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. 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).

Table 1: Overview of the constructed basic parts. Gene BioBrick No. forward Primer Reverse Primer SpyTag_His-tag BBa_K2273016 TM4487 iG17P068 Mini. SpyCatcher_His-Tag BBa_K2273017 TM4487 iG17P067 sfGFP_His-tag BBa_K2273021 TM4487 iG17P065 mCherry_His-tag BBa_K2273022 TM4487 iG17P066

In order to identify the best combination of SpyTag/SpyCatcher and FP fusion, we constructed all N- and C-terminal combinations (Table 2).

Table 2: List of SpyTag/SpyCatcher and FP composite parts. pSB1C3-mCherry-SpyTag-His BBa_K2273035 pSB1C3-mCherry-SpyCatcher-His BBa_K2273036 pSB1C3-SpyTag-mCherry-His BBa_K2273037 pSB1C3-SpyCatcher-mCherry-His BBa_K2273038 psB1C3-sfGFP-SpyTag-His BBa_K2273039 psB1C3-sfGFP-SpyCatcher-His BBa_K2273040 psB1C3-SpyTag-sfGFP-His BBa_K2273041 psB1C3-SpyCatcher-sfGFP-His BBa_K2273042

For the final construct we added the signal peptide sequence of amyE (BBa_K2273023) upstream of all constructs (Table 2) and cloned them into the single copy integrative B. subtilis vector pBS2EP_xylA. In brief, the vector has the following features for cloning in E.coli: an ori of replication and the bla gene mediating resistance against ampicillin. The B. subtilis specific part of the vector contains the multiple cloning site (MCS) in RFC10 standard with a P_xylA promoter upstream of the BioBrick prefix, a erm cassette providing resistance against erythromycin /lincomycin and flanking regions needed for integration into the lacA locus.

The sequenced plasmids were transformed into B. subtilis strain WB800N, a protease deficient strain. Fluorescence measurements of the supernatants derived from strains harbouring our constructs (Table 2) in the B. subtilis vector, was performed according to the protocol below.