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

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 

This manuscript describes an in vitro Cas9 based method to edit the genes of type I PKSs. After 
cutting by Cas9, Gibson assembly is used to provide the desired engineered PKS gene, and the 
plasmid is transformed into a heterologous host and the target compound isolated. This is a very 
nice approach to mutating PKS genes that leverages the ability of Cas9 to site-selectively cut even 
very large BACs. This will surely be a nice addition to the PKS engineering toolbox. However, 
enthusiasm is reduced due to some caveats that are not made clear and the otherwise lack of 
novelty as currently presented. For example, the approach inherently relies on the abilities of the 
heterologous host to provide the necessary building blocks, and on the potential permissively of 
tailoring enzymes. In addition, It is not particularly clear which compounds accessed here are 
actually novel. It would be helpful to cite literature that has reported other rapamycin analogues 
for comparison. Further, the production of compounds 17-19 requires further explanation. How is 
the stereochemistry of the hydroxyl in 17 determined? Where does the methylation event in 18 
derive from? In general, types of exchanges similar to those described here have been reported 
many times before. Thus, the emphasis here is not on the rational design of modular PKSs as the 
title suggests, but on simply leveraging Cas9 to make the desired sequence changes in vitro. Very 
little has been learned with respect to the catalytic abilities of the PKS modules, especially given 
that the absolute or relative yields of each product has not been explicit described. In addition, 
nothing has been learned with respect to optimal boundaries for domain/module swapping. It 
seems that most the boundaries and mutagenesis was designed on the basis of prior literature 
precedent. While quite useful, this method will likely be insufficient to generate new analogs at-will 
unless problems with protein:protein interactions and substrate specificity/channeling have also be 
solved. Accordingly, I don’t recommend publication in its current form but would consider a heavily 
revised version of the manuscript. 
 
Other issues that need to be addressed: 
 
p2, Abstract: “Despite intense studies, the accurate targeting of the desired regions in the type-I 
modular polyketide synthase (PKS) gene has not been accomplished.” This is exaggerated or 
inaccurate. There are hundreds of examples in the literature and patent literature that 
demonstrate the successful manipulation of type I PKSs. 
 
p3, lines 1-2: “middle-weight molecular compounds have attracted increasing attention as new 
compounds to fill the gap between antibodies and low-molecular-weight compounds. This sentence 
needs to be edited to clarify what gap is being addressed, i.e. gap in molecular weight. 
 
p3, line 3: “PPI” should be written in full. 
 
p3, line 8: For clarity, please specify whether the difficulties cited are those of chemical synthesis 
and/or biosynthesis. The next sentence (“Therefore, various congeners will be produced from 
known useful natural compounds by genetic manipulation) doesn’t follow naturally. 
 
p3: “Relatively few experiments on gene modification in modular PKS genes have been reported.” 
As stated, this is untrue. 
 
p4: The introduction would benefit from including greater discussion on the mutagenesis and gene 
assembly methods available for type I PKSs and how this fits in with production of polyketides 
when the gene editing is done in vitro. This would better support the novelty of the CRISPR-Cas9 
approach reported. 
 
p4: “SUKA” needs writing in full or supporting with a citation. Please also further describe why 
these were chosen. 

p5: “If we can accurately edit the modular PKS of rapamycin, which shows very high homology, 
this technique will be applicable for most natural products.” This sentence needs clarifying. Is the 
Rap system selected here because the Rap modules show high homology to each other? Or do the 
Rap modules show high homology to other PKSs? In either case, some quantitative analysis would 
be helpful to support this argument. 
 
p5: There are several areas that need clarifying in the “AT domain-targeted module editing” 
section. For example, “Our first aim was to exchange the extender unit for a particular module of 
rapamycin PKS by swapping the AT domains from other modules”. It’s not possible to replace and 
extender unit (acyl-CoA) with a module. In another example, “We first targeted module 9 to 
methylate the triene moiety of rapamycin” doesn’t make sense. The AT domain does not methylate 
the triene. Rather, a malonyl-CoA is replaced with a methylmalonyl-CoA at this position in the 
polyketide. After this, please make it clearer that each AT domain targeted is being replaced with 
AT domains from the same Rap PKS, but different modules. Similarly, it is not clear what 
“targeted” means until SI Fig. 2 is viewed. It may be useful to the reader to define that Mod6AT 
and Mod8AT are being inserted at these positions. 
 
p15: “Therefore, we attempted to edit multiple modules”. Suggest adding “simultaneously” for 
clarification. 
 
p19. Table 1: Please include error bars and standard deviations. Please also unify the use of 
significant figures. 
 
p20: “we believe that the substrate specificities of the PKS modules are much broader than 
expected”. I don’t see any evidence in this manuscript that supports this statement, particularly 
without yields or quantification of the each product. Please clarify or remove. There are plenty of 
other published examples that do support PKS promiscuity. 
 
SI Figure 6: Why are the HPLC peaks for compound 8 and 17 not visible? 
 
 
 
Reviewer #2: 
Remarks to the Author: 
This manuscript by Kudo et al. describes the combined application of in vitro CRIPS-Cas9 and 
Gibson assembly technology to edit type 1 modular PKSs. Authors demonstrated that the method 
developed can be applied for the engineering AT domain, module deletion/insertion, reductive 
domains, stereochemistry of beta-hydroxyl group, and double modification (ER and KR) using 
rapamycin PKS as a model system. Although in vitro CIRPR/Cas9 system for refactoring of 
biosynthetic gene clusters in Streptomyces has been reported previously (mBio 6(6):e01714-15), 
this manuscript may be the first report to use CIRPR/Cas9 technology to edit the large modular 
PKSs. 
 
Although the method used in this study seems promising to edit PKS efficiently, authors should 
present the complete structural data (e.g. 1D and 2D NMR) of all compounds generated in this 
study to prove the accuracy and efficiency of the method as claimed in the manuscript. Some 
compounds might be produced in a small amount. However, authors should show that this method 
at least produces a reasonable amounts of the designed polyketide for structure determination to 
prove their claims that this in vitro editing method can produce almost all the designed compounds 
and modular PKSs have broader substrate specificities than expected. The structures of many of 
the new compounds generated in this study were predicted only by MS fragmentation pattern, 
which may not be acceptable in a high-profile journal such as Nature Communications. 
 
1. Abstract is too general to provide the actual outcome of this study. 
2. Page 3, line 4: What is PPI? Please give the full name. 
3. Page 3, last two lines: “Relatively few experiments on gene modification in modular PKS genes 
have been reported.” This sentence is not true. There are a large number of papers on the 
engineering modular PKS. The main problem in PKS engineering is that it is difficult to predict the 
results due to the complex structure of giant PKS protein and there is no rule to design the 
recombinant PKS. 
4. Page 4, the second line from the bottom: Please give the reference of “SUKA” strain here where 
it appears first. 
5. Page 5, line 10: Please describe the quantitative production level of SUKA33::rapH or 
SUKA34::rapH. 
6. Page 5, last line: “We first targeted module 9 to methylate the triene moiety of rapamycin which 
is reported to play a significant role in the biological activity.” Please give reference of this fact. 
7. Page 7: Please describe the quantitative production levels of compounds 2, 3, 4, 6, and 6 in the 
main text, although they were described in the SI. Please do the same for other compounds. 
Importantly, NMR data of compounds 2 and 6 were provided in the SI. It is critical to validate the 
chemical structure of all analogs obtained in this study for proving the efficiency of the method 
developed in this study. Although Supplementary Figure 4 shows the MS fragmentation patterns of 
compounds 3, 4, and 5, all structure should be confirmed by NMR. 
8. Page 9: The NMR data of compound 8 should be provided. 
9. Page 10: The NMR data of compounds 10, 11, 12, and 13 should be provided. 
10. Page 13: The NMR data of compounds 15 and 16 should be provided. 
11. Page 16: The NMR data of compounds 18 and 19 should be provided. 
12. Page 17: “Biological activities”. Unfortunately, all bioactivities of new compounds were 
reduced. It is strongly suggest to test their immunosuppressive activity to see if some of new 
analogs showing applicable antifungal activity (e.g. compound 17) can have potential to develop 
as antifungal agents with reduced immunosuppressive activity. 
13. Authors should describes how many independent experiments were carried out to present all 
the chromatograms in SI. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
The manuscript describes a strategy for using Cas9 and guide RNAs to site-specifically modify a 
polyketide synthase gene cluster in vitro. Domains/modules were replaced by desiging guide RNAs 
that flank these regions, using Cas9 to impart double strand breaks on either side, and a 
replacement sequence was added using PCR/gibson assembly. 
 
These modified PKS clusters, housed within BAC heterologous expression vectors, were then 
transformed into a heterologous host and the resulting compounds were analyzed. The changes 
induced at the genetic level resulted in expected modifications to the chemical structures of the 
products. The authors thoroughly characterized the resulting products using mass spectrometry, 
multi-dimensional NMR, and UV-VIs spectroscopy. This reviewer appreciated the explanations of 
which key signals in the NMR spectra support their conclusions that were in provided in addition to 
full spectral assignments in the SI. 
 
In general, the authors provide convincing data to support their conclusions. One exception to this 
is the experiments targeting inversion of stereochemistry (pg.15). If the full NMR characterization 
of this derivative is still forthcoming, perhaps it would be best to report that in a subsequent 
manuscipt. 
 
Genetic modification of the rapamycin gene cluster to produce derivative compounds is not a novel 
achievement, but the exact methodology employed here, using cas9 in vitro on clusters in BACs, is 
novel as far as this reviewer knows. The work presented will be of interest in the field. 
 
One suggested improvement would be to provide a detailed/quantitative comparison of the 
mutation incidence rate of this methodology compared with previously reported techniques that 
are described in the introduction of this manuscript. Another suggested improvement would be to 
provide information about the titer/yield of the various rapamycin derivatives. The authors provide 
convincing evidence that they induced the expected changes in chemical structures of products, 
but if the genetic modifications result in a significant decrease in yield of derivatives compared to 
"wild-type" rapamycin, this information is highly relevant.