Combinatorial InVitroFlow-assisted Mutagenesis (CombIMut) yields a 41-fold improved CelA2 cellulase

19/05/2022
 

Congratulations Georgette Körfer and Volkan Besirlioglu on their recent publication!

Combining diversity generation methods and ultrahigh-throughput screening (uHTS) technologies is an essential factor in efficiently exploring nature’s sequence space and elucidating the structure-function relationships of enzymes. In this study, flow cytometer-based ultrahigh-throughput screening with cell-free compartmentalization technology using double emulsions (InVitroFlow) were combined, which is providing analysis capabilities of up to 107 events per hour. The InVitroFlow technology platform has been validated for exploring the natural diversity of amino acids with coverage greater than 95% with simultaneous saturation at four positions by OmniChange at rationally selected positions of CelA2 cellulase. Screening of more than 36 million events revealed a significantly improved cellulase variant CelA2-M3 with an 8-fold increase in specific activity compared with the parent CelA2-H288F and a 41-fold increase in specific activity compared to wild-type CelA2 for the substrate 4-methylumbelliferyl-β-D-cellobioside.

This work was financially supported by the German Federal Ministry of Education and Research (BMBF) under the Basic Technologies for the Next Generation of Biotechnological Processes program (FKZ 031A571A).

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Körfer, G., Besirlioglu, V., Davari, M. D., Martinez, R., Vojcic, L., and Schwaneberg, U. (2022), Combinatorial InVitroFlow-assisted mutagenesis (CombIMut) yields a 41-fold improved CelA2 cellulase. Biotechnol. Bioeng. Accepted Author Manuscript. https://doi.org/10.1002/bit.28110

  Overview of the screening principle using the InVitroFlow technology platform. Copyright: © Wiley Periodicals LLC. Overview of the screening principle using the InVitroFlow technology platform.