title: The nucleotide composition of the spacer sequence influences the expression yield of heterologously expressed genes in Bacillus subtilis.
abstract: 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.
authors.affiliation: BRAIN AG, Darmstädter Strasse 34-36, 64673 Zwingenberg, Germany. Electronic address: kl@brain-biotech.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Liebeton
authors.affiliation: Institute of Biochemistry, Department of Biology, ETH Zurich, Otto-Stern-Weg 3, 8093 Zurich, Switzerland.
authors.forename: Jette
authors.initials: J
authors.lastname: Lengefeld
authors.affiliation: BRAIN AG, Darmstädter Strasse 34-36, 64673 Zwingenberg, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Eck
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D054892:Metagenome; D009711:Nucleotides / Q000235:genetics; D010447:Peptide Hydrolases / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D021903:DNA, Ribosomal Spacer; D009711:Nucleotides; D012336:RNA, Ribosomal, 16S; D010447:Peptide Hydrolases
keywords: Bacillus subtilis; Folding of transcript; Heterologous expression; Spacer sequence; Translation initiation