Improving the Synthesis Efficiency of Amino Acids by Analyzing the Key Sites of Intracellular Self-Assembly of Artificial Cellulosome

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1 Comments

Point 1: There is a mix of modelling technics that is poorly explained. For example, different forcefields are used for MM calculations and MD simulations. AMBER is a reliable all-atom protein forcefield, while GROMOS is a united-atom forcefield far less reliable for protein modelling. Furthermore, the use of united atoms deprives the model from hydrogen atoms and therefore from the formation of hydrogen bonds critical for protein stability and cohesin-dockerin binding.

Response 1: According to the reviewer’s comments, the revised manuscript retains only part of the MD simulation, and the GROMOS force field is a carefully designed combined atomic and molecular force field that can improve the computational efficiency of the model. In the simulation process, we have supplementary hydrogenation operations to form a simulated environment for DocA and Coh interaction.

Point 2: The use of MM calculations is not properly discussed, and it is hard to figure what was their role in this study.

Response 2: According to the reviewer’s comments, we used MM calculations to analyze changes in the properties of DocA mutants interacting with the adhesion protein Coh, but the discussion was insufficient. After comprehensive consideration, this part of the calculation and analysis was simplified and removed, focusing on the strength and stability of interaction between DocA mutants and adhesion protein Coh.

Point 3: The details of the MD simulations are lacking. Whether it is the exact content of the simulation box, or the MD parameters more extensive details are needed for reproducibility.

Response 3: According to the reviewer’s comments, We have added details of MD simulations such as " The net charge of the system was neutralized using sodium ions. The protein is placed in a square box, the edge of the box is 1.5 nm away from the protein, and 15,000 water molecules are added to solvate the protein. " in the" 2.4. Molecular Dynamics (MD) Simulations "section, including data from the simulation box and MD parameters, to make this section more complete.

Point 4: The MD simulations are too short. 10 ns simulations for protein as small as dockerin and cohesin is insufficient by today’s standard. Any recent desktop equipped with a high-end CPU (Intel i9) and GPU (Nvidia RTX4090) would deliver a microsecond worth of simulation in a single day.

Response 4: According to the reviewer’s comments, this part mainly selects the mutation sites of key amino acids of Ca²⁺ binding of DocA. Therefore, GROMACS 4.5 software was used for molecular dynamics simulation of 10 ns to preliminatively determine the impact on the structural stability of DocA, and the specific activity verification should be combined with parts “2.7. Detection of the binding affinity of DocA, and Coh proteins” and “2.8. Application of bimolecular fluorescence complementation-flow cytometric sorting (BIFC-FC)”, which was further discussed in Part 3.3.

Point 5: I am also concerned by the use of Gromacs 4.5.4. It is more than 10 years old. A new release of Gromacs is made available every year with several updates in between. The software is free, open-source and readily available on Linux distributions. The use of such an old version raises questions.

Response 5: According to the reviewer’s comments, the release of the new version is conducive to the efficiency of update operation and the optimization of update instructions, but the principle and basic steps remain unchanged. Gromacs 4.5.4 belongs to the classic version of the software, and the conclusion can still support the research. In this study, GROMACS 4.5 software was used for molecular dynamics simulation to preliminatively determine the stability of DocA protein structure.

Point 6: Two binding modes have been identified between cohesin and dockerin but only one has been investigated here. Also, the second one is a minor binding mode, the effects of mutating dockerin and removing the ionic bridges on this complex should also be examined.

Response 6: According to the reviewer’s comments, as for the effect of the removal of ionic bridges by mutant dockerin, the docking protein DocA and its mutants were treated in a calcium-free environment in the protein simulation section, which was discussed in Section 3.2. In terms of intracellular and extracellular activity verification, we mainly aimed to obtain docking protein mutants that can interact with adhesin in an intracellular environment with low calcium ions, so as to control the ionic environment. Relevant discussions are in parts 3.3 and 3.4.

Point 7: The only significant MD analysis is the RMSD which is insufficient to characterize the protein stability. Other descriptors like the secondary structure, the native contacts, the H-bond pattern, the RMSF, the radius of gyration, should also be examined. Furthermore, the RSMD plots should have lines rather than scattered points which make it hard to read the figure. The RMSD is not converged, the simulations must be extended. The histogram plot of the RMSD provide little added value and information and should only be generated on the converged section of the data.

Response 7: According to the reviewer’s comments, for the experimental part of MD analysis, root-mean-square fluctuation (RMSF) was used to measure the average amino acid flexibility of DocA mutants from the initial frame. RMSF changes of DocA mutated proteins were added in Part 3.5 and discussed and analyzed, as shown in Figure 9. In addition, Figure 2 and Figure 9 change the RSMD change graph to a line graph.

Point 8: For the dockerin-cohesin complexes more structural quantitative analyses must be provided. The binding free energy is also necessary to evaluate the stability of the complex. It must be compared to the native protein with its two calcium ions.

Response 8: According to the reviewer’s comments, for dockerin-cohesin complexes, the stability of natural proteins with two calcium ions is discussed in “3.2. Simulation Calculation and Selection of Docking Protein DocA Mutation Site”. In parts 3.3 and 3.4, the intracellular and extracellular experiments were conducted with different calcium ion concentrations.

Point 9: This also applies to experimental data. There is a lack of comparison with the native system in its native environment (calcium-rich). In fine, the aim is to produce a mutant as good as the native one (with calcium) and not doing better than what we know is an unstable protein (native dockerin without the calcium ions).

Response 9: According to the reviewer’s comments, in this study, the stable assembly of cellulosomes requires the participation of a certain concentration of calcium ions, and the concentration of intracellular calcium ions is too low, and the key components cannot be stably assembled. Therefore, the goal is to reduce the dependence of the docking protein and adhesion protein assembly on calcium ions and obtain complex elements that can be assembled in the cell. Although the intracellular interaction between DocA mutant and Coh is still weaker than that of the natural complex (containing calcium), according to the discussion in section 3.6, the application of this mutant complex in L-lysine fermentation can improve the transfer efficiency of intermediate metabolites between different catalytic active centers and improve the fermentation intensity by virtue of the proximity effect..

Point 10: The spectroscopic analysis is insufficient to demonstrate that some mutants dockerin binds cohesins as well as the native one. The affinity of the mutants might differ from that of the native protein and lead to binding more strongly with other cohesins leading to completely different cellulosomes.

Response 10: According to the reviewer’s comments, in order to verify the affinity between protein DocA mutants and Coh, extracellular interaction tests were performed by purifying extracellular active proteins. Through BIFC-FC intracellular experiment, only when DocA mutants and Coh interact, combined with fluorescence intensity changes to determine the complex activity. The mutant complex was applied to L-lysine fermentation and its activity expression was measured by increasing fermentation intensity.

Point 11: Activity experiments must be carried out to show that functional cellulosome are expressed and whether the action of cellulose is the same and comparable in magnitude to the wild species.

Response 11: According to the reviewer’s comments, the expression of cellulosomes was determined through activity experiments, which focused on the research of key sites of intracellular self-assembly activity of cellulosomes. The activity of DocA key site mutants mainly includes three experiments: (1) Extracellular interaction test through extracellular active protein purification; (2) The activity of the complex was determined by BIFC-FC intracellular experiment combined with the change of fluorescence intensity; (3) The mutant complex was applied to L-lysine fermentation, and its activity expression was measured by increasing fermentation intensity.

Point 12: In Table 2, like in Table 3, the mutations must be indicated by the underlying the mutated amino acid.

Response 12: According to the reviewer’s comments, the mutant amino acids of DocA mutants in Table 2 were added for annotation. The mutation site is denoted in underlined text.

Thanks.
Yours sincerely,

Junqing Wang.

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript describes the application of DocA-S3 to the assembly of key enzymes of L-lysine biosynthesis, in which the intracellular assembly of DapA and DapB increased the L-lysine production.

1.      English and manuscript writing must be significantly improved by the native speaker.

2.      The strain names must be in italic throughout the text.

3.      More detailed table and figure captions should be given for self-understood.

4.      Statistical analysis method should be given in Materials and method section, and any comparison of the data must be made based on statistical analysis. Otherwise no statistically meaningful discussion can be made.

5.      More detailed 3D structure with relevant amino acid residues should be shown in Figs.1 and 7.

6.      The letters/characters in Fig.2 are difficult to see and understand. More magnified figures should be given.

7.      Likewise, letters/characters are difficult to see in Fig. 4A, 4B, 4C, and 4D.

8.      Molecular docking simulation must be verified by in vitro activity data in general.

9.      Many papers have already been published on the channeling via enzyme complex such as the enzymes from GAP to PEP in the glycolysis to enhance the glycolytic flux to improve the glucose consumption rate, and the cell growth etc. It the present study, it is not clear how the Coh-DocA assembly (as well as DapA and DapB) increased the glucose consumption rate and the cell growth (and in turn L-lysine synthesis) as shown in Figs.9 and 10.

10.  More detailed genotype for the L-lysine producing E. coliQDE should be explained.

11.  Metabolic pathways showing the related enzymes should be shown.

English must be improved by the native speaker.

Author Response

Response to Reviewer 2 Comments

Point 1: English and manuscript writing must be significantly improved by the native speaker.

Response 1: According to the reviewer’s comments, the writing of this manuscript has been reviewed and refined by native English speakers.

Point 2: The strain names must be in italic throughout the text.

Response 2: According to the reviewer’s comments, we performed a writing check on the full text, and the strain names are shown in italics.

Point 3: More detailed table and figure captions should be given for self-understood.

Response 3: According to the reviewer’s comments, we have made important corrections to “Figure 2 RMSD change map and frequency statistics of 16 unit point mutant proteins”, “Figure 4 Determination of Ca²⁺ dependence of DocA point mutation protein binding to adhesion protein Coh”, and “Figure 8 RMSD change map and frequency statistics of DocA mutant proteins” , and added “Figure 9 RMSF value curves of of DocA mutant proteins.” to make the full text more full.

Point 4: Statistical analysis method should be given in Materials and method section, and any comparison of the data must be made based on statistical analysis. Otherwise no statistically meaningful discussion can be made.

Response 4: According to the reviewer’s comments, we added statistical analysis methods in the materials and methods section to ensure the stability and repeatability of experimental data for further discussion.

Point 5: More detailed 3D structure with relevant amino acid residues should be shown in Figs.1 and 7.

Response 5: According to the reviewer’s comments, we supplemented and labeled key amino acid sites of DocA mutants in Figure. 1 and Figure. 7.

Point 6: The letters/characters in Fig.2 are difficult to see and understand. More magnified figures should be given.

Response 6: According to the reviewer’s comments, we redrew Figure 2 and adjusted the image resolution (> 300dpi) to better understand the experimental results.

Point 7: Likewise, letters/characters are difficult to see in Fig. 4A, 4B, 4C, and 4D.

Response 7: According to the reviewer’s comments, we redrew Figure 4 and adjusted the image resolution (> 300dpi) to better understand the experimental results.

Point 8: Molecular docking simulation must be verified by in vitro activity data in general.

Response 8: According to the reviewer’s comments, the expression of cellulosomes was determined through activity experiments, which focused on the research of key sites of intracellular self-assembly activity of cellulosomes. The activity of DocA key site mutants mainly includes three experiments: (1) Extracellular interaction test through extracellular active protein purification; (2) The activity of the complex was determined by BIFC-FC intracellular experiment combined with the change of fluorescence intensity; (3) The mutant complex was applied to L-lysine fermentation, and its activity expression was measured by increasing fermentation intensity.

Point 9: Many papers have already been published on the channeling via enzyme complex such as the enzymes from GAP to PEP in the glycolysis to enhance the glycolytic flux to improve the glucose consumption rate, and the cell growth etc. It the present study, it is not clear how the Coh-DocA assembly (as well as DapA and DapB) increased the glucose consumption rate and the cell growth (and in turn L-lysine synthesis) as shown in Figs.9 and 10.

Response 9: According to the reviewer’s comments, as for the research on intracellular self-assembly of key enzymes for L-lysine synthesis based on key components of cellulosomes, intracellular cellulosomes were constructed by DocA mutant-Coh and the key enzymes for L-lysine synthesis were assembled. The fermentation results were shown in Figure. 9 and 10. It is further discussed in part 3.6 that the strategy of bacterial construction can exert the "proximity effect" among multi-enzyme complexes, improve the transfer efficiency of intermediate metabolites between different catalytic active centers, and indirectly improve the catalytic rate of each key enzyme.

Point 10: More detailed genotype for the L-lysine producing E. coliQDE should be explained.

Response 10: According to the reviewer’s comments, we added the genotype characteristics of L-lysine-producing Escherichia coli QDE in Table 2, which was applied to the intracellular self-assembly study of key enzymes for L-lysine synthesis based on key elements of cellulosomes.

Point 11: Metabolic pathways showing the related enzymes should be shown.

Response 11: According to the reviewer’s comments, in part 2.9, for L-lysine producing bacteria E.coli QDE, we added the contents of L-lysine synthesis pathway and key enzymes.

Thanks.

Yours sincerely,

Junqing Wang.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

No specific comments.