Performance of Recombinant Komagataella phaffii in Plant-Based Meat Flavor Compound-Leghemoglobin (LegH) Production through Fed-Batch Fermentations
, Anastasija Suleiko 1,2, Janis Edmunds Daugavietis 4, Elina Didrihsone 1, Janis Liepins 4, Emils Bolmanis 3, Oskars Grigs 1,2 and Juris Vanags 1,2,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript entitled " Production of plant-based meat flavor compound leghemoglobin (LegH) in recombinant Komagataella phaffii fed-batch fermentations" evaluated the potential of the alga as a candidate for antioxidant applications. This is an interesting topic, and the manuscript provided some interesting results. There are other commentsas follows:
Introduction: this part should be more focused according to the topic of the manuscript.
Line 344: why the glucose con. Was set at 5g/L, not 10 or others, in the next step? And at line 370-372, it is not reasonable the glucose con. is 10 g/L. I suggest an initial glucose con. experiment should be carried out.
The writings need to be revised, such as Line 145-147: g should be in italics; 200 mg should be “Two hundred”; ml should be mL.
Line 539-540: the LegH productivity in the published papers should be given such as in a table.
Line 674-788 Refernces: The first letter of every word in the referenced title names, upper or lower case, should be consistent.
Comments on the Quality of English LanguageThat's OK. Some errors should be correct.
Author Response
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Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe paper of Seleiko et al presents a very low degree of novelty, since the same Authors recently published a similar paper (Processes 2023, 11, 3215) where fed bach fermentations were used to express recombinant Soy leghemoglobin in Pichia pastoris (i.e Komagataella phaffii..it is the same organism !!) with better productivity (1.77 mg/g of WCW, and a total up to 600 mg/Liter) that those reported here ( 0.51 mg/g DCW and a total of about 60 mg/L..). It is noteworthing that others produced up to 3 gr/Liter of leghemoglobin in Pichia (Shao et al. Biores Technol 2022, 363, 127884). The use of glucose instead of glycerol did not improved the amount of recombinant protein !
Additional questions?
1) Why the Authors choose to use glucose instead of glycerol for batch and fed-batch? What is the advantage?
2) In Mat e Methods please explain what is the X-33 strain used (genotype..etc,) and which vector was used for the expression of Soy Leghemoglobin. In a previous paper they used the pPICZC vector (that contains also the alpha -factor leader peptide for secretion). It is the same plamid? if so why there is no secretion of the rec protein in the present paper?
3) Which is the sequence use for LegHemoglobin? (source, reference etc.?)
The cited Ref 35 is a preprint, please use the published paper ( Scientific Report 13, 15816, 2023)
4) In Fig. 3a why glucose increases again after 30 h ?
The reference list contains some errors:
-What is the meaning of the word Dostupné z present in all the citations??
-Ref 27 is an US-Patent and the Authors are : SHANKAR Smita and HOYT Martin Andrews
-Ref 30 the Journal is lacking
-Ref 35 change with Scientific Report
-Ref 40 the number of paper is lacking ( Processes (2023) 11, 3215)
Author Response
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Reviewer 3 Report
Comments and Suggestions for Authors1. In this manuscript, the author only provided the data of LegH production in modified medium, but did not provide the data of LegH production in BSM medium under the same conditions. It is suggested that the authors should provide data as the control.
2. For the optimization of glucose concentration, the author only did 20 g/L,10 g/L and 5 g/L. The author can try other glucose concentrations to increase the reliability of the data.
3. For the methanol phase, what is the reason for the authors to choose 1 g/L as the feeding rate?
4. The component of the medium used in this manuscript is complex, which is not suitable for large-scale industrial application. How can the author optimize its formula in the future?
5. In the result part, the authors only describe the rise and fall of the parameters in the diagram, and should also point out the reasons behind the changes.
6. The language of this manuscript should be polished further to make it coherent and concise.
Comments on the Quality of English LanguageThere are so many short paragraphs in the current manuscript.
Author Response
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Reviewer 4 Report
Comments and Suggestions for AuthorsIn the reviewed original research article, Suleiko et al. optimize fed-batch fermentations for the production of LegH with Komagataella phaffii (previously Pichia pastoris). Accordingly, the authors focused on the use of an ammonium hydroxide-free medium and the optimization of data-driven feeding feeding strategies. As a general remark, the article is professionally written, and the overall appearance is entirely satisfactory. Moreover, the work fits the journal's scope well and might be of interest to a broad readership of the journal. Nonetheless, some questions and unclarities arose while reviewing the manuscript, which should be addressed and/or answered by the authors before the article might be accepted (minor revision). I hope my comments below are helpful and will contribute to improving the quality of the manuscript.
General comments:
1. The overall quality of the manuscript is quite satisfactory. However, the use of some connectives (especially “although”) appears out of place in some cases. Hence, it is highly recommended that the authors finalise the manuscript with the help of an English native speaker.
2. During most of the experiments, the authors tracked off-gas composition and derived the OUR and RQ-values. Please add the corresponding equations to the manuscript. Furthermore, were also carbon balances calculated? If so, please add them to the manuscript and discuss them, even when they might not close perfectly. If not, why were the balances not calculated? Please commend and/or amend.
3. In general, during the discussion part, the obtained results from the optimized production process are briefly compared with data from the literature. However, for an improved overview, it would be much appreciated if the authors added a table comparing their process performance (regarding µmax, Yps and product titers) with data from the literature, listing the exact numbers.
Specific comments:
1. Line 256: what was the diameter of the used glas filter? What was the purpose of filtering the sample here? Was it to get rid of the cells and measure only the turbidity of the remaining medium salts/compounds? Please comment and amend.
2. Figure 1 (and the following): the units provided on the y-axis contain a dot “.” as a separator. Please remove this or format them to be one level higher, representing a multiplication sign.
3. Line 315: before stating the ethanol concentration a hypen “-“ is stated (this is also the case in other parts of the manuscript). This might be misinterpreted as a minus. Please remove the hyphens here and in other parts of the manuscript.
4. Lines 327-335: how were the biomass from substrate yield and the specific growth rate obtained? By linear regression from multiple data points or only from two data points? Please add this information to the Material and Methods section. Furthermore, for the specific growth rate µ, please state only the unit 1/h, g/g/h is correct, but g is biomass in both cases and can be shortened away.
5. Figure 2 and experiment 2: why are no OUR and RQ values provided here?
6. Table 2: why is qs,max (second row = sigma_max) provided with 2 decimals, while the other two values are provided with three? Please correct.
7. Line 394: it is said that the OUR reached >6 kg/m³/h. Can this value even be achieved in conventional large-scale fermenters with limited maximal power input (assuming maximally 3 kW/m³)? Please discuss issues in this context that might occur during scale-up with the optimized process in this regard. What would be strategies to tackle this?
8. Figure 4: Why is no OUR displayed (the RQ values suggest that off-gas data were available for this experiment)?
9. Lines 483-485: The authors write that “Furthermore, the amount of biomass produced at the end of the pre-feeding phase is enough to implement continuous feeding techniques, e.g., without posing significant technical constraints on the liquid supply equipment (peristaltic pump productivity).” This sentence is difficult to understand. Please explain and rephrase.
Comments on the Quality of English Language
See general comment no. 1
Author Response
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Round 2
Reviewer 1 Report
Comments and Suggestions for Authorsno more comments.
Comments on the Quality of English Languageno more comments.
Author Response
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Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsI am still convinced that this paper presents a very low degree of novelty. The only novelty is the changes of primary carbon source (glucose instread of glycerol) for fed-batch fermentation and a better description of the fermentation process, but the productivity of recombinant protein (soy leghemoglobin.) indicated in the Title is low and also lower that that reported previously by the same authors!
The introduction is too long and deals essentially with the relevance of expressing Soy Leghemoglobin, but if this is not the goal of this paper, the text should be modifyed and also the Title also should be changed..
The glucose was not measured on line, (in a continuous way) but at-line using a colorimetric analyzer with specific strips ( it is not clear in which way the blood sugar analyzer was adapted to this specific purpose...and this should be explained better), so the advantage to use glucose is not so relevant
The source of Leghemoglobin cDNA used should be clearly indicated and This phrase should be enclosed in Mat and Meth
"An artificial gene encoding the soy legHemoglobin sequence (GenBank Acc. NP_001235248.2) was designed by GenScript and synthesized by BioCat GmbH (Heidelberg, Germany)"
Author Response
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