Dataset Information

Residue-Specific Incorporation of the Non-Canonical Amino Acid Norleucine Improves Lipase Activity on Synthetic Polyesters.

ABSTRACT: Environmentally friendly functionalization and recycling processes for synthetic polymers have recently gained momentum, and enzymes play a central role in these procedures. However, natural enzymes must be engineered to accept synthetic polymers as substrates. To enhance the activity on synthetic polyesters, the canonical amino acid methionine in Thermoanaerobacter thermohydrosulfuricus lipase (TTL) was exchanged by the residue-specific incorporation method for the more hydrophobic non-canonical norleucine (Nle). Strutural modelling of TTL revealed that residues Met-114 and Met-142 are in close vicinity of the active site and their replacement by the norleucine could modulate the catalytic activity of the enzyme. Indeed, hydrolysis of the polyethylene terephthalate model substrate by the Nle variant resulted in significantly higher amounts of release products than the Met variant. A similar trend was observed for an ionic phthalic polyester containing a short alkyl diol (C5). Interestingly, a 50% increased activity was found for TTL [Nle] towards ionic phthalic polyesters containing different ether diols compared to the parent enzyme TTL [Met]. These findings clearly demonstrate the high potential of non-canonical amino acids for enzyme engineering.

SUBMITTER: Haernvall K

PROVIDER: S-EPMC8826565 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

Residue-Specific Incorporation of the Non-Canonical Amino Acid Norleucine Improves Lipase Activity on Synthetic Polyesters.

Haernvall Karolina K Fladischer Patrik P Schoeffmann Heidemarie H Zitzenbacher Sabine S Pavkov-Keller Tea T Gruber Karl K Schick Michael M Yamamoto Motonori M Kuenkel Andreas A Ribitsch Doris D Guebitz Georg M GM Wiltschi Birgit B

Frontiers in bioengineering and biotechnology 20220126

Environmentally friendly functionalization and recycling processes for synthetic polymers have recently gained momentum, and enzymes play a central role in these procedures. However, natural enzymes must be engineered to accept synthetic polymers as substrates. To enhance the activity on synthetic polyesters, the canonical amino acid methionine in <i>Thermoanaerobacter thermohydrosulfuricus</i> lipase (TTL) was exchanged by the residue-specific incorporation method for the more hydrophobic non-c ...[more]

PMID: 35155387

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Project description:Synthetic biology holds promise to revolutionize the life sciences and biomedicine via expansion of macromolecular diversity outside the natural chemical space. Use of non-canonical amino acids (ncAAs) via codon reassignment has found diverse applications in protein structure and interaction analysis, introduction of post-translational modifications, production of constrained peptides, antibody-drug conjugates, and novel enzymes. However, simultaneously encoding multiple ncAAs in vivo requires complex engineering and is sometimes restricted by the cell's poor uptake of ncAAs. In contrast the open nature of cell-free protein synthesis systems offers much greater freedom for manipulation and repurposing of the biosynthetic machinery by controlling the level and identity of translational components and reagents, and allows simultaneous incorporation of multiple ncAAs with non-canonical side chains and even backbones (N-methyl, D-, β-amino acids, α-hydroxy acids etc.). This review focuses on the two most used Escherichia coli-based cell-free protein synthesis systems; cell extract- and PURE-based systems. The former is a biological mixture with >500 proteins, while the latter consists of 38 individually purified biomolecules. We delineate compositions of these two systems and discuss their respective advantages and applications. Also, we dissect the translational components required for ncAA incorporation and compile lists of ncAAs that can be incorporated into polypeptides via different acylation approaches. We highlight the recent progress in using unnatural nucleobase pairs to increase the repertoire of orthogonal codons, as well as using tRNA-specific ribozymes for in situ acylation. We summarize advances in engineering of translational machinery such as tRNAs, aminoacyl-tRNA synthetases, elongation factors, and ribosomes to achieve efficient incorporation of structurally challenging ncAAs. We note that, many engineered components of biosynthetic machinery are developed for the use in vivo but are equally applicable to the in vitro systems. These are included in the review to provide a comprehensive overview for ncAA incorporation and offer new insights for the future development in cell-free systems. Finally, we highlight the exciting progress in the genomic engineering, resulting in E. coli strains free of amber and some redundant sense codons. These strains can be used for preparation of cell extracts offering multiple reassignment options.

Dataset Information

Residue-Specific Incorporation of the Non-Canonical Amino Acid Norleucine Improves Lipase Activity on Synthetic Polyesters.

Publications

Residue-Specific Incorporation of the Non-Canonical Amino Acid Norleucine Improves Lipase Activity on Synthetic Polyesters.

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