Fig. 4
Analysis of cutinase secretion by B. subtilis with Cut11 carrying three different signal peptides and varying the length of the ribosome binding site spacers. B. subtilis PAL5 strains harboring expression plasmids pBS-Xnt-SPPel-Cut11 (SPPel), pBS-Xnt-SPEpr-Cut11 (SPEpr) or pBS-Xnt-SPBsn-Cut11 (SPBsn) were cultivated in a BioLector microbioreactor system at 30 °C and 1100 rpm for 24 h. The plasmid series differ in the length of the ribosome binding site spacer (4–12 nucleotides, indicated as Xnt in the plasmid name) between the Shine-Dalgarno sequence and the cut11 start codon resulting in a gradual change of translation efficiency as described by Volkenborn et al., 2020. A Relative enzymatic activities (grey bars) were measured with the substrate pNPP. B Relative amounts of secreted Cut11 (green bars) determined by the in vivo split GFP assay, where a solution of 3% (v/v) GFP1-10 was directly added into the culture broth after 16 h of cultivation. C The change of secretion stress was determined as relative fluorescence of mCherry (red bars). All cutinase activities and fluorescence values of split GFP and mCherry were measured after 24 h of cultivation, normalized to the backscatter and compared to the 4nt-SPPel variant. Cultivations were performed in biological triplicates and activity measurements were additionally performed in technical triplicates. Error bars indicate the standard deviations