Directed evolution to re-adapt a co-evolved network within an enzyme

• Published in: Journal of biotechnology Vol. 157; no. 1; pp. 237 - 245
• Main Authors: Strafford, John, Payongsri, Panwajee, Hibbert, Edward G., Morris, Phattaraporn, Batth, Sukhjeet S., Steadman, David, Smith, Mark E.B., Ward, John M., Hailes, Helen C., Dalby, Paul A.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.01.2012; Elsevier; Elsevier Science Publishers
• Subjects: Acetaldehyde - analogs & derivatives; Acetaldehyde - metabolism; active sites; Aldehydes - metabolism; Biocatalysis; Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; Catalytic Domain - genetics; Coevolution; Directed evolution; Directed Molecular Evolution - methods; enzyme activity; Enzyme engineering; evolution; functional properties; Fundamental and applied biological sciences. Psychology; Gene Library; inclusion bodies; Kinetics; Methods. Procedures. Technologies; Models, Molecular; mutagenesis; Mutagenesis, Site-Directed - methods; mutants; Mutation; protein depletion; Protein engineering; Protein Stability; protein synthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Alignment; solubility; Stereoisomerism; Substrate Specificity; Transketolase; Transketolase - chemistry; Transketolase - genetics; Transketolase - isolation & purification; Transketolase - metabolism; PP; Directed evolution; Biocatalysis; Enzyme engineering; Transketolase; Pyr; Coevolution; SCA; PA; TPP; TK; HPA; GA; Directed molecular evolution; Engineering; Enzyme; Transferases
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2011.11.017

► Previous single mutants of transketolase improved activity on new substrates. ► Recombination to form double mutants led to critical loss of functional expression. ► Mutated sites were found to be in a structural network of co-evolved residues. ► The network was re-adapted around previous mutants for kinetic synergy and functional expression. We have previously used targeted active-site saturation mutagenesis to identify a number of transketolase single mutants that improved activity towards either glycolaldehyde (GA), or the non-natural substrate propionaldehyde (PA). Here, all attempts to recombine the singles into double mutants led to unexpected losses of specific activity towards both substrates. A typical trade-off occurred between soluble expression levels and specific activity for all single mutants, but many double mutants decreased both properties more severely suggesting a critical loss of protein stability or native folding. Statistical coupling analysis (SCA) of a large multiple sequence alignment revealed a network of nine co-evolved residues that affected all but one double mutant. Such networks maintain important functional properties such as activity, specificity, folding, stability, and solubility and may be rapidly disrupted by introducing one or more non-naturally occurring mutations. To identify variants of this network that would accept and improve upon our best D469 mutants for activity towards PA, we created a library of random single, double and triple mutants across seven of the co-evolved residues, combining our D469 variants with only naturally occurring mutations at the remaining sites. A triple mutant cluster at D469, E498 and R520 was found to behave synergistically for the specific activity towards PA. Protein expression was severely reduced by E498D and improved by R520Q, yet variants containing both mutations led to improved specific activity and enzyme expression, but with loss of solubility and the formation of inclusion bodies. D469S and R520Q combined synergistically to improve kcat 20-fold for PA, more than for any previous transketolase mutant. R520Q also doubled the specific activity of the previously identified D469T to create our most active transketolase mutant to date. Our results show that recombining active-site mutants obtained by saturation mutagenesis can rapidly destabilise critical networks of co-evolved residues, whereas beneficial single mutants can be retained and improved upon by randomly recombining them with natural variants at other positions in the network.