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Enhancement of Activity and Thermostability of Keratinase From Pseudomonas aeruginosa CCTCC AB2013184 by Directed Evolution With Noncanonical Amino Acids

ABSTRACT: A keratinase from Pseudomonas aeruginosa (KerPA), which belongs to the M4 family of metallopeptidases, was characterised in this study. This enzyme was engineered with non-canonical amino acids (ncAAs) using genetic code expansion. Several variants with enhanced activity and thermostability were identified and the most prominent, Y21pBpF/Y70pBpF/Y114pBpF, showed an increase in enzyme activity and half-life of approximately 1.3-fold and 8.2-fold, respectively. Considering that keratinases usually require reducing agents to efficiently degrade keratin, the Y21pBpF/Y70pBpF/Y114pBpF variant with enhanced activity and stability under reducing conditions may have great significance for practical applications. Molecular Dynamics (MD) was performed to identify the potential mechanisms underlying these improvements. The results showed that mutation with pBpF at specific sites of the enzyme could fill voids, form new interactions, and reshape the local structure of the active site of the enzyme.

SUBMITTER: Pan X

PROVIDER: S-EPMC8558439 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 CGPV3pMeoF/K190pMeoF and CGPG20pMeoF/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.ImportanceNoncanonical 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.

Dataset Information

Enhancement of Activity and Thermostability of Keratinase From Pseudomonas aeruginosa CCTCC AB2013184 by Directed Evolution With Noncanonical Amino Acids

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