Peniapyrones A-I, Cytotoxic Tricyclic-Fused α-Pyrone Derivatives from an Endophytic Penicillium brefeldianum F4a.

Five cyclopenta[d]pyrano[4,3-b]pyran-1,7(6H)-dione 6/6/5-fused tricyclic ring system containing metabolites peniapyrones A-E (1-5), and four previously undescribed cyclopenta[4,5]furo[3,2-c]pyran-1-one 6/5/5-fused tricyclic ring system containing compounds peniapyrones F-I (6-9), were isolated from the endophytic Penicillium brefeldianum F4a. Their structures, including absolute configurations, were determined through spectroscopic analysis and quantum chemical calculations. Peniapyrones D (4) and E (5) were a pair of diastereoisomers. Compounds 1, 3, and 5-9 showed cytotoxic activity against AsPC-1, CRL-2234, and MCF-7 cancer cell lines. Compounds 1, 3, 6, 8, and 9 inhibited the Kirsten rat sarcoma viral oncogene homologue (KRAS) mutant AsPC-1 cell line.

Sensitive and Direct Analysis of Pseudomonas aeruginosa through Self-Primer-Assisted Chain Extension and CRISPR-Cas12a-Based Color Reaction.

Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic Gram-negative pathogen that may cause infections to immunocompromised patients. However, sensitive and reliable analysis of P. aeruginosa remains a huge challenge. In this method, target recognition assists the formation of a self-primer and initiates single-stranded chain production. The produced single-stranded DNA chain is identified by CRISPR-Cas12a, and consequently, the trans-cleavage activity of the Cas12a enzyme is activated to parallelly digest Ag+ aptamer sequences that are chelated with silver ions (Ag+). The released Ag+ reacted with 3,3',5,5'-tetramethylbenzidine (TMB) for coloring. Compared with the traditional color developing strategies, which mainly rely on the DNA hybridization, the color developing strategy in this approach exhibits a higher efficiency due to the robust trans-cleavage activity of the Cas12a enzyme. Consequently, the method shows a low limit of detection of a wide detection of 5 orders of magnitudes and a low limit of detection of 21 cfu/mL, holding a promising prospect in early diagnosis of infections. Herein, we develop a sensitive and reliable method for direct and colorimetric detection of P. aeruginosa by integrating self-primer-assisted chain production and CRISPR-Cas12a-based color reaction and believe that the established approach will facilitate the development of bacteria-analyzing sensors.

Genome Mining and Molecular Networking Guided Isolation of Antimycin Analogs with Antifeedant Activities from the Deep-Sea-Derived Streptomyces sp. NA13.

Polyphagous insects could affect agricultural production, which leads to serious economic losses. Due to the negative effects of synthesized insecticides, finding eco-friendly and new biopesticides is emergent. To develop natural origin insecticides, an integrative approach combining antifeedant activity screening, genome mining, and molecular networking has been applied to discover antifeedant secondary metabolites from Streptomyces sp. NA13, which leads to the isolation of a novel antimycin Q (1) and six known antimycin analogs (antimycins A1a, A2a, A3a, A4a, A7a, and N-formylantimycic acid methyl ester, 2-7). Their structures were identified by high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopic. The absolute configuration of 1 was elucidated by the comparison of coupling constant, electronic circular dichroism (ECD) analysis, and NMR calculations. 1-6 exhibited different levels of antifeedant activities against Helicoverpa armigera, especially 1-4. At the same time, the antifeedant activity of antimycin was reported firstly.

Penipentenone A and brefeldin A derivatives potently inhibit KRAS mutant cancer cells from an endophytic fungus Penicillium brefeldianum F4a.

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation is one of the most important carcinogenic factors in many solid tumors, which leads to the poor prognosis and therapy resistance of cancer. In order to develop direct or indirect KRAS inhibitors, one unique asymmetric dicyclopentenone penipentenone A, three undescribed brefeldin A (BFA) derivatives, and five known BFA derivatives were discovered from the endophytic fungus Penicillium brefeldianum guided by LC-MS/MS and cytotoxic activities. Their structures were elucidated by optical rotation, mass spectrometry, and NMR spectroscopic data. The absolute configurations of four undescribed compounds were elucidated by comparison of the experimental and calculated ECD spectra. The antiproliferative activities of obtained compounds against three KRAS mutant tumor cell lines and two BFA derivative-sensitive cell lines were evaluated. Besides 4-epi-15-epi-brefeldin A, the other compounds showed significant inhibitory activities against those tumor cell lines with IC50 values ranging from 0.82 to 18.87 µM. Intriguingly, penipentenone A selectively inhibited KRAS mutant cancer cells SW620 (KRASG12V) and ASPC-1 (KRASG12D). BFA and four derivatives showed potent cytotoxic activities against all selected tumor cell lines H358 (KRASG12C), SW620 (KRASG12V), ASPC-1 (KRASG12D), PC-3, and HepG-2. These findings will provide undescribed lead compounds for developing drugs that target KRAS mutations.

Nigirpexin E, a new azaphilone derivative with anti-tobacco mosaic virus activity from soil-derived fungus Trichoderma afroharzianum LTR-2.

A new compound classified as one new azaphilone derivative, nigirpexin E (1), was obtained from the soil-derived fungus Trichoderma afroharzianum LTR-2, together with seven known compounds (2-8). The structures of 1-8 were determined by their HRESIMS, optical rotation, and NMR spectroscopic data. The absolute configuration of nigirpexin E (1) was determined on the basis of comparisons of experimental and theoretically calculated ECD spectra. Compound 3 was firstly isolated from Trichoderma. Bioactivities of the isolated compounds were assayed their anti-tobacco mosaic virus (anti-TMV) activities. The results showed that compound 1 exhibited significant inactivation effect against TMV with an inhibition rate of 67.25% (0.5 mg ml-1), which was higher than that of positive control ribavirin (56.74%). This is the first report of the anti-TMV activity of azaphilone derivatives.

Novel Antioxidants and α-Glycosidase and Protein Tyrosine Phosphatase 1B Inhibitors from an Endophytic Fungus Penicillium brefeldianum F4a.

Oxidative stress plays a very important role in the progression of diabetes and its complications. A therapeutic agent that is both antidiabetic and antioxidant would be the preferred choice for the treatment of diabetes. The crude extract of the endophytic fungus Penicillium brefeldianum F4a has significant antioxidant and α-glycosidase and protein tyrosine phosphatase 1B (PTP1B) inhibition activities. Chemical investigation of P. brefeldianum F4a using an activity-guided isolation led to the discovery of three new compounds called peniorcinols A-C (1-3) along with six known compounds: penialidins A (4), penialidin F (5), myxotrichin C (6), riboflavin (7), indole-3-acetic acid (8), and 2-(4-hydroxy-2-methoxy-6-methylphenyl) acetic acid (9). Their chemical structures were established by their NMR and HRESIMS. The absolute configurations of 1 and 3 were determined by experimental and calculated electronic circular dichroism (ECD). Their antioxidant activities were evaluated by DPPH• and ABTS•+ scavenging assays. Compounds 1-6 and 8-9 showed moderate to strong free radical scavenging activities. Significantly, 4-6 exhibited more potent ABTS•+ scavenging activity than that of the positive control. Their α-glycosidase and PTP1B inhibition activities were tested. Among them, compound 3 showed α-glucosidase inhibition activity, and compounds 7 and 8 showed PTP1B inhibitory activity for the first time. It is worth noting that 3 and 8 displayed both antioxidant and α-glycosidase or PTP1B inhibition activities. These finding suggest that compounds 3 and 8 could be used as lead compounds to generate new potent drugs for the treatment of oxidative stress-related diabetes.

Structure-based molecular networking for the target discovery of novel germicidin derivatives from the sponge-associated streptomyces sp. 18A01.

Four new α-pyrone derivatives, named germicidins P-S (1-4) along with nine known analogues (5-13) were discovered from the sponge-associated Streptomyces sp. 18A01 guided by Global Natural Products Social (GNPS) molecular networking, the LC-DAD-MS profile, and hexokinase II (HK2) inhibitory activity. The structures of 1-13 were elucidated by analysis of their HRMS, optical rotation, and NMR spectroscopic data. The absolute configurations of germicidin P (1) and germicidin Q (2) were determined on the basis of comparisons of experimental and theoretically calculated ECD spectra. Bioactivities of the isolated compounds were assayed against human HK2. The bioassay results showed that compounds 1-4 and 11-13 exhibited significant inhibitory activities against HK2, with IC50 values ranging from 5.1 to 11.0 µM. A molecular docking simulation demonstrated that these germicidins were docked in the inner active site tunnel of HK2. Interestingly, the amino residue Arg91 has a better binding affinity and efficacy than the amino residue Asn89 in the process of HK2 binding to the ligands, resulting in better hexokinase inhibitory activity. This result provided a valuable perspective for better understanding their HK2 inhibition activity.

Cytotoxic and antimicrobial activities of secondary metabolites isolated from the deep-sea-derived Actinoalloteichus cyanogriseus 12A22.

A new deep-sea-derived actinomycete 12A22 was isolated from the sediment of the South China Sea which showed potential cytotoxic and antimicrobial activities. The actinomycete was identified as Actinoalloteichus cyanogriseus by investigating morphological characteristics and phylogenetic analyses based on its 16S rRNA gene sequence. Two compounds, cyclo-(L-Pro-D-Pro-L-Tyr-L-Tyr) (1) and 2-hydroxyethyl-3-methyl-1,4-naphthoquinone (2), were isolated and characterized from the fermentation broth of the strain 12A22. Compound 2 exhibited significant inhibitory activities against a variety of phytopathogenic fungi (Fusarium oxysporum f. sp. cucumerinum, Setosphaeria turcica, and Botrytis cinerea) and Gram-positive bacterium (Bacillus subtilis). In particular, this compound showed better antifungal activity against Botrytis cinerea than positive control amphotericin B. Besides, compound 2 showed moderate cytotoxic activity against human breast cancer MDA-MB-435 cells with IC50 10.59 µM, weaker than the positive control diaminedichloroplatinum with 5.91 µM. Our results suggested that this naphthoquinone could be used as a potential antimicrobial and antitumor agent. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02846-0.

Isolation and characterization of a new cyclic lipopeptide surfactin from a marine-derived Bacillus velezensis SH-B74.

A marine-sediment-derived bacterium Bacillus velezensis SH-B74 can produce cyclic lipopeptides (CLPs). This study presented the isolation, characterization, and activity evaluation of a new CLP from the bacterial cultures of the strain SH-B74. Multiple chromatographic methods (solid-phase extraction and reversed-phase high-performance liquid chromatography) were applied to the purifying procedure of CLP, and the structural characterization of the new CLP was conducted by various spectroscopy (1D and 2D nuclear magnetic resonance together with Fourier transform infrared spectroscopy) and spectrometry (liquid chromatography-mass spectrometry, high-resolution mass spectrometry and tandem mass spectrometry) techniques as well as Marfey's method. The results displayed that the new CLP (anteiso-C15 Ile2,7 surfactin, 1) consists of a peptidic backbone of L-Glu1, L-Ile2, D-Leu3, L-Val4, L-Asp5, D-Leu6, L-Ile7, and an anteiso-C15 type saturated fatty acid chain. Further activity assay showed that the new CLP displays activity on the inhibition of the appressoria formation of rice blast causal pathogen Magnaporthe oryzae. To sum up, the results presented the perspective of potential application of the new CLP as a green agrichemical to control M. oryzae.

Isolation and characterization of a new cyclic lipopeptide orfamide H from Pseudomonas protegens CHA0.

A new cyclic lipopeptide (CLP) orfamide H (1) was purified and identified from the cultural broth of the bacterial strain Pseudomonas protegens CHA0. The crude extract of the strain CHA0 was obtained by an acid-aided precipitation process, then the compound 1 was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). Subsequently, the chemical structure of orfamide H was determined by 1D and 2D nuclear magnetic resonance (NMR) and mass spectrometry (MS). Further biological assays indicate that the new CLP orfamide H shows the activity on inhibiting the appressoria formation of the fungus Magnaporthe oryzae, the causal agent of the blast disease in rice. Taken all together, these results indicated that the new CLP orfamide H has the capacity to be developed as an agrichemical to control blast disease in rice.

Complete genome sequence of a marine-sediment-derived bacterial strain Bacillus velezensis SH-B74, a cyclic lipopeptides producer and a biopesticide.

A marine-sediment sample-derived strain Bacillus velezensis SH-B74 has the capacity to produce cyclic lipopeptides (CLPs), and these CLPs secreted by the strain show biological activities against various pests under both in vitro and in planta conditions, such evidence has supported that the strain SH-B74 is a biopesticide. To get a better insight into the mechanisms on the control of the pesticides by the strain, a genome sequencing project has been applied to the genomic DNA of the strain SH-B74. The results show that the strain SH-B74 has a chromosome size of 4,042,190 bp, with a GC content of 46.5%, in addition, the strain contains a 61,634 bp plasmid pSH-B74, with a GC content of 40.8%. Data from bioinformatic analysis reveal that the strain SH-B74 has genes with the capacity to increase environmental adaptation, promote the rhizosphere fitnesses and secrete a spectrum of antibiotics, including nonribosomal peptide synthetases (NRPSs)-derived CLPs bacillopeptin, plipastatin, and surfactin. The presence of CLPs in the bacterial cultures of the strain SH-B74 was confirmed further by LC-MS analysis. Thus, genome sequencing and analyses together with chemical analysis reveal the promising perspectives of the strain SH-B74 that are of spectacular importance to its trait as a plant beneficial microbe to be used in agriculture practices.

Genome mining and metabolic profiling illuminate the chemistry driving diverse biological activities of Bacillus siamensis SCSIO 05746.

Bacillus spp. are important producers of bioactive natural products with potential applications in medicine and agriculture. Bacillus sp. SCSIO 05476 from a deep-sea sediment exhibits broad-spectrum antimicrobial activities and strong cytotoxic activity. Here, an integrative approach combining genome mining and metabolic profiling has been applied to decipher the chemical origins of this strain's varied and significant biological activities. First, genome mining revealed 19 candidate gene clusters encoding the biosynthesis of diverse secondary metabolites. Then, a series of bacillibactins, fengycins, bacillomycins, surfactins, bacillaenes, macrolactins, and related species were found by LC-DAD-MS. Finally, three new linear bacillibactins, linbacillibactins A-C (1-3), along with 11 known secondary metabolites, bacillibactin (4), normal-C13 Val7 surfactin (5), anteiso-C13 Leu7 surfactin (6), iso-C14 Leu7 surfactin (7), normal-C14 Leu7 surfactin (8), anteiso-C14 Leu7 surfactin (9), macrolactin D (10), normal-C14 bacillomycin D (11), iso-C16 bacillomycin D (12), normal-C17 bacillomycin D (13), and iso-C17 bacillomycin D (14), were obtained and elucidated by bioactivity and structure-guided isolation from the fermentation of strain SCSIO 05746. Among them, new compounds 1-3 show significant siderophore activities comparable to that of bacillibactin (4), compounds 13 and 14 exhibit strong cytotoxic activity. At the same time, the strain classification status was confirmed by genomic analyses, and the complete genome sequence of Bacillus siamensis was presented firstly. This study provides a foundation for understanding the mechanisms driving SCSIO 05746's multiple bioactivities and demonstrates a successful way of discovering bioactive metabolites using a combination of genome mining and metabolic profiling methods.

Characterization and abolishment of the cyclopiazonic acids produced by Aspergillus oryzae HMP-F28.

Extracellular alkalinization and H2O2 production are important early events during induced resistance establishment in plants. In a screen for metabolites as plant resistance activators from 98 fungal isolates associated with marine sponge Hymeniacidon perleve, the cyclopiazonic acids (CPAs) produced by Aspergillus oryzae HMP-F28 induced significant extracellular alkalinization coupled with augmented H2O2 production in tobacco cell suspensions. Bioassay-guided fractionation led to the isolation and structural elucidation of a new CPA congener (4, 3-hydroxysperadine A) and three known ones (1-3). To construct a mutasynthetic strain to generate unnatural CPA analogues, a hybrid pks-nrps gene (cpaS) was disrupted to abolish the production of the critical precursor of cyclo-acetoacetyl-L-tryptophan (cAATrp) and all the downstream CPA products. Elimination of cAATrp will allow cAATrp mimics being processed by the CPA biosynthetic machinery to produce CPA derivatives with designed structural features.

Plipastatin A1 produced by a marine sediment-derived Bacillus amyloliquefaciens SH-B74 contributes to the control of gray mold disease in tomato.

Certain Bacillus species have the capacity to produce cyclic lipopeptides and these lipopeptides are promising determinants contributing to the biocontrol of plant diseases. In the current study, a cyclic lipopeptide plipastatin A1 was isolated from the fermentation broth of a marine sediment-derived Bacillus amyloliquefaciens SH-B74 by the combination of solid-phase extraction and reversed-phase high-performance liquid chromatography, and its structure was identified by tandem mass spectrometry, high-resolution electro-spray ionization mass spectrometry, and gas chromatography-mass spectrometry together with nuclear magnetic resonance analysis. Moreover, data from activity evaluation revealed that plipastatin A1 has excellent in vitro activity on the suppression of the conidia germination of B. cinerea, the causal agent of gray mold disease in tomato. Furthermore, plipastatin A1 can successfully decrease the incidence of gray mold disease on tomato leaves at 50 µM concentration. This study indicates that B. amyloliquefaciens SH-B74 appears to be a potentially sustainable pesticide to control gray mold disease in tomato plants, and its cyclic lipopeptide plipastatin A1 plays an important role in the in vitro and in planta biocontrol of B. cinerea.

The complete genome sequence of Bacillus velezensis 9912D reveals its biocontrol mechanism as a novel commercial biological fungicide agent.

A Bacillus sp. 9912 mutant, 9912D, was approved as a new biological fungicide agent by the Ministry of Agriculture of the People's Republic of China in 2016 owing to its excellent inhibitory effect on various plant pathogens and being environment-friendly. Here, we present the genome of 9912D with a circular chromosome having 4436 coding DNA sequences (CDSs), and a circular plasmid encoding 59 CDSs. This strain was finally designated as Bacillus velezensis based on phylogenomic analyses. Genome analysis revealed a total of 19 candidate gene clusters involved in secondary metabolite biosynthesis, including potential new type II lantibiotics. The absence of fengycin biosynthetic gene cluster is noteworthy. Our data offer insights into the genetic, biological and physiological characteristics of this strain and aid in deeper understanding of its biocontrol mechanism.

Production and characterization of surfactin-type lipopeptides as bioemulsifiers produced by a Pinctada martensii-derived Bacillus mojavensis B0621A.

Bacillus mojavensis B0621A was isolated from the mantle of a pearl oyster Pinctada martensii collected from South China Sea. Semi-purified surfactins (225 mg L(-1)) were obtained by acid precipitation and vacuum flash chromatography. The component of the semi-purified surfactins was preliminarily analyzed by liquid chromatography mass spectrometer system, and the results showed that all these surfactins could be a group of homologues. Eight surfactin homologues were isolated and afforded by reversed phase high-performance liquid chromatography. Furthermore, their structure was characterized by mass spectrometry analysis combined with nuclear magnetic resonance spectroscopy techniques. These surfactins shared seven amino acids as peptide backbone and a saturated ß-hydroxy fatty acid chain residue (from C13 to C15), differed each other from peptide sequence in the position of Leu7 or Val7. All these surfactins had significant activity and stability of emulsification under various pH (from 7.0 to 12.0), temperature range (from 20 to 115 °C) and sodium chloride concentration (from 2.5 to 20.0 %, w/v). Taken all together, these results indicated that B. mojavensis B0621A have potential to be an alternative source as a biological-derived emulsifying agent.

Nocardiopsis oceani sp. nov. and Nocardiopsis nanhaiensis sp. nov., actinomycetes isolated from marine sediment of the South China Sea.

Two actinomycete strains, designated 10A08AT and 10A08BT, were isolated from marine sediment samples of the South China Sea and their taxonomic positions were determined by a polyphasic approach. The two Gram-stain-positive, aerobic strains produced branched substrate mycelium and aerial hyphae, and no diffusible pigment was produced in the media tested. At maturity, spore chains were formed on aerial hyphae and all mycelium fragmented with age. Whole-cell hydrolysates of both strains contained meso-diaminopimelic acid and no diagnostic sugars. Their predominant menaquinones (>10 %) were MK-9(H4), MK-9(H6) and MK-10(H6) for strain 10A08AT and MK-9(H4), MK-9(H6), MK-10(H4) and MK-10(H6) for strain 10A08BT. The polar lipids detected from the two strains were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and unknown phosphoglycolipids and phospholipids. The major fatty acids (>10 %) of both strains were iso-C16 : 0 and summed feature 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B). The genomic DNA G+C contents of strains 10A08AT and 10A08BT were 70.9 and 71.6 mol%, respectively. On the basis of 16S rRNA gene sequence similarities, the two strains were shown to be most closely related to species of the genus Nocardiopsis. DNA­DNA hybridization relatedness values of < 70 % between these two isolates and their closest neighbour, Nocardiopsis terrae YIM 90022T, and between the two strains supported the conclusion that they represent two novel species. Based on phylogenetic analysis and phenotypic and genotypic data, it is concluded that the two isolates belong to the genus Nocardiopsis, and the names Nocardiopsis oceani sp. nov. (type strain 10A08AT = DSM 45931T = BCRC 16951T) and Nocardiopsis nanhaiensis sp. nov. (type strain 10A08BT = CGMCC 47227T = BCRC 16952T) are proposed.

Draft genome sequence of the novel strain Pseudomonas sp. 10B238 with potential ability to produce antibiotics from deep-sea sediment.

Pseudomonas sp. 10B238 was a putatively novel species of Pseudomonas, isolated from a deep-sea sediment of the South China Sea, which had the genetic potential to produce secondary metabolites related to nonribosomal peptides (NRPs), as well as showed moderate antimicrobial activities. Here we report a high quality draft genome of Pseudomonas sp. 10B238, which comprises 4,933,052bp with the G+C content of 60.23%. A total of 11 potential secondary metabolite biosynthetic gene clusters were predicted, including a NRP for new peptide siderophore. And many anaerobic respiratory terminal enzymes were found for life in deep-sea environments. Our results may provide insights into biosynthetic pathway for antimicrobial bioactive compounds and be helpful to understand the physiological characteristic of this species.

Nine new diterpenes from the leaves of plantation-grown Cunninghamia lanceolata.

Nine new diterpenes named lanceolatanol hydroperoxide (1), epilanceolatanol hydroperoxide (2), lanceolatanoic acid hydroperoxide (3), epilanceolatanoic acid hydroperoxide (4), lanceolatanol (5), lanceolatanoic acid (6), 11-acetoxylanceolatanoic acid (7), 11-acetoxylanceolatanoic acid methyl ester (8) and epoxyhinokiol (13) were characterized from the leaves of plantation-grown Cunninghamia lanceolata along with twelve known compounds. The compounds were evaluated for their growth inhibitory activities against the human prostate cell line (PC-3).

Cyclic dipeptides produced by fungus Eupenicillium brefeldianum HMP-F96 induced extracellular alkalinization and H2O 2 production in tobacco cell suspensions.

Extracellular alkalinization and H2O2 production are important early events during induced systemic resistance (ISR) establishment in plants. In a screen for metabolites as potential ISR activators from 98 fungal isolates associated with marine sponge Hymeniacidon perleve, the crude metabolites of fungus Eupenicillium brefeldianum HMP-F96 induced significant extracellular alkalinization coupled with H2O2 production in tobacco cell suspensions. A combined bioactivity and (1)H NMR-guided fractionation approach was used to disclose the chemical determinants responsible for the activities. Eight cyclic dipeptides were purified from the fermentation broth of the strain and were structurally characterized by NMR and MS experiments. This study represents the first report of the occurrence of cyclic dipeptides in E. brefeldianum and of their activities of inducing extracellular alkalinization and H2O2 production in tobacco cell suspensions.