Screening and genetic engineering of marine-derived Aspergillus terreus for high-efficient production of lovastatin.

BACKGROUND: Lovastatin has widespread applications thanks to its multiple pharmacological effects. Fermentation by filamentous fungi represents the major way of lovastatin production. However, the current lovastatin productivity by fungal fermentation is limited and needs to be improved. RESULTS: In this study, the lovastatin-producing strains of Aspergillus terreus from marine environment were screened, and their lovastatin productions were further improved by genetic engineering. Five strains of A. terreus were isolated from various marine environments. Their secondary metabolites were profiled by metabolomics analysis using Ultra Performance Liquid Chromatography-Mass spectrometry (UPLC-MS) with Global Natural Products Social Molecular Networking (GNPS), revealing that the production of secondary metabolites was variable among different strains. Remarkably, the strain of A. terreus MJ106 could principally biosynthesize the target drug lovastatin, which was confirmed by High Performance Liquid Chromatography (HPLC) and gene expression analysis. By one-factor experiment, lactose was found to be the best carbon source for A. terreus MJ106 to produce lovastatin. To improve the lovastatin titer in A. terreus MJ106, genetic engineering was applied to this strain. Firstly, a series of strong promoters was identified by transcriptomic and green fluorescent protein reporter analysis. Then, three selected strong promoters were used to overexpress the transcription factor gene lovE encoding the major transactivator for lov gene cluster expression. The results revealed that compared to A. terreus MJ106, all lovE over-expression mutants exhibited significantly more production of lovastatin and higher gene expression. One of them, LovE-b19, showed the highest lovastatin productivity at a titer of 1512 mg/L, which represents the highest production level reported in A. terreus. CONCLUSION: Our data suggested that combination of strain screen and genetic engineering represents a powerful tool for improving the productivity of fungal secondary metabolites, which could be adopted for large-scale production of lovastatin in marine-derived A. terreus.

Novel anti-inflammatory diketopiperazine alkaloids from the marine-derived fungus Penicillium brasilianum.

Diketopiperazine alkaloids have proven the most abundant heterocyclic alkaloids up to now, which usually process diverse scaffolds and rich biological activities. In our search for bioactive diketopiperazine alkaloids from marine-derived fungi, two novel diketopiperazine alkaloids, penipiperazine A (1) and its biogenetically related new metabolite (2), together with a known analogue neofipiperzine C (3), were obtained from the strain Penicillium brasilianum. Their planar structures and absolute configurations were elucidated by extensive spectroscopic analyses, 13C NMR calculation, Marfey's, ECD, and ORD methods. Compound 1 featured a unique 6/5/6/6/5 indole-pyrazino-pyrazino-pyrrolo system, and its plausible biogenetic pathway was also proposed. Additionally, compounds 1-3 have been tested for their inflammatory activities. 1 and 2 significantly inhibited the release of NO and the expression of related pro-inflammatory cytokines on LPS-stimulated RAW264.7 cells, suggesting they could be attracting candidate for further development as anti-inflammatory agent. KEY POINTS: • A novel diketopiperazine alkaloid featuring a unique 6/5/6/6/5 indole-pyrazino-pyrazino-pyrrolo system was isolated from the marine fungus Penicillium brasilianum. • The structure of 1 was elucidated by detailed analysis of 2D NMR data, 13C NMR calculation, Marfey's, ECD, and ORD methods. • Compounds 1 and 2 significantly inhibited the release of NO and the expression of related pro-inflammatory cytokines on LPS-stimulated RAW264.7 cells.

An unexpected role of EasDaf: catalyzing the conversion of chanoclavine aldehyde to chanoclavine acid.

Ergot alkaloids (EAs) are a diverse group of indole alkaloids known for their complex structures, significant pharmacological effects, and toxicity to plants. The biosynthesis of these compounds begins with chanoclavine-I aldehyde (CC aldehyde, 2), an important intermediate produced by the enzyme EasDaf or its counterpart FgaDH from chanoclavine-I (CC, 1). However, how CC aldehyde 2 is converted to chanoclavine-I acid (CC acid, 3), first isolated from Ipomoea violacea several decades ago, is still unclear. In this study, we provide in vitro biochemical evidence showing that EasDaf not only converts CC 1 to CC aldehyde 2 but also directly transforms CC 1 into CC acid 3 through two sequential oxidations. Molecular docking and site-directed mutagenesis experiments confirmed the crucial role of two amino acids, Y166 and S153, within the active site, which suggests that Y166 acts as a general base for hydride transfer, while S153 facilitates proton transfer, thereby increasing the acidity of the reaction. KEY POINTS: • EAs possess complicated skeletons and are widely used in several clinical diseases • EasDaf belongs to the short-chain dehydrogenases/reductases (SDRs) and converted CC or CC aldehyde to CC acid • The catalytic mechanism of EasDaf for dehydrogenation was analyzed by molecular docking and site mutations.

Semi-Synthesis and Biological Evaluation of 25(R)-26-Acetoxy-3ß,5α-Dihydroxycholest-6-One.

Previously, we identified a series of steroids (1-6) that showed potent anti-virus activities against respiratory syncytial virus (RSV), with IC50 values ranging from 3.23 to 0.19 µM. In this work, we first semi-synthesized and characterized the single isomer of 5, 25(R)-26-acetoxy-3ß,5α-dihydroxycholest-6-one, named as (25R)-5, in seven steps from a commercially available compound diosgenin (7), with a total yield of 2.8%. Unfortunately, compound (25R)-5 and the intermediates only showed slight inhibitions against RSV replication at the concentration of 10 µM, but they possessed potent cytotoxicity activities against human bladder cancer 5637 (HTB-9) and hepatic cancer HepG2, with IC50 values ranging from 3.0 to 15.5 µM without any impression of normal liver cell proliferation at 20 µM. Among them, the target compound (25R)-5 possessed cytotoxicity activities against 5637 (HTB-9) and HepG2 with IC50 values of 4.8 µM and 15.5 µM, respectively. Further studies indicated that compound (25R)-5 inhibited cancer cell proliferation through inducing early and late-stage apoptosis. Collectively, we have semi-synthesized, characterized and biologically evaluated the 25R-isomer of compound 5; the biological results suggested that compound (25R)-5 could be a good lead for further anti-cancer studies, especially for anti-human liver cancer.

Sclerotioloids A-C: Three New Alkaloids from the Marine-Derived Fungus Aspergillus sclerotiorum ST0501.

Alkaloids, as one of the largest classes of natural products with diverse structures, are an important source of innovative medicines. Filamentous fungi, especially those derived from the marine environment, are one of the major producers of alkaloids. In this study, three new alkaloids, sclerotioloids A-C (1-3), along with six known analogs (4-9), were obtained under the guidance of the MS/MS-based molecular networking from the marine-derived fungus, Aspergillus sclerotiorum ST0501, collected from the South China Sea. Their chemical structures were elucidated by comprehensive analysis of the spectroscopic data, including 1D and 2D NMR and HRESIMS. Additionally, the configuration of compound 2 was unambiguously determined by X-ray single crystal diffraction, and that of compound 3 was determined by the TDDFT-ECD approach. Sclerotioloid A (1) represents the first example of 2,5-diketopiperazine alkaloid with a rare terminal alkyne. Sclerotioloid B (2) showed the inhibition of NO production induced by lipopolysaccharide (LPS), with an inhibition rate of 28.92% higher than that of dexamethasone (25.87%). These results expanded the library of fungal-derived alkaloids and further prove the potential of marine fungi in the generation of alkaloids with new scaffolds.

Trends of antimalarial marine natural products: progresses, challenges and opportunities.

Covering 1972 to 2021Malaria remains a significant public health problem in some regions of the world. The great efforts to control malaria have been severely compromised due to the widespread resistance of Plasmodium falciparum to nearly all frontline drugs. Pursuit of novel molecules from the sea will potentially result in new interventions against malaria, which are urgently needed to combat the increase of resistance. Focusing on the strategy of the "Blue Drug Bank", the molecules highlighted here can serve as an inspiration for future medicinal chemistry campaigns. This review covers the developments in the field of antimalarial marine lead compounds reported between 1972 and July 2021, and offers a comprehensive overview on their progresses and potentials. We selected 60 representative potential candidate molecules from 361 marine natural products, and highlighted their structure-activity relationships, molecular mechanisms of targets, and drug-like properties in order to assess their full potential to be developed. We summarized 107 clinically proven or potential antimalarial targets and their subcellular locations in the relevant target proteins, which linked the molecules to the target proteins at the subcellular level. Hence, it could be expected that natural products targeting different mechanisms may prove to be an effective strategy in antimalarial drug research and development in the future.

Cytotoxic and Antibacterial Isomalabaricane Terpenoids from the Sponge Rhabdastrella globostellata.

Nine new isomalabaricane terpenoids (1-9) were isolated from the sponge Rhabdastrella globostellata of Ximao Island, together with 13 known ones (10-22). The structures were established by spectroscopic data interpretation and chemical calculations, as well as by comparison with spectroscopic data of known compounds. Notably, of the new isolates, hainanstelletin A (5) is the first representative of a nitrogenous isomalabaricane. The isolated compounds were evaluated against several cancer cell lines and two bacterial pathogens. In addition, moderate to strong antibacterial activities against Streptococcus pyogenes were also detected among geometric isomers 1, 2, and 10-12, with minimum inhibitory concentrations of 0.1-1.8 µg/mL.

Xishaeleganins A-D, Sesquiterpenoid Hydroquinones from Xisha Marine Sponge Dactylospongia elegans.

Four new sesquiterpene hydroquinones, xishaeleganins A-D (6-9), along with eleven known related ones (12 and 14-23) were isolated from the Xisha marine sponge Dactylospongia elegans (family Thorectida). Their structures were determined by extensive spectroscopic analysis, ECD calculations, and by comparison with the spectral data reported in the literature. Compounds 7, 15, 20, and 21 showed significant antibacterial activity against Staphylococcus aureus, with minimum inhibitory concentration values of 1.5, 2.9, 5.6, and 5.6 µg/mL, which are comparable with those obtained for the positive control vancomycin (MIC: 1.0 µg/mL).

Anti-inflammatory Polyketides from the Marine-Derived Fungus Eutypella scoparia.

Three new polyketides, eutyketides A and B (1 and 2) and cytosporin X (3), along with four known compounds (4-7), were obtained from the marine-derived fungus Eutypella scoparia. The planar structures of 1 and 2 were elucidated by extensive HRMS and 1D and 2D NMR analyses. Their relative configurations of C-13 and C-14 were determined with chemical conversions by introducing an acetonylidene group. The absolute configurations of 1-3 were determined by comparing their experimental electronic circular dichroism (ECD) data with their computed ECD results. All of the isolated compounds were tested for their anti-inflammatory activities on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages. Compounds 5 and 6 showed stronger anti-inflammatory activities than the other compounds, with the inhibition of 49.0% and 54.9% at a concentration of 50.0 µg/mL, respectively.

Discovery of Marine Natural Products as Promising Antibiotics against Pseudomonas aeruginosa.

Pseudomonas aeruginosa, one of the most intractable Gram-negative bacteria, has become a public health threat due to its outer polysaccharide layer, efflux transporter system, and high level of biofilm formation, all of which contribute to multi-drug resistance. Even though it is a pathogen of the highest concern, the status of the antibiotic development pipeline is unsatisfactory. In this review, we summarize marine natural products (MNPs) isolated from marine plants, animals, and microorganisms which possess unique structures and promising antibiotic activities against P. aeruginosa. In the last decade, nearly 80 such MNPs, ranging from polyketides to alkaloids, peptides, and terpenoids, have been discovered. Representative compounds exhibited impressive in vitro anti-P. aeruginosa activities with MIC values in the single-digit nanomolar range and in vivo efficacy in infectious mouse models. For some of the compounds, the preliminary structure-activity-relationship (SAR) and anti-bacterial mechanisms of selected compounds were introduced. Compounds that can disrupt biofilm formation or membrane integrity displayed potent inhibition of multi-resistant clinical P. aeruginosa isolates and could be considered as lead compounds for future development. Challenges on how to translate hits into useful candidates for clinical development are also proposed and discussed.

Targeted Isolation of Asperheptatides from a Coral-Derived Fungus Using LC-MS/MS-Based Molecular Networking and Antitubercular Activities of Modified Cinnamate Derivatives.

Under the guidance of MS/MS-based molecular networking, four new cycloheptapeptides, namely, asperheptatides A-D (1-4), were isolated together with three known analogues, asperversiamide A-C (5-7), from the coral-derived fungus Aspergillus versicolor. The planar structures of the two major compounds, asperheptatides A and B (1 and 2), were determined by comprehensive spectroscopic data analysis. The absolute configurations of the amino acid residues were determined by advanced Marfey's method. The two structurally related trace metabolites, asperheptatides C and D (3 and 4), were characterized by ESI-MS/MS fragmentation methods. A series of new derivatives (8-26) of asperversiamide A (5) were semisynthesized. The antitubercular activities of 1, 2, and 5-26 against Mycobacterium tuberculosis H37Ra were also evaluated. Compounds 9, 13, 23, and 24 showed moderate activities with MIC values of 12.5 µM, representing a potential new class of antitubercular agents.

17-Hydroxybrevianamide N and Its N1-Methyl Derivative, Quinazolinones from a Soft-Coral-Derived Aspergillus sp. Fungus: 13S Enantiomers as the True Natural Products.

Under the guidance of MS/MS-based molecular networking and HPLC-UV, two new alkaloid racemates, (±)-17-hydroxybrevianamide N (1) and (±)-N1-methyl-17-hydroxybrevianamide N (2), featuring a rare o-hydroxyphenylalanine residue and an imide subunit, were isolated from a soft-coral-derived Aspergillus sp. fungus. The true natural products (+)-1 and (+)-2 were further monitored and obtained from the freshly prepared EtOAc extracts, while (-)-1 and (-)-2 are artifacts generated during extraction and purification processes. Simultaneously, the structures including absolute configurations of (+)-13S-1, (-)-13R-1, (+)-13S-2, and (-)-13R-2 were elucidated on the basis of comprehensive spectroscopic analysis, ECD calculations, and X-ray diffraction data. Interestingly, basic solution promotes the racemization of (+)-1 and (-)-1, whereas acidic solution suppresses the transformation. The current research was concerned with the true natural products and their artifacts, providing critical insight into the isolation and identification of natural products.

Semisynthesis, Antiplasmodial Activity, and Mechanism of Action Studies of Isocoumarin Derivatives.

In this study, eight natural isocoumarins (1-8) were isolated from a marine-derived Exserohilum sp. fungus. To explore their structure-activity relationship and discover potent antimalarial leads, a small library of 22 new derivatives (1a-1n, 2a, 3a-3c, 4a-4c, and 7a) were semisynthesized by varying the substituents of the aromatic ring and the aliphatic side chains. The natural compound (1) and three semisynthetic derivatives (1d, 1n, and 2a), possessing an all-cis stereochemistry, exhibited strong antiplasmodial activity with IC50 values of 1.1, 0.8, 0.4, and 2.6 µM, respectively. Mechanism studies show that 1n inhibits hemozoin polymerization and decreases the mitochondrial membrane potential but also inhibits P. falciparum DNA gyrase. 1n not only combines different mechanisms of action but also exhibits a high therapeutic index (CC50/IC50 = 675), high selectivity, and a notable drug-like profile.

Naphthalene derivatives and halogenate quinoline from the coral-derived fungus Trichoderma harzianum (XS-20090075) through OSMAC approach.

OSMAC approach was performed on the soft coral-derived fungus Trichoderma harzianum (XS-20090075) leading to the significant changes of its secondary metabolites by using two different cultures. A new naphthalene derivative, trichoharzin B (1) and a new natural product, methyl-trichoharzin (2) were isolated by using rice medium. Whereas, a new natural product, ethyl 2-bromo-4-chloroquinoline-3-carboxylate (9) was obtained by using Czapek's medium. Their structures were established by extensive spectroscopic investigation. The absolute configuration of 5 was determined by single-crystal X-ray diffraction. Compound 9 was the first halogenate quinoline derivative isolated from the genus of Trichoderma.

Microketides A and B, Polyketides from a Gorgonian-Derived Microsphaeropsis sp. Fungus.

Microketides A and B (1 and 2), a pair of new C-11 epimeric polyketides, were obtained from the gorgonian-derived fungus Microsphaeropsis sp. RA10-14 collected from the South China Sea. The absolute configurations of 1 and 2 were assigned by the modified Mosher's method, TDDFT-ECD, and NMR calculations. Compounds 1 and 2 were evaluated for antibacterial, antifungal, and growth inhibition of marine phytoplankton activities. Microketide A (1) exhibited promising inhibitory activity against Pseudomonas aeruginosa, Nocardia brasiliensis, Kocuria rhizophila, and Bacillus anthraci with the same MIC value as ciprofloxacin (0.19 µg/mL).

The oxygenated products of cryptotanshinone by biotransformation with Cunninghamella elegans exerting anti-neuroinflammatory effects by inhibiting TLR 4-mediated MAPK signaling pathway.

Cryptotanshinone (1), a major bioactive constituent in the traditional Chinese medicinal herb Dan-Shen Salvia miltiorrhiza Bunge, has been reported to possess remarkable pharmacological activities. To improve its bioactivities and physicochemical properties, in the present study, cryptotanshinone (1) was biotransformed with the fungus Cunninghamella elegans AS3.2028. Three oxygenated products (2-4) at C-3 of cryptotanshinone (1) were obtained, among them 2 was a new compound. Their structures were elucidated by comprehensive spectroscopic analysis including HRESIMS, NMR and ECD data. All of the biotransformation products (2-4) were found to inhibit significantly lipopolysaccharide-induced nitric oxide production in BV2 microglia cells with the IC50 values of 0.16-1.16 µM, approximately 2-20 folds stronger than the substrate (1). These biotransformation products also displayed remarkably improved inhibitory effects on the production of inflammatory cytokines (IL-1ß, IL-6, TNF-α, COX-2 and iNOS) in BV-2 cells via targeting TLR4 compared to substrate (1). The underlying mechanism of 2 was elucidated by comparative transcriptome analysis, which suggested that it reduced neuroinflammatory mainly through mitogen-activated protein kinase (MAPK) signaling pathway. Western blotting results revealed that 2 downregulated LPS-induced phosphorylation of JNK, ERK, and p38 in MAPK signaling pathway. These findings provide a basal material for the discovery of candidates in treating Alzheimer's disease.

Annular oxygenation and rearrangement products of cryptotanshinone by biotransformation with marine-derived fungi Cochliobolus lunatus and Aspergillus terreus.

Structural modification of natural products by biotransformation with fungi is an attractive tool to obtain novel bioactive derivatives. In the present study, cryptotanshinone (1), a quinoid abietane diterpene from traditional Chinese medicine Salvia miltiorrhiza (Danshen), was transformed by two marine-derived fungi. By using Cochliobolus lunatus TA26-46, one new oxygenated and rearranged product (2), containing a 5,6-dihydropyrano[4,3-b]chromene moiety, together with one known metabolite (10), were obtained from the converted broth of cryptotanshinone (1) with the isolated yields of 1.0% and 2.1%, respectively. While, under the action of Aspergillus terreus RA2905, seven new transformation products (3-9) as well as 10 with the fragments of 2-methylpropan-1-ol and oxygenated p-benzoquinone were produced and obtained with the isolated yields of 0.1%-1.3%. The structures of the new compounds were elucidated by comprehensive spectroscopic analysis including High Resolution Electrospray Ionization Mass Spectroscopy (HRESIMS), Nuclear Magnetic Resonance (NMR) and Electronic Circular Dichroism (ECD). The metabolic pathways of cryptotanshinone by these two fungi were presumed to be the opening and rearrangement of furan ring, and/or oxygenation of cyclohexane ring. Cryptotanshinone (1) and its metabolites displayed anti-inflammatory activities against NO production in LPS-stimulated BV-2 cells and antibacterial activities towards methicillin-resistant Staphylococcus aureus. These findings revealed the potential of marine fungi to transform the structures of natural products by biotransformation.

Antimicrobial and Antioxidant Polyketides from a Deep-Sea-Derived Fungus Aspergillus versicolor SH0105.

Fifteen polyketides, including four new compounds, isoversiol F (1), decumbenone D (2), palitantin B (7), and 1,3-di-O-methyl-norsolorinic acid (8), along with 11 known compounds (3-6 and 9-15), were isolated from the deep-sea-derived fungus Aspergillus versicolor SH0105. Their structures and absolute configurations were determined by comprehensive spectroscopic data, including 1D and 2D NMR, HRESIMS, and ECD calculations, and it is the first time to determine the absolute configuration of known decumbenone A (6). All of these compounds were evaluated for their antimicrobial activities against four human pathogenic microbes and five fouling bacterial strains. The results indicated that 3,7-dihydroxy-1,9-dimethyldibenzofuran (14) displayed obvious inhibitory activity against Staphylococcus aureus (ATCC 27154) with the MIC value of 13.7 µM. In addition, the antioxidant assays of the isolated compounds revealed that aspermutarubrol/violaceol-I (15) exhibited significant 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity with the IC50 value of 34.1 µM, and displayed strong reduction of Fe3+ with the ferric reducing antioxidant power (FRAP) value of 9.0 mM under the concentration of 3.1 µg/mL, which were more potent than ascorbic acid.

New Thiodiketopiperazine and 3,4-Dihydroisocoumarin Derivatives from the Marine-Derived Fungus Aspergillus terreus.

Aspergillus terreus has been reported to produce many secondary metabolites that exhibit potential bioactivities, such as antibiotic, hypoglycemic, and lipid-lowering activities. In the present study, two new thiodiketopiperazines, emestrins L (1) and M (2), together with five known analogues (3-7), and five known dihydroisocoumarins (8-12), were obtained from the marine-derived fungus Aspergillus terreus RA2905. The structures of the new compounds were elucidated by analysis of the comprehensive spectroscopic data, including high-resolution electrospray ionization mass spectrometry (HRESIMS), one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR), and electronic circular dichroism (ECD) data. This is the first time that the spectroscopic data of compounds 3, 8, and 9 have been reported. Compound 3 displayed antibacterial activity against Pseudomonas aeruginosa (minimum inhibitory concentration (MIC) = 32 µg/mL) and antifungal activity against Candida albicans (MIC = 32 µg/mL). In addition, compound 3 exhibited an inhibitory effect on protein tyrosine phosphatase 1 B (PTP1B), an important hypoglycemic target, with an inhibitory concentration (IC)50 value of 12.25 µM.

Structure-Activity Relationships and Potent Cytotoxic Activities of Terphenyllin Derivatives from a Small Compound Library.

A small library of 120 compounds was established with seventy new alkylated derivatives of the natural product terphenyllin, together with 45 previous reported derivatives and four natural p-terphenyl analogs. The 70 new derivatives were semi-synthesized and evaluated for cytotoxic activities against four cancer cell lines. Interestingly, 2',4''-diethoxyterphenyllin, 2',4,4''-triisopropoxyterphenyllin, and 2',4''-bis(cyclopentyloxy)terphenyllin showed potent activities with IC50 values in a range from 0.13 to 5.51 µM, which were similar to those of the positive control, adriamycin. The preliminary structure-activity relationships indicated that the introduction of alkyl substituents including ethyl, allyl, propargyl, isopropyl, bromopropyl, isopentenyl, cyclopropylmethyl, and cyclopentylmethyl are important for improving the cytotoxicity.