Novel In Vitro Multienzyme Cascade for Efficient Synthesis of d-Tagatose from Sucrose

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Page 1. Please provide the full name of the abbreviation MCTS.

Page 2. Wrong link 1615 instead of 16. Same for 2222 link. Same for 2929 link on page 4 and 3030 link on page 5.

Page 3. Table 1. A very small confidence interval value of 0.03 for activity 112.79.

Page 6. Fig 6. “ADP/Sucrose concentration” in fig 6e axis title, but “e) ADP concentrations” in the title of the figure 6.

Page 8. Section 3.4. “[Error! Reference source not found.]” at the end of the section.

Page 9. There is no concentration of ATP in the method of determining FRK activity.

Author Response

#Reviewer 1

Comment 1. Page 1. Please provide the full name of the abbreviation MCTS.

Response: Done. Thank you for the reviewer’s constructive suggestion. We have added the full name of the abbreviation MCTS in the revised manuscript: multienzyme cascade route for d-tagatose synthesis from sucrose (MCTS) (page 1, line 10)

Comment 2. Page 2. Wrong link 1615 instead of 16. Same for 2222 link. Same for 2929 link on page 4 and 3030 link on page 5.

Response: Done. We have changed [1615] to [15,16], [2222] to [22], [2929] to [29], [3030] to [30], as seen in page 2 (line 51 and 62) and page 4 (line 137 and 144) in the revised manuscript.

Comment 3. Page 3. Table 1. A very small confidence interval value of 0.03 for activity 112.79.

Response: Done. We are sorry for that we calculated a wrong error value. We have corrected the significant figure and error values in Table 1 in the revised manuscript.

Enzyme name	NCBI accession number	Specific activity(U·mg−1)
BaSP CaFRK CaF6PE MmT6PP	WP_011742626.1 KHD36265.1 WP_014433578 WP_013296249.1	84 ± 2 113 ± 3 (2 ± 0.5) × 10–2 (1 ± 0.2) × 10–1

 

Comment 4. Page 6. Fig 6. “ADP/Sucrose concentration” in fig 6e axis title, but “e) ADP concentrations” in the title of the figure 6.

Response: Done. Thanks for the reviewer’s constructive suggestion. We have changed the “e) ADP concentrations” to “f) ADP:sucrose ratio” in Fig. 6 in the revised manuscript (Page 6, line 178).

Comment 5. Page 8. Section 3.4. “[Error! Reference source not found.]” at the end of the section.

Response: Addressed. We have added the literature [37] at the end of the section 3.4 in the revised manuscript (Page 8, line 305). The reference [37] is as follows:

37. Gao, H.; Li, M.; Wang, Q.; Liu, T.; Zhang, X.; Yang, T.; Xu, M.; Rao, Z. A high-throughput dual system to screen polyphosphate kinase mutants for efficient ATP regeneration in L-theanine biocatalysis. Biotechnol Biofuels Bioprod. 2023, 16(1), 122.

Comment 6. Page 9. There is no concentration of ATP in the method of determining FRK activity.

Response: Done. We have added the concentration of ATP (10 mM) in the method of determining CaFRK activity in the revised manuscript (Page 9, line 318).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors suggest a new, five-enzyme system to produce D-tagatose from a natural substrate, sucrose in this case. They convincingly show that this system shows definite advantages over the known ones and promising results for biotechnological application. The major remark is that some results look like preliminary.

Optimization of the buffer composition seems incomplete. It remains unclear whether a different  combination of PBS and Tris buffers could result in a higher yield. Different concentrations should be tested.

Some metal-activated enzymes show enhanced activity with a combination of metal cofactors, e. g. Mn + Mg. Were such combinations tested?

It remains somewhat unclear whether the yields reported refer to their limiting values at infinite time or to a fixed time point. In the latter case, effects on yield may result from changed kinetics of the reaction.

Minor remarks:

Abscissa axis ticks are not applicable in Figs 4b, 5, 6c-e and should be removed.

Numerical values should be rounded off to significant digits: 84.48±1.69 à 84 ± 2. The error value in  112.79 ± 0.03 is unrealistic. Rates cannot be measured with such a precision.

Units are separated by a space from the numerical values.

Comments on the Quality of English Language

Ther are only minor problems with English.

Author Response

#Reviewer 2

The authors suggest a new, five-enzyme system to produce D-tagatose from a natural substrate, sucrose in this case. They convincingly show that this system shows definite advantages over the known ones and promising results for biotechnological application. The major remark is that some results look like preliminary.

Comment 1. Optimization of the buffer composition seems incomplete. It remains unclear whether a different combination of PBS and Tris buffers could result in a higher yield. Different concentrations should be tested.

Response: Done. Thanks for the reviewer’s constructive suggestion. As shown in Fig. 6c, we have supplemented the investigation of the different mixed buffers (50 mM Tris-HCl + different concentrations of PBS buffer (0, 5, 10, 20, 30, and 50 mM), and the optimal buffer is 10 mM PBS buffer + 50 mM Tris-HCl buffer (pH 7.5). We also discussed the possible reason, as seen in section 2.4.2 in the revised manuscript (Page 6, line 176-177, 197-200).

Comment 2. Some metal-activated enzymes show enhanced activity with a combination of metal cofactors, e. g. Mn + Mg. Were such combinations tested?

Response: Done. We have tested the effects of the combinations of metal cofactors (e.g., Mn²⁺ + Mg²⁺, Mn²⁺ + Co²⁺, and Mg²⁺ + Co²⁺). As shown in Fig. 6d in the revised manuscript, because of better effects of some bivalent metal ions (e.g., Mn²⁺, Mg²⁺, and Co²⁺,), the synergistic effect of mixed metal ions (e.g., Mn²⁺ + Mg²⁺, Mn²⁺ + Co²⁺, and Mg²⁺ + Co²⁺, each ion concentration was 2 mM) was investigated. However, mixed ions showed lower d-tagatose yields than that with single ion Mn²⁺ (4 mM) (Page 6, line 208-211).

Comment 3. It remains somewhat unclear whether the yields reported refer to their limiting values at infinite time or to a fixed time point. In the latter case, effects on yield may result from changed kinetics of the reaction.

Response: Done. Thanks for the reviewer’s constructive suggestion. In the revised manuscript, we have supplemented the time course of the MCTS route for d-tagatose yield using 10 mM sucrose as substrate. As shown in Fig. 7, the d-tagatose yield dramatically increased to 52% in the first two hours, then slowly increased to the highest (82.3%) at 24 h. However, there was approximately 13% unutilized fructose in the mixture. As shown in Fig. S2, in addition to fructose, other intermediate product (e.g., F6P) was also detected in the reaction mixture, suggesting the cascade enzymes (e.g., CaFRK, and CaF6PE) need to be further improved in our future work. (Page 7, line 226-238).

Minor remarks:

Comment 4. Abscissa axis ticks are not applicable in Figs 4b, 5, 6c-e and should be removed.

Response: Done. Thank you very much for pointing out the irregular writing of the graphs. We have removed the axis ticks in Figs 4b, 5, 6c-e in the revised manuscript.

Comment 5. Numerical values should be rounded off to significant digits: 84.48±1.69 à 84 ± 2. The error value in  112.79 ± 0.03 is unrealistic. Rates cannot be measured with such a precision.

Response: Done. Thanks for the reviewer’s constructive suggestion. We are sorry for that we calculated a wrong error value of “112.79 ± 0.03”. We have corrected the significant figure and error values in Table 1 in the revised manuscript (as follows).

Enzyme name	NCBI accession number	Specific activity(U·mg−1)
BaSP CaFRK CaF6PE MmT6PP	WP_011742626.1 KHD36265.1 WP_014433578 WP_013296249.1	84 ± 2 113 ± 3 (2 ± 0.5) × 10–2 (1 ± 0.2) × 10–1

 

Comment 5. Units are separated by a space from the numerical values.

Response: Done. Thank you very much for pointing out the irregular writings. We have corrected these writings throughout the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

 

In this manuscript, the authors report a new biocatalytic pathway to produce D-tagatose from sucrose using 4 distinct recombinant enzymes: a sucrose phosphorylase, a fructokinase, a D-fructose 6-phosphate 4-epimerase and a D-tagatose 6-phosphate phosphatase. In addition to this enzymatic cascade, authors implemented a regeneration system from ATP using polyphosphate kinase. The full enzymatic system allowed the authors to reach a 72.4% conversion rate with a productivity of 0.27 g.L^-1.h^-1. The proof of concept was made at 10 mM and 50 mM at a milliliter scale.

Overall, the experiments were carefully performed, the methods are well described, and the results were well presented. However, major revision must be addressed to improve the quality of the manuscript:

1.      Parts of the manuscript are difficult to understand (specially in the introduction) because of incorrect grammar which makes it unpublishable as is. Therefore, the manuscript must be read and corrected by a native English speaker.

2.      Some references are not reported well in the text: "the generation of ATP from ADP using acetyl phosphate as a phosphate donor [2222].", "cascade routes for the biosynthesis of D-tagatose were attempted [15,1615]." or " NADPH has a characteristic absorption peak at 340 nm, and ATP concentration is proportional to NADPH content [Error! Reference source not found.]."

3.      Authors should not use the term "Yield" as they are not isolating D-tagatose. Therefore, they should use the term "conversion".

4.      Page 3, authors say "As shown in Fig 2, the SDS-PAGE results showed that the molecular weights of BaSP, CaFRK, CaF6PE, MmT6PP were 60.79 kDa, 36.30 kDa, 50.32 kDa, and 28.59 kDa, respectively.". However, SDS PAGE is not precise enough to measure molecular weight but it can confirm an expected molecular weight. This sentence should therefore be rephrased.

5.      Page 6, did the authors check if the cascade is producing acidity or basicity? It could explain why PBS + Tris is a good buffer combo as the pH coverage is better (in addition to their conclusions). 

6.      The authors should show the HPLC chromatogram of the reaction after optimization in order to determine what are the remaining 20% byproducts. Also, the authors should perform the kinetic of the reaction over the 24 hours periods as they are reporting a productivity. A single point is not enough to report such information. 

 

Comments on the Quality of English Language

Parts of the manuscript are difficult to understand (specially in the introduction) because of incorrect grammar which makes it unpublishable as is. Therefore, the manuscript must be read and corrected by a native English speaker. 

Author Response

# Reviewer 3

In this manuscript, the authors report a new biocatalytic pathway to produce D-tagatose from sucrose using 4 distinct recombinant enzymes: a sucrose phosphorylase, a fructokinase, a D-fructose 6-phosphate 4-epimerase and a D-tagatose 6-phosphate phosphatase. In addition to this enzymatic cascade, authors implemented a regeneration system from ATP using polyphosphate kinase. The full enzymatic system allowed the authors to reach a 72.4% conversion rate with a productivity of 0.27 g.L^-1.h^-1. The proof of concept was made at 10 mM and 50 mM at a milliliter scale.

Overall, the experiments were carefully performed, the methods are well described, and the results were well presented. However, major revision must be addressed to improve the quality of the manuscript:

Comment 1. Parts of the manuscript are difficult to understand (specially in the introduction) because of incorrect grammar which makes it unpublishable as is. Therefore, the manuscript must be read and corrected by a native English speaker.

 

Response: Done. The manuscript has been corrected by a native English speaker (by shinewrite company). The incorrect grammar and sentence have been corrected.

Comment 2. Some references are not reported well in the text: "the generation of ATP from ADP using acetyl phosphate as a phosphate donor [2222].", "cascade routes for the biosynthesis of D-tagatose were attempted [15,1615]." or " NADPH has a characteristic absorption peak at 340 nm, and ATP concentration is proportional to NADPH content [Error! Reference source not found.]."

Response: Done. Thank you very much for pointing out the irregular writings. We have change “1615” to “15,16”, “2929” to “29”, “3030” to “30”. In addition, we have added the literature [37] at the end of the section 3.4 in the revised manuscript (Page 8, line 305). The reference [37] is as follows:

37. Gao, H.; Li, M.; Wang, Q.; Liu, T.; Zhang, X.; Yang, T.; Xu, M.; Rao, Z. A high-throughput dual system to screen polyphosphate kinase mutants for efficient ATP regeneration in L-theanine biocatalysis. Biotechnol Biofuels Bioprod. 2023, 16(1), 122.

Comment 3. Authors should not use the term "Yield" as they are not isolating D-tagatose. Therefore, they should use the term "conversion".

Response: We have supplemented the calculated formula for D-tagatose in the revised manuscript (Page 8-9, line 306-309).

 

Actually, herein the yield refers that the amount of produced d-tagatose divides that of initial sucrose. But the “conversion” generally means that the amount of consumed sucrose divides that of initial sucrose. Therefore, we think it’s more appropriate to use “yield” here.

Comment 4. Page 3, authors say "As shown in Fig 2, the SDS-PAGE results showed that the molecular weights of BaSP, CaFRK, CaF6PE, MmT6PP were 60.79 kDa, 36.30 kDa, 50.32 kDa, and 28.59 kDa, respectively.". However, SDS PAGE is not precise enough to measure molecular weight but it can confirm an expected molecular weight. This sentence should therefore be rephrased.

Response: Done. Thanks for the reviewer’s constructive suggestion. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the molecular weights of BaSP, CaFRK, CaF6PE, MmT6PP were approximately 61, 36, 50, and 29 kDa, respectively (Fig. 2).” (Page 3, line 106-108)

Comment 5. Page 6, did the authors check if the cascade is producing acidity or basicity? It could explain why PBS + Tris is a good buffer combo as the pH coverage is better (in addition to their conclusions).

Response: Done. The pH value of the whole reaction process was monitored, and the reaction mixture was basically maintained at pH 7.5. As shown in Fig. 6c, we have supplemented the investigation of the different mixed buffers (50 mM Tris-HCl + different concentrations of PBS buffer (0, 5, 10, 20, 30, and 50 mM), and the optimal buffer is 10 mM PBS buffer + 50 mM Tris-HCl buffer (pH 7.5). Actually, in the combination buffer (PBS + Tris-HCl), PBS can provide phosphate ions as the donor for sucrose phosphorylase, and promote the synthesis of fructose. However, excess phosphate ions may inhibit the activity of MmT6PP for T6P dephosphorylation to generate d-tagatose. Therefore, a mixed buffer (10 mM PBS and 50 mM Tris-HCl) showed best effect on the d-tagatose yield in our study. (Page 6, line 200-203)

Comment 6. The authors should show the HPLC chromatogram of the reaction after optimization in order to determine what are the remaining 20% byproducts. Also, the authors should perform the kinetic of the reaction over the 24 hours periods as they are reporting a productivity. A single point is not enough to report such information.

Response: Done. Thanks for the reviewer’s constructive suggestion. We have supplemented the HPLC chromatogram of the reaction after optimization in the revised manuscript. As shown in Fig. S2, the remaining 20% byproducts include about 13% fructose and 7% other byproducts (e.g., F6P). In addition, as shown in Fig. 7, we have investigated the time course of d-tagatose yield using 10 mM sucrose as substrate, and the reaction reached the highest d-tagatose yield at 24 h.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors adequately addressed previous remarks and substantially improved the manuscript.

Comments on the Quality of English Language

There are only minor problems with English.

Author Response

Comment 1. Comments and Suggestions for Authors

The authors adequately addressed previous remarks and substantially improved the manuscript.

Response: Thanks for the reviewer’s approval of our revised manuscript.

Comment 2. Comments on the Quality of English Language: there are only minor problems with English.

Response: Done. We have further edited our English language in our revised manuscript 2.