Addressing the Numbers Problem in Directed Evolution
作者： Manfred T. Reetz , Daniel Kahakeaw , Renate Lohmer
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摘要: Our previous contribution to increasing the efficiency of directed evolution is iterative saturation mutagenesis (ISM) as a systematic means generating focused libraries for control substrate acceptance, enantioselectivity, or thermostability enzymes. We have now introduced crucial element knowledge-guided targeted in general that helps solve numbers problem evolution. show choice amino acid (aa) alphabet, specified by utilized codon degeneracy, provides experimenter with powerful tool designing "smarter" randomized require considerably less screening effort. A comparison two different degeneracies was made examining relative quality identically sized enzyme relation degree oversampling required process. The specific example our case study concerns conventional NNK degeneracy (32 codons/20 aa) versus NDT (12 codons/12 aa). model reaction hydrolytic kinetic resolution chiral trans-disubstituted epoxide, catalyzed epoxide hydrolase from Aspergillus niger. library proves be much higher quality, measured dramatically frequency positive variants and magnitude catalyst improvement (enhanced rate enantioselectivity). provide statistical analysis constitutes useful guide optimal design generation libraries. This type approach accelerates process laboratory can expected broadly applicable when engineering functional proteins general.
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