Fluorescent proteins (FP) are an integral part of modern biology due to its diverse biochemical and photophysical properties. The boundaries of FP have been extended through conventional mutagenesis and directed evolution approaches. Engineering of FP based on the standard genetic code consisting of 20 amino acids with limited functional groups restrict its diversification. Degeneracy of genetic code has helped in covering this substantial gap through genetic code engineering, wherein introduction of unnatural amino acid (UAA) analogues resulted in a collection of FP with varying properties. This review features the work carried till date in the area of FP incorporated with UAAs and explores strategies employed for incorporation, impact of UAAs in chromophore and surrounding residues and changes in inherent properties of FP. The long-standing association of FP as a tool for high throughput screening of orthogonal aaRS/tRNA pairs used in site specific incorporation of UAAs is expounded. Insertion of UAAs in FP has enabled their use in contemporary fields such as biophotovoltaics, bioremediation, biosensors, biomaterials and imaging of acidic vesicles. Thus, expansion of genetic code of FP is envisaged to rejig the existing spectra of colors and future research initiative in this direction is expected to glow brighter and brighter.

荧光蛋白 (FP) 由于其多样化的生化和光物理特性而成为现代生物学不可或缺的一部分。FP 的界限已经通过传统的诱变和定向进化方法得到了扩展。基于由 20 个氨基酸组成的标准遗传密码的 FP 工程限制了其多样化。遗传密码的简并性通过遗传密码工程帮助弥补了这一巨大差距，其中引入非天然氨基酸 (UAA) 类似物导致了具有不同特性的 FP 的集合。本综述介绍了迄今为止在与 UAA 结合的 FP 领域开展的工作，并探讨了用于掺入的策略、UAA 在生色团和周围残基中的影响以及 FP 固有特性的变化。阐述了 FP 作为用于高通量筛选正交 aaRS/tRNA 对的工具的长期关联，用于 UAA 的位点特定掺入。在 FP 中插入 UAA 使其能够用于现代领域，如生物光伏、生物修复、生物传感器、生物材料和酸性囊泡成像。因此，设想扩展 FP 的遗传密码以重新调整现有的颜色光谱，并且未来在这个方向上的研究计划预计会越来越亮。