The nucleotide composition of the spacer sequence influences the expression yield of heterologously expressed genes in Bacillus subtilis

• A strong influence of the spacer sequence on the expression yield of GOIs is shown.
• Changing the nucleotide composition of the spacer changes the expression level.
• A polyadenylate-moiety as spacer increased the yield, but only moderately.
• The effect of the spacer sequence is specific to the gene of interest.
• The effects of the spacer might be exerted via the folding of the transcript.

Bacillus subtilis is a commonly used host for the heterologous expression of genes in academia and industry. Many factors are known to influence the expression yield in this organism e.g. the complementarity between the Shine–Dalgarno sequence (SD) and the 16S-rRNA or secondary structures in the translation initiation region of the transcript. In this study, we analysed the impact of the nucleotide composition between the SD sequence and the start codon (the spacer sequence) on the expression yield. We demonstrated that a polyadenylate-moiety spacer sequence moderately increases the expression level of laccase CotA from B. subtilis. By screening a library of artificially generated spacer variants, we identified clones with greatly increased expression levels of two model enzymes, the laccase CotA from B. subtilis (11 fold) and the metagenome derived protease H149 (30 fold). Furthermore, we demonstrated that the effect of the spacer sequence is specific to the gene of interest. These results prove the high impact of the spacer sequence on the expression yield in B. subtilis.