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Publisher DOI: 10.1007/s00253-022-12062-2
Title: A host-vector toolbox for improved secretory protein overproduction in bacillus subtilis
Language: English
Authors: Krüger, Anna  
Welsch, Norma 
Dürwald, Alexandra 
Brundiek, Henrike 
Wardenga, Rainer 
Piascheck, Henning 
Mengers, Hendrik G. 
Krabbe, Jana 
Beyer, Sandra 
Kabisch, Johannes 
Popper, Lutz 
Hübel, Tanno 
Antranikian, Garabed 
Schweder, Thomas 
Keywords: Bacillus subtilis; Expression system; Fed batch; Human interleukin-1β; Overproduction; Protease deficiency; Protein secretion; Yeast sulfhydryl oxidase
Issue Date: 8-Jul-2022
Publisher: Springer
Source: Applied Microbiology and Biotechnology 106 (13/16): 5137-5151 (2022-08-01)
Abstract (english): 
Abstract: Target proteins in biotechnological applications are highly diverse. Therefore, versatile flexible expression systems for their functional overproduction are required. In order to find the right heterologous gene expression strategy, suitable host-vector systems, which combine different genetic circuits, are useful. In this study, we designed a novel Bacillus subtilis expression toolbox, which allows the overproduction and secretion of potentially toxic enzymes. This toolbox comprises a set of 60 expression vectors, which combine two promoter variants, four strong secretion signals, a translation-enhancing downstream box, and three plasmid backbones. This B. subtilis toolbox is based on a tailor-made, clean deletion mutant strain, which is protease and sporulation deficient and exhibits reduced autolysis and secondary metabolism. The appropriateness of this alternative expression platform was tested for the overproduction of two difficult-to-produce eukaryotic model proteins. These included the sulfhydryl oxidase Sox from Saccharomyces cerevisiae, which forms reactive hydrogen peroxide and undesired cross-linking of functional proteins, and the human interleukin-1β, a pro-inflammatory cytokine. For the best performing Sox and interleukin, overproducing and secreting variants of these new B. subtilis toolbox fermentation strategies were developed and tested. This study demonstrates the suitability of the prokaryotic B. subtilis host-vector system for the extracellular production of two eukaryotic proteins with biotechnological relevance. Key points: • Construction of a versatile Bacillus subtilis gene expression toolbox. • Verification of the toolbox by the secretory overproduction of two difficult-to-express proteins. • Fermentation strategy for an acetoin-controlled overproduction of heterologous proteins.
DOI: 10.15480/882.4538
ISSN: 1432-0614
Journal: Applied microbiology and biotechnology 
Institute: Technische Mikrobiologie V-7 
Document Type: Article
More Funding information: This study was financially supported by the German Federal Ministry of Education and Research (BMBF; reference 0315400A/B) in the frame of the BIOCATALYSIS2021 network and by the Ministry of Economy, Labour and Tourism of Mecklenburg Vorpommern and the European Fund for Regional Development (V-630-S-177–2013/213; V-630-F-177–2013/212; V630-VB305-2013/211).
License: CC BY 4.0 (Attribution) CC BY 4.0 (Attribution)
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