Combining Xylose Reductase from Spathaspora arborariae with Xylitol Dehydrogenase from Spathaspora passalidarum to Promote Xylose Consumption and Fermentation into Xylitol by Saccharomyces cerevisiae
and Boris U. Stambuk 1,*Round 1
Reviewer 1 Report
This manuscript discusses research into genetically modifying Saccharomyces cerevisaie for xylose consumption and xylitol generation. The researchers achieved this by introducing genes for xylose reductase and xylitol dehydrogenase from two different yeast species of the Spathaspora genus. The introduction of these genes allowed S. cerevisiae to efficiently consume xylose and produce xylitol, which is a high value chemical that is of great interest for production in biorefineries. The manuscript is well written and research design is appropriate. This reviewer recommends that a few comments be addressed prior to acceptance for publciation:
- Although the work seems promising, can the authors comment on what level of xylitol titers or yields would make production economical in a biorefinery?
- This reviewer recommends including a brief explanation in the "Materials/Methods" section on how Km, Vmax, µmax, product yield, and productivity were calculated. An included list of equations would be beneficial.
Author Response
Response to reviewer # 1.
We thanks the comments and suggestions raised by the reviewer, which we think contributed to improve the manuscript.
Point 1. Although the work seems promising, can the authors comment on what level of xylitol titers or yields would make production economical in a biorefinery?
Response 1: This is an important issue regarding commercial xylitol production. Unfortunately nowadays almost all industrial xylitol produced in the world follows the chemical route, through catalytic hydrogenation of purified xylose from hemicellulosic hydrolysates, a process considered costly, non-environmental friendly, and energy-consuming. Approximately 80% of the total cost of xylitol production involves the expensive xylose purification and xylitol crystallization process, and the high price of xylitol in the market is probably directly related to the high cost of the chemical production process. Very few companies are adopting biotechnological routes for xylitol production, and thus our knowledge regarding costs and bottlenecks from an industrial perspective are not totally known. However, from previous work (mostly laboratory or small scale) it is obvious that high xylitol titers are needed in order to allow the fermented broths containing xylitol to be clarify, concentrated (>750 g/L), and cooled in order to favor xylitol crystallization, all contributing significantly to the overall costs of the product. This has been briefly discussed in lines 331-334 of the revised manuscript, including a couple of relevant references to the topic.
Point 2. This reviewer recommends including a brief explanation in the "Materials/Methods" section on how Km, Vmax, µmax, product yield, and productivity were calculated. An included list of equations would be beneficial.
Response 2: We agree with the reviewer, and a brief explanation on how µmax (lines 104-105 of revised manuscript), Km and Vmax (lines 179-181), and product yield and productivity (lines 187-191) were calculated, were included in the revised version of the manuscript.
All introduced changes are highlighted in yellow in the revised version of the manuscript.
Reviewer 2 Report
General comments:
The manuscript addresses some relevant aspect related to the potential of yeasts to metabolize both pentoses and hexoses. It is rather short but shows some novel data.
Some additional experiment on pentose/hexose co-fermentation would be necessary, since this is the main claimed goal of the study. This can easily be done as such experiment will take only about 2-3 days (as also seen in Fig. 1 and Fig.2).
Materials and Methods:
For the batch assays (bottles), I miss some information about the initial pH and if the pH value remained constant. Some acids (acetic acid) were produced; also other parameters could affect the pH value.
Results and Discussion:
The sentence on L. 189-192 has no meaning. Please revise and rewrite. Several other sentences have grammatical errors (e.g., L. 242-245, L. 295, etc.) that need to be revised.
I miss some mass balance and brief discussion on the product recovery; e.g. 20 g/L xylose is fed (Figure 1) as substrate and consumed but only 2 g/L xylose is obtained. Is that a reasonable yield or expected theoretical value?
References:
Although this is not a review paper, 30% of the manuscript are references (>80 references). This is not justified and should be reduced by at least 1/3.
For example:
L.49-51: There is no beed to cite 4 references just to explain that an economically feasible process needs to be developed.
or
L.298: 8 references cited just to say that the present study reaches high(er) productivities, while those references are not further used at all in the Discussion.
or
L.299-301: there is no need to cite as much as five (randomly selected?) references just to say that further research is required on this topic.
Etc.
Author Response
Response to reviewer # 2.
We thank the comments and suggestions raised by the reviewer, which we think certainly contributed to improve the manuscript!
Point 1. Some additional experiment on pentose/hexose co-fermentation would be necessary, since this is the main claimed goal of the study. This can easily be done as such experiment will take only about 2-3 days (as also seen in Fig. 1 and Fig. 2).
Response 1: Although we had some problems due to the current COVID-19 pandemic and all the security and restrictions imposed to enter into the university and laboratory, we were able to perform the requested experiment, and the results of 2% glucose/xylose co-fermentation by our best yeast strain are presented in the new Figure 3 and lines 316-331 (as well as lines 32-34 of the Abstract) of the revised version of the manuscript.
Point 2. For the batch assays (bottles), I miss some information about the initial pH and if the pH value remained constant. Some acids (acetic acid) were produced; also other parameters could affect the pH value.
Response 2: The initial pH of the medium (pH 5.0) was already described in line 97 of our original manuscript. The reviewer is right, the pH of the medium dropped a little (to pH 4.5) when no acetate was produced during fermentation (lines 296-297 of our revised version of the manuscript), but changed more and dropped into pH 3.5 when acetate was produced. This information is now in lines 289-290 (fermentation of 2% xylose by strain ASY-2 transformed with the pPGK-SaXYL1 and pTEF‑SpXYL2.2 plasmids), and in lines 327-328 (co-fermentation of 2% glucose plus 2% xylose by the same strain) of our revised version of the manuscript.
Point 3. The sentence on L. 189-192 has no meaning. Please revise and rewrite. Several other sentences have grammatical errors (e.g., L. 242-245, L. 295, etc.) that need to be revised.
Response 3: These sentences, as well as several other parts of the new version of the manuscript, have been revised and improved (lines 77-79, 81-82, 193-199, 236, 242-246, 256-259, 296) in our new version of the manuscript.
Point 4. I miss some mass balance and brief discussion on the product recovery; e.g. 20 g/L xylose is fed (Figure 1) as substrate and consumed but only 2 g/L xylose is obtained. Is that a reasonable yield or expected theoretical value?
Response 4: We belief that the reviewer wanted to say “but only 2 g/L xylitol is obtained”. In the case of cell growth on xylose (the data shown in Figure 1), obviously a lot of carbon is directed into biomass formation (probably by respiration of the carbon source) during incubation in aerobic conditions with shaking (160 rpm) in cotton plugged Erlenmeyer flasks filled to 1/5 of the volume with medium, and that is the reason why the xylitol yields under this conditions are low and corresponding to just 17-25% of the maximal theoretical yield. However, latter on we show that under fermentation conditions (Figure 2) the best strain reached almost 70% of the theoretical yield (a value similar or even higher to many other publications in the field, as stated in lines 303-306). We hope that we have cleared this issue by indicating what is the expected maximal theoretical yield (lines 306-310) and also the fact that biomass was formed during incubation in 20 g/L xylose shown in Figure 1 (lines 259-260). All other metabolite yields (xylitol, glycerol, acetate, ethanol) based on the amount of sugar consumed are shown in Table 6 and lines 320-326 (for the co-fermentation).
Point 5. Although this is not a review paper, 30% of the manuscript are references (>80 references). This is not justified and should be reduced by at least 1/3.
For example:
L.49-51: There is no beed to cite 4 references just to explain that an economically feasible process needs to be developed....... etc..... etc....etc.....
Response 5: As requested by this reviewer, we made a significant reduction in the number of references, from the original 81 to just 56 (31% reduction), but we had to include 3 new references to answer the request of reviewer # 1 (the 3 last references in the list, see also lines 306-310 and 331-334 in our revised version of the manuscript). Consequently, all references numbering in the text was changed accordingly.
All introduced changes are highlighted in yellow in the revised version of the manuscript.
Round 2
Reviewer 2 Report
Most comments have been suitably addressed.
