Fluorescence

Application Study: Screening for increased recombinant protein titer with 21st BIO

Objective

Identify high producers of recombinant protein using classical mutagenesis in Aspergillus oryzae

Introduction

Classical mutagenesis remains a potent method to generate diverse mutations, especially when working with cell factories. This capability enables identification of numerous and less intuitive combinations of mutations, often unattainable with modern targeted approaches. In this study, UV-mutagenesis was employed to generate a mutant library of Aspergillus oryzae containing an expression cassette for fluorescent protein. Continuous assessment of fluorescence signal and identification of strains with improved protein titer was enabled using the Reshape Imaging System (RIS) in combination with automated image analysis.

Results

Mutant Aspergillus oryzae strains display increased fluorescence. Pools of UV-mutagenized A. oryzae spores were plated on induction medium and development of fluorescence was monitored with Reshape technology. AI-powered image analysis facilitated discrimination between enhanced fluorescence stemming from strain improvement and that from confluent growth of overlapping colonies through time course evaluation (Fig. 1).

Enhanced fluorescent strains identified with Reshape. Replating of selected candidate strains and tailored image analysis allowed identification of colonies displaying enhanced fluorescence. Fungal colonies were detected in bright field and the fluorescence was quantified over time in the corresponding areas (Fig. 2).

Increased fluorescence correlates to improved protein yield. To correlate the level of detected fluorescence with protein yield, culture supernatants of the parental strain and selected strains (E1 and E2) were examined by SDS-PAGE. In comparison to the parental A. oryzae strain, the derivatives E1 and E2 significantly improved yield of the recombinant protein (Fig. 3). These results demonstrate the effectiveness of mutagenesis in combination with Reshape technology to increase protein titers in industrial cell factories.

Conclusion

This study validates UV-mutagenesis combined with Reshape Imaging System and tailored image analysis for identification of enhanced protein yield. The results underscore the effectiveness of this combined approach in boosting protein titers in industrial cell factories, offering promising prospects for further advancements in bioengineering applications.

‍