- Production of bacterial cellulose in large scale via the fermentation of G.xylinus
- Purification of SpyCatcher003 with Cellulose Binding Modules
- Purification of SpyTag003 with structural proteins
Considering that G. xylinus is not a model organism and the transformation method of G. xylinus has not been thoroughly investigated, we did not engineer G. xylinus for a higher production of bacterial cellulose, so the research for gene expressions will not be involved. However, we did consider other ways to increase its production rate.
Because we are not modifying the strain, then we must search for the best condition where G. xylinus can be most productive. From two aspects, we decided to approach this problem: culture medium and culture device. We designed a dynamic bioreactor where G. xylinus can get both enough nutrients and sufficient oxygen for growth. When selecting the culture medium, we first tested the HS medium.
HS medium, short for Hestrin-Shramm medium, is a traditional culture medium for G. xylinus. Unfortunately, it cannot provide G. Xylinus with enough nutrients for growth when G. xylinus is dynamically cultured in a large quantity1.
Here is where the YME medium (Yamanaka Mathematically optimized Ethanol Media) is introduced; it can provide a lot more nutrients than HS (Table1). However, these nutrients can not come from nowhere; it is due to the extremely large amount of yeast extract in the medium. To conserve energy and reduce waste, we considered many alternatives for yeast extract and locked on beer dregs. More details available on our Human Practices page.HUMAN PRACTICE
A good piece of leather should be tensile, strong, and flexible. To achieve these standards, we crosslinked recombinant proteins critical to these facets between cellulose layers, the place where it is the weakest (Figure 1).Figure 1. Interaction ietween recombinant proteins (created with BioRender.com)
Collagen is the main component of natural leather. So, we chose collagen-like proteins to imitate the properties of collagen2,3. We also realized that leather should be wear-resistant and robust, and we synthesized spider fibroin to achieve this4.
These functional proteins will be linked with elastin when synthesized. Proteins synthesized separately will be connected, trying to reach infinity affinity to speed the reaction up. Infinity affinity is achieved with SpyTag003 and SpyCatcher003; they are specialized proteins that can link together through isopeptide bonds5. Now we constructed a network of proteins. To combine this network with the network of cellulose, we need cellulose-binding modules (CBMs). They hold on to the cellulose fibers, providing the fulcrums for other proteins6.
We choose the AOX1 promoter for its high level of expression of recombinant proteins when induced by methanol for expression in P. pastoris. While the cellulose-binding modules clamp onto cellulose, the SpyCatcher003 are ready to connect with SpyTag003 through peptide bonds; this can significantly approach infinity affinity and accelerate the reaction. SpyCatcher003 and SpyTag003 are the elastin-like polypeptides; they connect neighboring proteins and enhance the BC's performance. mRFP is added to indicates successful production.
Similar to the production of CBM. The spider fibroins and collagens are both expressed twice and connected to Spy-Tag with the elastin-like polypeptide. Notice the part that codes for collagen have V domain: V domain is responsible for stabilizing the downstream protein folding when collagen-like protein is within the ORF7,8. As the recombinant proteins get 'sandwiched' between cellulose layers, they quickly connect to the cellulose-binding modules through interactions between SpyTag003 and SpyCatcher003, fixed into position. These recombinant proteins also carry an indicator, sfGFP, to identify and distinguish it.
Unfortunately, it’s troublesome for us to engineer P. pastoris because of its low transformation efficiency. Therefore, we decided to adopt an E. coli protein expression system as our backup. The proteins expressed are same but we slightly adjusted how it will be expressed. The T7 promoter can combine with T7 RNA synthase. When inhibition at lac operator is being relieved by IPTG, it can help express the proteins and increase expression rate significantly.
For protein Synthesis in Pichia Pastoris, these shuttle plasmids are first transformed into competent E. coli cells, where they are massively replicated. Then the plasmids are collected and transformed into Pichia pastoris for gene expression under methanol conditions. For protein Synthesis in Escherichia coli, we transform the backbone PET30 with proper CDS into E. coil BL21D3. 6*His tags are expressed with the recombinant proteins for protein purification through nickel affinity chromatography.
1. Krystynowicz, A. et al. Factors affecting the yield and properties of bacterial cellulose. J Ind Microbiol Biotechnol 29, 189-195, doi:10.1038/
2. Peng, Y. Y. et al. Towards scalable production of a collagen-like protein from Streptococcus pyogenes for biomedical applications. Microb Cell
3. Xu, Y., Keene, D. R., Bujnicki, J. M., Hook, M. & Lukomski, S. Streptococcal Scl1 and Scl2 proteins form collagen-like triple helices. J Biol Chem 277, 27312-27318, doi:10.1074/jbc.M201163200 (2002).
4. Xu, M. & Lewis, R. V. Structure of a protein superfiber: spider dragline silk. Proc Natl Acad Sci U S A 87, 7120-7124, doi:10.1073/pnas.87.18.7120 (1990).
5. Keeble, A. H. et al. Approaching infinite affinity through engineering of peptide-protein interaction. Proc Natl Acad Sci U S A, doi:10.1073/ pnas.1909653116 (2019).
6. Mohammadi, P. et al. Biomimetic composites with enhanced toughening using silk-inspired triblock proteins and aligned nanocellulose reinforcements. Sci Adv 5, eaaw2541, doi:10.1126/sciadv.aaw2541 (2019).
7. Parmar, P. A. et al. Temporally degradable collagen-mimetic hydrogels tuned to chondrogenesis of human mesenchymal stem cells. Biomaterials 99, 56-71, doi:10.1016/j.biomaterials.2016.05.011 (2016).
8.Peng, Y. Y., Stoichevska, V., Schacht, K., Werkmeister, J. A. & Ramshaw, J. A. Engineering multiple biological functional motifs into a blank collagen-like protein template from Streptococcus pyogenes. J Biomed Mater Res A 102, 2189-2196, doi:10.1002/jbm.a.34898 (2014).