Heterologous Production of the Structurally Complex Diterpenoid Forskolin in Synechocystis sp. PCC. 6803

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Forskolin is a pharmaceutical compound naturally produced by Plectranthus barbatus. The
present study utilized cyanobacteria for the first time to produce this complex diterpenoid.
The manuscript is very well written in an easy-to-understand form. Some of the concerns are
as follows, which need to be rectified before the acceptance of the manuscript for publication.
1. The abstract does not effectively summarise the key findings, like growth and
chlorophyll content. It talks more about the basic introduction of forskolin, which
should be shifted to the introduction section.
2. The introduction section does not focus much on forskolin.
3. Since literature shows various studies on microbial production of forskolin, a few line
related to this and their production titer should be added in the introduction section.
This will give readers an insight into how much gap there is which needs to be
addressed in future studies to reach the level of heterotrophic production of forskolin.
4. Page 3, line 130: I think that will not be Fig. 1. It will be Fig. 2. Just check it once.
5. For the chemicals (like internal standard, forskolin standard, etc), the provider should
be given, stating the company name, city, and country.
6. The manuscript lacks the results to show the confirmation of the complete segregation
of the constructed strain.
7. Since the manuscript is a communication type and short, in Fig. 2(A) the plasmid
constructs can be made a bit detailed. Like on arrows, the promoter’s name can be
mentioned, what is the semicircle (I guess RBS), if the terminator is there, mention
that. The gel image confirming the presence of extracellular gene(s) engineered can
be added.
8. Is forskolin intracellular or extracellular?
9. The conclusion section can be elaborated, indicating the future direction of the work.
For instance, checking the generated strain for genetic stability, etc.

Author Response

Forskolin is a pharmaceutical compound naturally produced by Plectranthus barbatus. The present study utilized cyanobacteria for the first time to produce this complex diterpenoid. The manuscript is very well written in an easy-to-understand form. Some of the concerns are as follows, which need to be rectified before the acceptance of the manuscript for publication.
1. The abstract does not effectively summarise the key findings, like growth and
chlorophyll content. It talks more about the basic introduction of forskolin, which
should be shifted to the introduction section.

Very good suggestion! We have added more text about the key findings but also kept the text about forskolin as we find this briefly introduces the compound and its potential.

2. The introduction section does not focus much on forskolin.

Agree, we have deliberately kept this part short as the focus has been on the fact that the six gene-pathway is expressed and active in a photosynthetic bacterium. In the revised version we have added a sentence.

3. Since literature shows various studies on microbial production of forskolin, a few line
   related to this and their production titer should be added in the introduction section.
   This will give readers an insight into how much gap there is which needs to be
   addressed in future studies to reach the level of heterotrophic production of forskolin.

In the discussion of the revised manuscript, we discuss this: A Saccharomyces cerevisiae (yeast) strain expressing the entire forskolin biosynthetic pathway was able to produce 40 mg of forskolin per liter of yeast culture using fed batch fermentation (Ref 6 Pateraki et al., 2017. Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii. eLife 6. https://doi.org/10.7554/eLife.23001).

4. Page 3, line 130: I think that will not be Fig. 1. It will be Fig. 2. Just check it once.

Correct! This has been corrected!

5. For the chemicals (like internal standard, forskolin standard, etc), the provider should
   be given, stating the company name, city, and country.

Yes, this has been corrected!

 

6. The manuscript lacks the results to show the confirmation of the complete segregation of the constructed strain.

We are very sorry for the confusion that has arisen! The two diterpene synthases PbTPS2 and PbTPS3 are in an operon which was genome integrated as reported in Reference # 40 ‘Sutradja et al (2024). Modulation of the MEP Pathway for Overproduction of 13-R-manoyl Oxide in Cyanobacteria. Synthetic Biology and Engineering 2, 10005. https://doi.org/10.35534/sbe.2024.10005’. Therefore, we do not show the complete segregation in the current manuscript. The 3 cytochrome p450s and the acetyl transferase whereas expressed from a replicative plasmid (pDF-trc) and therefore does not require segregation. We have clarified this in the caption for Figure 2 and in the Materials and Methods, Cloning and Transformation. Thank you for pointing this out.

7. Since the manuscript is a communication type and short, in Fig. 2(A) the plasmid
   constructs can be made a bit detailed. Like on arrows, the promoter’s name can be
   mentioned, what is the semicircle (I guess RBS), if the terminator is there, mention
   that. The gel image confirming the presence of extracellular gene(s) engineered can
   be added.
   
   

Thanks! We have modified the figure accordingly. However, we do not understand what is meant by “The gel image confirming the presence of extracellular gene(s) engineered can be added.

8. Is forskolin intracellular or extracellular?

In the current study expressing the pathway enzymes in Synechocystis, forskolin is exclusively found extracellular. In the manuscript we write: “Both the cell-free supernatant and the cell pellet were extracted to localize where the product accumulated. Forskolin was exclusively found in the cell-free supernatant suggesting that all is secreted from the cells.”

9. The conclusion section can be elaborated, indicating the future direction of the work.
   For instance, checking the generated strain for genetic stability, etc.

We have added a few lines of what we think is the most urgent future directions.

Overall we are grateful for the comments and suggestions from this reviewer as this has improved and clarified the manuscript!

 

Reviewer 2 Report

Comments and Suggestions for Authors

There is growing interest and motivation in the use of cyanobacteria as a synthetic biology chassis, although cyanobacteria are much easier to manipulate and have greater potential for industrialization than higher plants are; however, cyanobacteria are less productive than heterotrophic microbes, such as E. coli and yeast, because of their self-feeding ability. The study is still encouraged from three different aspects. First, the author showed that under the current conditions, the production of forskolin had no negative effect on the growth of Synechocystis cells, which provide chance to further optimize to improve productivity. Second, although cyanobacteria do not have cytochrome P450 oxidoreductases, the three introduced P450s are still functional, which is likely because the native photoproduced ferredoxin donates electrons. Third, all synthesized forskolin is secreted into the medium, which facilitates the downstream purification steps of the produced compounds. Although the authors did not design an enzyme candidate pool to explore how to reassemble to optimize, it is fair to publish in esteem journal from my side.

Author Response

There is growing interest and motivation in the use of cyanobacteria as a synthetic biology chassis, although cyanobacteria are much easier to manipulate and have greater potential for industrialization than higher plants are; however, cyanobacteria are less productive than heterotrophic microbes, such as E. coli and yeast, because of their self-feeding ability. The study is still encouraged from three different aspects. First, the author showed that under the current conditions, the production of forskolin had no negative effect on the growth of Synechocystis cells, which provide chance to further optimize to improve productivity. Second, although cyanobacteria do not have cytochrome P450 oxidoreductases, the three introduced P450s are still functional, which is likely because the native photoproduced ferredoxin donates electrons. Third, all synthesized forskolin is secreted into the medium, which facilitates the downstream purification steps of the produced compounds. Although the authors did not design an enzyme candidate pool to explore how to reassemble to optimize, it is fair to publish in esteem journal from my side.

 

We are happy for the constructive feedback! And indeed, we would love to engage into ways to improve and optimize productivity by manipulating the pool of the 6 enzymes! There are lessons to be learned from work performed in yeast.

Reviewer 3 Report

Comments and Suggestions for Authors

I left my comments on the attached document.

Comments for author File: Comments.pdf

Author Response

Summary: In this communication, Nadia and colleagues report the first heterologous production of forskolin in cyanobacteria, which are good heterologous production hosts as they merely require atmospheric CO2, inorganic elements, and light to grow and produce organic compounds. The authors express all six biosynthetic genes related to forskolin in Synechocystis sp. PCC. 6803 and successfully detect the production of forskolin by LC-MS. Although the titer of forskolin is not very high, 25 µg/L, this report is very meaningful and provides a successfully example for heterologous production of structurally complex diterpenoids in cyanobacteria. Therefore, I recommend this paper for publication and a minor revision is warranted. I explain my concerns in more detail below.

Comments for author:

1.1 Page 1, line 36: Till now the number of terpenoids is already more than 100,000. Please update it and cite the newest literature.

We have not been able to find a reference specifically stating that more than 100,000 terpenoids have been identified in nature. However, we found a reference stating that the number identified is now 80,000. This figure is now included in the main text. If the reviewer could provide the reference, we would be grateful!

 

1.2 Page 2, line 66: Only soluble transferase can decorate the terpene skeleton? If so, please provide some literature to support.

Wrt “soluble” transferase. We have deleted soluble to avoid confusion although the term is correct in the context used. The problem is that transferases can be many different classes of enzymes (glycosyl transferases, acyl transferases etc) and the substates can be small molecules like forskolin but also proteins. In plants the acyltransferases and glycosyltransferases working on natural products are soluble (as far as we know) but in humans some are membrane bound. So, the use of the word soluble needs to be linked to a specific function to avoid doubts.

1.3 Page 5, line 212: The author said: No forskolin could be detected in a corresponding strain containing the empty pDF-trc vector. How about 13-RMO? Can author see it by HPLC or GCMS?

Yes, the two terpene synthases result in the synthesis of 13-R-MO as reported in Sutardja et al., https://doi.org/10.35534/sbe.2024.10005. 13-R-MO is detected by GC-MS analysis.  In the context of the current manuscript the purpose of this statement was to indicate that in the absence of the 4 genes on the pDF-FSK vector, no forskolin is produced.

1.4 Page 5, line 227: There is a typo mistake for the unit of titer, should be µg/L?

Thanks for noting this! Corrected!

 

 

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript titled " Heterologous production of the structurally complex diterpenoid forskolin in Synechocystis sp. PCC. 6803," submitted to Bioengineering-MDPI, presents a novel and noteworthy advancement. The work is scientifically sound, highly relevant to the field of photosynthetic biomanufacturing, and fits well within the journal's scope. However, minor issues were identified during the review of this manuscript that require attention for improvement.

1. How did the authors confirm the expression of transformants’ genes?
2. Line 27: “Forskolin titers reached 25.0 ±4 µg/L, all of which was secreted…” should be rephrased to improve the readability. E.g., “...and was entirely secreted into the medium.”
3. Line 109: “50 µ µmol photons” has an extra “µ” symbol.
4. Line 218: “OD around 4” should be “OD730 around 4” for clarity and consistency.
5. Line 227 states 25 µg/mL, which contradicts the earlier value of 25 µg/L in Line 27. Kindly recheck the units throughout the manuscript for consistency.
6. Kindly add a limitation section before the conclusion of this study.
7. Figure 2A lacks clarity. No schematic of constructs (promoters, genes, resistance markers, etc.) is shown. Improve this figure for clarity and annotation.
8. Figure 3: On the y-axis of 3B, units must be after the chlorophyll a, while on the y-axis of 3C, the peak area is not labeled with units.
9. Scientific names in the references are not italic. Kindly improve them.

Author Response

The manuscript titled " Heterologous production of the structurally complex diterpenoid forskolin in Synechocystis sp. PCC. 6803," submitted to Bioengineering-MDPI, presents a novel and noteworthy advancement. The work is scientifically sound, highly relevant to the field of photosynthetic biomanufacturing, and fits well within the journal's scope. However, minor issues were identified during the review of this manuscript that require attention for improvement.

1. How did the authors confirm the expression of transformants’ genes?

We have not confirmed the expression of the heterologously expressed pathway!

As we write in the manuscript: “Independent antibiotic-resistant colonies were selected and screened for forskolin production. Measuring the activity of individual enzymes in the pathway was not possible because the required intermediates are complex, challenging to synthesize, and difficult to isolate from natural sources due to their structural similarity to other intermediates, making purification impractical for enzymatic assays. Instead, we decided to analyse the production of the end-product forskolin, the compound previously documented to be synthesized by the six genes here introduced in Synechocystis sp. PCC 6803”

1. Line 27: “Forskolin titers reached 25.0 ±4 µg/L, all of which was secreted…” should be rephrased to improve the readability. E.g., “...and was entirely secreted into the medium.”

Corrected!

1. Line 109: “50 µ µmol photons” has an extra “µ” symbol.

Well spotted! Corrected!

1. Line 218: “OD around 4” should be “OD730 around 4” for clarity and consistency.

Corrected!

1. Line 227 states 25 µg/mL, which contradicts the earlier value of 25 µg/L in Line 27. Kindly recheck the units throughout the manuscript for consistency.

Corrected!

1. Kindly add a limitation section before the conclusion of this study.

We are not quite sure what is meant with this request. We have added a few lines on future work in the revised version of the manuscript.

1. Figure 2A lacks clarity. No schematic of constructs (promoters, genes, resistance markers, etc.) is shown. Improve this figure for clarity and annotation.

Fig. 2A has been improved as requested!

 

1. Figure 3: On the y-axis of 3B, units must be after the chlorophyll a, while on the y-axis of 3C, the peak area is not labeled with units.

Corrected!

Scientific names in the references are not italic. Kindly improve them.

Done!

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The raise queries has been satisfactorily added by the authors. No more concern from my side.