Optimization of a Recombinant Lectin Production in Pichia pastoris Using Crude Glycerol in a Fed-Batch System

Round 1

Reviewer 1 Report

Comment and suggestion for authors:

I miss biological activity assay of purified rTBL-1 in this study. Could authors add any information about  biological properties of purified rTBL-1, e.g. into discussion (if any such research was performed)?

Author Response

Reviewer 1. Comment and suggestion for authors:

I miss biological activity assay of purified rTBL-1 in this study. Could authors add any information about biological properties of purified rTBL-1, e.g. into discussion (if any such research was performed)?

Thank you for your comment. Yes, we added two results (lines495-533), including also the materials and methods details (lines 211-228). Figure 6 shows the comparison between native lectin and the rTBL-1 genetic sequences. And Figure 7 shows the cytotoxic effect of rTBL1 and the comparison respect to the lethal concentration of the native lectin.

We thank very much to the reviewer for the comments and suggestions that helped to improve this work in an important way.

Reviewer 2 Report

The manuscript entitled: “Optimization of a recombinant lectin production in Pichia pastoris using crude glycerol in a fed-batch system”, reference: 1193429

General comments:

In my opinion, this manuscript clearly encompasses an enormous amount of work, and depicts important findings. However, there is a serious issue on the clarity of the presented results. In the Materials and Methods section, seems that several types of glycerol underwent the same analysis, but the Results and Discussion section shows otherwise correct? An example is Figure 1, 3 and 4 which encompass different results for a single type of glycerol each, correct? It almost feels like that the authors intended to gather different and non-correlative experiments in a single manuscript, which clearly hinders its clarity. Please correct me if I am wrong, but I expected all figures to be like Figure 2, were all the types of glycerol were accessed (or at least two). A clear separation of each glycerol analysed, and an adequate explanation to support it, would in my opinion, help to improve its quality.

The Materials and Methods section is, in my understanding very difficult to follow. It has several subheadings that could be merged or preferably, rearranged. For instance, instead of “jumping” between the flask, bioreactor, batch and feed-batch, in my opinion it would be clearer if the authors clearly explain the details of flask fermentation, and of bioreactor fermentation.

The authors provide insufficient information of the design of experiments used. What were the tested ranges and corresponding minimal and maximum values? Moreover, and the more grievous, I could not find any statistical data of the obtained models. Were the models statistically significant? Which parameters were significant? What was the lack of fit value, the adequate precision and the coefficient of determination? Without them I am not able adequately evaluate the obtained models, therefore this information must not be published.

Point-by-point suggestions:

Line 81, please define all the acronyms consistently, including YPD (line 102). Please carefully revise the entire manuscript.

Line 96, please provide additional details. What was the sodium hydroxide concentration used, and what do the authors mean with simple centrifugation?

Line 98, how did the authors evaporate the ethanol? Please provide detailed information of the process, so it can be replicated by the readers.

Section 2.2, has in my opinion some clarity issues. Were the cultures performed in static conditions? If not, what was the shaking speed? In addition, please insert a space between different units, as an example: g L-1. Furthermore, percentage and degrees Celsius are units, thus they should be separated from its numerical value.

I beg your pardon but I am confused, how was the bioreactor inoculated: using the procedure described in the beginning of section 2.2 or 2.3?

Line 123, how was the “30% of saturation" maintained? Higher shaking speed? Increased airflow? Injection of oxygen? Please provide additional details of the bioreactor process.

Line 128, the correct unit for g force is not "x g" but a g italicized (so it can be distinguished from grams). Please also revise in line 172.

Line 166, “A second order polynomial model provided the best fit to the experimental data obtained” in my opinion this information should be in the Results section.

Line 176, just curious, so rTBL-1 has a histidine tag so it can be purified through Ni Sepharose, correct? Is all this detailed in reference [6]? If so, I think it should be mentioned and referenced in this section also.

Line 181, how were the samples stored after freeze-dry?

Line 190, ImageJ software can, and should be adequately cited, please see: https://imagej.net/Citing

Line 222, please define the unit: WCWL. In addition, can the author please explain what does the symbol “»” mean?

Statistical analysis details must be adequately described in the Materials and Methods section.

Line 239, “Small letters” or different letters represent significant differences? I am confused, please help me to understand.

Subsequently to the first appearance in the manuscript text of the full species name (i.e. Pichia pastoris), its abbreviated from must be consistently used (i.e. P. pastoris), including figure captions.

Figure 4, I beg your pardon, but a quick assessment of these figures shows that the design performed by the authors does not completely cover the values spectrum, in other words, the maximum value was not observed. Please comment.

Author Response

Reviewer 2. Comments and Suggestions for Authors

The manuscript entitled: “Optimization of a recombinant lectin production in Pichia pastoris using crude glycerol in a fed-batch system”, reference: 1193429

General comments:

1. In my opinion, this manuscript clearly encompasses an enormous amount of work, and depicts important findings. However, there is a serious issue on the clarity of the presented results. In the Materials and Methods section, seems that several types of glycerol underwent the same analysis, but the Results and Discussion section shows otherwise correct? An example is Figure 1, 3 and 4 which encompass different results for a single type of glycerol each, correct? It almost feels like that the authors intended to gather different and non-correlative experiments in a single manuscript, which clearly hinders its clarity. Please correct me if I am wrong, but I expected all figures to be like Figure 2, were all the types of glycerol were accessed (or at least two). A clear separation of each glycerol analyzed, and an adequate explanation to support it, would in my opinion, help to improve its quality.

Thank you very much. The first two experiments were done in batch cultures in order to find: 1) the optimal glycerol concentration using only pure glycerol (G99) (Figure 1) and, 2) the comparison between the four glycerol types using the same concentration each one (Figure 2). As we can observe, there were no differences for wet cell weight between the four glycerol types, therefore, we used in the subsequent assays only pure (G99) and crude (G65) glycerol for the bioreactor level. In Figure 3 we assayed the effect of pH using only pure glycerol (G99) at the bioreactor level in order to optimize such parameter, and finally, a parameter analysis was carried out in a fed-batch system with both G99 and G65. It should be noted that the analysis was performed comparing G99 and G65 in all experiments (Tables 4 and 5).

2. The Materials and Methods section is, in my understanding very difficult to follow. It has several subheadings that could be merged or preferably, rearranged. For instance, instead of “jumping” between the flask, bioreactor, batch and feed-batch, in my opinion it would be clearer if the authors clearly explain the details of flask fermentation, and of bioreactor fermentation.

Thank you for the observation, we changed the Materials and methods subtitles and explanation in order to achieve a better understanding. Changes are shown in lines 102-168.

3. The authors provide insufficient information of the design of experiments used.
4. What were the tested ranges and corresponding minimal and maximum values?

We added a Table (Table 2) in order to be clearer about the range levels for the operational variables, line 168.

1. Moreover, and the more grievous, I could not find any statistical data of the obtained models. Were the models statistically significant?

Statistical analyzes are described in lines 229-239. Results only for the polynomial model for rTBL-1 is described in lines 356-375.

rTBL-1 = 234.29 + 4.43(X1) + 27.32(X2) - 10.62(X3) - 3.86(X4) - 47.72(X1)² - 13.62(X2)² + 29.74(X3)² - 12.99(X4)² + 0.11(X1 * X2) - 4.72(X1 * X3) - 4.90(X1 * X4)  - 3.90(X2*X3) + 5.30(X2*X4) - 11.08(X3*X4)

Other two models were obtained (not shown in the manuscript):

Dry cell weight = 96.35 + 0.79(X1) + 12.58(X2) - 1.94(X3) - 1.40(X4) - 15.87(X1)² + 2.80(X2)² + 9.11(X3)² - 14.32(X4)² + 0.03(X1*X2) - 2.29(X1*X3) + 0.28(X1*X4) - 3.94(X2*X3) + 0.63(X2*X4) - 4.74(X3 * X4)

Kla = 81.47 + 1.24(X1) + 5.49(X2) - 3.55(X3)- 3.97(X4)- 9.64(X1)² + 0.11(X2)² + 2.76(X3)² - 3.68(X4)²+ 1.86(X1*X2)- 3.71(X1*X3)+ 0.511(X1*X4)- 2.25(X2*X3) + 0.68(X2*X4) - 2.66(X3*X4)

1. Which parameters were significant?

The significant parameters from the Pareto diagram of the statistical analysis are described in lines 370-375.

For the production of rTBL-1, the significant parameters expressed by the Pareto diagram were agitation and temperature. On the other hand, for biomass production the significant parameter continued to be agitation, as well as temperature and pH for the oxygen transfer coefficient. The following figures show the four parameters but are not shown in the manuscript.

Figures are shown in the attached file.

1. What was the lack of fit value, the adequate precision and the coefficient of determination? Without them I am not able adequately evaluate the obtained models, therefore this information must not be published.

According to our statistical analysis and the experimental design carried out in this study, the data of adequate precision and the coefficient of determination for each model according Fisher's tests for both models determined that statistically describe the experimental data. However, the correlation coefficients of both models, the one that presented the greatest adjustment was the quadratic model with a value of 0.84 for rTBL-1. Values for rTBL-1 are shown in Table 3, line 368. The results for dry cell weight and Kla are also shown here but not in the manuscript.

Model Lack-of-Fit Statistics (rTBL-1)

Model Lack-of-Fit Statistics (DCW)

Model Lack-of-Fit Statistics (kla)

4. Point-by-point suggestions:

1. Line 81, please define all the acronyms consistently, including YPD (line 102). Please carefully revise the entire manuscript.

All non-common acronyms were defined.

1. Line 96, please provide additional details. What was the sodium hydroxide concentration used, and what do the authors mean with simple centrifugation?

The answer is shown in lines 95-96.

1. Line 98, how did the authors evaporate the ethanol? Please provide detailed information of the process, so it can be replicated by the readers.

Conditions are described in line 98.

2. Section 2.2, has in my opinion some clarity issues. Were the cultures performed in static conditions? If not, what was the shaking speed? In addition, please insert a space between different units, as an example: g L-1. Furthermore, percentage and degrees Celsius are units, thus they should be separated from its numerical value.

Cultures were performed under shaking conditions (line 200 rpm). Corrections were done along the text. Sections 2.2 and 2.3 were modified in order to be clearer. Is described in line 103-169.

2. I beg your pardon but I am confused, how was the bioreactor inoculated: using the procedure described in the beginning of section 2.2 or 2.3?

The inoculum for the bioreactor was prepared as mentioned in Section 2.3, lines 139-143.

1. Line 123, how was the “30% of saturation" maintained? Higher shaking speed? Increased airflow? Injection of oxygen? Please provide additional details of the bioreactor process.

Through a cascade of air flow and agitation, described in line 143.

172. Line 128, the correct unit for g force is not "x g" but a g italicized (so it can be distinguished from grams). Please also revise in line 172.

Corrections were made, thank you.

1. Line 166, “A second order polynomial model provided the best fit to the experimental data obtained” in my opinion this information should be in the Results section.

Thank you, it was considered and included in results section, lines 366-370.

1. Line 176, just curious, so rTBL-1 has a histidine tag so it can be purified through Ni Sepharose, correct? Is all this detailed in reference [6]? If so, I think it should be mentioned and referenced in this section also.

Addition was made in line 174.

1. Line 181, how were the samples stored after freeze-dry?

Description is shown in line 180.

1. Line 190, ImageJ software can, and should be adequately cited, please see: https://imagej.net/Citing

Reference 19 was included in lines 609-610.

1. Line 222, please define the unit: WCWL. In addition, can the author please explain what does the symbol “»” mean?

WCW L^-1 is wet cell weight for liter, WCW was defined properly in line 146. The symbol “»” is major or equal that, we changed for >.

1. Statistical analysis details must be adequately described in the Materials and Methods section.

Statistical analyzes are described in lines 230-240.

1. Line 239, “Small letters” or different letters represent significant differences? I am confused, please help me to understand.

Different letters represent significant differences, the text was corrected in lines 267, 329, 416.

1. Subsequently to the first appearance in the manuscript text of the full species name (i.e. Pichia pastoris), its abbreviated from must be consistently used (i.e. P. pastoris), including figure captions.

Corrections were made along the manuscript.

1. Figure 4, I beg your pardon, but a quick assessment of these figures shows that the design performed by the authors does not completely cover the values spectrum, in other words, the maximum value was not observed. Please comment.

Figure 4 was changed and the maximum and minimum values for rTBL-1, DCW and Kla under the optimal operating conditions of the experimental design (26 runs) is showed.

We thank very much to the reviewer for the comments and suggestions that helped to improve this work in an important way.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The manuscript entitled: “Optimization of a recombinant lectin production in Pichia pastoris using crude glycerol in a fed-batch system”, reference: processes-1193429

General comments:

I would like to congratulate the authors for the clear improvements performed on the manuscript. In particular, the Material and Methods section is now much easier to follow and understand the manuscript rational, and additional important results are presented. Therefore, the manuscript impact and clarity were considerably improved.

Nevertheless, and please correct me if am wrong interesting results depicted in Figure 7 are not discussed at all. Please comment.

Point by point suggestions:

Line 218, please always define the %, this is, if it is (w/v), (w/w) or (v/v), when applicable.

Line 222 and 226, please define acronyms DMEM and EDTA.

Figure 7, in my understanding the authors should keep consistency, thus if the authors use different letters to define statistical differences, the authors should not use asterisks for the same purpose. In other words, please choose asterisks or different letters, not both.