Evolution of fluorinated enzymes: An emerging trend for biocatalyst stabilization

Abstract

Nature uses remarkably limited sets of chemistries in its repertoire, especially when compared to synthetic organic chemistry. This limits both the chemical and structural diversity that can ultimately be achieved with biocatalysis, unless the powers of chemical synthesis are merged with biological systems by integrating nonnatural synthetic chemistries into the protoplasma of living cells. Of particular interest, here is the fluorous effect that has recently established the potential to generate enzymes with an increased resistance toward both high temperature and organic solvents. For these reasons, we are witnessing a rapid development of efficient methodologies for the incorporation of fluorinated amino acids in protein synthesis, using both in vivo and in vitro strategies. In this review, we highlight relevant and trendsetting results in the design and engineering of stable fluorinated proteins and peptides along with whole‐cell biocatalysis as an economically attractive and convenient application with exclusive focus on industrial biocatalysis. Finally, we envision new strategies to improve current achievements and enable the field to progress far beyond the current state‐of‐the‐art.

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

“…At a molecular level, incorporating a ncAA at multiple sites throughout the target protein promotes the synergistic effects exhibited by some chemical groups, for example, fluorine‐containing side chains. These residue‐specific methods have been used in some biotechnological applications, such as protein fluorination, to increase the thermal stability and solvent tolerance of industrial biocatalysts …”

Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids

“…The list of expected tryptic digests of mDHFR was obtained using the online server ExPASy SIB Bioinformatics Resource Portal. The peptide fragment "NGDLPWPPLRNEFK" (residues [19][20][21][22][23][24][25][26][27][28][29][30][31][32], containing the Phe residue at position 31 served as the reference. The expected monoisotopic mass of the reporter peptide is 1682.9 (m/z).…”

“…Several techniques using non-natural amino acids have developed [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26]. Residue-specific incorporation of non-natural amino acids is recently demonstrated as a very useful tool for redesign of industrial enzymes [27], or bio-catalysts in general [28], and even useful to perform enzyme reactions in hostile environments [29]. Alternatively, site-specific incorporation in vivo techniques including stop codon suppression in vivo (SCS) shows great potential for enzyme engineering.…”

Fluorine-Rich Planetary Environments as Possible Habitats for Life

“…These considerations might be of particular importance in assessing whether life might be able to exist in hydrophobic solvents [ 9 , 32 ] under “reverse phase” conditions. Figure modified from [ 33 ].…”