Study On The Construction Of The Intracellular Self-assembly System Imitating Cellulosome

Cellulosome is a kind of natural multi-enzyme complex attached to the cell walls of some anaerobic biological cells.It has the function of organizing and coordinating multiple enzyme components.It can efficiently catalyze the degradation of lignocellulose.Its structural stability is strong and flexible.At present,imitating cellulosome to achieve efficient polymerization of multiple enzyme systems on the surface of a variety of microorganisms has great application potential in improving the synergistic efficiency of multiple enzymes,but it has never been able to achieve self-assembly in cells.In this study,cellulosome was selected as the basic model of the intracellular self-assembly system,and its key components?cohesion and dockerin?were selected as the main research objects.E.coli was used as the platform to focus on the exploration and analysis of its self-assembly mechanism.On the one hand,we used a rational design method to directional modification of dockerin,combined with experiments to find the mutation direction suitable for intracellular assembly.On the other hand,we constructed a flow-through screening system suitable for high-throughput screening,which would help us screen for successful mutations.These research results are divided into the following three aspects:?i?The type I dockerin Doc A and cohesin Coh were selected as the research objects.The pictures of the 3D structure model were downloaded and analyzed with software such as Py MOL.We found that the dockerin has two Ca²⁺binding sites.After that,the two proteins were expressed in E.coli,and then the molecular interaction instrument was used to preliminary explore the binding mechanism of the two proteins.The study found that the stable binding of dockerin and cohesin requires the participation of Ca²⁺concentration of about 5×10^-4 mol/L.When the Ca²⁺concentration was 0?5×10^-3 mol/L,the higher the Ca²⁺concentration,the higher the binding capacity.The change of Ca²⁺concentration within 5×10^-4?10^-2 mol/L had the most significant effect on the binding ability.However,the binding capacity was close to the binding capacity at a Ca²⁺concentration of 0 mol/L when the Ca²⁺concentration was lower than 10^-4 mol/L,indicating that the two original proteins cannot assemble inside the cell.?ii?With the more manipulable protein Doc A,the main experimental subject,the amino acid in the range of Ca²⁺4?was used as the key point for targeted transformation.Firstly,we used Gromacs and other software to analyze the docking of14 single-point mutants with the protein Coh.It was found that the difference between the RMSD value of the mutant D39R and the original protein was a minimum of 0.174,indicating that the mutation could increase the rigidity of Loop D39-E55.The Biacore T200 was used to test the binding capacity of the selected mutant to the protein Coh at different Ca²⁺concentrations.After analyzing and testing,we found the D39R performed best.D39R and protein Coh had the strongest binding capacity when the Ca²⁺concentration was 5×10^-4 mol/L,6.71 times the original protein.It still had a certain binding ability at low Ca²⁺concentration.Moreover,four kinds of double-site mutations experiments were carried out.With the help of Rosetta@home computing website analysis and experimental investigation,it was found that the optimal double-site mutant 36864 had the strongest binding capacity at a Ca²⁺concentration of 5×10^-4M,5.88 times the original protein.It had a certain binding capacity at low Ca²⁺concentration,and its binding capacity was 1.19 times the mutant D39R when the Ca²⁺concentration was 0 mol/L.Through comparison,it was found that the effect of the two-site mutant protein in intracellular assembly was significantly better than that of the single-point mutant protein.It was also preliminarily concluded that the two Ca²⁺binding sites affected the stable binding of the two proteins.?iii?To improve the screening efficiency of dockerin random mutants,a screening system based on fluorescent proteins was initially constructed in this study after analyzing the influence of the connection mode of fluorescent protein species on the dockerin mutants and flow cytometry screening.Firstly,four fusion proteins constructed by PCR technology,including‘protein INP-EGFP-Doc A?green?',‘protein EBFP-Coh?blue?',‘protein m Cherry-Coh?Red?',‘protein INP-oc A'and‘protein EGFP-Coh?Green?'.Then the fusion proteins were expressed in E.coli respectively and tested the fluorescence signals.According to the results,the green fluorescent protein EGFP-Doc A and EGFP-Coh signal was strong,indicating that EGFP was highly compatible with Doc A and Coh.It laid the foundation for efficient screening by flow cytometry in the later stage.