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Lariatin A, an 18-residue lasso peptide encoded by the five-gene cluster larABCDE, displays potent and selective anti-mycobacterial activity. The structural feature is an N-terminal macrolactam ring, through which the C-terminal passed to form the rigid lariat-protoknot structure. In the present study, we established a convergent expression system...
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Context 1
... A in R. jostii; larA encodes a precursor peptide consisting of 46 amino acid residues, larB and larD encode maturation enzymes that convert the precursor peptide into the mature lariatin B, unspecific peptidase are probably relevant to hydrolyze the C terminus of lariatin B to yield lariatin A, and larE encodes an exporter of lariatin A 23 (Fig. S1). In this study, we focused on the larA deletion mutant from the lariatin-producing bacterium as a host for the production of lariatin variants. To construct the host, the larA gene in R. jostii K01-B0171 chromosomal DNA was disrupted by insertional inactivation with pk18mob derived from none replication E. coli plasmid (pΔ larA). ...
Context 2
... of determinants for the maturation and production of lariatin A. The first step in the maturation of lariatin A is the peptide bond cleavage reaction between Ala26 and Gly27 of the precursor protein LarA catalyzed by LarD (Fig. S1). Critical positions of this maturation reaction in the LarA were determined by an alanine scan at the positions − 4 to − 2 close to the protease cleavage site. When the mutation had no effects on the production of lasso peptides, lariatin A or the related lariatin variants were anticipated to be detectable by HPLC analysis. As a ...
Citations
... Interestingly, the structural genes of lassomycin share high homology with the associated genes of lariatin A, another anti-tuberculosis lasso peptide with no tailoring modification [33,34]. Previous studies showed that the C-terminus of lariatin A significantly affected its anti-mycobacterial activity [35,36], which raises the question of whether the C-terminal methylation of lassomycin is equally necessary for its physiological functionality. ...
... It seems that this acetyltransferase is unrelated to ulleungdin. affected its anti-mycobacterial activity [35,36], which raises the question of whether the Cterminal methylation of lassomycin is equally necessary for its physiological functionality. ...
Lasso peptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs) and feature the threaded, lariat knot-like topology. The basic post-translational modifications (PTMs) of lasso peptide contain two steps, including the leader peptide removal of the ribosome-derived linear precursor peptide by an ATP-dependent cysteine protease, and the macrolactam cyclization by an ATP-dependent macrolactam synthetase. Recently, advanced bioinformatic tools combined with genome mining have paved the way to uncover a rapidly growing number of lasso peptides as well as a series of PTMs other than the general class-defining processes. Despite abundant reviews focusing on lasso peptide discoveries, structures, properties, and physiological functionalities, few summaries concerned their unique PTMs. In this review, we summarized all the unique PTMs of lasso peptides uncovered to date, shedding light on the related investigations in the future.
... For example, newly identified lasso peptides such as capistruin, astexin, caulosegnin, and paeninodin have been extensively studied through mutagenesis experiments (Knappe et al., 2009;Hegemann et al., 2013a;Li et al., 2015). The structure-activity relationship of the anti-mycobacterial lasso peptide lariatin A was also investigated through mutational analysis, revealing crucial roles for Tyr6, Gly11, and Asn14 in the compound's antibacterial activity, and essential roles for Val15, Ile16, and Pro18 in enhancing that activity (Inokoshi et al., 2016). Several point mutants of lariatin A showed improved anti-mycobacterial activity relative to the wild-type sequence, suggesting that genetic engineering is a promising method for the efficient design of lariatin analogs to counter tuberculosis (Inokoshi et al., 2016). ...
... The structure-activity relationship of the anti-mycobacterial lasso peptide lariatin A was also investigated through mutational analysis, revealing crucial roles for Tyr6, Gly11, and Asn14 in the compound's antibacterial activity, and essential roles for Val15, Ile16, and Pro18 in enhancing that activity (Inokoshi et al., 2016). Several point mutants of lariatin A showed improved anti-mycobacterial activity relative to the wild-type sequence, suggesting that genetic engineering is a promising method for the efficient design of lariatin analogs to counter tuberculosis (Inokoshi et al., 2016). ...
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of natural products that exhibit a range of structures and bioactivities. Initially assembled from the twenty proteinogenic amino acids in a ribosome-dependent manner, RiPPs assume their peculiar bioactive structures through various post-translational modifications. The essential modifications representative of each subfamily of RiPP are performed on a precursor peptide by the so-called processing enzymes; however, various tailoring enzymes can also embellish the precursor peptide or processed peptide with additional functional groups. Lasso peptides are an interesting subfamily of RiPPs characterized by their unique lariat knot-like structure, wherein the C-terminal tail is inserted through a macrolactam ring fused by an isopeptide bond between the N-terminal amino group and an acidic side chain. Until recently, relatively few lasso peptides were found to be tailored with extra functional groups. Nevertheless, the development of new routes to diversify lasso peptides and thus introduce novel or enhanced biological, medicinally relevant, or catalytic properties is appealing. In this review, we highlight several strategies through which lasso peptides have been successfully modified and provide a brief overview of the latest findings on the tailoring of these peptides. We also propose future directions for lasso peptide tailoring as well as potential applications for these peptides in hybrid catalyst design.
... To date, most mutational investigations of lasso peptides have been carried out in E. coli, 16,42,[55][56][57][58][59] with the single exception being a mutational analysis of lariatin A peptides in the Gram-positive bacteria Rhodococcus jostii. 60 In the present study, our single-mutation analysis of the 15 positions of the core peptide revealed functional impacts of residues on stlassin peptide formation (including post-translational processing by StlaB1/B2/C), including Leu1, Asp8, Ala7, and Trp9 (related to the macrolactam ring formation), Gly13 (in the middle of the macrolactam ring), and Trp14 and Phe15 (related to the C-terminal steric lock formation). ...
Lasso peptides are a unique family of natural products whose structures feature a specific threaded fold, which confers these peptides the resistance to thermal and proteolytic degradation. This stability gives lasso peptides excellent pharmacokinetic properties, which together with their diverse reported bioactivities have garnered extensive attention because of their drug development potential. Notably, the threaded fold has proven quite inaccessible by chemical synthesis, which has hindered efficient generation of structurally diverse lasso peptides. We herein report the discovery of a new lasso peptide stlassin (1) by gene activation based on a Streptomyces heterologous expression system. Site-directed mutagenesis on the precursor peptide-encoding gene is carried out systematically, generating 17 stlassin derivatives (2-17 and 21) with residue-replacements at specific positions of 1. The solution NMR structures of 1, 3, 4, 14 and 16 are determined, supporting structural comparisons that ultimately enabled the rational production of disulfide bond-containing derivatives 18 and 19, whose structures do not belong to any of the four classes currently used to classify lasso peptides. Several site-selective chemical modifications are first applied on 16 and 21, efficiently generating new derivatives (20, 22-27) whose structures bear various decorations beyond the peptidyl monotonicity. The high production yields of these stlassin derivatives facilitate biological assays, which show that 1, 4, 16, 20, 21 and 24 possess antagonistic activities against the binding of lipopolysaccharides to toll-like receptor 4 (TLR4). These results demonstrate proof-of-concept for the combined mutational/chemical generation of lasso peptide libraries to support drug lead development.
... Among the lasso peptides of actinomycetes, homologous expression of the lasso peptide lariatin was accomplished using the larA (lariatin precursor encoding gene) mutant of actinomycete Rhodococcus jostii [41]. High production of the lasso peptide lariatin (105 μg/mL) was reported in this system. ...
Lasso peptides produced by bacteria have a very unique cyclic structure (“lasso” structure) and are resistant to protease. To date, a number of lasso peptides have been isolated from proteobacteria and actinobacteria. Many lasso peptides exhibit various biological activities, such as antibacterial activity, and are expected to have various applications. Based on study of genome mining, large numbers of biosynthetic gene cluster of lasso peptides are revealed to distribute over genomes of proteobacteria and actinobacteria. However, the biosynthetic gene clusters are cryptic in most cases. Therefore, the combination of genome mining and heterologous production is efficient method for the production of lasso peptides. To utilize lasso peptide as fine chemical, there have been several attempts to add new function to lasso peptide by genetic engineering. Currently, a more efficient lasso peptide production system is being developed to harness cryptic biosynthetic gene clusters of lasso peptide. In this review, the overview of lasso peptide study is discussed.
... This family of natural products is biosynthesized via extensive modifications of the precursor peptides, which imply that significant chemical changes can be introduced by simple site-directed mutagenesis (Velásquez and van der Donk 2011). Previously, a lariatin library was quickly generated, and several analogs with enhanced antitubercular activity were identified (Fig. 5a) (Inokoshi et al. 2016). These strategies could be adopted by the lassomycin system, which has some recently uncovered biosynthetic pathways . ...
... Owing to their remarkable antitubercular activity and short biosynthetic pathway, the lassomycin and lariatin lasso peptides are ideal candidates (Gavrish et al. 2014;Inokoshi et al. 2012). Currently, the heterologous expression system for lariatin and the biosynthetic pathway orthologs for lassomycin are under investigation (Inokoshi et al. 2016;Su et al. 2019). Specific secondary metabolites, such as the diketopiperazine natural product, mycocyclosin, specifically secreted by M. tuberculosis could be developed as a quorum sensing molecule ( Fig. 6) (Dornevil et al. 2017). ...
More than 100 years have passed since the discovery of Mycobacterium tuberculosis, in 1882, as the pathogen that causes tuberculosis (TB). However, globally, TB is still one of the leading causes of death by infectious diseases. In 2018, approximately 10.0 million people were diagnosed with TB owing to the development of advanced strategies by M. tuberculosis to resist antibiotics, including the development of a dormant state. The World Health Organization (WHO) and the Sustainable Development Goals (SDGs) are dedicated to ending TB by 2030. However, the development of strategies to discover new TB drugs and new therapies is crucial for the achievement of this goal. Unfortunately, the rapid occurrence of multidrug-resistant strains of M. tuberculosis has worsened the current situation, thereby warranting prioritized discovery of new anti-TB drugs and the development of new treatment regimens in academia and the pharmaceutical industry. In this mini review, we provide a brief overview of the current research and development pipeline for new anti-TB drugs and present our perspective of TB drug innovation. The data presented herein may enable the introduction of more effective medicines and therapeutic regimens into the market.Key Points
• The Updated Global New TB Drug Pipelines are briefly summarized.
• Novel strategies for the discovery of new TB drugs, including novel sources, bioinformatics, and synthetic biology strategies, are discussed.
• New therapeutic options, including living therapeutics and phage therapy, are proposed.
... To date this hypothesis remains untested [54]. Recent work has shed light on which amino acids in lariatin A are important for anti-mycobacterial activity: Tyr-6 (Phe and Trp can be substituted), Gly-11, and Asn-14 were determined to be essential [69]. ...
Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large class of natural products produced across all domains of life. The lasso peptides, a subclass of RiPPs with a lasso-like structure, are structurally and functionally unique compared to other known peptide antibiotics in that the linear peptide is literally “tied in a knot” during its post-translational maturation. This underexplored class of peptides brings chemical diversity and unique modes of action to the antibiotic space. To date, eight different lasso peptides have been shown to target three known molecular machines: RNA polymerase, the lipid II precursor in peptidoglycan biosynthesis, and the ClpC1 subunit of the Clp protease involved in protein homeostasis. Here, we discuss the current knowledge on lasso peptide biosynthesis as well as their antibiotic activity, molecular targets, and mechanisms of action.
... Shortly thereafter, microcin J25 (MccJ25), the most well-studied lasso peptide, was discovered in a search for new antimicrobial compounds in infant fecal bacteria [51]. Another compelling example of an antimicrobial lasso peptide discovered by activity-guided approaches is lariatin, which has activity against mycobacteria including M. tuberculosis [23][24][25]. Lasso peptides that have been discovered by activity-guided approaches have been reviewed elsewhere extensively [20,40,49]. Here, instead, we focus on genomics-guided approaches for lasso peptide discovery. ...
Over the course of roughly a decade, the lasso peptide field has been transformed. Whereas new compounds were discovered infrequently via activity-driven approaches, now, the vast majority of lasso peptide discovery is driven by genome-mining approaches. This paper starts with a historical overview of the first genome-mining approaches for lasso peptide discovery, and then covers new tools that have emerged. Several examples of novel lasso peptides that have been discovered via genome mining are presented as are examples of new enzymes found associated with lasso peptide gene clusters. Finally, this paper concludes with future directions and unsolved challenges in lasso peptide genome mining.
... However, progress in the biosynthesis of lasso peptides has significantly expedited the process of obtaining new analogs (Piscotta et al. 2015). The repertoire of lasso peptide analogs could be significantly expanded by simple site-directed mutagenesis on the precursor since lasso peptides are of ribosomal origin (Hegemann et al. 2013a(Hegemann et al. , 2014Inokoshi et al. 2016;Pan and Link 2011). Currently, a convergent expression system for the precursor larA was established in the larA-deficient R. jostii strain (Inokoshi et al. 2016). ...
... The repertoire of lasso peptide analogs could be significantly expanded by simple site-directed mutagenesis on the precursor since lasso peptides are of ribosomal origin (Hegemann et al. 2013a(Hegemann et al. , 2014Inokoshi et al. 2016;Pan and Link 2011). Currently, a convergent expression system for the precursor larA was established in the larA-deficient R. jostii strain (Inokoshi et al. 2016). The larA-carrying plasmid was readily used to generate up to 36 variants and enabled parallel biosynthesis of lariatin mutants for structure-activity relationship (SAR) studies. ...
... The larA-carrying plasmid was readily used to generate up to 36 variants and enabled parallel biosynthesis of lariatin mutants for structure-activity relationship (SAR) studies. Firstly, the mutational study identified four residues (Gly1, Arg7, Glu8, and Trp9) in the core peptide that are critical for the biosynthesis of lariatin A (Inokoshi et al. 2016). Furthermore, all the generated lariatin A analogs were used for systematic SAR studies, which demonstrated that three residues (Tyr6, Gly11, and Asn14) are important for anti-TB activity (Fig. 4a) (Inokoshi et al. 2016). ...
Lasso peptides are ribosomally synthesized and post-translationally modified natural products with a characteristic slipknot-like structure, which confers these peptides remarkable stability and diverse pharmacologically relevant bioactivities. Among all the reported lasso peptides, lassomycin and lariatins are unique lasso peptides that exhibit noticeable anti-tuberculosis (TB) activity. Due to the unique threaded structure and the unusual bactericidal mechanism toward Mycobacterium tuberculosis, these peptides have drawn considerable interest, not only in the field of total synthesis but also in several other fields including biosynthesis, bioengineering, and structure-activity studies. During the past few years, significant progress has been made in understanding the biosynthetic mechanism of these intriguing compounds, which has provided a solid foundation for future work. This review highlights recent achievements in the discovery, structure elucidation, biological activity, and the unique anti-TB mechanism of lasso peptides. Moreover, the discovery of their biosynthetic pathway has laid the foundation for combinatorial biosynthesis of their analogs, which provides new perspectives for the production of novel anti-TB lasso peptides.
... Blue asterisks mark methyltransferase-containing clusters acquired through horizontal gene transfer (Inokoshi et al. 2012). It is also worth mentioning that both lassomycin and lariatin showed anti-TB activity, further demonstrating that they are closely related; however, whether they have similar antibacterial mechanisms is still in question (Inokoshi et al. 2016). ...
Lasso peptides belong to a peculiar family of ribosomally synthesized and post-translationally modified peptides (RiPPs)—natural products with an unusual isopeptide-bonded slipknot structure. Except for assembling of this unusual lasso fold, several further post-translational modifications of lasso peptides, including C-terminal methylation, phosphorylation/poly-phosphorylation, citrullination, and acetylation, have been reported recently. However, most of their biosynthetic logic have not been elucidated except the phosphorylated paeninodin lasso peptide. Herein, we identified two novel lassomycin-like lasso peptide biosynthetic pathways and, for the first time, characterized a novel C-terminal peptide carboxyl methyltransferase involved in these pathways. Our investigations revealed that this new family of methyltransferase could specifically methylate the C terminus of precursor peptide substrates, eventually leading to lassomycin-like C-terminal methylated lasso peptides. Our studies offer another rare insight into the extraordinary strategies of chemical diversification adopted by lasso peptide biosynthetic machinery and predicated two valuable sources for methylated lasso peptide discovery.
... 29,31,124 The biosynthetic enzymes investigated thus far show a strict preference for either Glu or Asp for macrolactam formation, depending on whatever the native precursor peptide contains. 26,29,31,34,39,50,55,125,126 Most investigated systems also seem to be fixed with regard to the size of the ring formed. In this matter, all isolated naturally occurring lasso peptides have rings consisting of 7 (only with Glu), 8, or 9 amino acids and precursor variants with shifted positions of these residues are not processed by the enzymes. ...
... 34 Most lasso peptide biosynthetic machineries also show a strong preference for the residue naturally occurring at position 1 of the core peptide, i.e. if the lasso peptide contains a Gly at position 1, an exchange to Ala leads to significant decreases in lasso peptide production, and vice versa. 26,28,31,39,50,55,125,126 The only known case where this is different is again the fusilassin machinery, which was shown to tolerate amino acids of all kinds at position 1 with exception of Pro. 34 As the native precursor peptide features a Trp at position 1 of the core region, it is not surprising that processing is most efficient for other large residues (e.g., Tyr, Phe, Lys, and Leu), but less so for smaller and charged residues (e.g., His, Glu, Ala). ...
... 28,47 3. The Thr(-2) residue in all so far investigated lasso peptide precursors. 26,28,29,31,35,39,50,55,124,125 4. The position 1 residue of many core peptides. 26,28,29,31,34,35,42,50,55,125,126 5. ...
