The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

Anna A Toymentseva,Karen Schrecke,Thorsten Mascher,Margarita R Sharipova

doi:10.1186/1475-2859-11-143

Anna A Toymentseva, Karen Schrecke + Show 2 more

Open Access

https://doi.org/10.1186/1475-2859-11-143

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Abstract

BackgroundBacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (PliaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min.ResultsBased on these traits of PliaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the “LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the PliaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the PliaI promoter.ConclusionsThe LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of PliaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available, and affordable inducers and (v) the convenient conversion of LIKE-derived inducible expression strains into strong constitutive protein production factories.

Highlights

Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins
Features of the native liaI promoter (PliaI) Previously, we have characterized the cell envelope stress-inducible promoter PliaI, which controls the expression of the liaIH operon in B. subtilis
In the presence of suitable inducers such as the cell wall antibiotic bacitracin, it strongly responds in a concentration-dependent manner, resulting in a more than 100-fold increased activity already 5–10 min after the addition of bacitracin

Summary

Introduction

Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (PliaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. The nisin-controlled gene expression (NICE) system was developed for different species of Lactococcus and Lactobacillus and allows the production of the desired proteins in high amounts (comparable to other expression systems), reaching a maximum 3 h after nisin induction [15,16]. A very similar subtilin-regulated expression system (SURE) was recently constructed for B. subtilis [9] Both systems enable the controlled overexpression of a variety of homologous and heterologous proteins and enzymes and show a number of advantages to other control elements, such as the strict control of gene expression, no leakage of the promoter regulation under non-inducing conditions, high levels of expression upon induction and almost no limitations in the choice of sugar-containing media [9,15]. For the use in B. subtilis, the SURE system has several advantages over the NICE system: (i) The SURE system only requires a single plasmid, thereby ensuring a stable expression platform; (ii) the expression levels achieved by the SURE system are significantly higher; and (iii) it requires lower concentrations of the inducer molecule [9,17]

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