原位
荧光
氨基酸
定向进化
荧光蛋白
生物
生物化学
定向分子进化
化学
绿色荧光蛋白
计算生物学
生物物理学
遗传学
物理
基因
有机化学
量子力学
突变体
作者
Y.B. Yang,Jing Zhang,Jian Yang,Huiwen Luo,Yongjun Sun,Famin Ke,Qin Wang,Xiaowei Gao
摘要
ABSTRACT The incorporation of noncanonical amino acids (ncAAs) into proteins can enhance their function beyond the abilities of canonical amino acids and even generate new functions. However, the ncAAs used for such research are usually chemically synthesized, which is expensive and hinders their application on large industrial scales. We believe that the biosynthesis of ncAAs using metabolic engineering and their employment in situ in target protein engineering with genetic code expansion could overcome these limitations. As a proof of principle, we biosynthesized four ncAAs, O-L-methyltyrosine, 3,4-dihydroxy-L-phenylalanine, 5-hydroxytryptophan, and 5-chloro-L-tryptophan using metabolic engineering and directly evolved the fluorescent consensus green protein (CGP) by combination with nine other exogenous ncAAs in Escherichia coli . After screening a TAG scanning library expressing 13 ncAAs, several variants with enhanced fluorescence and stability were identified. The variants CGP V3pMeoF/K190pMeoF and CGP G20pMeoF/K190pMeoF expressed with biosynthetic O-L-methyltyrosine showed an approximately 1.4-fold improvement in fluorescence compared to the original level, and a 2.5-fold improvement in residual fluorescence after heat treatment. Our results demonstrated the feasibility of integrating metabolic engineering, genetic code expansion, and directed evolution in engineered cells to employ biosynthetic ncAAs in protein engineering. These results could further promote the application of ncAAs in protein engineering and enzyme evolution. IMPORTANCE Noncanonical amino acids (ncAAs) have shown great potential in protein engineering and enzyme evolution through genetic code expansion. However, in most cases, ncAAs must be provided exogenously during protein expression, which hinders their application, especially when they are expensive or have poor cell membrane penetration. Engineering cells with artificial metabolic pathways to biosynthesize ncAAs and employing them in situ for protein engineering and enzyme evolution could facilitate their application and reduce costs. Here, we attempted to evolve the fluorescent consensus green protein (CGP) with biosynthesized ncAAs. Our results demonstrated the feasibility of using biosynthesized ncAAs in protein engineering, which could further stimulate the application of ncAAs in bioengineering and biomedicine.
科研通智能强力驱动
Strongly Powered by AbleSci AI