Analysis of protein secretion in Bacillus subtilis by combining a secretion stress biosensor strain with an in vivo split GFP assay

Microbial Cell Factories

Research

Roland Freudl¹  Patrick J. Bakkes²  Marco Oldiges²  Carolin Müller²  Karl-Erich Jaeger³  Marzena Malek³  Thomas Drepper³  Andreas Knapp⁴  Patrick Lenz⁵ 

[1] Institute of Bio- and Geoscience IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany;Institute of Biotechnology, RWTH Aachen University, 52074, Aachen, Germany;Institute of Bio- and Geoscience IBG-1: Biotechnology, Forschungszentrum Jülich, 52425, Jülich, Germany;Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich, 52425, Jülich, Germany;Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich, 52425, Jülich, Germany;Castrol Germany GmbH, 41179, Mönchengladbach, Germany;Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich, 52425, Jülich, Germany;Institute of Biotechnology, RWTH Aachen University, 52074, Aachen, Germany;

关键词: Bacillus subtilis;    Protein secretion;    Split GFP assay;    Secretion stress biosensor;    Online detection;   

DOI  :  10.1186/s12934-023-02199-8

 received in 2023-07-18, accepted in 2023-09-06,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundBacillus subtilis is one of the workhorses in industrial biotechnology and well known for its secretion potential. Efficient secretion of recombinant proteins still requires extensive optimization campaigns and screening with activity-based methods. However, not every protein can be detected by activity-based screening. We therefore developed a combined online monitoring system, consisting of an in vivo split GFP assay for activity-independent target detection and an mCherry-based secretion stress biosensor. The split GFP assay is based on the fusion of a target protein to the eleventh β-sheet of sfGFP, which can complement a truncated sfGFP that lacks this β-sheet named GFP1-10. The secretion stress biosensor makes use of the CssRS two component quality control system, which upregulates expression of mCherry in the htrA locus thereby allowing a fluorescence readout of secretion stress.ResultsThe biosensor strain B. subtilis PAL5 was successfully constructed by exchanging the protease encoding gene htrA with mCherry via CRISPR/Cas9. The Fusarium solani pisi cutinase Cut fused to the GFP11 tag (Cut11) was used as a model enzyme to determine the stress response upon secretion mediated by signal peptides SPPel, SPEpr and SPBsn obtained from naturally secreted proteins of B. subtilis. An in vivo split GFP assay was developed, where purified GFP1-10 is added to the culture broth. By combining both methods, an activity-independent high-throughput method was created, that allowed optimization of Cut11 secretion. Using the split GFP-based detection assay, we demonstrated a good correlation between the amount of secreted cutinase and the enzymatic activity. Additionally, we screened a signal peptide library and identified new signal peptide variants that led to improved secretion while maintaining low stress levels.ConclusionOur results demonstrate that the combination of a split GFP-based detection assay for secreted proteins with a secretion stress biosensor strain enables both, online detection of extracellular target proteins and identification of bottlenecks during protein secretion in B. subtilis. In general, the system described here will also enable to monitor the secretion stress response provoked by using inducible promoters governing the expression of different enzymes.

【 授权许可】

CC BY   
© BioMed Central Ltd., part of Springer Nature 2023

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