Minimal lactazole scaffold for in vitro thiopeptide bioengineering

Vinogradov, Alexander A.; Shimomura, M; Goto, Yuki; Ozaki, Taro; Asamizu, Shumpei; Sugai, Yoshinori; Suga, Hiroaki; Onaka, Hiroyasu

doi:10.1038/s41467-020-16145-4

Cited by 58 publications

(95 citation statements)

References 69 publications

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“…LazBF dehydrates 4 out of 6 Ser in the CP of LazA wt , and, as we previously found, 36 depending on the order of enzyme addition, over-and underdehydrated thiopeptides "Local" action also characterizes other Laz enzymes (Fig. 6a).…”

Section: Discussionsupporting

confidence: 74%

“…Furthermore, our previous findings 36 According to our aims, we intercepted biosynthetic intermediates and reconstructed the order of events leading up to the final macrocyclization reaction during the lactazole A assembly process at a single PTM resolution. When supplemented with substrate preference studies for individual enzymes, these results help in rationalizing the roots of cooperation between Laz enzymes, and establish the basis for selective lactazole A production.…”

Section: Main Textmentioning

confidence: 98%

“…First, we investigated in vitro modification of LazA min utilizing the FIT-Laz system. 36 Synthetic DNA bearing lazA min gene was in vitro transcribed and translated, generating the precursor peptide, which was incubated with a mixture of recombinantly produced Accordingly, the captured intermediates were further analyzed by CID tandem mass spectrometry (MS/MS) in a data-dependent acquisition (DDA) mode (S.I. 2.4-2.5).…”

Section: Biosynthetic Timingmentioning

confidence: 99%

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Promiscuous enzymes cooperate at the substrate level en route to lactazole A

Vinogradov

Shimomura

Kano

et al. 2020

Preprint

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18Enzymes involved in ribosomally synthesized and post-translationally modified peptide 19 (RiPP) biosynthesis often have relaxed specificity profiles and are able to modify diverse 20 substrates. When several such enzymes act together during precursor peptide maturation, 21 a multitude of products can form, and yet usually, the biosynthesis converges on a single 22 natural product. For the most part, the mechanisms controlling the integrity of RiPP 23 assembly remain elusive. Here, we investigate biosynthesis of lactazole A, a model 24 thiopeptide produced by five promiscuous enzymes from a ribosomal precursor peptide. 25Using our in vitro thiopeptide production (FIT-Laz) system, we determine the order of 26 biosynthetic events at the individual modification level, and supplement this study with 27 substrate scope analysis for participating enzymes. Combined, our results reveal a 28 dynamic thiopeptide assembly process with multiple points of kinetic control, intertwined 29 enzymatic action, and the overall substrate-level cooperation between the enzymes. This 30 work advances our understanding of RiPP biosynthesis processes and facilitates 31 thiopeptide bioengineering. 32 3 Main text 33 Ribosomally synthesized and post-translationally modified peptides (RiPPs) are 34 structurally and functionally diverse natural products united by a common biosynthetic 35 logic. 1 Usually during RiPP maturation, biosynthetic enzymes utilize the N-terminal 36 sequence of a ribosomally produced precursor peptide as a recognition motif (leader 37 peptide; LP) and install post-translational modifications (PTMs) in the C-terminal section of 38 the same substrate (core peptide; CP). This mode of action leads to relaxed substrate 39 requirements around the modification sites, which is often exemplified by one RiPP 40 enzyme introducing multiple PTMs in a single substrate. In one extreme case, a single 41 enzyme epimerizes 18 out of 49 amino acids in polytheonamide A precursor peptide 42 during its biosynthesis. 2,3 Unique enzymology of RiPP biosynthetic enzymes has come 43 under intense scrutiny in the recent years, which explained observed substrate specificities 44 in many cases. 4,5,14,15,6-13 During biosynthesis of complex RiPPs, when multiple enzymes 45 capable of differentially modifying their substrate act together, a multitude of products can 46 often form, and yet usually the biosynthetic pathway manages to produce a single natural 47

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Section: Discussionsupporting

confidence: 74%

Section: Main Textmentioning

confidence: 98%

Section: Biosynthetic Timingmentioning

confidence: 99%

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Promiscuous enzymes cooperate at the substrate level en route to lactazole A

Vinogradov

Shimomura

Kano

et al. 2020

Preprint

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“…Some examples include 2,3-butanediol, 45 diverse glycan motifs, 46 and a cyclic dipeptide produced from two non-ribosomal peptide synthetases. 47 Several mature RiPPs have been produced using cell-free methods, such as goadsporin, 48 nisin, 49 thiocillin, 50 lactazole 51 . To the best of our knowledge, lasso peptides have not previously been produced in the manner.…”

Section: Introductionmentioning

confidence: 99%

Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme-Substrate Tolerance

Kretsch

Daigh

et al. 2020

Preprint

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Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like structure. Owing to a rigid topology, lasso peptides are unusually stable towards heat and proteolytic degradation. Some lasso peptides have been shown to bind human cell-surface receptors and exhibit anticancer properties, while others display antibacterial or antiviral activities. Known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent RiPP class; however, the discovery, isolation, and characterization of lasso peptides are constrained by the lack of an efficient production system. In this study, we employ a cell-free biosynthesis (CFB) strategy to address the longstanding challenges associated with lasso peptide production. We report the successful formation of a diverse array of lasso peptides that include known examples as well as a new predicted lasso peptide from Thermobifida halotolerans. We further demonstrate the utility of CFB to rapidly generate and characterize multisite precursor peptide variants in order to evaluate the substrate tolerance of the biosynthetic pathway. We show that the lasso-forming cyclase from the fusilassin pathway can produce millions of sequence-diverse lasso peptides via CFB with an extraordinary level of sequence permissiveness within the ring region of the lasso peptide. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.

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“…Cell-free systems have been used to synthesize other ribosomally synthesized and post-translationally modified peptide (RiPP) natural products, however only precursor peptides (or analogues) were synthesized by CFE. [51][52][53][54][55][56] These initial examples of natural product pathways established in cellfree systems mark the starting point of a field that will improve metabolic engineering efforts for a wide variety of natural product classes.…”

Section: High-throughput Pathway Assembly In the Cell-free Environmentmentioning

confidence: 99%

Cell‐Free Exploration of the Natural Product Chemical Space

et al. 2020

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Natural products and secondary metabolites comprise an indispensable resource from living organisms that have transformed areas of medicine, agriculture, and biotechnology. Recent advances in high-throughput DNA sequencing and computational analysis suggest that the vast majority of natural products remain undiscovered. To accelerate the natural product discovery pipeline, cell-free metabolic engineering approaches used to develop robust catalytic networks are being repurposed to access new chemical scaffolds, and new enzymes capable of performing diverse chemistries. Such enzymes could serve as flexible biocatalytic tools to further expand the unique chemical space of natural products and secondary metabolites, and provide a more sustainable route to manufacture these molecules. Herein, we highlight select examples of natural product biosynthesis using cell-free systems and propose how cell-free technologies could facilitate our ability to access and modify these structures to transform synthetic and chemical biology.

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Minimal lactazole scaffold for in vitro thiopeptide bioengineering

Cited by 58 publications

References 69 publications

Promiscuous enzymes cooperate at the substrate level en route to lactazole A

Promiscuous enzymes cooperate at the substrate level en route to lactazole A

Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme-Substrate Tolerance

Cell‐Free Exploration of the Natural Product Chemical Space

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