Appartient à l’ensemble documentaire : MethMusi

Atze van Stralen (4773390)

Elrike Frenzel (4773387)

Jelmer Legebeke (4773384)

Oscar Kuipers (3504497)

Richard van Kranenburg (107687)

Biotechnology for Biofuels

Frenzel, Elrike

Legebeke, Jelmer

van Stralen, Atze

van Kranenburg, Richard

Kuipers, Oscar P.

University of Groningen

English

In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria

Elrike Frenzel

Jelmer Legebeke

Atze van Stralen

Richard van Kranenburg

Oscar P. Kuipers

Crossref

Background: Fluorescent reporter proteins (FP) have become an indispensable tool for the optimization of microbial cell factories and in synthetic biology per se. The applicability of the currently available FPs is, however, constrained by species-dependent performance and misfolding at elevated temperatures. To obtain functional reporters for thermophilic, biotechnologically important bacteria such as Parageobacillus thermoglucosidasius, an in vivo screening approach based on a mutational library of superfolder GFP was applied.Results: Flow cytometry-based benchmarking of a set of GFPs, sfGFPs and species-specific codon-optimized variants revealed that none of the proteins was satisfyingly detectable in P. thermoglucosidasius at its optimal growth temperature of 60 degrees C. An undirected mutagenesis approach coupled to fluorescence-activated cell sorting allowed the isolation of sfGFP variants that were extremely well expressed in the chassis background at 60 degrees C. Notably, a few nucleotide substitutions, including silent mutations, significantly improved the functionality and brightness. The best mutant sfGFP(N39D/A179A) showed an 885-fold enhanced mean fluorescence intensity (MFI) at 60 degrees C and is the most reliable reporter protein with respect to cell-to-cell variation and signal intensity reported so far. The in vitro spectral and thermostability properties were unaltered as compared to the parental sfGFP protein, strongly indicating that the combination of the amino acid exchange and an altered translation or folding speed, or protection from degradation, contribute to the strongly improved in vivo performance. Furthermore, sfGFP(N39D/A179A) and the newly developed cyan and yellow derivatives were successfully used for labeling several industrially relevant thermophilic bacilli, thus proving their broad applicability.Conclusions: This study illustrates the power of in vivo isolation of thermostable proteins to obtain reporters for highly efficient fluorescence labeling. Successful expression in a variety of thermophilic bacteria proved that the novel FPs are highly suitable for imaging and flow cytometry-based studies. This enables a reliable cell tracking and single-cell-based real-time monitoring of biological processes that are of industrial and biotechnological interest.</p

ARTS repository - University of Groningen

Additional file 4. Workflow for in vivo enrichment of thermostable sfGFP mutants and single variant screening in P. thermoglucosidasius

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MOESM4 of In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria

Proceedings - University of Groningen

Dissertations of the University of Groningen

Background: Fluorescent reporter proteins (FP) have become an indispensable tool for the optimization of microbial cell factories and in synthetic biology per se. The applicability of the currently available FPs is, however, constrained by species-dependent performance and misfolding at elevated temperatures. To obtain functional reporters for thermophilic, biotechnologically important bacteria such as Parageobacillus thermoglucosidasius, an in vivo screening approach based on a mutational library of superfolder GFP was applied. Results: Flow cytometry-based benchmarking of a set of GFPs, sfGFPs and species-specific codon-optimized variants revealed that none of the proteins was satisfyingly detectable in P. thermoglucosidasius at its optimal growth temperature of 60 °C. An undirected mutagenesis approach coupled to fluorescence-activated cell sorting allowed the isolation of sfGFP variants that were extremely well expressed in the chassis background at 60 °C. Notably, a few nucleotide substitutions, including silent mutations, significantly improved the functionality and brightness. The best mutant sfGFP(N39D/A179A) showed an 885-fold enhanced mean fluorescence intensity (MFI) at 60 °C and is the most reliable reporter protein with respect to cell-to-cell variation and signal intensity reported so far. The in vitro spectral and thermostability properties were unaltered as compared to the parental sfGFP protein, strongly indicating that the combination of the amino acid exchange and an altered translation or folding speed, or protection from degradation, contribute to the strongly improved in vivo performance. Furthermore, sfGFP(N39D/A179A) and the newly developed cyan and yellow derivatives were successfully used for labeling several industrially relevant thermophilic bacilli, thus proving their broad applicability. Conclusions: This study illustrates the power of in vivo isolation of thermostable proteins to obtain reporters for highly efficient fluorescence labeling. Successful expression in a variety of thermophilic bacteria proved that the novel FPs are highly suitable for imaging and flow cytometry-based studies. This enables a reliable cell tracking and single-cell-based real-time monitoring of biological processes that are of industrial and biotechnological interest

Van Stralen, Atze

Van Kranenburg, Richard

NARCIS 

Additional file 5. Table listing examples of thermostable sfGFP variants isolated from FACS enrichment in P. thermoglucosidasius DSM 2542 at 60Â Â°C

MOESM5 of In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria