Background

Efficient and low cost production of valuable natural compounds, like proteins, has developed into a leading industry. Starting, by choosing a suitable production host followed by establishing a profitable downstream process, every step is constantly optimized to increase overall yields.

When it comes to choosing a production host, Bacillus subtilis is particularly interesting: the Gram-positive model organism can be easily genetically modified and has powerful secretion capacities. [1]

In this part of EncaBcillus we aimed at making use of the B. subtilis native advantages and combined them with Peptdiosomes – a new innovative cultivation platform for functional co-cultivation. Multiple Peptidosomes, with each encapsulating one specific strain that secrets one protein of interest. By doing so, the production of multi-complex proteins cloud be achieved by separated subpopulations all in one reaction hub.

To ensure the assembly of proteins outside of the Peptidosomes we further characterized the SpyTag/SpyCatcher system. Theses functional units can be attached to any protein of interest and upon secretion will result in a covalent isopeptide bond between the SpyTag/SpyCatcher partners [2]. The system originates from Streptococcus pyogenes and since it’s discovery it has been under constant development [3]. In our project we applied codon-adapted B. subtilis specific tags and reduced the SpyCatcher in length to enhance it´s usability when translationally fused to a protein of interest. Thus, decreasing the chances of the tag interfering with overall protein folding. [4]

To demonstrate the applicability of both tags we fused them to a green (sfGFP) and a red (mCherry) fluorescent protein, enabling an easy detectable output. (For more details please check our Design section) Since a core part of this project involves secretion, we included a signal peptide in front of all our constructs. (click here to learn more about our Signal Peptide Toolbox). To evaluate the efficiency of the secretion process we monitored the fluorescence in supernatants harvested from B. subtilis strains carrying our constructs and compared them to supernatants obtained from the wild type. In order to prove the functionality of the SpyTag/SpyCatcher system we co-incubated supernatants derived from different strains and performed a SDS-Page where we could demonstrate the formation of a fusion protein.

Design

All of the composite parts necessary for the genetic constructs were equipped with the RFC 25 standard, cloned into pSB1C3 backbone and submitted to the registry. All cloning was done according to standard protocols and the plasmids were stored in Escherichia coli DH10β. All constructs were verified by sequencing.

The gene encoding the mini. SpyCatcher (BBa_K2273015) was chemically synthesized. The codon optimized SpyTag (BBa_K2273014) was generated via overlapping primers iG17P049 and G17P050 and amplified using the primers TM4487 and iG17P039 (Protocol for LFH-PCR). We used a sfGFP (BBa_K2273033) that was codon optimized for Streptococcus pneumoniae, which has been demonstrated to work best in Bacillus subtilis [5]. The used mCherry (BBa_K2273034) was codon adapted for B. subtilis (Popp et al., 2017, accepted). The His-tag, necessary for protein purification was included in the reverse primers (table 1).

References

[1]	Nijland, Reindert & Kuipers, Oscar. (2008). Optimization of Protein Secretion by Bacillus subtilis. Recent patents on biotechnology
[2]	Gilbert et. all (2017) Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis. ACS Synth. Biol.
[3]	Zakeri et. All (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Applied Microbiology and Biotechnology.
[4]	Li et. All (2013) Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag . J. Mol. Biol.
[5]	Overkamp, W. et al. Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging. Appl. Environ. Microbiol. 79, 6481–6490 (2013).