Directed evolution of the fluorescent protein CGP with in situ biosynthesized noncanonical amino acids

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...

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Bibliographic Details
Published in:Applied and environmental microbiology 2024-04, Vol.90 (4), p.e0186323
Main Authors: Yang, Yanhong, Zhang, Jing, Yang, Jian, Luo, Huiwen, Sun, Yingjie, Ke, Famin, Wang, Qin, Gao, Xiaowei
Format: Article
Language:English
Subjects:
Amino acids
Amino Acids - metabolism
Biosynthesis
Biotechnology
Consensus
Directed evolution
E coli
Engineering
Enzymology and Protein Engineering
Evolution
Fluorescence
Genetic code
Heat treatment
Heat treatments
Metabolic engineering
Metabolism
Methyltyrosines - genetics
Phenylalanine
Protein engineering
Protein Engineering - methods
Proteins
Proteins - metabolism
Tryptophan
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Summary: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 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 . After screening a TAG scanning library expressing 13 ncAAs, several variants with enhanced fluorescence and stability were identified. The variants CGP and CGP 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. 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 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.
ISSN:0099-2240
1098-5336
1098-5336
DOI:10.1128/aem.01863-23