Engineering an Artificial Pathway to Improve the Bioconversion of Lysine into Chiral Amino Alcohol 2-Hydroxycadaverine Using a Semi-Rational Design
, Shujian Xiao 1,†, Qing Luo 2, Bangxu Wang 1, Rumei Zeng 1, Liming Zhao 3 and Jiamin Zhang 1Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript entitled "Engineering an artificial pathway for improving the bioconversion of lysine into an chiral amino alcohol 2-hydroxycadaverine by semi-rational design" proposes a whole-cell 2-step biosynthetic pathway towards 2-hydroxycadaverine. The substrate, lysine, is first converted into 3-hydroxylysine by a lysine hydroxylase (KDO), then 3-hydroxylysine is decarboxylated into 2-hydroxycadaverine by a yridoxal phosphate dependent decarboxylase (DC), both expressed by E. coli. Keypoints in the study are 1) the suppression of the expression of lysine decarboxilase cadA, which would convert lysine into useless 1,5-pentanediamine; 2) engineering the yiridoxal phosphate dependent decarboxylase DC to increase its affinity for 3-hydroxylysine.
I have some points of major concern:
- the reported production of the target compound seems low, although higher when using the engineered enzyme vs the wild type: its value is 195 mg/L after 12 h, starting from a concentration of substrate of 4 g/L (Table 2 in the manuscript). What explanation could the authors provide for such low conversion? Especially when compared to the high values reported in the cited literature (e.g., refs 21 and 27). Does the claimed improvement in terms of avoided enzyme purification justify such significant drop in the yield?
- the results reported in Table 2 are not completely clear. Does the reported concentration of 3-hydroxylysine refer to the concentration after 12 h of enzymatic reaction? If so, it would be of use to report the concentration of remaining lysine as well (since it was analysed, as reported in paragraph 2.7), to understand. The amount of remaining lysine could allow to understand the reasons for the low conversion into the desired 2-hydroxycadaverine: in particular, have the authors verified that the enginereed decarboxylase DC is actually selective towards 3-hydroxylysine, and no decarboxylation of lysine occurs?
- in the materials and methods section, the biocatalytic conversion of lysine (the main reaction of the paper) is not described. One paragraph should be dedicated to the employed procedure.
- as claimed in the title, 2-hydroxycadaverine is a chiral amino alcohol. However, no enantiomeric excess is reported, neither for the final product, nor for the intermediate 3-hydroxycadaverine. The authors should include chiral HPLC analysis, or optical rotation, to assess the enantiomeric purity of the product.
Moreover, there are some minor issues:
- in Figure 1, the position of the OH group in 2-hydroxycadaverine is wrong (it is in position 1, should be in position 2). Furthermore, stereogenic markers should be employed to specify the configuration of chiral centres.
- why the docking of KDO and lysine has been performed, if KDO is not an engineered enzyme? Is it because it has never been studied with lysine as substrate? In any case, the authors should provide stronger justification for this part of the study.
- at page 10, lines 318-319, the authors claim that the artificial pathway is "greener and healthier". On what basis is this claim substantiated? A quantitative or semi-quantitative ground should be provided, through comparison of the methodology with previously reported ones, by applying at least one green metric. Otherwise, the sentence should be removed.
the authors should justify why the
Author Response
Reviewer #1: The manuscript entitled "Engineering an artificial pathway for improving the bioconversion of lysine into an chiral amino alcohol 2-hydroxycadaverine by semi-rational design" proposes a whole-cell 2-step biosynthetic pathway towards 2-hydroxycadaverine. The substrate, lysine, is first converted into 3-hydroxylysine by a lysine hydroxylase (KDO), then 3-hydroxylysine is decarboxylated into 2-hydroxycadaverine by a yridoxal phosphate dependent decarboxylase (DC), both expressed by E. coli. Keypoints in the study are 1) the suppression of the expression of lysine decarboxilase cadA, which would convert lysine into useless 1,5-pentanediamine; 2) engineering the yiridoxal phosphate dependent decarboxylase DC to increase its affinity for 3-hydroxylysine.
Comment 1: The reported production of the target compound seems low, although higher when using the engineered enzyme vs the wild type: its value is 195 mg/L after 12 h, starting from a concentration of substrate of 4 g/L (Table 2 in the manuscript). What explanation could the authors provide for such low conversion? Especially when compared to the high values reported in the cited literature (e.g., refs 21 and 27). Does the claimed improvement in terms of avoided enzyme purification justify such significant drop in the yield?
Response: Thank you for your comments. The determination of DCCp activity was carried out in this study. The wild-type DCCp showed a Km value of 20.72 mM, a Kcat value of 1.86 s−1 and a Kcat/Km value of 0.0898 mM−1s−1 when 3-hydroxylysine is used as the substrate. Due to low activity of DCCp toward 3-hydroxylysine, the conversion rate of 2-hydroxycadaverine is very low. This result is consistent with the results reported by Baud et al. (21), who also reported the conversion rate of DCCp was 29%.
The enzyme activity assay showed that the DCCp enzyme has no activity against lysine. Because the decarboxylase activity of CadA towards 3-hydroxylysine is much lower than towards L-lysine (27). So Li et al. conducted a 2-hydroxycadaverine cascade reaction (27).
- Baud D, Peruch O, Saaidi P-L, Fossey A, Mariage A, Petit J-L, et al. Biocatalytic Approaches towards the Synthesis of Chiral Amino Alcohols from Lysine: Cascade Reactions Combining alpha-Keto Acid Oxygenase Hydroxylation with Pyridoxal Phosphate-Dependent Decarboxylation. Advanced Synthesis & Catalysis. 2017;359(9):1563-9.
- Li Y, Zhang A, Hu S, Chen K, Ouyang P. Efficient and scalable synthesis of 1,5-diamino-2-hydroxy-pentane from L-lysine via cascade catalysis using engineered Escherichia coli. Microbial cell factories. 2022;21(1):142.
Comment 2: The results reported in Table 2 are not completely clear. Does the reported concentration of 3-hydroxylysine refer to the concentration after 12 h of enzymatic reaction? If so, it would be of use to report the concentration of remaining lysine as well (since it was analysed, as reported in paragraph 2.7), to understand. The amount of remaining lysine could allow to understand the reasons for the low conversion into the desired 2-hydroxycadaverine: in particular, have the authors verified that the enginereed decarboxylase DC is actually selective towards 3-hydroxylysine, and no decarboxylation of lysine occurs?
Response: Thank you for your comments. The determination of DCCp activity was carried out in this study. The enzyme activity assay showed that the DCCp enzyme has no activity against lysine. The wild-type DCCp showed a Kcat/Km value of 0.0898 mM−1s−1 when 3-hydroxylysine is used as the substrate.
In line 278 on page 9, we have added one sentence “The enzyme activity assay showed that the DCCp enzyme has no activity against lysine”.
Comment 3: In the materials and methods section, the biocatalytic conversion of lysine (the main reaction of the paper) is not described. One paragraph should be dedicated to the employed procedure.
Response: Thank you for your comments. We have made some revisions in the introduction.
In lines 131-145 on page 4：We have added one paragraph: "The production of 2HyC in recombinant E. coli ML101 and ML102 was conducted in 250 mL flasks. Strains ML101 and ML102 from glycerol stock were streaked onto LB agar plates supplemented with 100 μg/mL Amp and grown overnight in an incubator at 37 °C. Three colonies were selected from an agar plate and grown in 5 mL of LB medium supplemented with 100 μg/mL Amp in an orbital shaker operated overnight at 220 rpm and 37 °C. The culture was adopted as the seed inoculum to initiate fermentation in triplicate. The fermentation medium was inoculated with the seed broth at 1% dilution and grown in 40 mL LB medium (composing 10 g/L tryptone, 5 g/L yeast extract, 0.1 mM pyridoxal phosphate, 0.5 g/L K2PO4·3H2O, 3 g/L KH2PO4, 0.75 g/L FeSO4·7H2O, 15 g/L glucose, 20 mM α-ketoglutarate and 100 μg/mL Amp). The initial pH was adjusted to 7.0. After the OD600 reached 0.6, 4 g/L L-lysine was added to the fermentation medium as substrate at this time, IPTG was added to a final concentration of 0.5 mM to induce the protein expression. The flasks were placed in an orbital shaker operated at 220 RPM and 30 °C. Experiments were performed in triplicate and samples were taken at 12 and 24 h for 2HyC analysis.”
Comment 4: As claimed in the title, 2-hydroxycadaverine is a chiral amino alcohol. However, no enantiomeric excess is reported, neither for the final product, nor for the intermediate 3-hydroxycadaverine. The authors should include chiral HPLC analysis, or optical rotation, to assess the enantiomeric purity of the product.
Response: Thank you for your comments. We have made revisions based on your recommendation.
In line 158-160 on page 6, "Samples were analyzed by HPLC with a C18 column (4.6 × 250 mm) as described by Cheng et al. (35). ” was revised as “Samples were analyzed by HPLC with a C18 column (4.6 × 250 mm) and a Chirex®3126 (D)-penicillamine LC column (4.6 × 250 mm, Phenomenex, USA) as described by Cheng et al. (35).”
Comment 5: In Figure 1, the position of the OH group in 2-hydroxycadaverine is wrong (it is in position 1, should be in position 2). Furthermore, stereogenic markers should be employed to specify the configuration of chiral centres.
Response: Thank you for your comments. Thank you for your comments. Sorry, we made such a mistake. We have made revisions in Fig. 1A.
Fig. 1
Comment 6: Why the docking of KDO and lysine has been performed, if KDO is not an engineered enzyme? Is it because it has never been studied with lysine as substrate? In any case, the authors should provide stronger justification for this part of the study.
Response: Thank you for your comments. So far, there were no any reference reporting the molecular docking between lysine and KDOKr. To get better insight into the interaction between KDOKr and L-lysine, a molecular docking study was performed. As shown in Fig. 2C and Fig. 2D, the docking results of KDOKr and L-lysine showed that L-lysine formed four hydrogen bonds with the side chain E123, G124, S144 and R305. In addition, the binding of KDOKr with L-lysine could be attributed to the hydrophobic interaction because the strong hydrophobic interactions of L-lysine with N93, M132, Q143 and Q145 were formed. Molecular docking results showed that L-lysine could bind to the hydrophobic cavity of KDOKr due to the formation of hydrogen bonds and hydrophobic interactions with surrounding amino acid residues.
Comment 7: At page 10, lines 318-319, the authors claim that the artificial pathway is "greener and healthier". On what basis is this claim substantiated? A quantitative or semi-quantitative ground should be provided, through comparison of the methodology with previously reported ones, by applying at least one green metric. Otherwise, the sentence should be removed.
Response: Thank you for your comments. We have deleted this sentence in the revised manuscript.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript “Engineering an artificial pathway for improving the bioconversion of lysine into…” deals with the two-enzyme-step conversion of lysine to 2-hydroxycadaverine, focusing particularly on the modification of each of the two enzymes through rational enzyme engineering. Technically the contribution is fine. The major criticisms are 1) the confusion between the term “mutation” and “substitution” and 2) excessive significant digits (see detailed comments below). Both are easily rectified.
Line 21: italicize Escherichia coli
Lines 23, 89: change “yridoxal” to “pyridoxal”
Line 24: italicize Chitinophage pinensis
Lines 28, 97: The statement “0.63-fold” makes no sense. A fold difference less than one would imply that the value decreased. In other words, if the starting value is 100, then a 0.63-fold increase would mean the value is now 63. Please use percentage increase. For example, if the starting value is 100, and the new value is 125, then the percentage increase is (125-100)/100 = 25%.
Line 28: Very rarely are measurements made with sufficient precision to justify more than 2 or maybe 3 significant digits on any numeric value presented. Therefore, “358.64” is simply not statistically possible. The authors here should write “359” or “360” (3 or 2 significant digits). Similarly, “53.55%” should be written “54%”. The authors should follow the same rule elsewhere (lines 98, Table 2, lines 204-205, 267, 268, Table 3, etc.).
Line 42: change “its” to “their”
Line 43: I am not sure that the authors mean “medical aesthetics”. Actually, a whole lot of fields are listed that aren’t really necessary. It is reasonable to just state “…are widely used in diverse fields (13).”
Line 53: change “opens up new ideas for the production…” to “facilitated the production…”
Line 63: change “an important universal molecules used as an adjuvant or ligand…” to “important adjuvants and ligands…”
Line 73: change “amnio” to “amino”
Line 69: change “AAs have the characteristics of low toxicity, room temperature curing and low viscosity, and can also be used as corrosion inhibitors and carbon dioxide absorbents” to “AAs have low toxicity, low viscosity, inhibit corrosion, absorb carbon dioxide and cure at room temperature.”
Line 90: the statement “overexpression was successfully established in E. coli” is a result and does not belong in the Introduction section.
Figure 1A: The stoichiometric equation should include all substrates and products (including water, CO2, etc.)
Lines 112, 114: indicate EC number for each of the two enzymes.
Line 129: Indicate how much lysine was added. That is, “0.5 mM IPTG and ??? mM lysine were added….”
Lines 134: change “clone” to “clones”
Lines 179: change “hydroxycaverine” to “hydroxycadaverine”
Table 2 should not appear before the first text in the Results. This is a publisher’s problem, not an authors problem, but I ask the authors to insure that the final print version has Table 2 appearing after the first paragraph of the Results section.
Figure 1A. The authors note that the cadA gene, which codes for lysine decarboxylase, was knocked. It would be beneficial to include this knocked out step in Figure 1A (for example, showing that a red “X” through the conversion that is knocked out.
Many of the commas are misprinted somehow. For example, Line 228 after “Asn93” and “Met132” the commas are somehow off kilter.
Line 204: I suggest the word “demonstrated” rather than “proved”.
Line 208: change “the molecular docking…” to “a molecular docking…”
Line 238: some redundancy here. Delete “it can be seen that”
Line 239: change “molecules” to “residues”
The authors correctly use the term “saturation mutagenesis”, but the terminology is somewhat inaccurate elsewhere. Strictly speaking, modification of an amino acid on a protein is a “substitution” not a “mutation”. A strain containing a modified protein might be called a “variant” rather than a “mutant”. So, for example, the text “Most of the mutants of Arg53 and Val94 reduced the specific activity” (line 262) would be better written “Most of the substitutions at Arg53 and Val94”. Similarly, the text “The mutation DC cP (R53D/V94I) showed the…” (Line 267) would be better written “The DC cP variant R53D/V94I showed the…” Finally, along these same ideas, the authors interchange between the three letter designations for residues (e.g., Arg53) and the one letter designations for residues (e.g., R53). I suggest using the one letter designation throughout.
Line 298: change “hydrophobicavity” to “hydrophobic cavity”
Line 184 states “Results and Discussion” while Line 316 states “Discussion”. Line 316 should be changed to “Conclusions”. Actually the text after line 316 does not seem necessary since it does not present any new idea beyond what has been stated elsewhere.
Comments on the Quality of English Language
comments are made elsewhere
Author Response
Reviewer #2: The manuscript “Engineering an artificial pathway for improving the bioconversion of lysine into…” deals with the two-enzyme-step conversion of lysine to 2-hydroxycadaverine, focusing particularly on the modification of each of the two enzymes through rational enzyme engineering. Technically the contribution is fine. The major criticisms are 1) the confusion between the term “mutation” and “substitution” and 2) excessive significant digits (see detailed comments below). Both are easily rectified.
Comment 1: Line 21: italicize Escherichia coli.
Response: Thank you for your comments. We have made revisions based on your recommendation.
Comment 2: Lines 23, 89: change “yridoxal” to “pyridoxal”
Response: Thank you for your comments. Sorry, we made such a mistake. We have made revisions based on your recommendation.
Comment 3: Line 24: italicize Chitinophage pinensis
Response: Thank you for your comments. We have made revisions based on your recommendation.
Comment 4: Lines 28, 97: The statement “0.63-fold” makes no sense. A fold difference less than one would imply that the value decreased. In other words, if the starting value is 100, then a 0.63-fold increase would mean the value is now 63. Please use percentage increase. For example, if the starting value is 100, and the new value is 125, then the percentage increase is (125-100)/100 = 25%.
Response: Thank you for your comments and suggestions. We have made revisions in the revised manuscript.
Comment 5: Line 28: Very rarely are measurements made with sufficient precision to justify more than 2 or maybe 3 significant digits on any numeric value presented. Therefore, “358.64” is simply not statistically possible. The authors here should write “359” or “360” (3 or 2 significant digits). Similarly, “53.55%” should be written “54%”. The authors should follow the same rule elsewhere (lines 98, Table 2, lines 204-205, 267, 268, Table 3, etc.).
Response: Thank you for your comments and suggestions. We have made revisions in the revised manuscript.
Comment 6: Line 42: change “its” to “their”
Response: Thank you for your comments and suggestions. We have made revisions in the revised manuscript.
Comment 7: Line 43: I am not sure that the authors mean “medical aesthetics”. Actually, a whole lot of fields are listed that aren’t really necessary. It is reasonable to just state “…are widely used in diverse fields (13).”
Response: Thank you for your comments and suggestions. We have made revisions in the revised manuscript.
In line 43 on page 1, "These natural or non-natural chemicals and its derivatives are widely used in the fields of pharmaceutical, textile, pigment, cosmetics, additives, medical aesthetics, biofuels, food, and chemical industry (13)." was revised as “These natural or non-natural chemicals and their derivatives are widely used in diverse fields (13).”
Comment 8: Line 53: change “opens up new ideas for the production…” to “facilitated the production…”
Response: Thank you for your comments and suggestions. We have made revisions in the revised manuscript.
In line 53 on page 2, "The functionalization of amino acids and their derivatives has opens up new ideas for the production of compounds with multiple functions, thereby expanding their application fields and scope (20)." was revised as “The functionalization of amino acids and their derivatives has facilitated the production of compounds with multiple functions, thereby expanding their application fields and scope (20).”
Comment 9: Line 63: change “an important universal molecules used as an adjuvant or ligand…” to “important adjuvants and ligands…”
Response: Thank you for your comments and suggestions. We have made revisions in the revised manuscript.
In line 63 on page 2, "Amino alcohols (AAs) are an important universal molecules used as an adjuvant or ligand in organic synthesis and biology, with the dual chemical properties of amine and alcohol (26)" was revised as “Amino alcohols (AAs) are important adjuvants and ligands in organic synthesis and biology, with the dual chemical properties of amine and alcohol (26)”
Comment 10: Line 73: change “amnio” to “amino”
Response: Thank you for your comments. Sorry, we made such a mistake. We have made revisions in the revised manuscript.
Comment 11: Line 69: change “AAs have the characteristics of low toxicity, room temperature curing and low viscosity, and can also be used as corrosion inhibitors and carbon dioxide absorbents” to “AAs have low toxicity, low viscosity, inhibit corrosion, absorb carbon dioxide and cure at room temperature.”
Response: Thank you for your comments. We have made revisions in the revised manuscript.
In lines 69-71 on page 2, "In addition, AAs have the characteristics of low toxicity, room temperature curing and low viscosity, and can also be used as corrosion inhibitors and carbon dioxide absorbents." was revised as “In addition, AAs have low toxicity, low viscosity, inhibit corrosion, absorb carbon dioxide and cure at room temperature.”
Comment 12: Line 90: the statement “overexpression was successfully established in E. coli” is a result and does not belong in the Introduction section.
Response: Thank you for your comments. We have made revisions in the revised manuscript.
In lines 69-71 on page 2, "In this work, an artificial route for the production of AA 2-hydroxycadaverine with lysine hydroxylase from Kineococcus radiotolerans (KDOKr) and pyridoxal phosphate dependent decarboxylase from Chitinophage pinensis (DCCp) overexpression was successfully established in E. coli, as seen in Fig. 1A. " was revised as “In this work, an artificial route for the production of AA 2-hydroxycadaverine with lysine hydroxylase from Kineococcus radiotolerans (KDOKr) and pyridoxal phosphate dependent decarboxylase from Chitinophage pinensis (DCCp) overexpression was illustrated in Fig. 1A.”
Comment 13: Figure 1A: The stoichiometric equation should include all substrates and products (including water, CO2, etc.)
Response: Thank you for your comments. We have made revisions in Fig. 1A.
Comment 14: Lines 112, 114: indicate EC number for each of the two enzymes.
Response: Thank you for your comments. We have made revisions in the revised manuscript.
In lines 112-115 on page 3, "The nucleotide sequence of lysine hydroxylase (EC 1.14.11.4) gene from Kineococcus radiotolerans (KDOKr) is available in the GenBank with the accession number ABS05421.1. The protein sequence of pyridoxal phosphate-dependent decarboxylase (EC 4.1.1) from Chitinophaga pinensis (DCCp) is available in the GenBank with the accession number WP_012790490.1." was revised as “The nucleotide sequence of lysine hydroxylase (EC 1.14.11.4) gene from Kineococcus radiotolerans (KDOKr) is available in the GenBank with the accession number ABS05421.1. The protein sequence of pyridoxal phosphate-dependent decarboxylase (EC 4.1.1) from Chitinophaga pinensis (DCCp) is available in the GenBank with the accession number WP_012790490.1.”
Comment 15: Line 129: Indicate how much lysine was added. That is, “0.5 mM IPTG and ??? mM lysine were added….”
Response: Thank you for your comments. We have made revisions in the revised manuscript.
In lines 129-130 on page 4, "After the OD600 reached 0.6, 0.5 mM IPTG and lysine were added and continued culture at 30 ºC." was revised as “After the OD600 reached 0.6, 0.5 mM IPTG and 4 g/L lysine were added and continued culture at 30 ºC.”
Comment 16: Line 134: change “clone” to “clones”
Response: Thank you for your comments. We have made revisions in the revised manuscript.
Comment 17: Line 179: change “hydroxycaverine” to “hydroxycadaverine”
Response: Thank you for your comments. Sorry, we made such a mistake. We have made revisions in the revised manuscript.
Comment 18: Table 2 should not appear before the first text in the Results. This is a publisher’s problem, not an authors problem, but I ask the authors to insure that the final print version has Table 2 appearing after the first paragraph of the Results section.
Response: Thank you for your comments. We have made revisions in the revised manuscript.
Comment 19: Figure 1A. The authors note that the cadA gene, which codes for lysine decarboxylase, was knocked. It would be beneficial to include this knocked out step in Figure 1A (for example, showing that a red “X” through the conversion that is knocked out.
Response: Thank you for your comments. We have made revisions in Fig. 1A.
Comment 20: Many of the commas are misprinted somehow. For example, Line 228 after “Asn93” and “Met132” the commas are somehow off kilter.
Response: Thank you for your comments. We have made revisions in the revised manuscript.
In line 227 on page 7, " In addition, the binding of KDOKr with L-lysine could be attributed to the hydrophobic interaction because the strong hydrophobic interactions of L-lysine with Asn93、Met132、Gln143 and Gln145 were formed." was revised as “ In addition, the binding of KDOKr with L-lysine could be attributed to the hydrophobic interaction because the strong hydrophobic interactions of L-lysine with Asn93, Met132, Gln143 and Gln145 were formed. ”
Comment 21: Line 204: I suggest the word “demonstrated” rather than “proved”.
Response: Thank you for your comments. We have made revisions in the revised manuscript.
Comment 22: Line 208: change “the molecular docking…” to “a molecular docking…”
Response: Thank you for your comments. We have made revisions in the revised manuscript.
Comment 23: Line 238: some redundancy here. Delete “it can be seen that”
Response: Thank you for your comments. We have made revisions in the revised manuscript.
In line 239 on page 8, "As seen in Fig. 3C and 3D, it can be seen that 3-hydroxylysine forms hydrogen bonds with the five amino acid molecules around the DCCp pocket." was revised as “As seen in Fig. 3C and 3D, 3-hydroxylysine forms hydrogen bonds with the five amino acid residues around the DCCp pocket.”
Comment 24: Line 239: change “molecules” to “residues”
Response: Thank you for your comments. We have made revisions in the revised manuscript.
Comment 25: The authors correctly use the term “saturation mutagenesis”, but the terminology is somewhat inaccurate elsewhere. Strictly speaking, modification of an amino acid on a protein is a “substitution” not a “mutation”. A strain containing a modified protein might be called a “variant” rather than a “mutant”. So, for example, the text “Most of the mutants of Arg53 and Val94 reduced the specific activity” (line 262) would be better written “Most of the substitutions at Arg53 and Val94”. Similarly, the text “The mutation DCCp (R53D/V94I) showed the…” (Line 267) would be better written “The DCCp variant R53D/V94I showed the…” Finally, along these same ideas, the authors interchange between the three letter designations for residues (e.g., Arg53) and the one letter designations for residues (e.g., R53). I suggest using the one letter designation throughout.
Response: Thank you for your comments. We have made revisions in the revised manuscript.
In line 262 on page 9, "Most of the substitutions at Arg53 and Val94 reduced the specific activity" was revised as “Most of the mutants of R53 and V94 reduced the specific activity”
In line 267 on page 9, "The mutation DCCp (R53D/V94I) showed the greatest activity shown in Table. 3." was revised as “The DCCp variant R53D/V94I showed the greatest activity shown in Table. 3.”
Comment 26: Line 298: change “hydrophobicavity” to “hydrophobic cavity”
Response: Thank you for your comments. We have made revisions in the revised manuscript.
In line 299 on page 10, "V94 is located in the substrate pocket, so V94 may change the shape of the hydrophobicavity of the substrate pocket." was revised as “V94 is located in the substrate pocket, so V94 may change the shape of the hydrophobic cavity of the substrate pocket.”
Comment 27: Line 184 states “Results and Discussion” while Line 316 states “Discussion”. Line 316 should be changed to “Conclusions”. Actually the text after line 316 does not seem necessary since it does not present any new idea beyond what has been stated elsewhere.
Response: Thank you for your comments. We have made revisions in the revised manuscript.
Reviewer 3 Report
Comments and Suggestions for AuthorsIn the study, an artificial pathway was engineered in E. coli to produce 2-Hydroxycadaverine (2HyC), a novel chiral amino alcohol with applications in pharmaceutical, chemical, and polymer industries. To address the lower catalytic activity of decarboxylase (DCCp), a mutant library of DCCp was created, and the R53D/V94I mutant exhibited a 0.63-fold increase in Kcat/Km. In shake flasks, the mutant yielded 358.64 mg/L of 2HyC, representing a 53.55% increase over the control. This semi-rational design strategy enhances 2HyC production, establishing a foundation for sustainable industrial applications. However, certain details must be added before manuscript acceptance.
- Please include plasmid maps constructed in this study, preferably as supplementary material. Specify the promoter, RBS, and terminator on the plasmid.
- Include an HPLC chromatogram illustrating the target compound, intermediate, and substrate.
- Provide the sequences of all genes used in this study, including codon-optimized ones, in the supplementary data.
- Include information on the growth rate of E. coli in the manuscript
Check for grammar error, sentence construction, and choice of words.
Author Response
Reviewer #3: In the study, an artificial pathway was engineered in E. coli to produce 2-Hydroxycadaverine (2HyC), a novel chiral amino alcohol with applications in pharmaceutical, chemical, and polymer industries. To address the lower catalytic activity of decarboxylase (DCCp), a mutant library of DCCp was created, and the R53D/V94I mutant exhibited a 0.63-fold increase in Kcat/Km. In shake flasks, the mutant yielded 358.64 mg/L of 2HyC, representing a 53.55% increase over the control. This semi-rational design strategy enhances 2HyC production, establishing a foundation for sustainable industrial applications. However, certain details must be added before manuscript acceptance.
Comment 1: Please include plasmid maps constructed in this study, preferably as supplementary material. Specify the promoter, RBS, and terminator on the plasmid.
Response: Thank you for your comments and suggestions. We have added a plasmid map in the revised supplementary material (Fig. S1).
Comment 2: Include an HPLC chromatogram illustrating the target compound, intermediate, and substrate.
Response: Thank you for your comments. We have added the HPLC result in the revised supplementary material (Fig. S2). Thank you again for your valuable suggestion.
Comment 3: Provide the sequences of all genes used in this study, including codon-optimized ones, in the supplementary data.
Response: Thank you for your comments. We have provided the sequences of KDOKr and DCCp in the supplementary materials.
Comment 4: Include information on the growth rate of E. coli in the manuscript
Response: Thank you for your comments. We have added the information on the growth rate of E. coli in the revised manuscript.
In line 227 on page 7, we have added one sentence “The OD600 reached 4.05 after 8 h and 5.86 after 12 h.”.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsCompared to the previos version, the authors have addressed the concerns raised. From the methodological point of view, the authors must add in the supporting information all the chiral and non-chiral HPLC chromatograms cited in paragraph 2.7, to account for the purity and the conversion cited (e.g. 359 mg/L, etc...).
Once these data are reported, the paper can be accepted.
Author Response
Reviewer #1:
Comment 1: Compared to the previos version, the authors have addressed the concerns raised. From the methodological point of view, the authors must add in the supporting information all the chiral and non-chiral HPLC chromatograms cited in paragraph 2.7, to account for the purity and the conversion cited (e.g. 359 mg/L, etc...). Once these data are reported, the paper can be accepted.
Response: Thank you for your comments. The chiral and non-chiral HPLC chromatograms have been added as follows:
Fig. S2. The HPLC Results of the 2HyC standard (A) and Samples (B) by a Chirex®3126 (D)-penicillamine LC column.
Fig. S3. The HPLC Results of the 2HyC, 3-hydroxylysine and lysine by a C18 column.
Author Response File: 
Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors1. The authors should note the growth rates at specific times (0h, 2h, 3h, 6h, 12h, and 24h) for all strains in the study to get a complete picture of the growth rate.
2. Make sure to show all the plasmid maps (including the size) used in the study to give readers a clear understanding of the experimental setup.
3. Include an HPLC chromatogram of a sample with residual lysine and 3-hydroxylysine, preferably taken at 12 hours, to provide more insights into the experiment.
4. Explain why the two HPLC chromatograms look the same. It's important to understand if there's a reason behind this similarity in chromatogram patterns.
It's vital to address all the comments and suggestions outlined before finalizing the publication of this paper. Taking the time to incorporate these improvements will contribute to the overall quality of the paper.
Comments on the Quality of English LanguagePlease meticulously review the manuscript for any grammatical errors and ensure that sentence structures are accurate and well-constructed.
Author Response
Reviewer #3:
Comment 1: The authors should note the growth rates at specific times (0h, 2h, 3h, 6 h, 12 h, and 24 h) for all strains in the study to get a complete picture of the growth rate.
Response: Thank you for your comments. The OD600 value at specific times (0 h, 2 h, 3 h, 6 h, 12 h, and 24 h) of strains ML101 and ML102 have been added in Table. S1. Thank you again for your valuable suggestions.
Table. S1. The OD600 value at specific times of strains ML101 and ML102.
|
Time (h) |
The OD600 of ML101 |
The OD600 of ML102 |
|
0 |
0±0.00 |
0±0.00 |
|
2 |
0.41±0.03 |
0.38±0.02 |
|
3 |
0.82±0.04 |
0.84±0.05 |
|
6 |
2.13±0.11 |
2.24±0.12 |
|
12 |
5.36±0.42 |
5.57±0.44 |
|
24 |
5.78±0.46 |
5.84±0.43 |
Comment 2: Make sure to show all the plasmid maps (including the size) used in the study to give readers a clear understanding of the experimental setup.
Response: Thank you for your comments. We have added the plasmid maps in the revised supplementary material (Fig. S1). Thank you again for your valuable suggestions.
Fig. S1. The plasmid maps of pET22b-KDOKr-DCCp, pET22b-KDOKr-DCCp, and pET22b-KDOKr-DCCp,.
Comment 3: Include an HPLC chromatogram of a sample with residual lysine and 3-hydroxylysine, preferably taken at 12 hours, to provide more insights into the experiment.
Response: Thank you for your comments. We have added the HPLC result in the revised supplementary material (Fig. S3). Thank you again for your valuable suggestions.
Fig. S3. The HPLC Results of the 2HyC, 3-hydroxylysine and lysine by a C18 column after 12 h.
Comment 4: Explain why the two HPLC chromatograms look the same. It's important to understand if there's a reason behind this similarity in chromatogram patterns.
Response: Thank you for your comments. Fig. S2A and Fig. S2B are two different HPLC chromatograms, and it can be seen from the range of the vertical axis that they are different chromatograms. Thank you again for your comments.
Author Response File: Author Response.pdf
