Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells
Abstract:A compartmentalized tyramide labeling system (CoaTi) employing flow cytometry for sorting of yeast cells was developed as ultrahigh throughput screening for Glucose oxidase (GOx) from Aspergillus niger. CoaTi combines in vitro compartmentalization technology with the CARD reporter system which uses fluorescein tyramide labels for detection of peroxidase activity. Physical connection between cells and fluorescein tyramide radicals was achieved by compartmentalization of yeast cells inside microdroplets of single water-in-oil emulsions. After reaction cells were recovered from single emulsions and sorted by flow cytometry, an error prone PCR mutant library of Glucose oxidase (GOx) containing 107 cells and ∼105 of different GOx variants was screened. Mutagenic conditions of GOx mutant library were selected to generate <1% of active GOx population in order to explore influence of high mutation frequency on GOx activity. GOx variant Mut12 that contains 5 mutations (N2Y, K13E, T30V, I94V, K152R) showed a 1.2 times decreased Km (22.0 vs 18.1 mM) and a 2.7 fold increased kcat (150 s-1 vs 54.8 s-1) compared to wt GOx. Compared to the employed parent B11 GOx (16 mM, 80 s-1) it has a slightly increased Km and 1.8 times increased kcat.
Keywords: Applichem; Aspergillus niger; CoaTi Screening; CoaTi screening technology; Directed evolution; Escherichia coli; Flow Cytometry Screening; HRP; Horseradish; MICCRA D-1 dispenser; Mutant GOx; NanoDrop photometer; Pichia pastoris; SDS PAGE; Saccharomyces cerevisiae; Sigma-Aldrich Chemie; UV-VIS; emulsion; epPCR Library; ethanol fluorescein tyramide 2 mM; glucose oxidase; high throughput screening; spectroscopy
Document Type: Research Article
Publication date: 2011-01-01
- Combinatorial Chemistry & High Throughput Screening publishes full length original research articles and reviews describing various topics in combinatorial chemistry (e.g. small molecules, peptide, nucleic acid or phage display libraries) and/or high throughput screening (e.g. developmental, practical or theoretical). Ancillary subjects of key importance, such as robotics and informatics, will also be covered by the journal. In these respective subject areas, Combinatorial Chemistry & High Throughput Screening is intended to function as the most comprehensive and up-to-date medium available. The journal should be of value to individuals engaged in the process of drug discoveryand development, in the settings of industry, academia or government.