In vitro flow cytometry-based screening
One of the main bottlenecks of directed evolution for successful tailoring of biocatalysts for industrial applications is ultrahigh throughput screening uHTS. uHTS enables analysis of up to 107 events per hour by which a coverage of the generated protein sequence space is ensured. Combination of flow cytometry based uHTS and cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. This is the first report where the flow cytometry screening system in combination with cell-free enzyme expression within emulsion compartments, termed InVitroFlow, was used for directed cellulase evolution. The celA2 mutant library containing high mutational load was generated and encapsulated in double emulsion compartments together with fluorogenic substrate and cell-free expression mixture, see Figure 4 Steps 1-3. The compartmentalization enables a genotype-phenotype linkage through an encapsulation of the gene, enzyme it encodes and generated fluorescent product within the same compartment. Compartment containing active enzyme variants can be sorted on flow cytometer based on the generation of fluorescent product, see Figure 4 Step 4. The genes contained in the sorted compartment can be isolated by PCR and can subsequently be used as template for further iterative rounds of directed evolution and/or cloned into a vector with a subsequent transformation into an expression host for MTP screening, see Figure 4 Steps 5-7.Bio VI
Figure 4: InVitroFlow screening platform in 7 steps. Mutant library generation (1) and encapsulation into single (2) and double emulsion compartments (3) Analysis and sorting of the fluorescent compartments using flow cytometry (4). Isolation and amplification of DNA encoding for active enzyme variants using PCR (5) with subsequent cloning and transformation (6) for a fine characterization in MTP assay formats (7) or for a next iterative rounds of directed evolution.
The novel InVitroFlow screening platform was validated by screening a random mutant libraries and yielded improved cellulase variants, e.g. CelA2-H288F-M1, N273D/H288F/N468S, with 13.3-fold increased specific activity compared to CelA2 wildtype.
More detailed information on flow cytometry-based high-throughput screening can be found in the following publication:
Körfer, G., Pitzler, C., Vojcic, L., Martinez, R., and Schwaneberg, U. 2016. In vitro flow cytometry-based screening platform for cellulase engineering Sci Rep. 6:26128.
High-troughput screening with "Fur Shell" hydrogels
High-throughput screening formats play a pivotal role in directed evolution experiments and enzyme discovery. A high-throughput screening system based on formation of fluorescent hydrogels around E. coli cells expressing active enzyme - "Fur Shell" - was established for phytase and later on the screening platform was advanced into a general Fur Shell based screening toolbox for directed evolution of hydrolases i.e. cellulase, esterase, and lipase. Cells expressing active hydrolase generate ß-D-glucose from glucose derived substrates as depicted in Fig. 3A which is subsequently converted by glucose oxidase under hydrogen peroxide production as depicted in Fig. 3B. Hydrogen peroxide serves as a source of hydroxyl radicals which initiates a fluorescent hydrogel formation around E. coli cells expressing active hydrolase variants as depicted in Fig. 1C.
Figure 3: Principle of Fur Shell technology using a coupled enzyme/GOx reaction leading to a formation of fluorescent hydrogel
The screening platform was validated by screening epPCR libraries for phytase, cellulase, esterase, and lipase in a single round of directed evolution and identification of improved variants 1.3 – 7-fold for four hydrolases. The presented Fur Shell screening platform is valuable prescreening system in order to isolate active enzyme variants and to minimize screening efforts in a cost effective manner.
The Fur Shell screening platform is a general platform for directed hydrolase evolution and it is easy to use and time efficient when compared to other reported flow cytometry screening systems in directed evolution. The principle of the Fur Shell technology can be adapted to other enzyme classes and has a potential to become a standard screening format in directed enzyme evolution. High throughput enabled by this technology will allow exploring a novel mutagenesis strategies and sampling through a protein sequence space even for a very short peptides. In addition, the presented platform is attractive for application in bio based interactive materials since it offers E. coli directed polymer capsule formation.
More detailed information on the topic of high-throughput screening can be found in these publications
Lülsdorf*, N., Pitzler*, C., Biggel, M., Martinez, R., Vojcic, L. and Schwaneberg, U. 2015. A flow cytometer-based whole cell screening toolbox for directed hydrolase evolution through fluorescent hydrogels. Chem Commun. 51, 41:8679-8682.
Pitzler, C., Wirtz, G., Vojcic, L., Hiltl S., Böker, A., Martinez, R., and Schwaneberg, U. 2014. A fluorescent hydrogel-based flow cytometry high-throughput screening platform for hydrolytic enzymes. Chem Biol. 21, 12:1733-1742.