OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system


Despite a substantial amount of information about secretion mechanisms, types of signal peptides and the effect of signal peptides on the secretion of different enzymes in various expression systems (Degering et al. 2010; Mathiesen et al. 2008; Nakamura et al. 1989), little information on the structure of the N-termini of the secreted recombinant proteins in E. coli has been reported. Even though many online databases and software programs can be used to predict signal peptides and cleavage sites, these algorithms are not always accurate (Zhang and Henzel 2004), as demonstrated in this study.

It has been previously shown that in addition to the signal peptide, the protein domain adjacent to the signal sequence, called the export initiation domain, is critical for protein translocation across the inner membrane of E. coli (Andersson and von Heijne 1991). Bacillus Csn was selected as a model in this study because it is an extracellular enzyme and therefore contains an export initiation domain that favors secretion. As such, Bacillus Csn was well suited for the purpose of this study, where mainly the effect of the signal peptide was compared.

While native Bacillus signal peptide can be recognized by E. coli secretion machinery, the secretion efficiency was not as high as when the OmpA signal peptide was used. In the presence of the Bacillus signal peptide, the secretion efficiency was 33.8 % at 20 h after induction, indicating that two-thirds of the recombinant proteins were retained in the cell. However, the N-terminal signal peptide had identical cleavage sites as in native Bacillus strains, indicating that N-terminal signal peptide processing is very accurate. This result indicates that both E. coli OmpA and native Bacillus Csn signal peptides direct the export of proteins via similar Sec-dependent secretion machinery. In addition, our data suggests that Bacillus and E. coli signal peptidases are functionally similar, even though their cell wall structures are very different. These data support the previous observation that the major components of the Sec machinery, which are required for protein secretion in both Bacillus and E. coli, are quite similar (Yuan et al. 2010).

Both gram-positive and gram-negative employ a similar type I signal peptidase (SPase), a membrane-bound endopeptidase, to remove the signal peptide from exported pre-enzymes during the late stages of the transport process (Paetzel 2014; van Roosmalen et al. 2004). The consensus SPase recognition sequence is Ala-X-Ala at positions-1 and -3 relative to the cleavage site in pre-proteins (Paetzel 2014). B. subtilis proteomics analysis revealed that 71 % of the corresponding signal peptides contain the consensus Ala-X-Ala recognition sequence; while 18 % of the identified extracellular proteins contain a Val-X-Ala recognition sequence (van Roosmalen et al. 2004). Therefore, native Bacillus SP could be cleaved precisely after VFA sequence, generating precise N-terminus as indicated in Fig. 4. As for construct containing OmpA signal peptide, there were two Ala-X-Ala sequences, which was artificially created by genetic engineering, consequently we detected two cleavage sites after the two Ala-X-Ala sequences (cleavage site 2 and 3). The cleavage site 1 was unusual as it was situated in the middle of hydrophobic region of signal peptide, before a helix-breaking glycine residue. This cleavage might have occurred via a different secretory pathway due to the incompatibility between the export initiation domain of Bacillus Csn and Gram-negative OmpA signal peptide. This result suggested that, one should avoid creating more than one Ala-X-Ala sequence at the junction between signal peptide and mature protein, when engineering a recombinant protein for secretory production.

The two signal peptides had significantly different effects on the secretory production of recombinant proteins in E. coli. As shown in Table 1, the OmpA signal peptide led to significantly higher yields of recombinant enzyme, compared with the native signal peptide. Codon optimization analysis, using OPTIMIZER, an online application for improving expression levels, indicated that the codon adaptation index (CAI) based on codon usage of predicted highly expressed genes of the N-terminus of the native Bacillus signal peptide, was only 0.537 (1.0 is the highest) (Puigbo et al. 2007). Therefore one explanation for the higher expression yield could be because the codon of the homologous OmpA sequence located at the 5? end of the gene is more compatible with the E. coli translation machinery, especially at the initiation of translation step. Further study by codon optimization of native Csn signal peptide to mimic the codon usage of E. coli will be necessary to test if this could help improve expression levels without compromising the fidelity of signal peptide cleavage. If successful, this may have broader commercial applications in the future.

It is interesting to note that the ptac promoter was leaky (de Boer et al. 1983), resulting in low protein expression in the absence of induction with IPTG. At this low expression level, both signal peptides had 80 % secretion efficiency. It is possible that the secretion machinery was not fully occupied and still able to process the secretion of both signal peptides (Driessen et al. 2001). However, when IPTG was used to induce protein over-expression, secretion mediated by the native Bacillus signal peptide, which was less compatible with the E. coli translocase complex, became the rate-limiting step for secretory production much earlier than when OmpA was used (de Keyzer et al. 2003). Another possibility is that the signal peptides used different secretion pathways (Muller et al. 2001). Recently, it was shown that a short peptide could serve as a signal peptide and guide heterologous cellulose proteins across both the inner and outer membranes of E. coli (Gao et al. 2015). Taken together these results suggest that when the N-terminal sequence of a protein is not critical, the OmpA signal peptide is preferred for the secretion of recombinant proteins in E. coli-based systems.