- Construction of an efficient secretion system for recombinant proteins in Bacillus subtilis (2011)
- All proteins being translocated through the cytoplasmic membrane of bacteria cells as well as some proteins that are inserted into the cytoplasmic membrane contain a signal sequence at their N-terminus that is recognized by and targeted to the translocation machinery. Three translocation pathways have been described, so far in E. coli to allow secretion of proteins: The Sec, the Tat and the SRP (Signal Recognition Particle) pathway. While the Sec and the Tat pathway act post-translationally and accept unfolded and correctly folded polypeptides, respectively, the SRP pathway acts co-translationally. For proteins secreted through the cytoplasmic membrane via the Sec pathway, the ATP-dependent motor protein SecA is required for translocation. The translocation process of some proteins following the SRP pathway has also shown to be enhanced by the presence of SecA. The Sec and the SRP pathway share the heterotrimeric protein-conducting channel translocon complex composed of the SecYEG proteins. Based on the known characteristics of both pathways, the goal of this PhD project was to construct an efficient secretion system for recombinant proteins in Bacillus subtilis using an a-amylase as a reporter enzyme, which is secreted into the medium using the Sec pathway. Its gene amyQ was fused to an IPTG-inducible promoter. It turned out that increasing amounts of IPTG did not result in a concomitant increase of secreted a-amylase. Overproduction either formed aggregates within the cytoplasm or preproteins targeted to the translocon jammed the membrane. To release the accumulated protein within the cells two different experiments were carried out: i) a co-production and overexpression of SecA, and; ii) overexpression of an artificial secYEG operon. First, increased production of SecA showed significantly decrease in the total synthesis and secretion of a-amylase and did not reduce cytoplasmatic accumulation or membrane jamming. Second, the artificial operon enhanced expression of secY, secE and secG genes resulted in a higher amount of reporter enzyme secreted into the medium. Furthermore, two different experiments using the transposon mutagenesis strategy were carried out in order to screen for B. subtilis mutants able to increase secretion of α-amylase. Transposon mutagenesis was performed with the mariner-based transposon to inactivate gene(s) whose product might regulate directly or indirectly the secretion of α-amylase. No mutant strain presenting a higher secretion of α-amylase on indicator plates was found. In addition, I devised a modified transposon containing a xylose-expression cassette. To test the efficiency of the modified transposon, the promoter-less cat gene was used as a reporter gene and integrated into the B. subtilis chromosomal DNA. After transposon mutagenesis, mutants expressing the promoter-less cat gene were isolated. This result indicates that the modified transposon might lead to increased production of a gene in the presence of xylose and that this product might then enhance secretion of α-amylase to be detected on indicator plates. In the third part of my thesis, a terminator-test vector was constructed which should allow the identification of strong terminators acting as 5'-stabilizing element. This vector consists of an artificial bicistronic operon containing the two reporter genes bgaB and gfp allowing the insertion of the terminators between the two genes. Insertion of a terminator should lead to a reduction of the amount of GFP. The system was verified with the known sinIR transcriptional terminator. It turned out that the vector with the two reporter genes already exhibited instability in E. coli.