Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet

Abstract

The two main strategies for enzyme engineering, directed evolution and rational design, have found widespread applications in improving the intrinsic activities of proteins. Although numerous advances have been achieved using these ground-breaking methods, the limited chemical diversity of the biopolymers, restricted to the 20 canonical amino acids, hampers creation of novel enzymes that Nature has never made thus far. To address this, much research has been devoted to expanding the protein sequence space via chemical modifications and/or incorporation of noncanonical amino acids (ncAAs). This review provides a balanced discussion and critical evaluation of the applications, recent advances, and technical breakthroughs in biocatalysis for three approaches: (i) chemical modification of cAAs, (ii) incorporation of ncAAs, and (iii) chemical modification of incorporated ncAAs. Furthermore, the applications of these approaches and the result on the functional properties and mechanistic study of the enzymes are extensively reviewed. We also discuss the design of artificial enzymes and directed evolution strategies for enzymes with ncAAs incorporated. Finally, we discuss the current challenges and future perspectives for biocatalysis using the expanded amino acid alphabet.

Engineering Pyrrolysyl-tRNA Synthetase for the Incorporation of Non-Canonical Amino Acids with Smaller Side Chains

“…Research in the field of reprogrammed protein translation has now reached experimental and intellectual maturity: More than 200 non-canonical amino acids (ncAAs, i.e., a diversity that is an order of magnitude higher than that of the canonical amino acid repertoire) were introduced into proteins via various genetic code expansion routes: Selective pressure incorporation, stop codon suppression (SCS), fragment condensation, protein semisynthesis, and peptidomimetics [ 1 ]. It has been shown that AAs with non-proteinogenic functional groups can be used to manipulate, design, and elucidate protein structure, dynamics, function, allosterism, interactions, catalysis, folding, synthesis, trafficking, degradation, and aggregation [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ].…”

Synthesis of Sitagliptin Intermediate by a Multi-Enzymatic Cascade System Using Lipase and Transaminase With Benzylamine as an Amino Donor

“…Biocatalysts continue to be explored for consideration as green alternatives to alleviate the environmental issues encountered with the use of methods pertaining to traditional organic chemistry Pagar et al, 2021). Thus, multi-enzyme cascade reactions have garnered attention owing to their elegance (Sperl and Sieber, 2018;Hwang and Lee, 2019;Martínez-Montero et al, 2019), comprehensiveness, and involvement in the synthesis of a considerable variety of pharmaceuticals, agrochemicals, and other industrially important chemicals (Sung et al, 2018;Patil et al, 2019;Yoon et al, 2019).…”

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

“…Consequently, protein engineering is needed to improve the enzymatic properties of specific enzymes. Notably, protein engineering techniques, including rational design and directed evolution, mainly rely on mutual mutagenesis of the 20 canonical amino acids (cAAs) ( Pagar et al, 2021 ). The limited building blocks of only 20 cAAs inevitably restrict the improvement of protein properties during protein engineering ( Pagar et al, 2021 ).…”

“…Notably, protein engineering techniques, including rational design and directed evolution, mainly rely on mutual mutagenesis of the 20 canonical amino acids (cAAs) ( Pagar et al, 2021 ). The limited building blocks of only 20 cAAs inevitably restrict the improvement of protein properties during protein engineering ( Pagar et al, 2021 ). The function and properties of enzymes are determined by the composition and side chain interactions of the AAs, and the introduction of various noncanonical amino acids (ncAAs) into the field of protein engineering could provide more building blocks for the enzyme molecules ( Pagar et al, 2021 ).…”

“…The limited building blocks of only 20 cAAs inevitably restrict the improvement of protein properties during protein engineering ( Pagar et al, 2021 ). The function and properties of enzymes are determined by the composition and side chain interactions of the AAs, and the introduction of various noncanonical amino acids (ncAAs) into the field of protein engineering could provide more building blocks for the enzyme molecules ( Pagar et al, 2021 ). Furthermore, this would create new interactions between the side chains of the residues and form new local molecular structures of enzymes.…”