Ultrahigh Throughput Screening System for Directed Glucose Oxidase Evolution in Yeast Cells

Radivoje      Prodanovic; Raluca      Ostafe; Andreea      Scacioc; Ulrich      Schwaneberg

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: Directed evolution, high throughput screening, glucose oxidase, emulsion, Horseradish, HRP, Aspergillus niger, Sigma-Aldrich Chemie, Applichem, Escherichia coli, Saccharomyces cerevisiae, NanoDrop photometer, MICCRA D-1 dispenser, ethanol fluorescein tyramide 2 mM, epPCR Library, Flow Cytometry Screening, Mutant GOx, SDS PAGE, UV-VIS, spectroscopy, CoaTi Screening, Pichia pastoris, CoaTi screening technology