Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications

Abstract

The bioprocess engineering with biocatalysts broadly spans its development and actual application of enzymes in an industrial context. Recently, both the use of bioprocess engineering and the development and employment of enzyme engineering techniques have been increasing rapidly. Importantly, engineering techniques that incorporate unnatural amino acids (UAAs) in vivo has begun to produce enzymes with greater stability and altered catalytic properties. Despite the growth of this technique, its potential value in bioprocess applications remains to be fully exploited. In this review, we explore the methodologies involved in UAA incorporation as well as ways to synthesize these UAAs. In addition, we summarize recent efforts to increase the yield of UAA engineered proteins in Escherichia coli and also the application of this tool in enzyme engineering. Furthermore, this protein engineering tool based on the incorporation of UAA can be used to develop immobilized enzymes that are ideal for bioprocess applications. Considering the potential of this tool and by exploiting these engineered enzymes, we expect the field of bioprocess engineering to open up new opportunities for biocatalysis in the near future.

Rapid in vitro screening for the location‐dependent effects of unnatural amino acids on protein expression and activity

“…Such functionalization by uAA include click‐chemistry reactivity, post‐translational modification mimicry, unique spectroscopic signatures, light‐ or UV‐sensitivity, and modification of enzymatic activity (Montclare and Tirrell, ; Quast et al, ; Yoo et al, ). Consequently, proteins with uAA incorporation (uAA‐proteins) are increasingly used to develop biotherapeutics (Sun et al, ; Wals and Ovaa, ) and biocatalysts (Ravikumar et al, ). In such applications, the uAA needs to be positioned strategically to both functionalize the protein and preserve its structural and functional integrity.…”

Regioselective Biocatalytic Transformations Employing Transaminases and Tyrosine Phenol Lyases

“…Moreover, different enzyme engineering approaches have taken advantage of the activity shown by TPLs to successfully incorporate unnatural amino acids (UAAs) into recombinant proteins. While traditional mutagenesis is limited to the 'chemical space' of the 20 proteinogenic amino acids, the introduction of non-natural residues gives access to novel side-chain functionalities that can lead to more robust enzymes with enhanced catalytic properties [79]. For instance, a double variant of the TPL from Citrobacter freundii (M288S/F448C) has been used to catalyze the formation of the metal-chelating UAA 2-amino-3-(8hydroxyquinolin-5-yl)propanoic acid (HqAla, Scheme 6) from 8-hydroxyquinoline, ammonium chloride and pyruvate in high yield.…”

Tackling Achilles’ Heel in Synthetic Biology: Pairing Intracellular Synthesis of Noncanonical Amino Acids with Genetic‐Code Expansion to Foster Biotechnological Applications

“…Some of the challenges ahead of xenobiologists are the ethical implications, the development of a suitable biocontainment system, and sustained access to the necessary ncAAs …”