| Popis: |
Modern biotechnology has successfully launched synthesis routes that compete with traditional chemical processes for production of bulk chemicals and pharmaceuticals. In order to alter naturally occurring enzymes to perform under industrial conditions, directed evolution has become the predominant tool. Directed evolution mimics natural evolution through iterative rounds of mutagenesis and screening, yielding enzyme variants with i.e. improved activity, stability or tolerance towards organic solvents. The main bottleneck in directed evolution is to screen through the generated sequence space that can easily exceed hundred million clones in standard epPCR based approaches. A common directed evolution campaign lasts up to one year, whereby the screening for improved variants is the most time consuming part of the whole project. Novel screening formats that minimize sample size and increase throughput greatly reduce the cost and time burden of directed evolution campaigns. However, so far each of these formats lack a broad application potential, like being applicable to more than one specific enzymatic reaction or being limited to display technologies. Here, two ultra-high throughput screening platforms with broad utility were developed that use fluorescence activated cell sorting in order to isolate improved variants. The first developed flow cytometer-based technology for high throughput screening is based on a coupled reaction of a phytase from Yersinia mollaretii (YmPh) and a glucose oxidase, converting glucose-6-phosphate to glucono-delta-lactone and hydrogen peroxide. Fenton’s reaction produces hydroxyl radicals, acting as initiator of poly(ethyleneglycol)-acrylate-based polymerization incorporating a fluorescent monomer. As a consequence, a fluorescent hydrogel is formed around E. coli cells expressing active YmPh. Validation of the method was performed by screening model libraries with defined ratios of active and inactive YmPh with flow cytometry, resulting in a 5 fold enrichment of the active population. Further screening of in total 18 Mio events of an YmPh epPCR library yielded an enrichment of the active population from 40 % close to 90 % and a variant with 97 U/mg higher specific activity. The application of different glucose derivatives as substrates pursued the advancement of the technology into a general high-throughput screening toolbox for directed evolution of hydrolases. Libraries of lipolytic (Bacillus subtilis lipase A, BSLA) and cellulolytic enzymes (CelA2) were screened and yielded improved variants (BSLA: 1.3-fold increase in kcat, CelA2: 1.7-fold increase in kcat) after one round of directed evolution. Particularly noteworthy is the usage of a natural substrate (cellobiose) in a high throughput screening format enabling identification of cellulase variants with increased activity. The second flow cytometer-based technology developed during this work includes in vitro compartmentalization (IVC) of a cellulase DNA library inside polymersomes and subsequent sorting on flow cytometer. This technology represents the first ultra-high throughput screening platform for directed evolution that makes use of the beneficial properties of polymersomes. The strategy of IVC in polymersomes and subsequent flow cytometer sorting was validated by screening 39 Mio events of a celA2 library expressed in vitro. Remarkably, analysis of 400 single variants in microtiter plate before and after flow cytometer sorting yielded an impressive three times enrichment of the active population in one round of sorting. The developed technologies for high throughput flow cytometer screening allow due to their reliable, straightforward and fast implementation the realization of one round of directed evolution in less than one week. Consequently, these methods can solve demanding and challenging problems in directed evolution like revealing structure-function relationships and performing many rounds of directed evolution with high mutational load. |
