Part:BBa_K4275012
CipA1B2C
CipA1B2C, a cellulosome integrating protein A with one integral carbohydrate-binding module 3 (CBM3), two type I cohesins, and a type II dockerin, is the primary scaffoldin of a cellulosome complex. The CBM3 binds to a range of polysaccharides, including cellulose and PET, increasing the catalytic efficiency of the enzymes. Meanwhile, the type I cohesins interact with the type I dockerins of cellulosomal enzymes, enabling the enzymes to attach to CipA1B2C. CipA1B2C forms a complete cellulosome complex when it binds to outer layer protein B (OlpB), an anchoring protein attached to a cell surface[1]. CipA1B2C improves the efficiency of cellulosomal enzymes by increasing binding affinity to cellulose and PET through CBM and facilitating the spatial proximity among various enzymes, which allows enzymes to work in synergy. The ability to bind to various cellulosomal enzymes makes CipA a versatile tool for enhancing the efficiency of enzymes and conducting different enzyme synergies, which means CipA can promote the degradation rate of diverse materials (i.e. PET and cellulose)[1]. This is a part in a part collection where we enable efficient degradation of cellulose and PET in textile waste.
Figure 1 The 3D structure of the protein predicted by Alphafold2.
Usage and Biology
CipA1B2C is a scaffold protein with one carbohydrate-binding module 3 (CBM3) and two type I cohesins. Natural CipA is found in Clostridium thermocellum, a gram-positive thermophilic and anaerobic bacterium. Cellulosomal enzymes bind tightly to CipA1B2C via the high-affinity dockerin I: cohesin I noncovalent interaction. CipA1B2C is then attached to OlpB, the secondary scaffoldin of the cellulosome complex, through the interaction between the type II dockerin and type II cohesin. OlpB is anchored to the cell surface. The CipA1B2C, OlpB, and the cellulosomal enzymes together comprise a multiplex cellulosome. Utilizing the highly effective cellulosome complex, C.thermocellum is the most efficient microorganism for lignocellulosic biomass degradation.
Characterisation of the Cellulosome Complex
Mini-scaffold construction of cellulosome
We constructed E.coli expression vectors for the mini-scaffold protein subunits. The scaffoldin components of the wild-type cellulosome subunits are large protein scaffolds that would bring a massive protein burden to the bacterial host secreting them. We modified the coding sequences for the wild-type cellulosome protein scaffold, as shown in (Fig.3A and Fig.3B) The mini-scaffolds were successfully expressed by our host, verified by the SDS-PAGE analysis shown in (Fig.3C and Fig.3D)
Figure.2
Mini-scaffold expression in E. coli BL21. (A) Construction of primary scaffold CipA1B2C (i.e., 1 CBM3 and 2 type I cohesin) (B) Construction of anchorage scaffold OlpB-Ag3 (i.e., 3 type II cohesin). (C) SDS-page analysis for CipA1B2C. (D) SDS-page analysis for OlpB-Ag3.
In order to verify the three levels of protein-protein interaction that assembles our cellulosome complex, Ag3-eforRED, DocI-eforRED, and DocII-eforRED vectors were constructed and cultured for IPTG-inducible expression (Fig. 4D). SDS-page analysis was performed with lysed cells and all three targeted proteins were identified in both whole cell and supernatant (Fig. 5A and 5B).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 314
Illegal XhoI site found at 1237 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 543
Illegal AgeI site found at 325
Illegal AgeI site found at 669
Illegal AgeI site found at 868
Illegal AgeI site found at 1539 - 1000COMPATIBLE WITH RFC[1000]
References
1. Anandharaj, Marimuthu et al. "Constructing A Yeast To Express The Largest Cellulosome Complex On The Cell Surface". Proceedings Of The National Academy Of Sciences, vol 117, no. 5, 2020, pp. 2385-2394. Proceedings Of The National Academy Of Sciences, https://doi.org/10.1073/pnas.1916529117.
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