Peer review of: In vitro Cas9-assisted editing of modular polyketide synthase genes to produce desired natural pr (2020)


RR2
Reviewers' Comments:
 Reviewer #1:
 Remarks to the Author:
 The authors have addressed the majority of suggestions and criticisms of this reviewer. Revision of
 the introduction, title, and several other sections of the manuscript have shifted the focus towards
 how the edits were introduced, as opposed to focusing on PKS engineering itself. This is a welcome
 shift. With this in mind, I would still like to see a small change to the title - it should mention Cas9
 or CRISPR to emphasize more readily the actual technological advance that is being described. I
 believe this would increase readership significantly. In addition, I suggest mention of earlier works
 that similarly use Cas9 to achieve gene editing in vitro. This would better place the work in context
 and properly acknowledge previous advances that are relevant here.
 https://doi.org/10.1128/mBio.01714-15
 https://doi.org/10.1038/ncomms9101
 It might also be prudent to cite earlier works that describe employing Cas9 directly in producing
 strains so reader can decide for themselves whether carrying out editing in a heterologous host vs
 native strain is preferred.
 Reviewer #2:
 Remarks to the Author:
 Authors addressed some concerns raised by reviewers. However, there are a few issues that
 should be addressed more carefully.
 Complete NMR data of all compounds were requested, however only six of them were included in
 the revised version, which might be acceptable due to the low production and instability observed.
 Nevertheless, the determination of the stereochemistry of hydroxyl group at C16 position of
 compound 17, which was also reviewer #1�s concern, should have been addressed more carefully.
 In Supplementary Figure 9, authors chemically synthesized racemic mixture of (16S) and (16R)-
 16-O-demethyl rapamycin and compared their retention time with that of compound 17. Although
 Supplementary reference 6 showed the preparation of 16S) and (16R)-16-O-demethyl rapamycin,
 there are three methoxy groups in rapamycin and the chemical demethylation may not be so
 specific, the methoxy groups at C16 and C27 positions may be chemically equivalent. Authors
 should confirm the structure of 16-O-demethyl rapamycin, which was used as a standard for
 compound 17, by NMR.
 In addition, if authors can explain the possible cause of instability of compound 5, it will be better.
 For this reviewer, it is not easy to know the reason of instability of compound 5.
 New Supplementary Table 3 showing the yield of rapamycin derivatives obtained in this study
 clearly says that about half of the derivatives were produced in trace amounts. This might be due
 to the inadequate fusion sites between PKS domains. Although this kind of low production from the
 engineered PKS is not unusual, in order to prove the utility of the method developed in this study,
 I strongly suggest to improve the production levels of some derivatives by construction of several
 new engineered PKSs. This may not be successful to increase the yield, however authors can at
 least show the utility of the new method to engineer the large PKS. This should not be too difficult
 if the new method is efficient and promising as authors claimed.
 Finally, although authors modified the sentence on the previous studies on the engineering
 modular PKSs in introduction (line 48-50), this can still be misleading. It is suggested to review
 previous relevant studies more broadly but concisely and describe the advantage the method
 shown in this study.

 Reviewer #3:
 Remarks to the Author:
 The revisions presented by the authors have addressed this reviewer's critiques.