Minimal lactazole scaffold for in vitro thiopeptide bioengineering

Lactazole A is a cryptic thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8 kb biosynthetic gene cluster. Here, we establish a platform for in vitro biosynthesis of lactazole A, referred to as the FIT-Laz system, via a combination of the flexible in vitro translation (FIT) system with recombinantly produced lactazole biosynthetic enzymes. Systematic dissection of lactazole biosynthesis reveals remarkable substrate tolerance of the biosynthetic enzymes and leads to the development of the minimal lactazole scaffold, a construct requiring only 6 post-translational modifications for macrocyclization. Efficient assembly of such minimal thiopeptides with FIT-Laz opens access to diverse lactazole analogs with 10 consecutive mutations, 14- to 62-membered macrocycles, and 18 amino acid-long tail regions, as well as to hybrid thiopeptides containing non-proteinogenic amino acids. This work suggests that the minimal lactazole scaffold is amenable to extensive bioengineering and opens possibilities to explore untapped chemical space of thiopeptides.

: I don't know anyone who calls it a [2+4] cyclization. It's a [4+2] cycloaddition. Also, the authors show that tRNA is needed for Dha formation but they do not show that ATP/FMN are needed for azol(in)e formation. Why show one but not the other?
Reviewer #3 (Remarks to the Author): This is a tremendous body of work by Suga, Onaka, and coworkers. The technical aspect of the work top notch and will serve as the standard to which RiPP biosynthesis studies will be judged against.
The biggest negative aspect of the work continues to the lack of bioactivity of the lactazole molecule. The authors have done a commendable job of bringing this out in the introduction and the lack of biological testing of the products reported in this study cannot be held against the authors.
A major improvement in this work, as compared to authors' last submission has been the yield measurements. The reviewer recognizes that this is a good starting point and this system can be further improved upon.
All in all, tremendous work and extremely well polished presentation. The reviewer has no concerns in recommending publication.
This reviewer also commented on whether reviewer #1's concerns have been addressed: Reviewer 1 expressed two major reservations, first being the lack of yield measurements for the FIT-Laz system. This concern has been experimentally addressed by the authors now. Quantification of yields is now established.
The second concern was the lack of biological activity, and this concern was raised by other reviewers also. The reviewer had commented that library of lactazole derivatives could me made using the FIT-Laz system and that this should be formally explored here. I agree with the authors' answer to this concern that this is beyond the scope of this work. What is being shown here is the applicability of the FIT-Laz system to demonstrate the promiscuity of the RiPP biosynthetic system. Future work will undoubtedly involve optimizations on this system to generate libraries. The Editorial Note: This manuscript has been previously reviewed at another journal that is not operating a transparent peer review scheme. This document only contains reviewer comments and rebuttal letters for versions considered at Nature Communications.
experimental work and findings presented in this manuscript, in its present form, justifies its motivations.
All in all, I would say that the concerns expressed by Reviewer#1 have been adequately addressed by the authors.
Reviewer #1 (Remarks to the Author): While the authors have now included yields for their synthesis which has addressed a reviewer concern, this paper still falls short. As it stands, the paper is an investigation of the (surprising) promiscuity of the lactazole A biosynthetic machinery. This is a decent use of the FIT system, but the paper is still thin on impact. Doing an in vitro selection using their new machinery would bring it up to the next level. Another downstream issue is that any analogs they do find will be hampered by challenges in scale-up which requires significant effort. This effort would be justifiable if they had identified a potent hit. Without this, it is just an enzymatic pathway substrate tolerance story, worth publishing, but in a lower-tier journal.
Response to the reviewers of the manuscript " Minimal lactazole 1 scaffold for in vitro thiopeptide bioengineering" 2 Reviewer #2 3 > In this manuscript, Vinogradov and co-workers describe a remarkably tolerant 4 thiopeptide biosynthetic pathway. All of the major issues raised in an earlier round of 5 review have been corrected and the manuscript is significantly improved as a result. The 6 text is well written and the figures presented in a scholarly fashion. It's an enjoyable paper 7 to read and I recommend publication in Nat Commm, although some minor changes would 8 improve the manuscript further 9 Thank you so much for taking your time to thoroughly evaluate the manuscript! Please find 10 our responses to your suggestions below. 11

> Line 45: Staphylococcus typo 12
Lines 43-44 now read: "including methicillin resistant Staphylococcus aureus (MRSA)." 13 > Page 7-8, line 228-231: This is quite a tenuous claim without any substantive evidence. 14 Handling a large macrocycle is not the same as performing an intermolecular [4+2]. 15 Comparison of LazC to TbtD has been removed altogether. Also, the authors show that tRNA is needed for Dha formation but they do not show that 21 ATP/FMN are needed for azol(in)e formation. Why show one but not the other? 22

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> This is a tremendous body of work by Suga, Onaka, and coworkers. The technical 26 aspect of the work top notch and will serve as the standard to which RiPP biosynthesis 27 studies will be judged against. 28 The biggest negative aspect of the work continues to the lack of bioactivity of the lactazole 29 molecule. The authors have done a commendable job of bringing this out in the 30 introduction and the lack of biological testing of the products reported in this study cannot 31 be held against the authors. 32 A major improvement in this work, as compared to authors' last submission has been the 33 yield measurements. The reviewer recognizes that this is a good starting point and this 34 system can be further improved upon. 35 All in all, tremendous work and extremely well polished presentation. The reviewer has no 36 concerns in recommending publication. 37 This reviewer also commented on whether reviewer #1's concerns have been addressed: 38 Reviewer 1 expressed two major reservations, first being the lack of yield measurements 39 for the FIT-Laz system. This concern has been experimentally addressed by the authors 40 now. Quantification of yields is now established. 41 The second concern was the lack of biological activity, and this concern was raised by 42 other reviewers also. The reviewer had commented that library of lactazole derivatives 43 could me made using the FIT-Laz system and that this should be formally explored here. I 44 agree with the authors' answer to this concern that this is beyond the scope of this work.

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What is being shown here is the applicability of the FIT-Laz system to demonstrate the 46 promiscuity of the RiPP biosynthetic system. Future work will undoubtedly involve 47 optimizations on this system to generate libraries. The experimental work and findings 48 presented in this manuscript, in its present form, justifies its motivations. 49 All in all, I would say that the concerns expressed by Reviewer#1 have been adequately 50 addressed by the authors. 51 Thank you for the warm appraisal of our work! 52