Heterologous Production of Torularhodin, the Monocyclic Carotenoid with a Terminal Carboxyl Group, in Escherichia coli

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript is well written and results are presented clearly. There are some minor language corrections that might be taken, and in line 200 in the sentence ˝To know whether lycopene is the direct substrate of 3,4-dehydrolycopene, we introduced pUC-RtcrtI into an E. coli which has a plasmid…˝ syntax is not well. How can lycopene be substrate of 3,4-dehydrolycopen? This sentence has to be corrected.

I suggest that authors add a S3 figure in the main text instead in supplementary files because it would make following of material represented in Results chapter much easier for reader.

My opinion is that article is acceptable for publication after this minor corrections.

Author Response

Reviewer 1:

 

Thank you for your kind comments.

 

According to your comments, we revised the manuscript as follows.

 

1. There are some minor language corrections that might be taken

 

→We apologize for the errors in the English. The English errors have been corrected. (We won't list each revision individually, but they are marked in red.)

 

2. In line 200 in the sentence ˝To know whether lycopene is the direct substrate of 3,4-dehydrolycopene, we introduced pUC-RtcrtI into an E. coli which has a plasmid…˝ syntax is not well. How can lycopene be substrate of 3,4-dehydrolycopen? This sentence has to be corrected.

 

→We apologize for the unclear presentation. We wanted to know whether 3,4-dehydrolycopene was produced via lycopene or not. So, we revised the text to “To examine whether RgCrtI catalyzes the reaction from lycopene to 3,4-dehydrolycopene, we introduced pUC-RgcrtI into a lycopene-producing E. coli which has a plasmid pACHP-Lyc.” (lines 208-210).

 

3. I suggest that authors add a S3 figure in the main text instead in supplementary files because it would make following of material represented in Results chapter much easier for reader.

 

→Thank you for your suggestion. We moved the S3 figure into the main text and renamed “Figure 1”. As a result, we have also changed the numbers of several other figures.

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The study "Heterologous production of torularhodin, the monocyclic carotenoid with terminal carboxylic group, in Escherichia coli" presents the first successful heterologous production of torularhodin in E. coli, demonstrating that C30 carotenoid terminal oxidases from Planococcus maritimus can act on the fully conjugated acyclic end of C40 carotenoids. While the science is solid, I have several concerns regarding its presentation.

The introduction provides useful background on carotenoid diversity and torularhodin’s biological relevance but could more clearly articulate the specific motivation for moving production into a heterologous host. A concise description of why red yeast poses practical limitations and a clearer statement of the underlying biochemical knowledge gap (the absence of a known torulene-oxidizing enzyme) would strengthen the framing.

The statement from the Materials and Methods section (line 126) is never touched on again in the Results or discussion - at least some more explanation as to why the titer was lower in larger cultures that are (presumably) better aerated would be warranted. Other than that, the experimental design is logically structured and appropriate, and the results are scientifically sound but would benefit from additional quantitative rigor. Many conclusions are based on chromatographic peak presence rather than explicit numerical comparisons. Reporting actual torulene/torularhodin/β-carotene ratios (including number of replicates and variance) would substantially strengthen the robustness of interpretations. Figure clarity could be improved with regard to axis units, consistent peak labels, and more readable formatting. The identification of torularhodin is persuasive but would be even more convincing if the comparison with the authentic standard were integrated visually within the same figure panels.

The discussion is a bit thin at times - while the functional cross-reactivity of C30 oxidases with conjugated C40 substrates is correctly interpreted, it should further consider alternative explanations for incomplete torulene oxidation (e.g., aldehyde instability, flux bottlenecks, expression imbalance). Additionally, while Rtadh is shown to function as a terminal aldehyde dehydrogenase, the manuscript could acknowledge more explicitly that the native Rhodotorula terminal oxidase remains unidentified and that this represents a meaningful open question.

The conclusion requires the most substantive improvement. As written, it primarily restates earlier results. It should instead provide a genuine synthesis that highlights the significance of demonstrating heterologous torularhodin production, the functional implication of repurposing C30 oxidases for C40 carotenoids, the identification of Rtadh and its role, and the immediate next steps needed to enhance titers (pathway balancing, promoter and vector optimization, and identification of the missing fungal oxidase).

Below some further specific comments:

Abstract: "A limitation should be recognized to enhance its production levels using the red yeasts" - I don't understand what you're trying to say here (same in the intro in line 53-54).

Line 35: "contain" is certainly not the right word here - consider "comprises"

Line 116-118: "(Sequence registration by DDBJ is delayed due to high volume of submissions. Once registration is complete, we'll fill X1-4 out immediately.)" - statement is irrelevant in its current form, only mention if the sequences are registered and provide the accession number

Line 121: briefly define the composition of 2YT medium as it is not very common

Line 275: "and subjected to various analyses" that's very vague - clarify what analysies

Line 357: a concentration is not a yield, that's a titer

Figure S2: we've got three different terms here: "torularhodin", "torulardodin", "torularhorin" - only one is correct

Figure S5: what is "Finctional"?

Comments on the Quality of English Language

The English is generally understandable, but several sections contain awkward phrasing, tense inconsistencies, and unclear sentence structure. Clarifying grammar and improving flow would enhance readability.

Author Response

Reviewer 2

 

Thank you for your kind comments. We apologize for the unclear explanations and inadequate English. Your comments were extremely helpful in improving the content of the paper. According to your comments, we revised the manuscript as follows. We also carefully reviewed the English and made the necessary improvements which were not listed individually, but marked in red.

 

1. The introduction provides useful background on carotenoid diversity and torularhodin’s biological relevance but could more clearly articulate the specific motivation for moving production into a heterologous host. A concise description of why red yeast poses practical limitations and a clearer statement of the underlying biochemical knowledge gap (the absence of a known torulene-oxidizing enzyme) would strengthen the framing.

 

→ According to Reviewer’s comment, we improved the sentence as follows: “The unique structural property of torularhodin among carotenoids should generate hot research topics to some biologists and physiologists. Its strong antioxidant activities have been demonstrated, including activities for scavenging lipid peroxyl radicals and protecting cells from photooxidative damages [11-14]. Prevention from certain types of cancer has been reported for torularhodin [15]. This pigment is promising for use as food additives, nutritional supplements, or cosmetics [10,15,16]. Current levels of torularhodin in red yeasts (the torularhodin-producing yeasts) are insufficient for commercial production, and genetically engineered red yeast cells as well as knowledge on their carotenoid biosynthesis genes may be necessary to improve torularhodin production levels. Transformation of Rhodosporidium toruloides was shown to enhance the torularhodin yield [17]. As for biosynthesis, no genes mediating key steps of torularhodin biosynthesis from torulene have been reported (Fig. 1).

The purpose of this study is the production of torularhodin in E. coli, hopefully as the dominant pigment. This means that we must identify biosynthesis genes that can be utilized for its heterologous production, while oxidation genes that convert torulene to torularhodin are unknown. We here show the successful production of torularhodin in E. coli cells. By this work, a gene tool for torularhodin production with appropriate hosts should become available for researchers.”(lines 48-67)

 

2. The statement from the Materials and Methods section (line 126) is never touched on again in the Results or discussion - at least some more explanation as to why the titer was lower in larger cultures that are (presumably) better aerated would be warranted.

 

→As you pointed out, it was a mistake to describe in the method section what should have been explained in the results or discussion section. We deleted the text “This is because torularhodin production decreased when 2L Erlenmeyer flasks were used for culture.” (line 133). Then we revised the discussion (lines 377-381).

 

3. Other than that, the experimental design is logically structured and appropriate, and the results are scientifically sound but would benefit from additional quantitative rigor. Many conclusions are based on chromatographic peak presence rather than explicit numerical comparisons. Reporting actual torulene/torularhodin/β-carotene ratios (including number of replicates and variance) would substantially strengthen the robustness of interpretations. Figure clarity could be improved with regard to axis units, consistent peak labels, and more readable formatting. The identification of torularhodin is persuasive but would be even more convincing if the comparison with the authentic standard were integrated visually within the same figure panels.

 

→Thank you for your kind suggestions. First of all, we clarify the figures, Figure 2, 3, 4, 5 (previous names are Figure 1, 2, 3, 4, respectively) by stating the axis units, using consistent peak labels, and using more readable formatting. Furthermore, in Figure 4, we integrated the HPLC chromatograms of our sample and authentic torularhodin within the same figure panel for easy comparison.

 

→As you pointed out, reporting actual torulene/torularhodin/β-carotene ratios (including number of replicates and variance) would substantially strengthen the robustness of interpretations. However, our present study focused more on qualitative than quantitative aspects, so we did not repeat enough times to perform statistical analysis. Still, all analyses were performed at least twice, and similar results were confirmed. Therefore, we added the sentence “All analyses were performed at least twice, and similar results were confirmed” (lines 148-149). We also added the sentence “Max plots (280-560 nm) were monitored.”, to indicate what the vertical axis of the figure represents (line 146). Additionally, to make it easier for readers to understand, the ratio of peak areas was added (line 276). We also added the sentence “(only a few % of total carotenoid)” (line 364).

 

 

4. The discussion is a bit thin at times - while the functional cross-reactivity of C30 oxidases with conjugated C40 substrates is correctly interpreted, it should further consider alternative explanations for incomplete torulene oxidation (e.g., aldehyde instability, flux bottlenecks, expression imbalance). Additionally, while Rtadhis shown to function as a terminal aldehyde dehydrogenase, the manuscript could acknowledge more explicitly that the native Rhodotorula terminal oxidase remains unidentified and that this represents a meaningful open question.

 

→As you pointed out, the explanation regarding incomplete torulene oxidation was insufficient. We added the explanation of it (lines 345-348) (lines 374-377)

 

→According to your suggestion, we mentioned the important issues that need to be resolved (lines 359-362) (lines 371-383).

 

 

5. The conclusion requires the most substantive improvement. As written, it primarily restates earlier results. It should instead provide a genuine synthesis that highlights the significance of demonstrating heterologous torularhodin production, the functional implication of repurposing C30 oxidases for C40 carotenoids, the identification of Rtadhand its role, and the immediate next steps needed to enhance titers (pathway balancing, promoter and vector optimization, and identification of the missing fungal oxidase).

 

→ According to your suggestions, we added the sentences as follows. In the middle of the Conclusion section, we added “which was achieved by using the C30 carotenoid biosynthesis genes crtP/crtNb/cruO and aldH/crtNc, instead of unknown genuine genes.” (lines 388-390). In the last of the Conclusion section, we added “On the other hand, we were unable to detect a crtP/crtNb/cruO homolog besides RtcrtI (CAR1) in the red yeast genome, suggesting that the fungal torulene terminal oxidase gene may retain a structure distinct from the known corresponding enzyme.” (lines 393-395).

 

→Based on your suggestions, we added the sentence “We also identified the aldH/crtNc gene in R. toruloides.” in the Abstract section, because it was necessary to include the significance of identifying gene Rtadh (line 26).

 

Below some further specific comments:

 

6. Abstract: "A limitation should be recognized to enhance its production levels using the red yeasts" - I don't understand what you're trying to say here (same in the intro in line 53-54).

 

→As you suggested, this sentence was eliminated (line 15). Furthermore, the sentence was changes to “Current levels of torularhodin in red yeasts (the torularhodin-producing yeasts) are insufficient for commercial production, and genetically engineered red yeast cells as well as knowledge on their carotenoid biosynthesis genes may be necessary to improve torularhodin production levels.,” based on the comment of Reviewer 3 (lines 53-59).

Taking Reviewer’s suggestion into consideration, the Abstract section was changed to “The Rhodotorula toruloides crtI (CAR1) and crtYB (CAR2) genes, which were chemically synthesized, proved to lead to the complete conversion from phytoene to torulene, when they were introduced into an E. coli cell that carried the Pantoea ananatis crtE and Haematococcus pluvialis IDI genes. We found that the Planococcus maritimus genes coding for C30 carotenoid terminal oxidase (crtP/crtNb/cruO) and aldehyde dehydrogenase (aldH/crtNc), through their introduction into the E. coli transformant synthesizing torulene, mediated the efficient oxidations of torulene to torularhodin, and resulted in the production of torularhodin as the dominant carotenoid,” to emphasize results not only on C30 carotenoid genes but also on red yeast carotenoid genes (lines18-25).

 

7. Line 35: "contain" is certainly not the right word here - consider "comprises"

 

→ According to your suggestion, "contain" was changed to "comprises" (line 35).

 

8. Line 116-118: "(Sequence registration by DDBJ is delayed due to high volume of submissions. Once registration is complete, we'll fill X1-4 out immediately.)" - statement is irrelevant in its current form, only mention if the sequences are registered and provide the accession number

 

→We apologize for stopping at this halfway stage. After that, we received the accession numbers and filled them in, and we deleted the text in parentheses. (line 124).

 

9. Line 121: briefly define the composition of 2YT medium as it is not very common

 

→As you suggested, we defined the composition of 2YT medium (lines 128-129).

 

10. Line 275: "and subjected to various analyses" that's very vague - clarify what analysies

 

→As you suggested, we revised the text (line 283).

 

11. Line 357: a concentration is not a yield, that's a titer

 

→According to your suggestion, we revised it (line 367).

 

12. Figure S2: we've got three different terms here: "torularhodin", "torulardodin", "torularhorin" - only one is correct

 

→As you pointed out, we revised all to “torularhodin” (Figure S2)

 

13. Figure S5: what is "Finctional"?

 

→Sorry for the typo. We revised it to “Functional” (Figure S4).

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have constructed a strain of E. coli which produced torularhodin as the dominant carotenoid in these cells. 

Overall, the work is straightfoward and described well. It is also mentioned at the end of the Discussion that further work is required to increase the amount of torularhodin in these cells. So, while proof of concept, the research presented here is solid.

In general the ms. is written well. However, the scientific English in the Abstract and Introduction needs some attention. Some suggestions follow.

lines 228 and 268  I think the authors meant "torulene" rather than "toluene".

lines 11-12  Change to "double bonds, only synthesized in fungi called red (oleaginous) yeasts....".

line 15  "Red yeasts currently produce insufficient levels of torularhodin.".

line 25  "torularhodin was the dominant carotenoid produced....".

line 29  Similarly, "was the dominant carotenoid produced in....".

line 35  "which include bacteria....".

lines 36 and 37  Their carotenoids are thought to protect their cells from (photo)oxidative stress....".

lines 52-54  "However, current levels of torularhodin in red yeasts are too low for commercial production and, perhaps, engineered cells may be necessary for this.".

line 67  "also mediated the conversion of torulene to torularhodin.".

lines 138 and 151  Insert a space:  "5 min"; 1 ml/min".

 

Comments on the Quality of English Language

Addressed above.

 

Author Response

Reviewer 3

 

Thank you for your kind comments. We edited English according to the reviewer’s points.

 

According to your comments, we revised the manuscript as follows.

 

1. lines 228 and 268  I think the authors meant "torulene" rather than "toluene".

 

→We apologize for the typo. We corrected them to “torulene” (lines 237 and 275). There was also other typo, so we corrected it as well (line 201).

 

2. lines 11-12  Change to "double bonds, only synthesized in fungi called red (oleaginous) yeasts....".

 

→ The phrase was changed according to Review’s direction (lines 12-13).

 

3. line 15  "Red yeasts currently produce insufficient levels of torularhodin.".

 

→ The original sentence was eliminated based on the suggestion of Reviewer 2 (line 15). Instead, the recommended sentence was substantially written in the Introduction section (lines 53-56).

 

4. line 25  "torularhodin was the dominant carotenoid produced....".

 

→ The phrase was changed according to Review’s direction as follows: “and resulted in the production of torularhodin as the dominant carotenoid” (line 24-25).

 

5. line 29  Similarly, "was the dominant carotenoid produced in....".

 

→As you suggested, we corrected the text (line 29).

 

6. line 35  "which include bacteria....".

 

→ We changed it to “a part of non-photosynthetic microorganisms, which comprises bacteria”, taking both comments of Reviewers’ 2 and 3 into consideration (lines 34-35).

 

7. lines 36 and 37  Their carotenoids are thought to protect their cells from (photo)oxidative stress....".

 

→ The phrase was changed according to Review’s direction (line 37).

 

8. lines 52-54  "However, current levels of torularhodin in red yeasts are too low for commercial production and, perhaps, engineered cells may be necessary for this.".

 

→ The sentence was changes to “Current levels of torularhodin in red yeasts (the torularhodin-producing yeasts) are insufficient for commercial production, and genetically engineered red yeast cells as well as knowledge on their carotenoid biosynthesis genes may be necessary to improve torularhodin production levels.” based on Reviewer’s comment (lines 53-59).

 

9. line 67  "also mediated the conversion of torulene to torularhodin.".

 

→ The phrase was changed according to Review’s direction (line 75).

 

10. lines 138 and 151  Insert a space:  "5 min"; 1 ml/min".

 

→As you pointed, we inserted a space (lines 145 and 160).

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed all reviewer comments. The revised manuscript shows improved framing, clearer figures, and better alignment between methods, results, and discussion. Limitations are now appropriately acknowledged, and the conclusions are well supported. No further substantive revisions are required.