Directed evolution at high mutational loads

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In metabolic engineering low enzymatic activity is often a bottleneck in increasing a flux via pathways of interest. Therefore methods for diversity generation and screening of bottleneck enzymes from designed pathways will be improved to increase titers. In detail, the project deals with advancement of a novel flow cytometry screening principle based on fluorescent hydrogel formation on the surface of for example E. coli cells with a throughput of approximately 107 variants per hour for exploring a novel directed evolution strategy with high mutational loads. High mutational loads open the possibility to identify amino acid substitutions that depend on each other and that are not identified in standard directed evolution campaigns. The selected two enzymes will be identified upon pathways engineering and initial experiments to optimize the flow cytometer screening system will be performed with two pathway enzymes and related knock out strains to quantify the fluorescence background reaction.