Dynamic spatial organisation at all levels is a universal property of living systems [26]. Contemporary cells, both eukaryotes and prokaryotes, rely on compartmentalisation. In eukaryotes, compartmentalisation relies on intracytoplasmic membrane systems, for example, chloroplasts, mitochondria, vacuoles and the cell nucleus, allowing metabolic processes to be specifically localised and controlled [27][28]. For a very long time, bacterial cells were considered to be simple cells with hardly any spatial division. However, recent research has shown that they also exhibit subcellular differentiation [29]. The majority of prokaryotes use membrane-less, protein-based compartments to spatially control metabolism. Encapsulation can lead to the creation of unique microenvironments in regard to, redox state, pH and ionic strength [27]. Bacteria have to overcome many hurdles in the course of a pathway. First, the enzymes involved must be present in the correct stoichiometry. Secondly, intermediates can diffuse through the cell membrane. Third, intermediates produced in the biosynthetic pathway can be toxic to the cell. Furthermore, inhibitors of enzymes can occur, intermediates can be unstable, or intermediates can be used for other pathways [5][30].
To deal with these problems, nature has found a solution, compartmentalization. Enzymes can be brought together in the right amount, intermediates do not diffuse out of the cell as easily, toxicity is reduced by shielding and rapid conversion to the next product, inhibitors and competing enzymes are more likely to be excluded, in short, compartmentalization can improve the yield of several pathways [5][30][31].
For our project we worked with three different systems of compartmentalization (Figure 1).
Figure 1: Types of compartmentalization. Bacterial microcompartment shells derived from Haliangium ochraceum (HO-Shells) and encapsulin nanocompartments have rigid structures and Spd-5 proteins behave like liquid droplets when aggregated.
1. Bacterial microcompartments (BMCs) are self-organising organelles with a selectively permeable protein shell. All BMCs consist of three conserved families of proteins: BMC-H (forming hexamers), BMC-T (pseudohexamers) both with pores of different sizes in the middle and BMC-P (pentamers) [4][32]. Small molecules can enter the lumen of BMCs via the pores found within the BMC-H shell proteins (which vary in size from 4 - 7Å in diameter) or the larger pores (~12 - 14 Å in diameter) formed by BMC-T trimers which can have an open or closed confirmation [33][34]. For our project, we used the recently published synthetic BMCs from Kirst et al. [32] , which based on the shell system from the myxobacterium Haliangium ochraceum (HO-shell) (Figure 3A). The HO-shell is able to assemble without containing any cargo molecule inside [32][35] and is built by the shell proteins BMC-H, BMC-P and three BMC-T proteins (single-layer T1, and double-layer T2 and T3). The synthetic BMC shell, designed by the Kerfeld lab can form without the presence of the BMC-P proteins (Figure 3B) [29][36]. Without the pentamers, there are pores left that allow molecules to diffuse in/out of the lumen of the BMC. This form of the synthetic BMC is called full wiffleball. An even more simplified shell (minimal wiffleball) was designed to consist only two shell proteins, BMC-H and BMC-T1 (Figure 3C).
Synthetic BMCs serve as autonomous metabolic modules, which are decoupled from the regulatory mechanisms of the cell and are only connected to the metabolism of the cell via the engineered protein envelope [32].
Figure 2: Variations of the compartment structure showing the different components: (blue: P protein; yellow: H protein; pink: T1 protein; violet: T2/T3). The representation is based on the model of the Kerfeld group [32] (structure from PDB: 6MZX). Figure created with PyMOL.
In order to target enzymes into the shell, the SpyCatcher/SpyTag and SnoopCatcher/SnoopTag systems were used to covalently bind a cargo to the inside of the shell. The Spy/SnoopTag is a small peptide that spontaneously reacts with the protein, Spy/SnoopCatcher, to form an intermolecular isopeptide bond between the pair [37] . To precisely encapsulate two distinct cargo proteins into the lumen of a synthetic BMC, both, SpyTag and SnoopTag, are incorporated into a lumen-facing loop of the BMC-T1 shell protein. The respective catcher is attached to the N- or C-terminal region of distinct target proteins [32].
2. Encapsulins are nanocompartments, which similar to microcompartments, are self-assembled protein compartments, natively found in some bacteria and archaea [38]. They can be distinguished from microcompartments through the size of the compartments (20-42 nm) [38]. The encapsulins from Thermotoga maritima and Mycobacterium tuberculosis have outer diameters of 20–24 nm and are composed of 60 identical encapsulin protein subunits. The largest encapsulin compartment discovered to date is represented by that of Quasibacillus thermotolerans with a 42 nm outer diameter and 240 identical subunits [39]. Structural experiments showed that encapsulins are icosahedral shell-like protein compartments resembling viral capsids (Hong Kong 97‐like fold) [39][40]. The pore size of ~5 Å allows channelling small molecular substrates through the shell. Multiple encapsulins encapsulate cargo protein based on a short C-terminal peptide sequence, called the targeting peptide (TP) [38]. TPs often include a specific anchoring sequence, such as the Gly–Ser–Leu singlet or doublet motif and binding is mediated by hydrophobic and ionic interactions [41][42]. Encapsulins have attracted the attention of the synthetic biology community for the possibility of engineering small protein nanocages e.g. for drug delivery [43]. The encapsulins are highly suitable for such purposes given their high stability at high temperatures and various pH levels [44]. For our project, we decided to use an encapsulin derived from M. xanthus which is composed of the protein EncA, forming the shell (Figure 4) [40][45].
Figure 3: Encapsulin of the organism M. xanthus, which is composed of the protein EncA. Encapsulin compartments can provide stabilization and co-localization of cargo proteins and can also be engineered to encapsulate non-native enzymes, as previously shown in yeast (structure from PDB: 4PT2). Figure created with PyMOL.
In M. xanthus, the encapsulin is known to encapsulate three different cargo proteins, which play a role in iron storage [45]. This specific encapsulin was deemed a great fit for our team, as it has previously been engineered to encapsulate non-native enzymes in yeast [46].
3. Self-assembling proteins such as the spindle-deficient protein 5 (SPD-5) derived from Caenorhabditis elegans can form spontaneously, assembling dynamic organelles in vitro and in vivo [47]. Not only does SPD-5 reliably form droplet-like structures even in the highly crowded environment of the cytoplasm, but it also has been shown to naturally recruit enzymes and related molecules into the dynamic formation [48][49]. SPD-5 contains nine predicted coiled-coil domains, which make up ~40% of the protein. Studies have shown that the coiled-coil domains are involved in the formation of stoichiometric protein complexes and drive the formation of amorphous condensates followed by a rearrangement stage. SPD-5 condensates form as viscous liquids that quickly harden [48]. Even though SPD-5 is dynamic and does not permit exclusive entry and exit of specific molecules, it has been successfully used to enhance the efficiency of reactions, for example, by improving non-canonical amino acid incorporation with an orthogonal translation system ([50] and iGEM Freiburg 2019). In order to target cargos specifically to liquid droplets the SpyCatcher/SpyTag and SnoopCatcher/SnoopTag systems (explained above) was applied to enhance the property SPD-5 based compartments.
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iGEM Freiburg 2022 presents
chAMBER
