A host-vector toolbox for improved secretory protein overproduction in bacillus subtilis

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.

Subjects

Bacillus subtilis

Expression system

Fed batch

Human interleukin-1β

Overproduction

Protease deficiency

Protein secretion

Yeast sulfhydryl oxidase

DDC Class

610: Medizin

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).

Publication version

publishedVersion

Lizenz

https://creativecommons.org/licenses/by/4.0/

Name

Krüger2022_Article_AHost-vectorToolboxForImproved.pdf

Size

2.2 MB

Format

Adobe PDF

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A host-vector toolbox for improved secretory protein overproduction in bacillus subtilis