Elucidation of unusual biosynthesis and DnaN-targeting mode of action of potent anti-tuberculosis antibiotics Mycoplanecins.

DNA polymerase III sliding clamp (DnaN) was recently validated as a new anti-tuberculosis target employing griselimycins. Three (2 S,4 R)-4-methylproline moieties of methylgriselimycin play significant roles in target binding and metabolic stability. Here, we identify the mycoplanecin biosynthetic gene cluster by genome mining using bait genes from the 4-methylproline pathway. We isolate and structurally elucidate four mycoplanecins comprising scarce homo-amino acids and 4-alkylprolines. Evaluating mycoplanecin E against Mycobacterium tuberculosis surprisingly reveals an excitingly low minimum inhibition concentration at 83 ng/mL, thus outcompeting griselimycin by approximately 24-fold. We show that mycoplanecins bind DnaN with nanomolar affinity and provide a co-crystal structure of mycoplanecin A-bound DnaN. Additionally, we reconstitute the biosyntheses of the unusual L-homoleucine, L-homonorleucine, and (2 S,4 R)-4-ethylproline building blocks by characterizing in vitro the full set of eight enzymes involved. The biosynthetic study, bioactivity evaluation, and drug target validation of mycoplanecins pave the way for their further development to tackle multidrug-resistant mycobacterial infections.

Nobachelins, new siderophores from Nocardiopsisbaichengensis protecting Caenorhabditiselegans from Pseudomonasaeruginosa infection.

The biosynthetic potential of actinobacteria to produce novel natural products is still regarded as immense. In this paper, we correlated a cryptic biosynthetic gene cluster to chemical molecules by genome mining and chemical analyses, leading to the discovery of a new group of catecholate-hydroxamate siderophores, nobachelins, from Nocardiopsisbaichengensis DSM 44845. Nobachelin biosynthesis genes are conserved in several bacteria from the family Nocardiopsidaceae. Structurally, nobachelins feature fatty-acylated hydroxy-ornithine and a rare chlorinated catecholate group. Intriguingly, nobachelins rescued Caenorhabditiselegans from Pseudomonasaeruginosa-mediated killing.

Discovery and Characterization of a Myxobacterial Lanthipeptide with Unique Biosynthetic Features and Anti-inflammatory Activity.

The genomes of myxobacteria harbor a variety of biosynthetic gene clusters encoding numerous secondary metabolites, including ribosomally synthesized and post-translationally modified peptides (RiPPs) with diverse chemical structures and biological activities. However, the biosynthetic potential of RiPPs from myxobacteria remains barely explored. Herein, we report a novel myxobacteria lanthipeptide myxococin identified from Myxococcus fulvus. Myxococins represent the first example of lanthipeptides, of which the characteristic multiple thioether rings are installed by employing a Class II lanthipeptide synthetase MfuM and a Class I lanthipeptide cyclase MfuC in a cascaded way. Unprecedentedly, we biochemically characterized the first M61 family aminopeptidase MfuP involved in RiPP biosynthesis, demonstrating that MfuP showed the activity of an endopeptidase activity. MfuP is leader-independent but strictly selective for the multibridge structure of myxococin A and responsible for unwrapping two rings via amide bond hydrolysis, yielding myxococin B. Furthermore, the X-ray crystal structure of MfuP and structural analysis, including active-site mutations, are reported. Finally, myxococins are evaluated to exhibit anti-inflammatory activity in lipopolysaccharide-induced macrophages without detectable cytotoxicity.

The Disorazole Z Family of Highly Potent Anticancer Natural Products from Sorangium cellulosum: Structure, Bioactivity, Biosynthesis, and Heterologous Expression.

Myxobacteria serve as a treasure trove of secondary metabolites. During our ongoing search for bioactive natural products, a novel subclass of disorazoles termed disorazole Z was discovered. Ten disorazole Z family members were purified from a large-scale fermentation of the myxobacterium Sorangium cellulosum So ce1875 and characterized by electrospray ionization-high-resolution mass spectrometry (ESI-HRMS), X-ray, nuclear magnetic resonance (NMR), and Mosher ester analysis. Disorazole Z compounds are characterized by the lack of one polyketide extension cycle, resulting in a shortened monomer in comparison to disorazole A, which finally forms a dimer in the bis-lactone core structure. In addition, an unprecedented modification of a geminal dimethyl group takes place to form a carboxylic acid methyl ester. The main component disorazole Z1 shows comparable activity in effectively killing cancer cells to disorazole A1 via binding to tubulin, which we show induces microtubule depolymerization, endoplasmic reticulum delocalization, and eventually apoptosis. The disorazole Z biosynthetic gene cluster (BGC) was identified and characterized from the alternative producer S. cellulosum So ce427 and compared to the known disorazole A BGC, followed by heterologous expression in the host Myxococcus xanthus DK1622. Pathway engineering by promoter substitution and gene deletion paves the way for detailed biosynthesis studies and efficient heterologous production of disorazole Z congeners. IMPORTANCE Microbial secondary metabolites are a prolific reservoir for the discovery of bioactive compounds, which prove to be privileged scaffolds for the development of new drugs such as antibacterial and small-molecule anticancer drugs. Consequently, the continuous discovery of novel bioactive natural products is of great importance for pharmaceutical research. Myxobacteria, especially Sorangium spp., which are known for their large genomes with yet-underexploited biosynthetic potential, are proficient producers of such secondary metabolites. From the fermentation broth of Sorangium cellulosum strain So ce1875, we isolated and characterized a family of natural products named disorazole Z, which showed potent anticancer activity. Further, we report on the biosynthesis and heterologous production of disorazole Z. These results can be stepping stones toward pharmaceutical development of the disorazole family of anticancer natural products for (pre)clinical studies.

Bursts in biosynthetic gene cluster transcription are accompanied by surges of natural compound production in the myxobacterium Sorangium sp.

A better understanding of the genetic regulation of the biosynthesis of microbial compounds could accelerate the discovery of new biologically active molecules and facilitate their production. To this end, we have investigated the time course of genome-wide transcription in the myxobacterium Sorangium sp. So ce836 in relation to its production of natural compounds. Time-resolved RNA sequencing revealed that core biosynthesis genes from 48 biosynthetic gene clusters (BGCs; 92% of all BGCs encoded in the genome) were actively transcribed at specific time points in a batch culture. The majority (80%) of polyketide synthase and non-ribosomal peptide synthetase genes displayed distinct peaks of transcription during exponential bacterial growth. Strikingly, these bursts in BGC transcriptional activity were associated with surges in the net production rates of known natural compounds, indicating that their biosynthesis was critically regulated at the transcriptional level. In contrast, BGC read counts from single time points had limited predictive value about biosynthetic activity, since transcription levels varied >100-fold among BGCs with detected natural products. Taken together, our time-course data provide unique insights into the dynamics of natural compound biosynthesis and its regulation in a wild-type myxobacterium, challenging the commonly cited notion of preferential BGC expression under nutrient-limited conditions. The close association observed between BGC transcription and compound production warrants additional efforts to develop genetic engineering tools for boosting compound yields from myxobacterial producer strains.

Persicamidines-Unprecedented Sesquarterpenoids with Potent Antiviral Bioactivity against Coronaviruses.

A new family of highly unusual sesquarterpenoids (persicamidines A-E) exhibiting significant antiviral activity was isolated from a newly discovered actinobacterial strain, Kibdelosporangium persicum sp. nov., collected from a hot desert in Iran. Extensive NMR analysis unraveled a hexacyclic terpenoid molecule with a modified sugar moiety on one side and a highly unusual isourea moiety fused to the terpenoid structure. The structures of the five analogues differed only in the aminoalkyl side chain attached to the isourea moiety. Persicamidines A-E showed potent activity against hCoV-229E and SARS-CoV-2 viruses in the nanomolar range together with very good selectivity indices, making persicamidines promising as starting points for drug development.

Uncovering a Subtype of Microviridins via the Biosynthesis Study of FR901451.

Microviridins are a class of ribosomally synthesized and post-translationally modified peptides originally discovered from cyanobacteria, featured by intramolecular ω-ester and ω-amide bonds catalyzed by two ATP-grasp ligases. In this study, 104 biosynthetic gene clusters of microviridins from Bacteroidetes were bioinformatically analyzed, which unveiled unique features of precursor peptides. The analysis of core peptides revealed a microviridin-like biosynthetic gene cluster from Chitinophagia japonensis DSM13484 consisting of two potential precursors ChiA1 and ChiA2. Unexpectedly, the core peptide sequence of ChiA1 is consistent with the backbone of the elastase-inhibiting peptide FR901451, while ChiA2 is likely to be a precursor of an unknown product. However, an unusual C-terminal follower cleavage compared to the previously known microviridin pathways was observed and found to be dispensable for other modifications. To confirm the biosynthetic origin of FR901451, ATP-grasp ligases ChiC and ChiB were biochemically characterized to be responsible for the intramolecular ester and amide bond formation, respectively. In vitro reconstitution of the pathway showed the three-fold dehydrations of ChiA1 while unusual four-fold dehydrations were observed for ChiA2. Furthermore, in vivo gene coexpression facilitated the production of chitinoviridin A1 (FR901451) and two novel microviridin-class compounds chitinoviridin A2A and chitinoviridin A2B, with an extra macrolactone ring. All of these peptides showed potent inhibitory effects against elastase and chymotrypsin independently.

Dual-function chromogenic screening-based CRISPR/Cas9 genome editing system for actinomycetes.

Actinobacteria are one of the most important sources of pharmaceutically valuable and industrially relevant secondary metabolites. Modern genome mining reveals that the potential for secondary metabolite production of actinomycetes has been underestimated. Recently, the establishment of CRISPR/Cas9-based genetic manipulation approaches in actinomycetes opened a new era for genome engineering of this type of organism. Compared with the traditional methods, the application of CRISPR/Cas9 shows several advantages in actinomycetes including higher efficiency and ease of operation. However, the screening process for the correctly edited mutants and the plasmid curing are still time- and labor-intensive. To address this problem, we developed an updated version of the CRISPR/Cas9 genome editing system for actinomycetes, based on two chromogenic reporter systems (GusA and IdgS). Our system facilitates both processes of positive clone screening and plasmid curing. Here, we demonstrate by three case studies in both model actinomycetes and non-model actinomycetes that this system is faster and more efficient. We performed the deletion of one single gene, actIORFI (SCO5087 of the actinorhodin gene cluster) in Streptomyces coelicolor M145, one small-size (5.5 kb) gene cluster (orange-pigmented carotenoid gene cluster), and one relatively large-size (61 kb) gene cluster (abyssomicin gene cluster) in Verrucosispora sp. MS100137. The results presented in this study indicate that this updated CRISPR/Cas9 system employing chromogenic reporters is versatile and broadly applicable in genome engineering of actinomycetes, not only for the largest genus Streptomyces.

Heterologous expression of bacterial natural product biosynthetic pathways.

Covering: 2013 to June 2018 Heterologous expression of natural product biosynthetic pathways is of increasing interest in microbial biotechnology, drug discovery and optimization. It empowers not only the robust production of valuable biomolecules in more amenable heterologous hosts but also permits the generation of novel analogs through biosynthetic engineering. This strategy also facilitates the discovery of novel bioactive compounds following the functional expression of cryptic biosynthetic gene clusters (BGCs) from fastidious original producers or metagenomic DNA in surrogate hosts, thus facilitating genome mining in the post-genomic era. This review discusses recent advances and trends pertaining to the heterologous production of bacterial natural products, with an emphasis on new techniques, heterologous hosts, and novel chemistry since 2013.

Armeniaspirol Antibiotic Biosynthesis: Chlorination and Oxidative Dechlorination Steps Affording Spiro[4.4]non-8-ene.

Armeniaspirols are potent antibiotics containing an unusual spiro[4.4]non-8-ene moiety. Herein, we describe the cloning and functional analysis of the armeniaspirol biosynthetic gene cluster. Gene-inactivation studies and subsequent isolation of previously unknown biosynthetic intermediates shed light on intriguing biosynthetic details. Remarkably, deletion of ams15, which encodes a protein bearing a flavin-binding domain, led to the accumulation of several non-spiro intermediates with various numbers of chlorine substitutions on the pyrrole moiety. The di- and trichloropyrrole species were converted by Streptomyces albus expressing Ams15 into mono- and dichlorinated spiro derivatives, respectively. In addition, in vitro conversion of these non-spiro intermediates into des-N-methyl spiro intermediates by the cell lysate of the same recombinant strain proved Ams15 to be responsible for spiro formation through oxidative dehalogenation.

The natural product carolacton inhibits folate-dependent C1 metabolism by targeting FolD/MTHFD.

The natural product carolacton is a macrolide keto-carboxylic acid produced by the myxobacterium Sorangium cellulosum, and was originally described as an antibacterial compound. Here we show that carolacton targets FolD, a key enzyme from the folate-dependent C1 metabolism. We characterize the interaction between bacterial FolD and carolacton biophysically, structurally and biochemically. Carolacton binds FolD with nanomolar affinity, and the crystal structure of the FolD-carolacton complex reveals the mode of binding. We show that the human FolD orthologs, MTHFD1 and MTHFD2, are also inhibited in the low nM range, and that micromolar concentrations of carolacton inhibit the growth of cancer cell lines. As mitochondrial MTHFD2 is known to be upregulated in cancer cells, it may be possible to use carolacton as an inhibitor tool compound to assess MTHFD2 as an anti-cancer target.

Solving the Puzzle of One-Carbon Loss in Ripostatin Biosynthesis.

Ripostatin is a promising antibiotic that inhibits RNA polymerase by binding to a novel binding site. In this study, the characterization of the biosynthetic gene cluster of ripostatin, which is a peculiar polyketide synthase (PKS) hybrid cluster encoding cis- and trans-acyltransferase PKS genes, is reported. Moreover, an unprecedented mechanism for phenyl acetic acid formation and loading as a starter unit was discovered. This phenyl-C2 unit is derived from phenylpyruvate (phenyl-C3) and the mechanism described herein explains the mysterious loss of one carbon atom in ripostatin biosynthesis from the phenyl-C3 precursor. Through in vitro reconstitution of the whole loading process, a pyruvate dehydrogenase like protein complex was revealed that performs thiamine pyrophosphate dependent decarboxylation of phenylpyruvate to form a phenylacetyl-S-acyl carrier protein species, which is supplied to the subsequent biosynthetic assembly line for chain extension to finally yield ripostatin.

Isolation, Structure Elucidation, and (Bio)Synthesis of Haprolid, a Cell-Type-Specific Myxobacterial Cytotoxin.

Myxobacteria are well-established sources for novel natural products exhibiting intriguing bioactivities. We here report on haprolid (1) isolated from Byssovorax cruenta Har1. The compound exhibits an unprecedented macrolactone comprising four modified amino acids and a polyketide fragment. As configurational assignment proved difficult, a bioinformatic analysis of the biosynthetic gene cluster was chosen to predict the configuration of each stereocenter. In-depth analysis of the corresponding biosynthetic proteins established a hybrid polyketide synthase/nonribosomal peptide synthetase origin of haprolid and allowed for stereochemical assignments. A subsequent total synthesis yielded haprolid and corroborated all predictions made. Intriguingly, haprolid showed cytotoxicity against several cell lines in the nanomolar range whereas other cells were almost unaffected by treatment with the compound.

Interrogation of Streptomyces avermitilis for efficient production of avermectins.

The 2015 Nobel Prize in Physiology or Medicine has been awarded to avermectins and artemisinin, respectively. Avermectins produced by Streptomyces avermitilis are excellent anthelmintic and potential antibiotic agents. Because wild-type strains only produce low levels of avermectins, much research effort has focused on improvements in avermectin production to meet the ever increasing demand for such compounds. This review describes the strategies that have been widely employed and the future prospects of synthetic biology applications in avermectin yield improvement. With the help of genome sequencing of S. avermitilis and an understanding of the avermectin biosynthetic/regulatory pathways, synthetic and systems biotechnology approaches have been applied for precision engineering. We focus on the design and synthesis of biological chassis, parts, devices, and modules from diverse microbes to reconstruct and optimize their dynamic processes, as well as predict favorable effective overproduction of avermectins by a 4Ms strategy (Mine, Model, Manipulation, and Measurement).

Inactivation of SACE_3446, a TetR family transcriptional regulator, stimulates erythromycin production in Saccharopolyspora erythraea.

Erythromycin A is a widely used antibiotic produced by Saccharopolyspora erythraea; however, its biosynthetic cluster lacks a regulatory gene, limiting the yield enhancement via regulation engineering of S. erythraea. Herein, six TetR family transcriptional regulators (TFRs) belonging to three genomic context types were individually inactivated in S. erythraea A226, and one of them, SACE_3446, was proved to play a negative role in regulating erythromycin biosynthesis. EMSA and qRT-PCR analysis revealed that SACE_3446 covering intact N-terminal DNA binding domain specifically bound to the promoter regions of erythromycin biosynthetic gene eryAI, the resistant gene ermE and the adjacent gene SACE_3447 (encoding a long-chain fatty-acid CoA ligase), and repressed their transcription. Furthermore, we explored the interaction relationships of SACE_3446 and previously identified TFRs (SACE_3986 and SACE_7301) associated with erythromycin production. Given demonstrated relatively independent regulation mode of SACE_3446 and SACE_3986 in erythromycin biosynthesis, we individually and concomitantly inactivated them in an industrial S. erythraea WB. Compared with WB, the WBΔ3446 and WBΔ3446Δ3986 mutants respectively displayed 36% and 65% yield enhancement of erythromycin A, following significantly elevated transcription of eryAI and ermE. When cultured in a 5 L fermentor, erythromycin A of WBΔ3446 and WBΔ3446Δ3986 successively reached 4095 mg/L and 4670 mg/L with 23% and 41% production improvement relative to WB. The strategy reported here will be useful to improve antibiotics production in other industrial actinomycete.

Biosynthetic Studies of Telomycin Reveal New Lipopeptides with Enhanced Activity.

Telomycin (TEM) is a cyclic depsipeptide antibiotic active against Gram-positive bacteria. In this study, five new natural telomycin analogues produced by Streptomyces canus ATCC 12646 were identified. To understand the biosynthetic machinery of telomycin and to generate more analogues by pathway engineering, the TEM biosynthesis gene cluster has been characterized from S. canus ATCC 12646: it spans approximately 80.5 kb and consists of 34 genes encoding fatty acid ligase, nonribosomal peptide synthetases (NRPSs), regulators, transporters, and tailoring enzymes. The gene cluster was heterologously expressed in Streptomyces albus J1074 setting the stage for convenient biosynthetic engineering, mutasynthesis, and production optimization. Moreover, in-frame deletions of one hydroxylase and two P450 monooxygenase genes resulted in the production of novel telomycin derivatives, revealing these genes to be responsible for the specific modification by hydroxylation of three amino acids found in the TEM backbone. Surprisingly, natural lipopeptide telomycin precursors were identified when characterizing an unusual precursor deacylation mechanism during telomycin maturation. By in vivo gene inactivation and in vitro biochemical characterization of the recombinant enzyme Tem25, the maturation process was shown to involve the cleavage of previously unknown telomycin precursor-lipopeptides, to yield 6-methylheptanoic acid and telomycins. These lipopeptides were isolated from an inactivation mutant of tem25 encoding a (de)acylase, structurally elucidated, and then shown to be deacylated by recombinant Tem25. The TEM precursor and several semisynthetic lipopeptide TEM derivatives showed rapid bactericidal killing and were active against several multidrug-resistant (MDR) Gram-positive pathogens, opening the path to future chemical optimization of telomycin for pharmaceutical application.

A marine-derived Streptomyces sp. MS449 produces high yield of actinomycin X2 and actinomycin D with potent anti-tuberculosis activity.

In the course of our screening program for anti-Mycobacterium bovis bacillus Calmette-Guérin (BCG) and anti-Mycobacterium tuberculosis H37Rv (MTB H37Rv) agents from our marine natural product library, a newly isolated actinomycete strain, designated as MS449, was picked out for further investigation. The strain MS449, isolated from a sediment sample collected from South China Sea, produced actinomycin X(2) and actinomycin D in substantial quantities, which showed strong inhibition of BCG and MTB H37Rv. The structures of actinomycins were elucidated by nuclear magnetic resonance and mass spectrometric analysis. The strain MS449 was taxonomically characterized on the basis of morphological and phenotypic characteristics, genotypic data, and phylogenetic analysis. The 16S rRNA gene sequence of the strain was determined and a database search indicated that the strain was closely associated with the type strain of Streptomyces avermitilis (99.7 % 16S rRNA gene similarity). S. avermitilis has not been previously reported to produce actinomycins. The marine-derived strain of Streptomyces sp. MS449 produced notably higher quantities of actinomycin X(2) (1.92 mg/ml) and actinomycin D (1.77 mg/ml) than previously reported actinomycins producing strains. Thus, MS449 was considered of great potential as a new industrial producing strain of actinomycin X(2) and actinomycin D.

Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library.

A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues. A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K(m) of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that EstF was active in the temperature range of 0-60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited a high level of activity in the pH range of 7.0-10.0 showing the highest activity at pH 9.0.

Construction and preliminary analysis of a deep-sea sediment metagenomic fosmid library from Qiongdongnan Basin, South China Sea.

Preliminary characterization of the microbial phylogeny and metabolic potential of a deep-sea sediment sample from the Qiongdongnan Basin, South China Sea, was carried out using a metagenomic library approach. An effective and rapid method of DNA isolation, purification, and library construction was used resulting in approximately 200,000 clones with an average insert size of about 36 kb. End sequencing of 600 individual clones from the fosmid library generated 1,051 sequences with an average sequence length of 619 bp. Phylogenetic ascription indicated that this library was dominated by Bacteria, predominantly Proteobacteria, though Planctomycetes were also relatively abundant. Sulfate-reducing and anaerobic ammonium-oxidizing bacteria, which play important roles in the cycling of sedimentary nutrients, were abundant in the library. Cluster of orthologous groups category analysis showed that most of the genes contained in the end sequences were related to metabolism, and with cellular processes and signaling. Functional groups assigned by SEED (subsystems-based annotations) highlighted the existence of 'one-carbon' metabolism within this community as well as identifying functional genes involved in methanogenesis. Furthermore, diverse genes involved in the biodegradation of xenobiotics were found using Kyoto Encyclopedia of Genes and Genomes metabolic pathway analysis.