CompassR: A new rule for recombination of beneficial substitutions in directed evolution
Haiyang Cui, Hao Cao, Haiying Cai, Karl Erich Jaeger, Mehdi D. Davari, Ulrich Schwaneberg, Computer‐assisted Recombination (CompassR) teaches us how to recombine beneficial substitutions from directed evolution campaigns, Chemistry A European Journal, 2019, https://doi.org/10.1002/chem.201903994
The Computer-assisted Recombination – CompassR – strategy is a selection filter for experimentalists to recombine beneficial substitutions in order to gradually improve enzyme performance and maximize improvements through recombination.
Directed evolution has matured into a powerful method for improving enzymes in catalysis and medical applications as documented by the Nobel Prize in chemistry in 2018. A main remaining challenge is how to recombine beneficial substitutions. Systematic recombination studies show that poorly performing variants are usually obtained after recombination of 3 to 4 beneficial substitutions. The latter limits researchers to harness the benefits from beneficial substitutions that have been identified in directed evolution campaigns and to exploit nature’s potential in generating better enzymes. The Computer-assisted Recombination – CompassR – strategy provides a selection guide for beneficial substitutions that can be recombined to gradually improve enzyme performance by analysis of the relative free energy of folding. The performance of CompassR was evaluated by analysis of 84 recombinants located on 13 positions of the Bacillus subtilis lipase A. Analysis of their relative free energy of folding was used to deduct the CompassR rule that substitutions with a relative free energy of folding below +0.36 kcal/mol can be efficiently recombined, variants with a free energy of folding between +0.36 and +7.52 kcal/mol were active and inactive indicating unpredictable behavior, folding energies over +7.52 kcal/mol mostly yielded inactive variants. The finally obtained recombinant with four amino acid substitutions had a 2.7-fold improved specific activity in 18.3 % ionic liquid [BMIM][Cl]. Notably, the BSLA variants with 3 and 4 substitutions were better performing than the variants with 2 substitutions. In essence, the deducted CompassR allows to recombine beneficial substitutions in an iterative manner and empowers researchers to generate better enzymes in a time-efficient manner.
This work was realized in the division Computational Biology and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0187). Haiyang Cui is financially supported by the China Scholarship Council (CSC) scholarship.