Ensari Yunus, Dhoke Gaurao V, Davari Mehdi D, Ruff Anna Joëlle, Schwaneberg Ulrich
Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
Kafkas University, Faculty of Engineering and Architecture, Department of Bioengineering, 36100, Kars, Turkey.
Chembiochem. 2018 Jul 16;19(14):1563-1569. doi: 10.1002/cbic.201800159. Epub 2018 Jun 12.
Positions identified in directed evolution campaigns or by (semi)rational design can be recombined iteratively or simultaneously. Iterative recombination has yielded many success stories and is beneficially used if screening capabilities are limited (four iterative SSMs generate 20×4=80 different enzyme variants). Simultaneous site saturation mutagenesis offers significantly higher diversity (20 =160 000 variants) and enables greater improvements to be found, especially if the selected positions are in close proximity to each other (cooperative effects). Here we report a first comprehensive comparison of iterative and simultaneous saturation of four residues in Candida parapsilosis alcohol dehydrogenase 5 (cpADH5) with methyl 3-hydroxyhexanoate as substrate. Screening of 7200 clones from 33 site saturation mutagenesis libraries (exploring 17 recombination paths) yielded the cpADH5 W286A variant, with a 82-fold improved initial activity toward methyl 3-hydroxyhexanoate. Screening 3500 clones from a single OmniChange library with four positions (C57, W116, L119, and W286; 1.8 % of the generated sequence space) yielded the cpADH5 C57V/W286S variant, with a 108-fold improvement in initial activity toward methyl 3-hydroxyhexanoate. A 1.8 % coverage of the sequence space of the simultaneous multisite saturation library was, in comparison to the investigated 17 recombination paths, sufficient to identify a cpADH5 variant with improved activity.
在定向进化实验中或通过（半）理性设计确定的位点，可以进行迭代或同时重组。迭代重组已经产生了许多成功案例，并且在筛选能力有限时使用很有益（四次迭代的单点饱和突变产生20×4 = 80种不同的酶变体）。同时进行的位点饱和诱变提供了显著更高的多样性（20⁴ = 160000种变体），并且能够发现更大的改进，特别是如果所选位点彼此靠近（协同效应）。在此，我们报告了对近平滑念珠菌乙醇脱氢酶5（cpADH5）中四个残基进行迭代和同时饱和诱变的首次全面比较，以3-羟基己酸甲酯作为底物。从33个位点饱和诱变文库（探索17条重组路径）中筛选7200个克隆，得到了cpADH5 W286A变体，其对3-羟基己酸甲酯的初始活性提高了82倍。从一个具有四个位点（C57、W116、L119和W286；占产生的序列空间的1.8%）的单一全变文库中筛选3500个克隆，得到了cpADH5 C57V/W286S变体，其对3-羟基己酸甲酯的初始活性提高了108倍。与所研究的17条重组路径相比，同时多位点饱和文库的1.8%的序列空间覆盖率足以鉴定出具有改进活性的cpADH5变体。
