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Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i’s) in combination with chemotherapy have shown promising results in preclinical studies but minimal efficacy with substantial toxicity in clinical trials. To explore novel combinational strategies that can overcome these limitations, we performed an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identified thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a novel determinant of CHK1i sensitivity. We established a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR1 inhibitor auronafin, an anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, these findings identify a new pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity.

The ability to manipulate the chemical composition of proteins and peptides has been central to the development of improved polypeptide-based therapeutics and has enabled researchers to address fundamental biological questions that would otherwise be out of reach. Protein ligation, in which two or more polypeptides are covalently linked, is a powerful strategy for generating semisynthetic products and for controlling polypeptide topology. However, specialized tools are required to efficiently forge a peptide bond in a chemoselective manner with fast kinetics and high yield. Fortunately, nature has addressed this challenge by evolving enzymatic mechanisms that can join polypeptides using a diverse set of chemical reactions. Here, we summarize how such nature-inspired protein ligation strategies have been repurposed as chemical biology tools that afford enhanced control over polypeptide composition. [Figure not available: see fulltext.].

Histone H3 monoaminylations at glutamine(Q) 5 represent an important family of epigenetic markers in neurons that play critical roles in the mediation of permissive gene expression (1, 2). We previously demonstrated that H3Q5 serotonylation(ser) and dopaminylation(dop) are catalyzed by the Transglutaminase 2 (TGM2) enzyme and alter both local and global chromatin states (3, 4). Here, we found that TGM2 additionally functions as an “eraser” of H3 monoaminylations that is capable of “re-writing” these epigenetic marks in cells, including a new class of this modification, H3Q5 histaminylation(his), which displays dynamic diurnal expression in brain and contributes to neural rhythmicity. We found that H3Q5his inhibits binding of the MLL1 complex to the H3 N-terminus and attenuates its methyltransferase activity on H3 lysine(K) 4. We determined that H3Q5 monoaminylation dynamics are dictated by local monoamine concentrations, which are utilized by TGM2. Taken together, we present here a novel mechanism through which a single chromatin regulatory enzyme is capable of sensing chemical microenvironments to affect the epigenetic states of cells.One sentence summaryA single enzyme, TGM2, bidirectionally controls H3 monoaminylation dynamics, which, in turn, facilitate neural rhythmicity.

Bufadienolides are toxic components widely found in amphibious toads that exhibit a wide range of biological activities. Guided by UPLC-QTOF-MS analysis, several 3-epi-bufadienolides with unique structures were isolated from the bile of the Asiatic toad, Bufo gargarizans. However, the enzymatic machinery of this epimerization in toads and its significance in chemical ecology remains poorly understood. Herein, we firstly compared the toxicities of two typical bufadienolides, bufalin (featuring a 14β-hydroxyl) and resibufogenin (containing a 14, 15-epoxy group), with their corresponding 3-epi isomers in a zebrafish model. The results of the toxicology assays showed that the ratio of maximum non-toxic concentrations of these two pairs of compounds are 256 and 96 times, respectively, thereby indicating that 3-hydroxyl epimerization leads to a significant decrease in toxicity. Aiming to investigate the biotransformation of 3-epi bufadienolides in toads, we applied liver lysate to transform bufalin and found that it could stereoselectively catalyze the conversion of bufalin into its 3α-hydroxyl epimer. Following this, we cloned and characterized a short-chain dehydrogenase/reductase, HSE-1, from the toad liver cDNA library and verified its 3(β→α)-hydroxysteroid epimerization activity. To the best of our knowledge, this is the first hydroxyl epimerase identified from amphibians that regulates the toxicity of animal-derived natural products.

DJ-1 (also known as PARK7) is a multifunctional enzyme in human beings that is highly conserved and that has also been discovered in diverse species (ranging from prokaryotes to eukaryotes). Its complex enzymatic and non-enzymatic activities (such as anti-oxidation, anti-glycation, and protein quality control), as well as its role as a transcriptional coactivator, enable DJ-1 to serve as an essential regulator in multiple cellular processes (e.g., epigenetic regulations) and make it a promising therapeutic target for diverse diseases (especially cancer and Parkinson’s disease). Due to its nature as a Swiss army knife enzyme with various functions, DJ-1 has attracted a large amount of research interest, from different perspectives. In this review, we give a brief summary of the recent advances with respect to DJ-1 research in biomedicine and psychology, as well as the progress made in attempts to develop DJ-1 into a druggable target for therapy.

Reactive cellular metabolites can modify macromolecules and form adducts known as nonenzymatic covalent modifications (NECMs). The dissection of the mechanisms, regulation, and consequences of NECMs, such as glycation, has been challenging due to the complex and often ambiguous nature of the adducts formed. Specific chemical tools are required to directly track the formation of these modifications on key targets in order to uncover their underlying physiological importance. Here, we present the novel chemoenzymatic synthesis of an active azido-modified ribose analog, 5-azidoribose (5-AR), as well as the synthesis of an inactive control derivative, 1-azidoribose (1-AR), and their application toward understanding protein ribose-glycation in vitro and in cellulo. With these new probes we found that, similar to methylglyoxal (MGO) glycation, ribose glycation specifically accumulates on histones. In addition to fluorescent labeling, we demonstrate the utility of the probe in enriching modified targets, which were identified by label-free quantitative proteomics and high-resolution MS/MS workflows. Finally, we establish that the known oncoprotein and hexose deglycase, fructosamine 3-kinase (FN3K), recognizes and facilitates the removal of 5-AR glycation adducts in live cells, supporting the dynamic regulation of ribose glycation as well as validating the probe as a new platform to monitor FN3K activity. Altogether, we demonstrate this probe’s utilities to uncover ribose-glycation and deglycation events as well as track FN3K activity toward establishing its potential as a new cancer vulnerability.

Epigenetic modifications, including those on DNA and histones, have been shown to regulate cellular metabolism by controlling expression of enzymes involved in the corresponding metabolic pathways. In turn, metabolic flux influences epigenetic regulation by affecting the biosynthetic balance of enzyme cofactors or donors for certain chromatin modifications. Recently, non-enzymatic covalent modifications (NECMs) by chemically reactive metabolites have been reported to manipulate chromatin architecture and gene transcription through multiple mechanisms. Here, we summarize these recent advances in the identification and characterization of NECMs on nucleic acids, histones, and transcription factors, providing an additional mechanistic link between metabolism and epigenetics.

Because of their long half-lives and highly nucleophilic tails, histones are particularly susceptible to accumulating nonenzymatic covalent modifications, such as glycation. The resulting modifications can have profound effects on cellular physiology due to the regulatory role histones play in all DNA-templated processes; however, the complexity of Maillard chemistry on proteins makes tracking and enriching for glycated proteins a challenging task. Here, we characterize glyoxal (GO) modifications on histones using quantitative proteomics and an aniline-derived GO-reactive probe. In addition, we leverage this chemistry to demonstrate that the glycation regulatory proteins DJ-1 and GLO1 reduce levels of histone GO adducts. Finally, we employ a two-round pull-down method to enrich histone H3 GO glycation and map these adducts to specific chromatin regions.

Vegetated coastal ecosystems (VCEs; i.e., mangroves, saltmarshes, and seagrasses) represent important sources of natural methane emission. Despite recent advances in the understanding of novel taxa and pathways associated with methanogenesis in these ecosystems, the key methanogenic players and the contribution of different substrates to methane formation remain elusive. Here, we systematically investigate the community and activity of methanogens using publicly available metatranscriptomes at a global scale together with our in-house metatranscriptomic dataset. Taxonomic profiling reveals that 13 groups of methanogenic archaea were transcribed in the investigated VCEs, and they were predominated by Methanosarcinales. Among these VCEs, methanogens exhibited all the three known methanogenic pathways in some mangrove sediments, where methylotrophic methanogens Methanosarcinales/Methanomassiliicoccales grew on diverse methyl compounds and coexisted with hydrogenotrophic (mainly Methanomicrobiales) and acetoclastic (mainly Methanothrix) methanogens. Contrastingly, the predominant methanogenic pathway in saltmarshes and seagrasses was constrained to methylotrophic methanogenesis. These findings reveal different archaeal methanogens in VCEs and suggest the potentially distinct methanogenesis contributions in these VCEs to the global warming.

Metagenomic analysis has facilitated prediction of a variety of carbon utilization potentials by uncultivated archaea including degradation of protein, which is a wide-spread carbon polymer in marine sediments. However, the activity of detrital catabolic protein degradation is mostly unknown for the vast majority of archaea. Here, we show actively executed protein catabolism in three archaeal phyla (uncultivated Thermoplasmata, SG8-5; Bathyarchaeota subgroup 15; Lokiarchaeota subgroup 2c) by RNA- and lipid-stable isotope probing in incubations with different marine sediments. However, highly abundant potential protein degraders Thermoprofundales (MBG-D) and Lokiarchaeota subgroup 3 were not incorporating 13C-label from protein during incubations. Nonetheless, we found that the pathway for protein utilization was present in metagenome associated genomes (MAGs) of active and inactive archaea. This finding was supported by screening extracellular peptidases in 180 archaeal MAGs, which appeared to be widespread but not correlated to organisms actively executing this process in our incubations. Thus, our results have important implications: (i) multiple low-abundant archaeal groups are actually catabolic protein degraders; (ii) the functional role of widespread extracellular peptidases is not an optimal tool to identify protein catabolism, and (iii) catabolic degradation of sedimentary protein is not a common feature of the abundant archaeal community in temperate and permanently cold marine sediments.

Methylglyoxal (MGO) is a reactive dicarbonyl metabolite that modifies histones in vivo and induces changes in chromatin structure and function. Here we report the synthesis and application of a chemical probe for investigating MGO-glycation. A two-step synthesis of a Cu-click compatible alkynyl oxoaldehyde probe (AlkMGO) via sequential Dess–Martin and Riley oxidations is presented. This synthesis elevates the accessibility and utility of an important tool for tracking, enriching, and studying MGO-glycation to aid in understanding its underlying biochemical functions.

Cellular proteins continuously undergo non-enzymatic covalent modifications (NECMs) that accumulate under normal physiological conditions and are stimulated by changes in the cellular microenvironment. Glycation, the hallmark of diabetes, is a prevalent NECM associated with an array of pathologies. Histone proteins are particularly susceptible to NECMs due to their long half-lives and nucleophilic disordered tails that undergo extensive regulatory modifications; however, histone NECMs remain poorly understood. Here we perform a detailed analysis of histone glycation in vitro and in vivo and find it has global ramifications on histone enzymatic PTMs, the assembly and stability of nucleosomes, and chromatin architecture. Importantly, we identify a physiologic regulation mechanism, the enzyme DJ-1, which functions as a potent histone deglycase. Finally, we detect intense histone glycation and DJ-1 overexpression in breast cancer tumors. Collectively, our results suggest an additional mechanism for cellular metabolic damage through epigenetic perturbation, with implications in pathogenesis., Proteins continuously undergo non-enzymatic modifications such as glycation, which accumulate under physiological conditions but can be enhanced in disease. Here the authors characterise histone glycation, provide evidence that it affects chromatin, particularly in breast cancer, and identify DJ-1 as a deglycase.

Thiopeptins are a complex of thiopeptide antibiotics similar in structure to thiostrepton and harboring a thioamide, a rare moiety among natural products. Here, we illustrate through a series of in vivo experiments that the thioamide moiety of thiopeptins is generated posttranslationally by a TfuA-YcaO pair, encoded in the thiopeptin biosynthetic gene cluster, before the maturation of the thiopeptide bicyclic scaffold, enhancing the understanding of the biosynthetic logic of thioamide-containing thiopeptides.

The oncoprotein and Parkinson's disease-associated enzyme DJ-1/PARK7 has emerged as a promiscuous deglycase that can remove methylglyoxal-induced glycation adducts from both proteins and nucleotides. However, dissecting its structural and enzymatic functions remains a challenge due to the lack of potent, specific, and pharmacokinetically stable inhibitors targeting its catalytic site (including Cys106). To evaluate potential drug-like leads against DJ-1, we leveraged its deglycase activity in an enzyme-coupled, fluorescence lactate-detection assay based on the recent understanding of its deglycation mechanism. In addition, we developed assays to directly evaluate DJ-1's esterase activity using both colorimetric and fluorescent substrates. The resulting optimized assay was used to evaluate a library of potential reversible and irreversible DJ-1 inhibitors. The deglycase activity-oriented screening strategy described herein establishes a new platform for the discovery of potential anti-cancer drugs.

Bufospirostenin A (1) and bufogargarizin C (2), two novel steroids with rearranged A/B rings, were isolated from the toad Bufo bufo gargarizans. Compound 1 represents the first spirostanol found in animals. Compound 2 is an unusual bufadienolide with a cycloheptatriene B ring. Their structures were elucidated by spectroscopic analysis, single crystal X-ray diffraction analysis, and computational calculations.

Three pairs of bufadienolide l/d-lactate epimers (1-6) were isolated from the eggs of the toad Bufo bufo gargarizans. The structures were elucidated by using spectroscopic methods, X-ray diffraction analysis and a modified Mosher's method. Compounds 1-6 represent the first occurrence of lactate-conjugated bufadienolides in nature, and illustrate the existence of an enzyme-controlled epimerization from l- to d-lactate in amphibians. The biosynthetic pathways, in which two key enzymes might be involved (i.e., lactate racemase and acyltransferase), were proposed. In addition, the biological assays revealed that compounds 1-4 are potent cytotoxic agents against human gastric cancer cells BGC-823 and human lung cancer cells A549 with IC50 values in a range of 8.0 to 80.0 nM.

Thiopeptide antibiotics are a class of typical ribosomally synthesized and post-translationally modified peptides (RiPPs) with complex chemical structures that are difficult to construct via chemical synthesis. To date, more than 100 thiopeptides have been discovered, and most of these compounds exhibit remarkable biological activities, such as antibacterial, antitumor and immunosuppressive activities. Therefore, studies of the biosynthesis of thiopeptides can contribute to the development of new drug leads and facilitate the understanding of the complex post-translational modifications (PTMs) of peptides and/or proteins. Since the biosynthetic gene clusters of thiopeptides were first discovered in 2009, several research studies regarding the biochemistry and enzymology of thiopeptide biosyntheses have been reported, indicating that their characteristic framework is constructed via a cascade of common PTMs and that additional specific PTMs diversify the molecules. In this review, we primarily summarize recent advances in understanding the biosynthesis of thiopeptide antibiotics and propose some potential applications based on our insights into the biosynthetic logic and machinery.

We herein report the isolation and characterization of a key linear intermediate in the biosynthetic pathway of pyrroindomycins, the potent spirotetramate natural products produced by Streptomyces rugosporus. This polyene intermediate bears a γ-hydroxymethyl group that is exocyclic to the tetramate moiety, indicating that a serine residue serves as the three-carbon unit for tetramate formation and chain-elongation termination. The further conversion involves an acetylation-elimination of the exocyclic γ-hydroxymethyl group to generate a γ-methylene group, which is indispensable for intramolecular [4 + 2] cross-bridging to construct the characteristic pentacyclic core. The findings presented in this study provide new insights into the biosynthesis of pyrroindomycins, and thus suggest a common paradigm for both spirotetramates and spirotetronates in processing the exocyclic γ-hydroxymethyl group of the five-membered heterocycle.

Cells are continuously subjected to an array of reactive/toxic chemical species which are produced both endogenously through metabolic pathways and taken up exogenously by diet and exposure to drugs or toxins. As a result, proteins often undergo non-enzymatic covalent modifications (NECMs) by these species, which can alter protein structure, function, stability, and binding partner affinity. NECMs accumulate over time and are linked to various diseases such as Alzheimer's disease, cancer, and diabetes. In the cellular proteome, histones have some of the longest half-lives, making them prime targets for NECMs. In addition, histones have emerged as key regulators of transcription, a function that is primarily controlled by modification of their tails. These modifications are usually installed or removed enzymatically, but recent evidence suggests that some may also occur non-enzymatically. Despite the vast knowledge detailing the relationship between histone modifications and gene regulation, NECMs on histones remain poorly explored. A major reason for this difference stems from the fact that, unlike their enzymatically installed counterparts, NECMs are difficult to both control and test in vivo. Here, we review advances in our understanding of the effect non-enzymatic covalent modifications (NECMs) have on the epigenetic landscape, cellular fate, and their implications in disease. Cumulatively, this illustrates how the epigenetic code is directly toxified by chemicals and detoxified by corresponding eraser enzymes.

Many cellular processes as well as their associated pathologies are regulated by protein post-translational modifications (PTMs). Understanding the precise roles of these adducts hinges on the development of methods to robustly and site-specifically manipulate proteins in their physiological environments. Recently, ultrafast intein protein trans-splicing (PTS) was harnessed to incorporate site-specific modifications on cellular chromatin in live cells. In this chapter, we present the protocols for the generation of synthetic modifications on native chromatin as well as highlight the capabilities of this methodology.

Thiostrepton (TSR), an archetypal bimacrocyclic thiopeptide antibiotic that arises from complex posttranslational modifications of a genetically encoded precursor peptide, possesses a quinaldic acid (QA) moiety within the side-ring system of a thiopeptide-characteristic framework. Focusing on selective engineering of the QA moiety, i.e., by fluorination or methylation, we have recently designed and biosynthesized biologically more active TSR analogs. Using these analogs as chemical probes, we uncovered an unusual indirect mechanism of TSR-type thiopeptides, which are able to act against intracellular pathogens through host autophagy induction in addition to direct targeting of bacterial ribosome. Herein, we report the accumulation of 6'-fluoro-7', 8'-epoxy-TSR, a key intermediate in the preparation of the analog 6'-fluoro-TSR. This unexpected finding led to unveiling of the TSR maturation process, which involves an unusual dual activity of TsrI, an α/β-hydrolase fold protein, for cascade C-N bond cleavage and formation during side-ring system construction. These two functions of TsrI rely on the same catalytic triad, Ser72-His200-Asp191, which first mediates endopeptidyl hydrolysis that occurs selectively between the residues Met-1 and Ile1 for removal of the leader peptide and then triggers epoxide ring opening for closure of the QA-containing side-ring system in a regio- and stereo-specific manner. The former reaction likely requires the formation of an acyl-Ser72 enzyme intermediate; in contrast, the latter is independent of Ser72. Consequently, C-6' fluorination of QA lowers the reactivity of the epoxide intermediate and, thereby, allows the dissection of the TsrI-associated enzymatic process that proceeds rapidly and typically is difficult to be realized during TSR biosynthesis.

The quinaldic acid (QA) moiety in the side ring of thiostrepton (TSR), which can be modified regioselectively via precursor-directed mutational biosynthesis, was proven to be biologically relevant but tunable, affecting TSR's outstanding antibacterial activities. In this study, we sought to obtain TSR derivatives with varying amino acid residues connected to the QA moiety. The generation of these TSR derivatives relied on single “base”-based mutagenesis, and six new TSR-type compounds were obtained. Moreover, the simultaneous mutation of Ile1 and Ala2 in the TSR side ring resulted in a naturally occurring compound, siomycin (SIO), together with a new component, SIO-Dha2Ser. The anti-infection assays indicated that all of these new compounds could act as both antimicrobial agents and autophagy inducers, and these two kinds of activities can also be separated via regioselective modifications on the TSR side ring.

The double-mutant strain Streptomyces laurentii ΔtsrB/T was designed and constructed based on a recent understanding regarding the structure–activity relationship of thiostrepton (TSR) against prokaryotic pathogens. Five new C-terminally methylated TSR (CmTSR) derivatives that varied in the side-ring structure were obtained via the chemical feeding of quinaldic acid (QA) analogs. These derivatives provide new insights into the tolerance of QA incorporation in TSR biosynthesis. Certain members of the tested TSR derivatives, meanwhile, exhibited much better antibacterial activities than all currently known thiopeptide antibiotics.

天然产物一直是药物分子设计和开发过程中的重要灵感来源之一, 源自天然产物的临床用药目前也占据着难以替代的地位. 但是大多数天然来源的药物分子结构复杂、分离困难, 利用传统的合成化学和天然产物化学方法难以满足日益增长的市场需求. 天然产物在其产生物种中一定对应着一个由若干功能各异的基因元件所构成的生物合成基因群, 或成簇分布或离散分布. 合成生物学则旨在通过对不同基因元件的改造、组合、拼装而得到新的生物途径和体系. 本文主要将针对合成生物学在天然药物研究中的应用进行总结和展望, 并从基因元件以及合成生物学的角度重新认识和理解天然产物的生物合成.

Thiostrepton is a potent archetypal thiopeptide antibiotic. According to its mechanism known to target bacterial ribosome, we show here a rational design upon modeling of this molecule into the ribosome complex and an effective biosynthesis of new thiopeptide antibiotics through regioselective modifications. The resulting derivatives exhibit a series of anticipated and unanticipated pharmaceutical advantages, including improvement in activity against a number of drug-resistant pathogens and in water solubility that has largely affected the clinical use of thiostrepton.

Summary Here, we provide structural insights into PyrE3, a flavin-dependent [4 + 2] cyclase that catalyzes trans -decalin formation in the biosynthesis of pyrroindomycins. PyrE3 shares an architecture/domain organization head-to-tail similarity with the members of the family of para -hydroxybenzoate hydroxylase (pHBH)-fold monooxygenases, and possesses a flavin adenine dinucleotide (FAD)-binding domain, a middle domain, and a C-terminal thioredoxin-like domain. The FAD-binding domain forms a central hub of the protein structure, and binds with FAD in a "closed" conformation of pHBH-fold family monooxygenases known for their highly dynamic catalytic processes. FAD plays an essential structural role in PyrE3, where it is amenable to redox change; however, redox change has little effect on [4 + 2] cyclization activity. PyrE3 appears to selectively accommodate a tetramate-containing, linear polyene intermediate in a highly positively charged pocket, which is located at the interface between the FAD-binding domain and the middle domain, and can accelerate trans -decalin formation likely through an endo -selective [4 + 2] transition state.

Thiostrepton, a natural peptide macrocycle, is of great interest due to its structural complexity and numerous biological activities, including anti-bacterial, anti-tumor, and anti-plasmodial activities. The quinaldic acid (QA) moiety-containing side ring (loop 2) was proven to play an important role in carrying out these functions. Previously, we proposed biosynthetic logic for thiostrepton loop 2 and demonstrated the formation mechanism of QA. Herein, we report the discovery and efficient synthesis of a biologically active alkaloid, that is, a key intermediate involved in the thiostrepton biosynthetic pathway. A chemo-enzymatic method was performed to synthesize the molecule, and a series of analogs were prepared for bioassays, which included the examination of anti-bacterial and anti-tumor activities.

The ribosomal origin of thiopeptide antibiotics, a class of sulfur-rich and highly modified poly(thi)azolyl natural products, has recently been uncovered and features complex post-translational modifications (PTMs) of a precursor peptide. Based on molecular engineering and production improvement, we report insight into the biosynthesis of two bicyclic thiopeptide compounds, thiostrepton and nosiheptide. The PTMs of thiostrepton tolerate variations in the first two amino acids of the core peptide part of the precursor peptide: (1) the mutation of Ile1 to Val had no apparent effect on molecular maturation, suggesting that attachment of the quinaldic moiety at position 1 is not residue-dependent for the construction of the side ring system; and (2) the change of Ala2 to Ser led exclusively to the production of an analog that bears a corresponding dehydroamino acid residue, indicating that dehydration at position 2 is site-selective or that the oxazoline formed by cyclodehydration is inaccessible for maturation. For nosiheptide biosynthesis in particular, we provide the first structural evidence that construction of the specific side ring system precedes formation of the common central heterocycle domain and therefore propose that formation of a characteristic thiopeptide framework interweaves both common and specific PTMs that are interdependent. The above efforts benefited from the development of a uniform approach to examine the effectiveness for trans expression of gene encoding precursor peptides and associated PTM capacity. This approach is independent of knowledge regarding organism-specific regulatory mechanisms and potentially applicable to other systems that produce ribosomally synthesized peptide natural products.

Diversity-oriented biosynthesis of a library of antimycin-like compounds (380 altogether) was accomplished by using multiplex combinatorial biosynthesis. The core strategy depends on the use of combinatorial chemistry at different biosynthetic stages. This approach is applicable for the diversification of polyketides, nonribosomal peptides, and the hybrids that share a similar biosynthetic logic.

Side-ring-modified thiostrepton (TSR) derivatives that vary in their quinaldic acid (QA) substitution possess more potent biological activities and better pharmaceutical properties than the parent compound. In this work, we sought to introduce fluorine onto C-7′ or C-8′ of the TSR QA moiety via precursor-directed mutational biosynthesis to obtain new TSR variants. Unexpectedly, instead of the target product, the exogenous chemical feeding of 7-F-QA into the ΔtsrT mutant strain resulted in a unique TSR analog with an incomplete side-ring structure and an unoxidized QA moiety (1). Accordingly, two cytochrome P450 genes, tsrP and tsrR, were in-frame deleted to elucidate the candidate responsible for the monooxidation of the QA moiety in TSR. The unfluorinated analog of compound 1 that was thus isolated from ΔtsrP (2) and the abolishment of TSR production in ΔtsrR revealed not only the biosynthetic logic of the TSR side-ring but also the essential checkpoint in TSR maturation before macro-ring closure.

Mycobacterial infection has long been one of the most serious infectious diseases throughout the world. Since the abuse of antibiotics, and for other reasons, the emergence of bacterial drug-resistance is now one of the most urgent clinical problems. Nowadays, the speed of antibiotic development is actually far more slowly than that of the bacterial drug-resistance generation. Thereby, searching for new efficient antibiotics is a top priority in pharmaceutical studies. In this commentary, we summarized the recent advances regarding the development of new thiopeptide antibiotics via biosynthetic strategy and the discovery of a novel dual mechanism of action against Mycobacterium marinum-represented intracellular pathogens.