Short Description

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Background

Over the course of the last decades the quality, amount and spectrum of heterologous (and recombinant) proteins has drastically increased and therefore the need for techniques to easily express and purify these proteins has emerged. We find such proteins as ingredients of detergents (proteases), medical treatments (insulin) or food and beverage products (amylases). Simply put, heterologous proteins are ubiquitously present. [1]

In order to tackle this demand we chose to apply the model organism Bacillus subtilis. It is already one of the most frequently used hosts for overproduction of proteins throughout academia and industry because of its tremendous capacity to secret proteins, which can be exploited to increase the overall yields.

B. subtilis has four different secretion pathways, however the majority of proteins are being secreted via the general Sec pathway which has been identified as vital element of protein secretion among all domains of life [2]. In the Sec pathway the secretion of proteins into the surrounding supernatant is orchestrated by signal peptides (SP). These SPs are composed of approximately 120 to 180 nucleotides and they are located upstream of the protein to be secreted. Intracellularly, the SP is translationally fused to the specific protein but cut off during the membrane translocation process releasing the protein into the supernatant without the signal peptide attached to it. [3] Up to date, approximately 170 SPs belonging to the Sec-SRP pathway have been annotated in B. subtilis but unfortunately, no correlations on sequence levels have been identified that link efficient protein secretion with a distinct SP. [4]

As part of our project EncaBcillus we aimed at creating a platform for heterologous protein overexpression combined with their efficient secretion without releasing the cells of our chosen model organism B. subtilis into the environment. Hereby, we encapsulated B. subtilis in the Peptidosomes which serve as a new innovative method to keep the producing bacteria separated from the desired compounds. (For more details see Peptidosomes.)

To address the vision of creating this new platform, we first wanted to establish a fast and easy screening procedure to evaluate all combinational possibilities of each SP with a protein of interest (POI) – The Signal Peptide Toolbox.

As SPs of B. subtilis have been successfully adapted to GRAM-positive [5] and GRAM-negative bacteria [6], the Signal Peptide Toolbox can be applied to any bacterial host. Therefore, every future iGEM team will be able to use this tool to increase their protein secretion.

Design

AmyE	AspB	BglS	Bpr	CccA	CitH	Csn	DacB	DacE	DltD
AmyE	AmyE	AmyE	AmyE	AmyE	AmyE	AmyE	AmyE	AmyE	AmyE

1	Exchange of the promoter: Digest both, the provided EV and the new promoter using the restriction enzymes EcoRI and BsaI. Successfully transformed E. coli colonies on X-Gal containing agar plates stay blue. But on not X-Gal containing agar plates, successfully transformed E. coli colonies stay red.

Results

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References

[1]	van Dijl J. M. and Hecker M. (2013) Bacillus subtilis: from soil bacterium to super-secreting cell factory. Mircobial Cell Factories 12, 3 (1-6).
[2]	Papanikou E., Karamanou S. and Economou A. (2007) Bacterial protein secretion through the translocase nanomachine.. Nature Reviews Microbiology 5, 11 (839-851).
[3]	Fu L. L., Xu Z. R., Li W. F., Shuai J. B., Lu P. and Hu C. X. (2006) Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion. Biotechnology advances 25, 1 (1-12).
[4]	Brockmeier U., Caspers M., Freudl R., Jockwer A., Noll T. and Eggert T. (2013) Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. Journal of molecular biology 362, 3 (393-402).
[5]	Hemmerich J., Rohe P., Kleine B., Jurischka S., Wiechert W., Freudl R. and Oldiges M. (2016) Use of a Sec signal peptide library from Bacillus subtilis for the optimization of cutinase secretion in Corynebacterium glutamicum. Mircobial Cell Factories 15, 208.
[6]	Pechsrichuang P., Songsiriritthigul C., Haltrich D., Roytrakul S., Namvijtr P., Bonaparte N., Yamabhai M. (2016) OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system. Springerplus 5, 1200.