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

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