ß-Terrecyclene synthase constructs the quadrane backbone in terrecyclic acid biosynthesis.

Quadrane sesquiterpenes featuring a distinctive tricyclic skeleton exhibit potent antimicrobial and anticancer activities. Although extensive studies have attempted to reveal the multistep carbocation rearrangement involved in the formation of the tricyclic quadrane scaffold, the exact biosynthetic pathway and chemical logic to generate the quadrane structure remains mysterious. Here we identified a novel sesquiterpene synthase that is capable of generating ß-terrecyclene possessing the quadrane scaffold and characterized the biosynthetic pathway of a representative fungal quadrane terrecyclic acid. Further mutagenesis coupled with isotopically sensitive branching studies of this ß-terrecyclene synthase provided insight into the mechanism involved in the formation of the quadrane scaffold.

Metabolic Blockade-Based Genome Mining of Sea Anemone-Associated Streptomyces sp. S1502 Identifies Atypical Angucyclines WS-5995 A-E: Isolation, Identification, Biosynthetic Investigation, and Bioactivities.

Marine symbiotic and epiphyte microorganisms are sources of bioactive or structurally novel natural products. Metabolic blockade-based genome mining has been proven to be an effective strategy to accelerate the discovery of natural products from both terrestrial and marine microorganisms. Here, the metabolic blockade-based genome mining strategy was applied to the discovery of other metabolites in a sea anemone-associated Streptomyces sp. S1502. We constructed a mutant Streptomyces sp. S1502/Δstp1 that switched to producing the atypical angucyclines WS-5995 A-E, among which WS-5995 E is a new compound. A biosynthetic gene cluster (wsm) of the angucyclines was identified through gene knock-out and heterologous expression studies. The biosynthetic pathways of WS-5995 A-E were proposed, the roles of some tailoring and regulatory genes were investigated, and the biological activities of WS-5995 A-E were evaluated. WS-5995 A has significant anti-Eimeria tenell activity with an IC50 value of 2.21 µM. The production of antibacterial streptopyrroles and anticoccidial WS-5995 A-E may play a protective role in the mutual relationship between Streptomyces sp. S1502 and its host.

[Research progress on chemical constituents and pharmacological activities of small molecule compounds in scorpions].

Scorpions, a group of oldest animals with wide distribution in the world, have a long history of medicinal use. Scorpio, the dried body of Buthus martensii, is a rare animal medicine mainly used for the treatment of liver diseases, spasm, and convulsions in children in China. The venom has been considered as the active substance of scorpions. However, little is known about the small molecules in the venom of scorpions. According to the articles published in recent years, scorpions contain amino acids, fatty acids, steroids, and alkaloids, which endow scorpions with antimicrobial, anticoagulant, metabolism-regulating, and antitumor activities. This paper summarizes the small molecule chemical components and pharmacological activities of scorpions, with a view to providing valuable information for the discovery of new active molecules and the clinical use of scorpions.

Discrete Acyltransferases and Thioesterases in Iso-Migrastatin and Lactimidomycin Biosynthesis.

Iso-Migrastatin (iso-MGS) and lactimidomycin (LTM) are glutarimide-containing polyketide natural products (NPs) that are biosynthesized by homologous acyltransferase (AT)-less type I polyketide synthase (PKS) assembly lines. The biological activities of iso-MGS and LTM have inspired numerous efforts to generate analogues via genetic manipulation of their biosynthetic machinery in both native producers and model heterologous hosts. A detailed understanding of the MGS and LTM AT-less type I PKSs would serve to inspire future engineering efforts while advancing the fundamental knowledge of AT-less type I PKS enzymology. The mgs and ltm biosynthetic gene clusters (BGCs) encode for two discrete ATs of the architecture AT-enoylreductase (AT-ER) and AT-type II thioesterase (AT-TE). Herein, we report the functional characterization of the mgsB and ltmB and the mgsH and ltmH gene products, revealing that MgsB and LtmB function as type II thioesterases (TEs) and MgsH and LtmH are the dedicated trans-ATs for the MGS and LTM AT-less type I PKSs. In vivo and in vitro experiments demonstrated that MgsB was devoid of any AT activity, despite the presence of the conserved catalytic triad of canonical ATs. Cross-complementation experiments demonstrated that MgsH and LtmH are functionally interchangeable between the MGS and LTM AT-less type I PKSs. This work sets the stage for future mechanistic studies of AT-less type I PKSs and efforts to engineer the MGS and LTM AT-less type I PKS assembly lines for novel glutarimide-containing polyketides.

Semi-synthesis and structure-activity relationship study yield antibacterial vicenistatin derivatives with low cytotoxicity.

Vicenistatin (1) is a 20-membered polyketide macrocyclic antibiotic with potent antimicrobial and cytotoxic activities. In this study, to further explore the potential of 1 as candidates of antibacterial drug development, 4'-N-demethyl vicenistatin (2), a secondary metabolite obtained from the ∆vicG mutant strain of Monodonata labio-associated Streptomyces parvus SCSIO Mla-L010, was utilized as a starting material for modifications of 4'-amino group of vicenistatin. Six new vicenistatin derivatives (3-8) were semi-synthesized through a concise route of amino modification with various aliphatic and aromatic aldehydes. Our study reveals that the bioactivity of vicenistatin is closely related to amino modification in sugar moiety, which results from the length of alkyl side chain as well as the presence of electron withdrawing/denoting group on the benzene ring. Importantly, compounds 4 with a butyl group and 8 with a 3,5-dihydroxyl-benzyl group at 4'-amino group, respectively, exhibited good antimicrobial activities, with MIC values spanning 0.5-4 µg ml-1 to Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Micrococcus luteus and Bacillus subtilis, with low cytotoxicity. This research promotes the further exploration of structure-activity relationships of vicenistatin and provides new vicenistatin derivatives for development of future anti-infectious agents with reduced cytotoxicity.

Deciphering the Glycosylation Steps in the Biosynthesis of P-1894B and Grincamycin Isolated from Marine-Derived Streptomyces lusitanus SCSIO LR32.

Recently, we re-isolated the glycosylated angucycline antibiotics P-1894B (1) and grincamycin (1') from the marine-derived Streptomyces lusitanus SCSIO LR32 as potent antitumor agents and identified their biosynthesis gene cluster gcn. Both P-1894B (1) and grincamycin (1') possess a trisaccharide and a disaccharide moiety comprised of five deoxysugars. In this work, three genes encoding glycosyltransferases (GcnG1, GcnG2, and GcnG3) responsible for the assembly of deoxysugars into angucycline aglycone were identified from the biosynthesis gene cluster gcn. Gene inactivations of gcnG1, gcnG2, gcnG3, and gcnG1G2 by lambda-RED-mediated gene replacements led to the construction of four mutants, in which the glycosyltransferase genes were disrupted, respectively. The metabolites from the mutants were purified and identified, including two new analogues designated as grincamycin U (3a) and V (3'). The sequential glycosylation steps in the biosynthesis of P-1894B (1) and grincamycin (1') catalyzed by GcnG3, GcnG1, and GcnG2 were elucidated.

Efficient ilamycins production utilizing Enteromorpha prolifera by metabolically engineered Streptomyces atratus.

With the invasion of green tides and the increase of urban green areas worldwide, multimillion tons of Enteromorpha need to be reutilized. In this study, Enteromorpha prolifera powder is considered a promising biomass resource for the production of commercial chemical products production. Ilamycins, novel cyclic heptapeptides with significant anti-TB activities, are isolated from Streptomyces atratus SCSIO ZH16, a deep-sea-derived strain. Using EP powder as a nitrogen source, the production of ilamycins reached 709.97 mg/L through optimization of the nitrogen source using the engineered strain S. atratus SCSIO ZH16 ΔR. After mutant strain constructions and tests, strain S. atratus SCSIO ZH16 ΔR::bldD EP powder achieved a higher production titer of ilamycins. Furthermore, the production titer of ilamycins and ilamycin E reached 1561.77 mg/L and 745.44 mg/L, respectively, in a 5 L bioreactor. This study suggests that E. prolifera is a promising and eco-friendly nitrogen source for the production of ilamycins.

Characterization of the depsidone gene cluster reveals etherification, decarboxylation and multiple halogenations as tailoring steps in depsidone assembly.

Depsides and depsidones have attracted attention for biosynthetic studies due to their broad biological activities and structural diversity. Previous structure‒activity relationships indicated that triple halogenated depsidones display the best anti-pathogenic activity. However, the gene cluster and the tailoring steps responsible for halogenated depsidone nornidulin (3) remain enigmatic. In this study, we disclosed the complete biosynthetic pathway of the halogenated depsidone through in vivo gene disruption, heterologous expression and in vitro biochemical experiments. We demonstrated an unusual depside skeleton biosynthesis process mediated by both highly-reducing polyketide synthase and non-reducing polyketide synthase, which is distinct from the common depside skeleton biosynthesis. This skeleton was subsequently modified by two in-cluster enzymes DepG and DepF for the ether bond formation and decarboxylation, respectively. In addition, the decarboxylase DepF exhibited substrate promiscuity for different scaffold substrates. Finally, and interestingly, we discovered a halogenase encoded remotely from the biosynthetic gene cluster, which catalyzes triple-halogenation to produce the active end product nornidulin (3). These discoveries provide new insights for further understanding the biosynthesis of depsidones and their derivatives.

Chemical Constituents from Mycelia of Lepista sordida (Agaricomycetes) and Their ABTS Radical Scavenging Activity.

Lepista sordida is an edible mushroom possessing high nutritional value and high medicinal value. The artificial cultivation technology of L. sordida made a breakthrough and has been popularized in Yunnan, Guizhou, Sichuan province with good economic benefits. The secondary metabolites were investigated from ethyl EtOAc (acetate extract) of solid cultures of L. sordida. Silica gel column chromatography, semi-preparation HPLC, recrystallization, and medium pressure column chromatography were applied to obtain 15 compounds. Nine compounds were first isolated from genus Lepista and 11 compounds were first isolated from species L. sordida. Moreover, compounds 13 and 14 exhibited strong scavenging activity of ABTS.

Six Sets of Aromatic Polyketides Differing in Size and Shape Derive from a Single Biosynthetic Gene Cluster.

One new aromatic polyketide, prealnumycin B (1), and four known aromatic polyketides, K1115A (2), 1,6-dihydroxy-8-propylanthraquinone (DHPA, 3), phaeochromycin B (4), and (R)-7-acetyl-3,6-dihydroxy-8-propyl-3,4dihydronaphthalen-1(2H)-one (5), were isolated from the marine-derived Streptomyces sundarbansensis SCSIO NS01; these compounds represent four sets of aromatic polyketides differing in size and shape. A type II polyketide synthase (PKS) cluster, als, was identified by complete genome sequencing and was shown, by in vivo gene inactivation experiments in the wild-type (WT) NS01 strain and heterologous expression experiments, to encode the biosynthesis of compounds 1-5. Moreover, heterologous expression of the als cluster afforded three additional aromatic polyketides representing two different carbon skeletons, the new phaeochromycin L (6) and two known aromatic polyketides, phaeochromycins D (7) and E (8). These findings expand our knowledge of type II PKS machineries and their versatility in generating structurally diverse aromatic polyketides and highlight the power of type II PKSs in accessing new polyketides via ectopic expression in heterologous hosts.

Development of Marker Recycling Systems for Sequential Genetic Manipulation in Marine-Derived Fungi Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7.

Marine-derived fungi are emerging as prolific workhorses of structurally novel natural products (NPs) with diverse bioactivities. However, the limitation of available selection markers hampers the exploration of cryptic NPs. Recyclable markers are therefore valuable assets in genetic engineering programs for awaking silent SM clusters. Here, both pyrG and amdS-based recyclable marker cassettes were established and successfully applied in marine-derived fungi Aspergillus sp. SCSIO SX7S7 and Spiromastix sp. SCSIO F190, respectively. Using pyrG recyclable marker, a markerless 7S7-∆depH strain with a simplified HPLC background was built by inactivating a polyketide synthase (PKS) gene depH and looping out the pyrG recyclable marker after depH deletion. Meanwhile, an amdS recyclable marker system was also developed to help strains that are difficult to use pyrG marker. By employing the amdS marker, a backbone gene spm11 responsible for one major product of Spiromastix sp. SCSIO F190 was inactivated, and the amdS marker was excised after using, generating a relatively clean F190-∆spm11 strain for further activation of novel NPs. The collection of two different recycle markers will guarantee flexible application in marine-derived fungi with different genetic backgrounds, enabling the exploitation of novel structures in various fungi species with different genome mining strategies.

Sulfoxanthicillin from the deep-sea derived Penicillium sp. SCSIO sof101: an antimicrobial compound against Gram-positive and -negative pathogens.

Natural products along with their analogs have been intensively explored for their antimicrobial potential against 'ESKAPE' pathogens. Herein, we report a new natural product with strong antibacterial activity, sulfoxanthocillin (1), along with its decomposed product peniformamide (2), and the known compound xanthocillin X (3) from the deep-sea derived Penicillium sp. SCSIO sof101. The structures of compounds 1 and 2 were determined by extensive spectroscopic analysis. Compound 1 showed significant activity against series pathogens with MIC values ranging 0.06-8.0 µg mL-1. As an artificial unnatural product during the isolation process, compound 2 had lower antimicrobial activity than that of compound 1, which could be attributed to a change in structural modification from an isonitrile group in compound 1 to a formamide group in compound 2. In terms of cytotoxicity, 1 showed relatively low cytotoxicity against human tumor cell lines compared with xanthocillin X (3), suggesting that the sulfate group present in 1 should be a determinant of cytotoxic activities. Overall, sulfoxanthocillin (1) merits further attention as a potential lead compound for anti-infective interventions against Gram-negative and Gram-positive bacterial pathogens.

Antimicrobial Polyketides from the Marine-Derived Fungus Spiromastix sp. SCSIO F190.

Three diphenyl ethers (1-3) and a cyclopentenone (4), together with seven known compounds (5-11), were isolated from the fermentation broth of the marine sediment-derived fungus Spiromastix sp. SCSIO F190. Compounds 3 and 4 were found to exist as a pair of atropisomers (3a, 3b) and racemates (4a, 4b), respectively. The planar structures of compounds 1-4 were elucidated on the basis of NMR and HRESIMS data sets. The absolute configurations of 2 and 3 were determined by spectroscopic and single-crystal X-ray diffraction analyses, whereas the configuration of 4 was determined by spectroscopic and chiral analyses. All compounds, except for 4 and 11, displayed activities against various pathogenic bacteria. Notably, compounds 1-4, especially 1, exhibited strong activity against Gram-positive bacteria, including methicillin-resistant bacterial strains of Staphylococcus aureus (MRSA), Enterococcus faecalis ATCC 29212, and Bacillus subtilis BS01, with MIC values ranging from 0.5 to 4 µg/mL. Moreover, the structure-activity relationship analyses of the active compounds and their analogues revealed the critical structural features correlating to the observed antimicrobial activities, herein providing insights for antimicrobial drug development.

OSMAC-Based Discovery and Biosynthetic Gene Clusters Analysis of Secondary Metabolites from Marine-Derived Streptomyces globisporus SCSIO LCY30.

The one strain many compounds (OSMAC) strategy is an effective method for activating silent gene clusters by cultivating microorganisms under various conditions. The whole genome sequence of the marine-derived strain Streptomyces globisporus SCSIO LCY30 revealed that it contains 30 biosynthetic gene clusters (BGCs). By using the OSMAC strategy, three types of secondary metabolites were activated and identified, including three angucyclines, mayamycin A (1), mayamycin B (2), and rabolemycin (3); two streptophenazines (streptophenazin O (4) and M (5)); and a macrolide dimeric dinactin (6), respectively. The biosynthetic pathways of the secondary metabolites in these three families were proposed based on the gene function prediction and structural information. The bioactivity assays showed that angucycline compounds 1-3 exhibited potent antitumor activities against 11 human cancer cell lines and antibacterial activities against a series of Gram-positive bacteria. Mayamycin (1) selectively exhibited potent cytotoxicity activity against triple-negative breast cancer (TNBC) cell lines such as MDA-MB-231, MDA-MB-468, and Bt-549, with IC50 values of 0.60-2.22 µM.

Characterization of the Glycosyltransferase and Methyltransferase Encoded Remotely from the Actinopyrone Biosynthetic Gene Cluster Enables Access to Diverse Analogues.

The actinopyrone biosynthetic gene cluster (atpn) lacks glycosyl- and methyltransferase genes, yet its product clearly calls for such enzymes. Using bioinformatics and biochemical methods, we confirmed that the mt3913 and gt723 genes, well beyond the atpn cluster boundaries, encode methyltransferase and glycosyltransferase, respectively. Moreover, homologous protein GT1507 enabled us to produce 14 non-natural actinopyrone analogues. PM050463 (3) was found to display potent anti-Helicobacter pylori activity and no signs of cytotoxicity.

MGCEP 1.0: A Genetic-Engineered Marine-Derived Chassis Cell for a Scaled Heterologous Expression Platform of Microbial Bioactive Metabolites.

Marine microorganisms produce a variety of bioactive secondary metabolites, which represent a significant source of novel antibiotics. Heterologous expression is a valuable tool for discovering marine microbial secondary metabolites; however, marine-derived chassis cell is very scarce. Here, we build an efficient plug-and-play marine-derived gene clusters expression platform 1.0 (MGCEP 1.0) by the systematic engineering of the deep-sea-derived Streptomyces atratus SCSIO ZH16. For a proof of concept, four families of microbial bioactive metabolite biosynthetic gene clusters (BGCs), including alkaloids, aminonucleosides, nonribosomal peptides, and polyketides, were efficiently expressed in this platform. Moreover, 19 compounds, including two new angucycline antibiotics, were produced in MGCEP 1.0. Dynamic patterns of global biosynthetic gene expression in MGCEP 1.0 with or without a heterologous gene cluster were revealed at the transcriptome level. The platform MGCEP 1.0 provides new possibilities for expressing microbial secondary metabolites, especially of marine origin.

Halo- and Thiocarbazomycins from Coral- and Coral Reef Sands-Derived Actinomycetes.

Four actinomycete strains isolated from the coral Acropora austera and coral sand samples from the South China Sea, were found to produce a series of halogenated compounds baring similar ultraviolet absorption based on the analysis of HPLC and LC-MS. The production titers of halogenated compounds from Streptomyces diacarni SCSIO 64983 exceeded those of other similar strains leading us to focus on SCSIO 64983. Four new thiocarbazomycins A-B (1-2), chlocarbazomycin E (3), and brocarbazomycin A (4), together with three known chlocarbazomycins A-C (5-7) containing a carbazole core were identified, and their structures were determined using a combination of spectroscopic analysis including HRESIMS, 1D and 2D NMR. Structurally speaking, compounds 1 and 2 have the rare sulfur-containing carbazole nuclei, and 3 and 4 contain Cl and Br atoms, respectively. Although these compounds have not yet been found to have obvious biological activity, their discovery highlights the role of molecular libraries in subsequent drug discovery campaigns.

Development of the CRISPR-Cas9 System for the Marine-Derived Fungi Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7.

Marine-derived fungi are emerging as attractive producers of structurally novel secondary metabolites with diverse bioactivities. However, the lack of efficient genetic tools limits the discovery of novel compounds and the elucidation of biosynthesis mechanisms. Here, we firstly established an effective PEG-mediated chemical transformation system for protoplasts in two marine-derived fungi, Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7. Next, we developed a simple and versatile CRISPR-Cas9-based gene disruption strategy by transforming a target fungus with a single plasmid. We found that the transformation with a circular plasmid encoding cas9, a single-guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted and insertional gene mutations in both marine-derived fungal strains. In addition, the histone deacetylase gene rpd3 was mutated using the established CRISPR-Cas9 system, thereby activating novel secondary metabolites that were not produced in the wild-type strain. Taken together, a versatile CRISPR-Cas9-based gene disruption method was established, which will promote the discovery of novel natural products and further biological studies.

Metabolic Blockade-Based Genome Mining Reveals Lipochain-Linked Dihydro-ß-alanine Synthetases Involved in Autucedine Biosynthesis.

Continuously mining the Streptomyces olivaceus SCSIO T05 genome leads to the identification of new lipopeptides (autucedines A-C), constituting members of the 10th skeleton isolated from this strain. The corresponding biosynthetic gene cluster (aut) was verified by heterogeneous expression, and another two analogues (autucedines D and E) were isolated from the heterogeneous expression strain. Gene inactivation experiments revealed that construction of the unique "lipochain-linked dihydro-ß-alanine" unit takes place prior to the NRPS assembly line.

Secondary Metabolites and Biosynthetic Gene Clusters Analysis of Deep-Sea Hydrothermal Vent-Derived Streptomyces sp. SCSIO ZS0520.

Streptomyces sp. SCSIO ZS0520 is a deep-sea hydrothermal vent-derived actinomycete. Our previous metabolism investigation showed that Streptomyces sp. SCSIO ZS0520 is a producer of cytotoxic actinopyrones. Here, another four types of secondary metabolites were identified, including six salinomycin isomers (2-7), the macrolide elaiophylin (8), the triterpene N-acetyl-aminobacteriohopanetriol (9), and the pyrone minipyrone (10). Among them, compounds 2-6 and 10 are new compounds. To understand the biosynthetic pathway of these compounds, a bioinformatic analysis of the whole genome was carried out, which identified 34 secondary metabolite biosynthetic gene clusters. Next, the biosynthetic pathways responsive to four types of products were deduced on the basis of gene function predictions and structure information. Taken together, these findings prove the metabolite potential of ZS0520 and lay the foundations to solve the remaining biosynthetic issues in four types of marine natural products.