Investigation of magnetic nanosomes as multifunctional nanomachines
Microbial Ecology and Applied Microbiology
Metabolism, Biochemistry and Genetics of Microorganisms
Final Report Abstract
Enzymes are biocatalysts that allow chemical reactions to occur under ambient conditions and are widely used in the food, health and textile industry. Due to the high costs associated with the application of enzymes in industrial biotechnology the development of novel strategies to reduce the cost of enzymes is of significant interest. One approach is the immobilization of enzymes onto nanoparticles to combine their enzymatic activity with the physicochemical characteristics of the nanoparticle. Cellulosomes are natural occurring macromolecules produced by anaerobic cellulolytic bacteria mostly belonging to the Clostridaceae like: Clostridium cellulolyticum, Clostridium thermocellum, and Ruminococcus flavefaciens. These self-assembling systems consolidate multiple enzymes in close proximity for the synergistic deconstruction of cellulose and hemicellulose. The central scaffolding protein devoid of enzymatic activity that displays numerous cohesin modules bind the enzyme-borne dockerin modules with high affinity (KD ≤ 10^-9) in a species-dependent manner. This modular structure can be used to genetically combine individual modules from different organisms to create chimeric components to assemble designer cellulosomes. By adding cohesin domains from Clostridium josui and Acetivibrio cellulolyticus We created mono-, tri-, tetra-, and pentavalent designer scaffolds. Magnetosomes are biogenic nanoparticles produced by magnetotactic bacteria that can be functionalized through translational fusions of its anchor proteins. Through the translational fusion of designer scaffold proteins we created multivalent magnetic responsive nanosomes which can be used to immobilize up to 5 different enzymes. Furthermore, we investigated the enzymatic cocktail produce by an aerobic cellulolytic consortium which grew with ionic liquid pretreated switch grass as sole C-source. The well described cellulolytic microorganisms Rhodothermus marinus, Thermus thermophilus and Thermobispora bispora were identified by metagenomic analysis as the main organisms at the end of the cultivation. Surprisingly, the relative abundance of an uncultivated Paenibacillus correlated with the establishment of the main glycoside hydrolase (GH) activity in the supernatant at day 4 of the cultivation. Genome analysis of a near to complete draft genome of this organism revealed its potential in cellulose degradation and was tentatively named ‘Candidatus Reconcilibacillus cellulovorans’. Purification of cellulolytic enzymes was achieved by affinity digestion by taking advantage of cellulose binding motifs and the enzymatic activity against cellulose. Interestingly, native PAGE revealed the existence of large protein complexes with GH activity. In combination with 2D SDS PAGE and proteomics it was revealed that all subunits of the complex (CelA, Cel B and Celc) are expressed by ‘Candidatus Reconcilibacillus cellulovorans’ which is abundant at day 4 by is low abundant (~1%) at the end of the passage (day 14). This might indicate that the complexes may serve as public goods, releasing soluble glucans that support the other community members.The absence of cohesin dockerin motifs in all subunits points towards a differing interaction mechanism compared to cellulosomes produced by members of the Clostridaceae. Experimental results suggest that complex formation as well its unusual stability against proteolysis is connected to posttranslational modifications such as glycosylation.
Publications
- A Pioneer Population Produces Cellulase Complexes that Persist through Community Succession. (2017) Nature Microbiology
Kolinko S., Wu Y.-W., Tachea F., Denzel E., Hiras J., Bäcker N., Chan L. J., Eichorst S. A., Frey D., Adams P. A., Pray T., Tanjore D., Petzold C. J., Gladden J. M., Simmons B. A. & Singer S. W.
(See online at https://doi.org/10.1038/s41564-017-0052-z) - Society for Industrial Microbiology and Biotechnology (Denver, USA). A Pioneer Population Produces Cellulase Complexes that Persist through Community Succession. (2017)
Kolinko S., Wu Y.-W., Tachea F., Denzel E., Hiras J., Bäcker N., Chan L. J., Eichorst S. A., Frey D., Adams P. A., Pray T., Tanjore D., Petzold C. J., Gladden J. M., Simmons B. A. & Singer S. W.
(See online at https://doi.org/10.1038/s41564-017-0052-z)
