PONE-D-24-24922Strategies found not to be suitable for stabilizing high steroid hydroxylation activities of CYP450 BM3-based whole-cell biocatalystsPLOS ONE

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PLOS ONE

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Additional Editor Comments:

Dear Bruno

you will see the comments from the reviewers are supportive and I am looking forward to read the revision. And, as it was hard to appoint reviewers in time and thus we reached holiday saison I understand that it may take time. Still I hope you will improve and send a next version to make a decision.

Sincerely

Dirk

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

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Reviewer #1: In their manuscript “Strategies found not to be suitable for stabilizing high steroid hydroxylation activities of CYP450 BM3-based whole-cell biocatalysts”, Bertelmann and Bühler explore various strategies aimed to increase the stability of whole-cell steroid-hydroxylation by Escherichia coli equipped with heterologous cytochrome P450 monooxygenases (CYP450s). Approaches include biotransformation media/buffers with different nutrient compositions, growing cells vs. resting cells, different growth media, different expression systems (prompters, inducers, inducer concentrations), protein engineering, using Pseudomonas as a host, and testing different CYP450 enzymes.

In general, although the authors state that they did not achieve a better system (but see my comment on that below), the work seems to be carefully conducted and is of interest for applied studies.

However, the manuscript should be presented in a more reader-friendly way. The authors assume a lot of knowledge in the abstract and the introduction (see comments).

Also, although the results might indeed be a guideline for further studies, the article is scientifically somewhat unsatisfying, because likely reasons for decreasing activity are discussed, but no data are shown. For example, what about levels of O2 in the biotransforming cell cultures, heme content in CYP450s, or NAD(P)H:NAD(P)+ ratios? If there are values known from literature, I suggest mentioning them, perhaps in the introduction (see my comment Nr. 2).

Finally, I do not understand why the authors did not proceed with, and did not do more experiments on, the plac system (Fig. 4B), and particularly the 0.1 mM IPTG / 0.1 g CDW setup (see my comment Nr. 6). This should be commented on.

In summary, in my opinion, the article could be published without further experiments, but with a thorough editing of the text.

Comments in detail:

1. The abstract is a bit confusing for readers not familiar with this field and the preceding study, respectively. Perhaps the authors may shorten the description of results to make some room for briefly summarizing the former setup that they aimed to optimize.

2. The same holds true for the introduction. It would benefit from introducing CYP450s a bit, such as specifying their cofactors and redox partners, or what is meant by “multi-component nature” (line 48). Also, the previous system should be introduced better, e.g., what is meant by “self-sufficient” (line 51), what is uncoupling (line 62; although this is explained later), what is the KSA14m variant (line 64)… It would also help the readers a lot if you introduced possible optimization steps (or bottlenecks) before delving into the results.

3. Lines 167-168: When the cells are resuspended in potassium phosphate buffer, they are depleted for every nutrient (except K, P; and glucose). Could this influence catalysis and/or stability? For example, what happens with a CYP450 when iron becomes limiting? Why did you not use M9 without nitrogen?

4. Results: Specific activity does not correlate with the enzyme level (e.g., Fig. 1A; Fig. 2). Have you ever purified the enzyme from the cells and checked its cofactor load? Or else, could oxygen be a limiting factor? Since you add glucose, I suppose that the cells have a high respiration rate? The results obtained with low cell concentrations you present in Fig. 4 (and accompanying text) suggest that O2 might not be the limiting factor, but did you ever measure O2 levels in your biotransformation cultures? If you analyzed these things previously, it would be good to mention that. If not, I do not request these analyses for publication of this manuscript, but they might be worth a try in the future.

5. Depending on your response to 4., it might also be worth mentioning that E. coli strains with optimized heme uptake or production have been generated.

6. Also results, Fig. 4: why did you not follow up on the plac system (Fig. 4B), and the 0.1 mM IPTG / 0.1 g CDW setup in particular? It seems to me that it stabilized, although on a somewhat lower than maximally achievable level. Still, did you ever measure activity at later time points? Did you quantify product yields?

7. Do the results shown in Fig. 5B not argue for enzyme destabilization independent from substrate / product concentrations? This could be mentioned much earlier in the manuscript to avoid questions piling up.

8. Lines 495-496: It would be nice for the reader if you would explain what fhuA and todX mean. Also, I assume you want to refer to Fig. 6, not 4.

9. Fig. 6 and results with Pseudomonas, respectively: Obviously, CYP350 levels are not very high in this strain. Did you consider changing expression vectors before dismissing this species completely?

10. Line 545: To evaluate this system, it is important to know the putidaredoxin levels. At least for selected conditions, this should be done, e.g. by higher-percentage gels and/or a Tris/Tricine gel.

11. Conclusions: The manuscript is somewhat overwhelming. I suggest showing a simplified summarizing table.

Minor:

12. Fig. 2A, left panel: the product symbols look like the testosterone symbols after 1 h.

13. Results, Fig. 4 and Fig. 5: Please indicate which resting cell system was applied.

14. Line 440 and line 446: Is it position 87 or 82? Please specify.

15. Table 2 is hard to understand; I’d draw lines between the rows in the lower part.

Reviewer #2: Previously, the same group investigated a whole-cell E. coli-based biocatalyst co-expressing the outer membrane pore AlkL and the P450 BM3 variant KSA14m capable of hydroxylating testosterone primarily to the 15-b-hydroxylated product. During biotransformation, resting cells lose activity due to instability of BM3 monooxygenase. In the present study, the authors tested a number of approaches and varied several factors in an attempt to increase enzyme stability during the process, which could increase the duration of biotransformation and thus lead to higher overall product concentrations. Among other things, they compared resting and growing E. coli cells, varied nutritional conditions during gene expression, different media, promoters, and inducer concentrations. None of these changes resulted in improved cell activity or increased stability of BM3 KSA14m during biotransformation. Interestingly, culture medium and expression strategies influence the initial whole cell activity but not its stability. Furthermore, two additional stabilizing mutations were introduced into the monooxygenase gene, which ultimately did not change the product titer. The use of Pseudomonas instead of E. coli did not improve productivity. Two other steroid hydroxylating P450s, CYP154C5 and CYP106A2, were cloned in E. coli together with the two redox partner genes. While CYP106A2 could not be expressed at all, the biotransformation of testosterone by cells expressing CYP154C5 was found to be no more productive than cells with BM3 KSA14m.

In general, although no improvement could be achieved within this study, the "negative" results can be very helpful for further studies on optimization of whole cell biocatalysts for oxygenation reaction.

I recommend to publish the paper after some revisions:

1. As shown in Figure 2, growing cells continued to express KSA14m even after 24 h of biotransformation (compared to resting cells), but this did not lead to higher biocatalyst productivity. This was explained by a stronger competition of enzyme synthesis and metabolic demands of the reaction with the demands of cellular maintenance and biomass formation. It seems that cell activity (independently if growing or resting) is dependent on the P450 concentration per 1 g cww, as clearly shown in Fig. 2a and Fig. 2b. In the case when the induction was done simultaneously with substrate addion, the specific cell activity first increases with increasing P450 concentration (see the corresponding SDS gel) and then decreases with complete loss after 3.5 h. It would be interesting to measure P450 concentration (using CO difference spectrum). It will not change the situation with the loss of activity after a certain time of biotransformation, but it would allow a more concrete explanation that specific cell activities in growing cells were lower (even at the very beginning of the biotransformation) compared to the resting cells due to lower P450 concentration per cww.

2. It would be helpful for the readers if the genetic construct- AlkL and KSA14m expressed from the same operon – will be described in the introduction section.

3. L. 316-317: “Obviously, KSA14m experienced a similar inactivation in growing as in resting cells,which could not be alleviated by enzyme resynthesis in growing cells”. Compared to resting cells in which the P450 band disappeared after 24 biotransformation, the band corresponding to the P450 remains in growing cells. Could it be that the loss of activity is not only due to protein instability, but due to the loss of heme or FAD or FMN (the prosthetic groups of the reductase domain of P450 BM3)? None of these aspects have been checked in this study, but it is importnat to at least mention them in the discussion.

4. L. 375-377: Please mention the promoters used in this study and their strength compared to each other to make clearer their choice for this study.

5. Was CYP106A2 not active in whole cells or was cloning unsuccessful?

6. Fig.2 In both cases, 700 mkM product concertation was achieved after 2.5 – 4 h starting with 1 mM (=1000 mkM) testosterone. Was the productivity of resting cells used at the same cell density higher?

Reviewer #3: The manuscript presents negative results, however of important biotechnology field. The authors performed many rigorous, well documented and analyzed experiments but were not able to reach desired stability of whole cell oxidation of steroids.

I have only a few recomendations:

1. I am missing a discussion which would discuss more widely the impacts of the findings both to the scientific field as well as to the biotechnology praxis.

2. English is of high level, except of a few wrong articles:

- "the" missing in front of the "most" on several places.

- "a first approach" should be "the" (line 535).

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Josef Trögl

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Strategies found not to be suitable for stabilizing high steroid hydroxylation activities of CYP450 BM3-based whole-cell biocatalysts

PONE-D-24-24922R1

Dear Dr. Bühler,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Dirk Tischler

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear Prof. Bühler

the changes made improved the manuscript as suggested by the three reviewers and hence I accept the revised version.

Sincerely!

Reviewers' comments: