-directed saturation mutagenesis library based on MjYRS, we screened out a tyrosyl-tRNA synthetase (SeFRS) that specifically recognizes SeF. SDS-PAGE analysis confirmed that SeF was successfully incorporated into the target protein, and in-gel digestion mass spectrometry further verified the successful incorporation.

In recent years, research on photocatalytic reduction reactions has received extensive attention, especially artificial photoenzymes based on non-canonical amino acid cofactors that exhibit great potential. These photoenzymes can achieve carbon dioxide reduction and dehalogenation of haloarenes, and significantly improve catalytic efficiency and selectivity through the incorporation of non-canonical amino acids. Complementary to studies on screening non-canonical amino acid mutants, the development of artificial photoenzymes requires effective screening and evolution methods. To address this challenge, we adopted a method combining "bacterial surface display + photocatalytic proximity labeling + magnetic bead sorting/flow cytometry analysis" to optimize photosensitive elements, thereby exerting the core role of non-canonical amino acid cofactors in artificial photoenzyme catalysis.

In terms of the optimization and development of photosensitive and reduction elements, we performed DNA sequencing and validated the constructed photoenzyme mutant library. Through methods such as fluorescence detection and Western blot, we confirmed the performance of non-canonical amino acid-incorporated photosensitive enzymes in bacterial surface display and photocatalytic proximity labeling. Flow cytometry analysis was used to compare the reaction activities of different mutants, while ESI-MS mass spectrometry was used to further analyze the photosensitive reactions catalyzed by photoenzymes.