Protein engineering for metabolic engineering: Current and next‐generation tools

Abstract

Protein engineering in the context of metabolic engineering is increasingly important to the field of industrial biotechnology. As the demand for biologically-produced food, fuels, chemicals, food additives, and pharmaceuticals continues to grow, the ability to design and modify proteins to accomplish new functions will be required to meet the high productivity demands for the metabolism of engineered organisms. This article reviews advances of selecting, modeling, and engineering proteins to improve or alter their activity. Some of the methods have only recently been developed for general use and are just beginning to find greater application in the metabolic engineering community. We also discuss methods of generating random and targeted diversity in proteins to generate mutant libraries for analysis. Recent uses of these techniques to alter cofactor use, produce non-natural amino acids, alcohols, and carboxylic acids, and alter organism phenotypes are presented and discussed as examples of the successful engineering of proteins for metabolic engineering purposes.

Rational engineering of diol dehydratase enables 1,4-butanediol biosynthesis from xylose

“…Rational approaches to engineer protein have been largely pursued via manipulation of amino acid residues in the catalytic pocket achieved via site directed mutagenesis (Marcheschi et al, 2013). In order to promote non-native catalysis toward larger substrates, increasing the catalytic pocket has been the predominant strategy, generally achieved via replacement of target residues with smaller ones (Marcheschi et al, 2013).…”

“…Rational approaches to engineer protein have been largely pursued via manipulation of amino acid residues in the catalytic pocket achieved via site directed mutagenesis (Marcheschi et al, 2013). In order to promote non-native catalysis toward larger substrates, increasing the catalytic pocket has been the predominant strategy, generally achieved via replacement of target residues with smaller ones (Marcheschi et al, 2013). Additionally, substituting target residues via saturation mutagenesis (Savile et al, 2010) and/or directed evolution (Kan et al, 2016) have been shown to improve catalysis possibly due to changes in the above mentioned interactions altering catalysis.…”

Recent advances in engineering proteins for biocatalysis

“…Applications of protein engineering continue to grow, impacting research and development in technologies that include pharmaceuticals and therapeutics (Bommarius et al, ; Fisher and Tullman‐Ercek, ; and ), biosensing (Gredell et al, ), synthetic biology (Cirino and Qian, ), and industrial production of fuels and chemicals (Fisher and Tullman‐Ercek, ; Marcheschi et al, ). Mounting understanding of the native functional roles of proteins, and of protein sequence‐structure‐function relationships, offers new insights into how proteins may be engineered for specific applications.…”

Unfolding stabilities of two structurally similar proteins as probed by temperature-induced and force-induced molecular dynamics simulations

“…However, understanding structures-functions of proteins has not yet become a straightforward task: proteins arising out due to divergent evolutions are similar in their amino acid sequences and three-dimensional (3D) folds but drastically differing in their functions; in contrast, proteins arising out due to convergent evolutions are differing in their primary structures and biological activities but maintain similar 3D folds (Grishin, 2001;Murzin, 1998;Russell, Saqi, Sayle, Bates, & Sternberg, 1997;Sangar, Blankenberg, Altman, & Lesk, 2007). In these connections, figuring out residues that are essential for structural integrities and as well residues essential for functional activities of proteins are indispensable to manipulate stabilities and activities of those proteins through protein-engineering strategies from the view of biotechnological and biomedical industries standpoints (Brannigan & Wilkinson, 2002;Leisola & Turunen, 2007;Marcheschi, Gronenberg, & Liao, 2013;Shaw, 1987). Structure, folding and stabilities of cardiotoxin III (CTX III) and short-neurotoxin (SNTX or CBTX, cobrotoxin) belonging to three-finger toxin (TFT) superfamily of snake venom of Naja naja atra (Taiwan cobra) have been well characterized at molecular resolutions and as well at residue-level resolutions (Chang, Lu, Lin, & Yu, 2006;Kumar et al, 1995;Sivaraman, Kumar, & Yu, 1999a;Sivaraman et al, 1999b;Sivaraman, Kumar, Chang, Lin, & Yu, 1998;Sivaraman, Kumar, Jayaraman, Han & Yu, 1997;Sivaraman, Kumar, & Yu, 1996).…”