Combinatorial InVitroFlow‐assisted mutagenesis (CombIMut) yields a 41‐fold improved CelA2 cellulase

Biotechnology and Bioengineering, vol. 119, pp. 2076–2087

Abstract

Abstract The combination of diversity generation methods and ultrahigh‐throughput screening (uHTS) technologies is key to efficiently explore nature's sequence space and elucidate structure–function relationships of enzymes. Beneficial substitutions often cluster in a few regions and simultaneous amino acid substitutions at multiple positions (e.g., by OmniChange) will likely lead to further improved enzyme variants. An extensive screening effort is required to identify such variants, as the simultaneous randomization of four codons can easily yield over 105 potential enzyme variants. The combination of flow cytometer‐based uHTS with cell‐free compartmentalization technology using (w/o/w) double emulsions (InVitroFlow), provides analysis capabilities of up to 107 events per hour, thus enabling efficient screening. InVitroFlow is an elegant solution since diversity loss through a transformation of host cells is omitted and emulsion compartments provide a genotype‐phenotype linkage through a fluorescence readout. In this study, a multisite saturation mutagenesis and an OmniChange library with four simultaneously saturated positions in the active site of CelA2 cellulase were screened using InVitroFlow. Screening of over 36 million events, yielded a significantly improved cellulase variant CelA2‐M3 (H288F/H524Q) with an 8‐fold increase in specific activity compared to the parent CelA2‐H288F (83.9 U/mg) and a 41‐fold increased specific activity (674.5 U/mg) compared to wildtype CelA2 (16.6 U/mg) for the substrate 4‐MUC (4‐methylumbelliferyl‐β d‐cellobioside).

Authors 6

1. Georgette Körfer Aachen Lehrstuhl für Biotechnologie

   RWTH Aachen University

   Affiliation as printed

   Lehrstuhl für Biotechnologie RWTH Aachen University Aachen Germany

2. Volkan Besirlioglu Aachen Lehrstuhl für Biotechnologie

   RWTH Aachen University

   Affiliation as printed

   Lehrstuhl für Biotechnologie RWTH Aachen University Aachen Germany

3. Mehdi D. Davari

   Leibniz Institute of Plant Biochemistry

   Affiliation as printed

   Department of Bioorganic Chemistry Leibniz Institute of Plant Biochemistry Halle Germany

   Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle, Germany

4. Ronny Martínez

   University of La Serena

   Affiliation as printed

   Departamento de Ingeniería en Alimentos Universidad de La Serena La Serena Chile

5. Ljubica Vojcic Aachen Lehrstuhl für Biotechnologie

   Codexis (United States) · RWTH Aachen University

   Affiliation as printed

   Codexis Inc. Redwood City California USA

   Lehrstuhl für Biotechnologie RWTH Aachen University Aachen Germany

   Codexis Inc., Redwood City, California, USA

6. Ulrich Schwaneberg corresponding Aachen DWI - Leibniz Institute for Interactive Materials Lehrstuhl für Biotechnologie

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI – Leibniz Institute for Interactive Materials, Forckenbeckstrasse 50 Aachen Germany

   Lehrstuhl für Biotechnologie RWTH Aachen University Aachen Germany