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Actinobacteria, especially the genus Streptomyces, have been shown to be potential biocontrol agents for phytopathogenic bacteria. Bacteria spot disease caused by Xanthomonas spp. may severely affect chili pepper (Capsicum annuum) crops with a subsequent decrease in productivity. Therefore, the objective of the study was to isolate rhizospheric actinobacteria from soil samples treated by physical methods and evaluate the inhibitory activity of the isolates over Xanthomonas. Initially, soil samples collected from avocado tree orchards were treated by dry heat air and microwave irradiation; thereafter, isolation was implemented. Then, antibacterial activity (AA) of isolates was evaluated by the double-layer agar method. Furthermore, the positive/negative effect on AA for selected isolates was evaluated on three culture media (potato-dextrose agar, PDA; yeast malt extract agar, YME; and oat agar, OA). Isolates were identified by 16S rRNA sequence analysis. A total of 198 isolates were obtained; 76 (series BVEZ) correspond to samples treated by dry heat and 122 strains (series BVEZMW) were isolated from samples irradiated with microwaves. A total of 19 dry heat and 25 microwave-irradiated isolates showed AA with inhibition zones (IZ, diameter in mm) ranging from 12.7 to 82.3 mm and from 11.4 to 55.4 mm, respectively. An increment for the AA was registered for isolates cultured on PDA and YME, with an IZ from 21.1 to 80.2 mm and 14.1 to 69.6 mm, respectively. A lower AA was detected when isolates were cultured on OA media (15.0 to 38.1 mm). Based on the 16S rRNA gene sequencing analysis, the actinobacteria belong to the Streptomyces (6) and Amycolatopsis (2) genera. Therefore, the study showed that microwave irradiation is a suitable method to increase the isolation of soil bacteria with AA against Xanthomonas sp. In addition, Streptomyces sp. BVEZ 50 was the isolate with the highest IZ (80.2 mm). [ABSTRACT FROM AUTHOR]

Abstract The urgent need for sustainable agriculture has intensified the search for environmentally friendly alternatives to chemical herbicides. This study investigates the herbicidal potential of siderophores produced by Amycolatopsis lurida strain 407, focusing on its effects on the growth of ryegrass and redroot weeds. Strain 407 exhibited two distinct colony morphologies—red and white—when cultured under varying environmental conditions. The cell-free culture filtrate (CFCF) from both colony types significantly inhibited the growth of ryegrass and redroot. The concentration of siderophore produced in the iron-deficient medium was measured to be 613.4 ppm for 407 red and 388.5 ppm for 407 white, which indicates significant iron chelating activity. This study also showed a direct relationship between the presence of siderophore in plant culture medium and reduced growth. Also, analysis of fractions of the aqueous phase resulting from column chromatography revealed that all fractions from the 407 red reduced ryegrass shoot length by up to 45% and root length by 83–86%, while redroot seedling length decreased by up to 36%. Fractions from 407 white completely inhibited germination or reduced ryegrass root length by up to 94% and redroot seedling length by 52%. Fractions F4 W to F7 W and F2 R to F8 R, which showed iron chelating activity were most effective in reducing plant growth, suggesting that there are metabolites, alone or in company with siderophores, synergistically do herbicidal activity. The innovative application of siderophores as bioherbicide presents a promising environmentally friendly alternative to chemical herbicides.

A novel endophytic actinomycete, strain MEP2-6T, was isolated from scab tissues of potato tubers collected from Mae Fag Mai Sub-district, San Sai District, Chiang Mai Province, Thailand. Strain MEP2-6T is a gram-positive filamentous bacteria characterized by meso-diaminopimelic acid in cell wall peptidoglycan and arabinose, galactose, glucose, and ribose in whole-cell hydrolysates. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and hydroxy-phosphatidylethanolamine were the major phospholipids, of which MK-9(H6) was the predominant menaquinone, whereas iso-C16:0 and iso-C15:0 were the major cellular fatty acids. The genome of the strain was 10,277,369 bp in size with a G + C content of 71.7%. The 16S rRNA gene phylogenetic and core phylogenomic analyses revealed that strain MEP2-6T was closely related to Amycolatopsis lexingtonensis NRRL B-24131T (99.4%), A. pretoriensis DSM 44654T (99.3%), and A. eburnea GLM-1T (98.9%). Notably, strain MEP2-6T displayed 91.7%, 91.8%, and 87% ANIb and 49%, 48.8%, and 35.4% dDDH to A. lexingtonensis DSM 44653T (=NRRL B-24131T), A. eburnea GLM-1T, and A. pretoriensis DSM 44654T, respectively. Based on phenotypic, chemotaxonomic, and genomic data, strain MEP2-6T could be officially assigned to a novel species within the genus Amycolatopsis, for which the name Amycolatopsis solani sp. nov. has been proposed. The type of strain is MEP2-6T (=JCM 36309T = TBRC 17632T = NBRC 116395T). Amycolatopsis solani MEP2-6T was strongly proven to be a non-phytopathogen of potato scab disease because stunting of seedlings and necrotic lesions on potato tuber slices were not observed, and there were no core biosynthetic genes associated with the BGCs of phytotoxin-inducing scab lesions. Furthermore, comparative genomics can provide a better understanding of the genetic mechanisms that enable A. solani MEP2-6T to adapt to the plant endosphere. Importantly, the strain smBGCs accommodated 33 smBGCs encoded for several bioactive compounds, which could be beneficially applied in the fields of agriculture and medicine. Consequently, strain MEP2-6T is a promising candidate as a novel biocontrol agent and antibiotic producer. [ABSTRACT FROM AUTHOR]

Actinobacteria, especially the genus Streptomyces, have been shown to be potential biocontrol agents for phytopathogenic bacteria. Bacteria spot disease caused by Xanthomonas spp. may severely affect chili pepper (Capsicum annuum) crops with a subsequent decrease in productivity. Therefore, the objective of the study was to isolate rhizospheric actinobacteria from soil samples treated by physical methods and evaluate the inhibitory activity of the isolates over Xanthomonas. Initially, soil samples collected from avocado tree orchards were treated by dry heat air and microwave irradiation; thereafter, isolation was implemented. Then, antibacterial activity (AA) of isolates was evaluated by the double-layer agar method. Furthermore, the positive/negative effect on AA for selected isolates was evaluated on three culture media (potato-dextrose agar, PDA; yeast malt extract agar, YME; and oat agar, OA). Isolates were identified by 16S rRNA sequence analysis. A total of 198 isolates were obtained; 76 (series BVEZ) correspond to samples treated by dry heat and 122 strains (series BVEZMW) were isolated from samples irradiated with microwaves. A total of 19 dry heat and 25 microwave-irradiated isolates showed AA with inhibition zones (IZ, diameter in mm) ranging from 12.7 to 82.3 mm and from 11.4 to 55.4 mm, respectively. An increment for the AA was registered for isolates cultured on PDA and YME, with an IZ from 21.1 to 80.2 mm and 14.1 to 69.6 mm, respectively. A lower AA was detected when isolates were cultured on OA media (15.0 to 38.1 mm). Based on the 16S rRNA gene sequencing analysis, the actinobacteria belong to the Streptomyces (6) and Amycolatopsis (2) genera. Therefore, the study showed that microwave irradiation is a suitable method to increase the isolation of soil bacteria with AA against Xanthomonas sp. In addition, Streptomyces sp. BVEZ 50 was the isolate with the highest IZ (80.2 mm).

Nocardioform placentitis (NP) continues to result in episodic outbreaks of abortion and preterm birth in mares and remains a poorly understood disease. The objective of this study was to characterize the transcriptome of the chorioallantois (CA) of mares with NP. The CA were collected from mares with confirmed NP based upon histopathology, microbiological culture and PCR for Amycolatopsis spp. Samples were collected from the margin of the NP lesion (NPL, n = 4) and grossly normal region (NPN, n = 4). Additionally, CA samples were collected from normal postpartum mares (Control; CRL, n = 4). Transcriptome analysis identified 2892 differentially expressed genes (DEGs) in NPL vs. CRL and 2450 DEGs in NPL vs. NPN. Functional genomics analysis elucidated that inflammatory signaling, toll-like receptor signaling, inflammasome activation, chemotaxis, and apoptosis pathways are involved in NP. The increased leukocytic infiltration in NPL was associated with the upregulation of matrix metalloproteinase (MMP1, MMP3, and MMP8) and apoptosis-related genes, such as caspases (CASP3 and CASP7), which could explain placental separation associated with NP. Also, NP was associated with downregulation of several placenta-regulatory genes (ABCG2, GCM1, EPAS1, and NR3C1), angiogenesis-related genes (VEGFA, FLT1, KDR, and ANGPT2), and glucose transporter coding genes (GLUT1, GLUT10, and GLUT12), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could elucidate placental insufficiency accompanying NP. In conclusion, our findings revealed for the first time, the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during NP, which might lead to the development of efficacious therapies or diagnostic aids by targeting the key molecular pathways.

Lignin, a complex plant cell wall component, holds promise as a renewable aromatic carbon feedstock. p-Vanillin is a key product of lignin depolymerization and a precursor of protocatechuic acid (PCA) that has tremendous potential for biofuel production. While the GcoAB enzyme, native to Amycolatopsis sp., naturally catalyzes aryl-O-demethylation toward guaiacol, recent research introduced a single mutation, T296S, into the GcoAP450 enzyme, enabling it to catalyze aryl-O-demethylation of p-vanillin. This structural modification increases the efficiency of GcoAP450 for the natural substrate while being active for p-vanillin. This study reveals the increased flexibility of p-vanillin and its ability to adapt a favorable conformation by aligning the methoxy group in close proximity to Fe(IV) = O of Cpd I in the active site of the T296S variant. The QM/MM calculations in accordance with the experimental data validated that the rate-limiting step for the oxidation of p-vanillin is hydrogen atom abstraction and provided a detailed geometric structure of stationary and saddle points for the oxidation of p-vanillin. [ABSTRACT FROM AUTHOR]

Introduction: The increase in incidence of multidrug-resistant bacteria and the inadequacy of new antimicrobial drugs have led to a widespread outbreak of bacterial antimicrobial resistance. To discover new antibiotics, biodiversity, and novelty of culturable actinobacteria dwelled in soil of the Western Qinghai-Tibet Plateau were investigated. By integrating antibacterial assay with omics tools, Amycolatopsis sp. A133, a rare actinobacterial strain and its secondary metabolites were further studied. Method: Culture-dependent method was used to obtain actinobacterial strains from two soil samples collected from Ali region in Qinghai-Tibet Plateau. The cultural extractions of representative strains were assayed against "ESKAPE" pathogens by paper-disk diffusion method and the double fluorescent protein reporter "pDualrep2" system. An Amycolatopsis strain coded as A133 was prioritized and its secondary metabolites were further analyzed and annotated by omics tools including antiSMASH and GNPS (Global Natural Social Molecular Networking). The predicted rifamycin analogs produced by Amycolatopsis sp. A133 were isolated and identified by chromatographic separation, such as Sephadex LH-20 and HPLC, and spectral analysis, such as NMR and UPLC-HRESI-MS/MS, respectively. Results: A total of 406 actinobacteria strains affiliated to 36 genera in 17 families of 9 orders were isolated. Out of 152 representative strains, 63 isolates exhibited antagonistic activity against at least one of the tested pathogens. Among them, 7 positive strains were identified by the "pDualrep2" system as either an inhibitor of protein translation or DNA biosynthesis. The cultural broth of Amycolatopsis sp. A133 exhibited a broader antimicrobial activity and can induce expression of TurboRFP. The secondary metabolites produced by strain A133 was annotated as rifamycins and zampanolides by antiSMASH and GNPS analysis. Five members of rifamycins, including rifamycin W, protorifamycin I, rifamycin W-M1, proansamycin B, and rifamycin S, were purified and identified. Rifamycin W-M1, was found as a new member of the naturally occurring rifamycin group of antibiotics. Discussion: Assisted by omics tools, the successful and highly efficient discovery of rifamycins, a group of clinically used antibiotics from actinobacteria in Ali area encouraged us to devote more energy to explore new antibiotics from the soils on the Western Tibetan Plateau. [ABSTRACT FROM AUTHOR]

Though reactive flavin-N5/C4α-oxide intermediates can be spectroscopically profiled for some flavin-assisted enzymatic reactions, their exact chemical configurations are hardly visualized. Structural systems biology and stable isotopic labelling techniques were exploited to correct this stereotypical view. Three transition-like complexes, the α-ketoacid…N5-FMNox complex (I), the FMNox -N5-aloxyl-C'α- -C4α+ zwitterion (II), and the FMN-N5-ethenol-N5-C4α-epoxide (III), were determined from mandelate oxidase (Hmo) or its mutant Y128F (monooxygenase) crystals soaked with monofluoropyruvate (a product mimic), establishing that N5 of FMNox an alternative reaction center can polarize to an ylide-like mesomer in the active site. In contrast, four distinct flavin-C4α-oxide adducts (IV-VII) from Y128F crystals soaked with selected substrates materialize C4α of FMN an intrinsic reaction center, witnessing oxidation, Baeyer-Villiger/peroxide-assisted decarboxylation, and epoxidation reactions. In conjunction with stopped-flow kinetics, the multifaceted flavin-dependent reaction continuum is physically dissected at molecular level for the first time.

Actinomycetes are prolific producers of bioactive secondary metabolites. The prevalence of multidrug-resistant (MDR) pathogens has prompted us to search for potential natural antimicrobial agents. Herein, we report the isolation of rare actinobacteria from Egyptian soil. The strain was identified as Amycolatopsis keratiniphila DPA04 using 16S rRNA gene sequencing. Cultivation profiling, followed by chemical and antimicrobial evaluation of crude extracts, revealed the activity of DPA04 ISP-2 and M1 culture extracts against Gram-positive bacteria. Minimum inhibitory concentrations (MIC) values ranged from 19.5 to 39 µg/mL. Chemical analysis of the crude extracts using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF) led to the identification of 45 metabolites of different chemical classes. In addition, ECO-0501 was identified in the cultures with significant antimicrobial activity. Multidrug resistance in Staphylococcus aureus is reported to be related to the multidrug efflux pump (MATE). ECO-0501 and its related metabolites were subjected to molecular docking studies against the MATE receptor as a proposed mechanism of action. ECO-0501 and its derivatives (AK_1 and N-demethyl ECO-0501) had better binding scores (−12.93, −12.24, and −11.92 kcal/mol) than the co-crystallized 4HY inhibitor (−8.99 kcal/mol) making them promising candidates as MATE inhibitors. Finally, our work established that natural products from this strain could be useful therapeutic tools for controlling infectious diseases. [ABSTRACT FROM AUTHOR]

The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportionation reaction via an N5-alkanol-FMNred C'α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.

Fungus‐growing termites cultivate the fungal mutualist Termitomyces as their main food source; however, how fungus‐growing termites protect Termitomyces from threats is still unclear. In this study, we investigated the actinobacterial communities in Odontotermes formosanus individuals and their fungal combs.Moreover, the antifungal activities of the isolated actinobacteria were tested. 16 S rRNA gene sequencing results indicated that the actinobacteria in O. formosanus and its fungal combs belong to 5 classes, 17 orders, 40 families, and 84 genera.The relative abundance of Coriobacteriia in the nymphs, young workers, old workers, and soldiers was higher than that in the queens and fungal combs, and the relative abundance of class Actinobacteria in the queens and the fungal combs was higher than that in the nymphs, young workers, old workers, and soldiers.Based on antifungal bioassays, 3 strains of Amycolatopsis and 2 strains of Streptomyces isolated from old workers had strong inhibitory activity against Xylaria angulosa but weak inhibitory activity against Termitomyces sp.These results indicated that the actinobacteria of O. formosanus may contribute to protecting termite fungal food via their asymmetric antifungal activities. [ABSTRACT FROM AUTHOR]

Two new secondary metabolites, amycolatolides A (1) and B (2), along with twelve known compounds (3 − 14), were isolated from the fermentation products of lichen-derived Amycolatopsis sp. YIM 130923. The structures of 1 and 2 were elucidated on the basis of NMR spectroscopic and HRESI-MS analysis. Meanwhile, the absolute configuration of 1 was deduced by X-ray diffraction analysis. Furthermore, compound 3 was obtained from microbial resources for the first time, 13 and 14 were firstly found from the genus Amycolatopsis. Some of the isolated compounds were evaluated for their antimicrobial activities by broth microdilution method. The results showed that compound 14 possessed moderate antifungal activities against Fusarium graminearum and Aspergillus niger with MIC values of 16 and 32 μg/mL, respectively. [Display omitted] • Two new compounds were isolated from lichen-derived Amycolatopsis sp. YIM 130923. • The absolute configuration of 1 was determined by X-ray diffraction experiment. • Cadinane- and eudesmane-type sesquiterpenes were first obtained from Amycolatopsis. • Compound 14 showed moderate antimicrobial activities. [ABSTRACT FROM AUTHOR]

In the present work, Amycolatopsis sp. SND-1 (SND-1) was isolated from Cleome chellidonii Linn. (C. chellidonii) was performed as biocontrol and resistance elicitor in Vigna radiata (L.) R. Wilczek (mung bean) plants against Cercospora leaf spot causing pathogen Cercospora canescens (C. canescens). The SND-1 isolate showed 74% of inhibition against C. canescens in dual culture and GC–MS analysis revealed the presence of antifungal compounds. Molecular characterization through 16S rRNA showed that the isolated SND-1 belongs to Amycolatopsis sp. The in vitro plant growth trials exhibited production of indole acetic acid, gibberellic acid, cytokinin, ammonia, hydrogen cyanide, and siderophore and phosphate solubilization. In vivo study with talcum formulation of SND-1 revealed a significant increase in plant root length, shoots length, root and shoot fresh weight, and reduced the disease severity in treated mung bean plants. Triggering of resistance by SND-1 formulation was studied by histochemical depositions and biochemical defense enzymes that resulted in the acceleration in defense response in comparison with control plants. The bioactive endophytic Amycolatopsis sp. SND-1 enhanced the defense against C. canescens infection; hence, it can be used as a biological control agent in mung bean cultivars. [ABSTRACT FROM AUTHOR]

Amycolatopsis is an important source of diverse valuable bioactive natural products. The CRISPR/Cas-mediated gene editing tool has been established in some Amycolatopsis species and has accomplished the deletion of single gene or two genes. The goal of this study was to develop a high-efficient CRISPR/Cas9-mediated genome editing system in vancomycin-producing strain A. keratiniphila HCCB10007 and enhance the production of vancomycin by deleting the large fragments of ECO-0501 BGC. By adopting the promoters of gapdhp and ermE*p which drove the expressions of scocas9 and sgRNA, respectively, the all-in-one editing plasmid by homology-directed repair (HDR) precisely deleted the single gene gtfD and inserted the gene eGFP with the efficiency of 100%. Furthermore, The CRISPR/Cas9-mediated editing system successfully deleted the large fragments of cds13-17 (7.7 kb), cds23 (12.7 kb) and cds22-23 (21.2 kb) in ECO-0501 biosynthetic gene cluster (BGC) with high efficiencies of 81%–97% by selecting the sgRNAs with a suitable PAM sequence. Finally, a larger fragment of cds4-27 (87.5 kb) in ECO-0501 BGC was deleted by a dual-sgRNA strategy. The deletion of the ECO-0501 BGCs revealed a noticeable improvement of vancomycin production, and the mutants, which were deleted the ECO-0501 BGCs of cds13-17, cds22-23 and cds4-27, all achieved a 30%–40% increase in vancomycin yield. Therefore, the successful construction of the CRISPR/Cas9-mediated genome editing system and its application in large fragment deletion in A. keratiniphila HCCB10007 might provide a powerful tool for other Amycolatopsis species.

Unique biotopes can be a source of new plant growth promotion (PGP) bacteria with rare properties. The Antarctic habitat is an attractive location for research, as it is characterized by many stress factors, and the local microbiota is under permanent selective pressure. We believe that the rhizosphere bacteria of this habitat may have important PGP properties that can be used in agriculture. A variety of research methods were used in this work: the molecular genetic technique to establish the gene sequence, chemical to test the ability to produce nitrite, ammonia, and indole acetic acid, microbiological to investigate the cultured properties of the strain, as well as antagonistic and PGP activities. We found that the strain belongs to the genus Amycolatopsis. It showed antagonistic activity against phytopathogenic bacteria (Xanthomonas campestrispv. campestris IMB8003 and Bacillus subtilis ATCC 31324) and fungi (Alternaria alternata DSM 1102, Fusarium oxysporum IMB 54201, Aspergillus niger IMB 16706), and also demonstrated some PGP properties (solubilization of phosphorus and zinc and production of nitrite and ammonia). Inoculation of wheat seeds with spores of this strain promoted germination and growth of seedlings. The strain has demonstrated properties that make it a promising basis for developing biofertilizers that can be used in agriculture. [ABSTRACT FROM AUTHOR]

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g−1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l−1 in a second round of mutant selection. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products. • A general method for industrial strain development of actinomycetes. • Protocol for fluorescence-activated cell sorting of actinomycetes. • Metabolic engineering for increased production of cholesterol oxidase. • Activation of a silent mutaxanthene gene cluster and yield enhancement. [ABSTRACT FROM AUTHOR]

An extracellular lipase from Amycolatopsis mediteranei (AML) with potential applications in process biotechnology was recently cloned and examined in this laboratory. In the present study, the 3D structure of AML was elucidated by comparative modelling. AML lacked the ‘lid’ structure observed in most true lipases and shared similarities with plastic degrading enzymes. Modelling and substrate specificity studies showed that AML was a cutinase with a relatively exposed active site and specificity for medium chain fatty acyl moieties.AML rapidly hydrolysed the aliphatic plastics poly(ε-caprolactone) and poly(1,4-butylene succinate) extended with 1,6-diisocyanatohexane under mild conditions. These plastics are known to be slow to degrade in landfill. Poly(L-lactic acid) was not hydrolysed by AML, nor was the aromatic plastic Polyethylene Terephthalate (PET). The specificity of AML is partly explained by active site topology and analysis reveals that minor changes in the active site region can have large effects on substrate preference. These findings show that extracellular Amycolatopsis enzymes are capable of degrading a wider range of plastics than is generally recognised. The potential for application of AML in the bioremediation of plastics is discussed.

Recent studies in this laboratory showed that an extracellular cutinase from A. mediterranei (AmCut) was able to degrade the plastics polycaprolactone and polybutylene succinate. Such plastics can be slow to degrade in soils due to a lack of efficient polyester degrading organisms. AmCut also showed potential for the biocatalytic synthesis of esters by reverse hydrolysis. The gene for AmCut has an upstream leader sequence whose transcript is not present in the purified enzyme. In this study, we show using predictive modelling, that this sequence codes for an N-terminal signal peptide that directs transmembrane expression via the Sec secretion pathway. E. coli is a useful host for recombinant enzymes used in biocatalysis due to the ease of genetic manipulation in this organism, which allows tuning of enzymes for specific applications, by mutagenesis. When a truncated GST-tagged AmCut gene (lacking its signal peptide) was expressed in E. coli, all cutinase activity was observed in the cytosolic fraction. However, when GST-tagged AmCut was expressed in E. coli along with its native signal peptide, cutinase activity was observed in both the periplasmic space and the culture medium. This finding revealed that the native signal peptide of a Gram-positive organism (AmCut) was being recognised by the Gram-negative (E. coli) Sec transmembrane transport system. AmCut was transported into E. coli's periplasmic space from where it was released into the culture medium. Surprisingly, the presence of a bulky GST tag at the N-terminus of the signal peptide did not hinder transmembrane targeting. Although the periplasmic targeting was unexpected, it is not unprecedented due to the conservation of the Sec pathway across species. It was more surprising that AmCut was secreted from the periplasmic space into the culture medium. This suggests that extracellular AmCut translocation across the E. coli outer membrane may involve non-classical secretion pathways. This tuneable recombinant E. coli expressing extracellular AmCut may be useful for degradation of polyester substrates in the environment; this and other applications are discussed. [ABSTRACT FROM AUTHOR]

Actinobacteria of the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as bioconversion or bioremediation. Despite their importance, tools and methods for their genetic manipulation are less developed than in other actinobacteria such as Streptomyces. We report here the use of the pSAM2 site-specific recombination system to delete antibiotic resistance cassettes used in gene replacement experiments or to create large genomic deletions. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the integrase and the excisionase from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the attL and attR sites on each side of a large region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei by obtaining with high efficiency a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster. These robust and simple tools enrich the toolbox for genome engineering in Amycolatopsis. [ABSTRACT FROM AUTHOR]

Actinomycetes are prolific producers of bioactive secondary metabolites. The prevalence of multidrug-resistant (MDR) pathogens has prompted us to search for potential natural antimicrobial agents. Herein, we report the isolation of rare actinobacteria from Egyptian soil. The strain was identified as Amycolatopsis keratiniphila DPA04 using 16S rRNA gene sequencing. Cultivation profiling, followed by chemical and antimicrobial evaluation of crude extracts, revealed the activity of DPA04 ISP-2 and M1 culture extracts against Gram-positive bacteria. Minimum inhibitory concentrations (MIC) values ranged from 19.5 to 39 µg/mL. Chemical analysis of the crude extracts using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF) led to the identification of 45 metabolites of different chemical classes. In addition, ECO-0501 was identified in the cultures with significant antimicrobial activity. Multidrug resistance in Staphylococcus aureus is reported to be related to the multidrug efflux pump (MATE). ECO-0501 and its related metabolites were subjected to molecular docking studies against the MATE receptor as a proposed mechanism of action. ECO-0501 and its derivatives (AK_1 and N-demethyl ECO-0501) had better binding scores (−12.93, −12.24, and −11.92 kcal/mol) than the co-crystallized 4HY inhibitor (−8.99 kcal/mol) making them promising candidates as MATE inhibitors. Finally, our work established that natural products from this strain could be useful therapeutic tools for controlling infectious diseases.

A novel protease-producing and cellulose-degrading actinobacterium, designated strain NEAU-NG30 T , was isolated from a melon rhizosphere soil sample collected in Harbin, Heilongjiang Province, China, and established its status using a polyphasic taxonomic study. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NEAU-NG30 T was closely related to Amycolatopsis bullii DSM 45802 T (98.7%) and Amycolatopsis vancoresmycina DSM 44592 T (98.3%). The phospholipid profile contained diphosphatidylglycerol, phosphatidyl methylethanolamine, phosphatidylethanolamine and phosphatidylinositol. The diagnostic sugars in cell hydrolysates were determined to be galactose and arabinose. Cell walls contained meso -diaminopimelic acid as the diagnostic diamino acid. The predominant menaquinone was MK-9(H 4 ). The major fatty acids were iso-C 15:0 and iso-C 16:0 . Meanwhile, genome analysis of sequences revealed a genome size of 9 338 250 bp and a DNA G+C content of 71.6%. In addition, the average nucleotide identity values and the level of digital DNA-DNA hybridization between strain NEAU-NG30 T and its reference strains fall below the thresholds typically used for delineating prokaryote species. According to phenotypic, chemotaxonomic and genotypic studies, it is indicated that strain NEAU-NG30 T is considered to be a novel species of the genus Amycolatopsis , for which the name Amycolatopsis melonis sp. nov. is proposed, with NEAU-NG30 T (=MCCC 1K08677 T =JCM 35654 T ) as the type strain.

Two-novel filamentous actinobacteria designated strains 2-2 T and 2-15 T were isolated from soil of a coal mining site in Mongolia, and their taxonomic positions were determined using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that each of the strains formed a distinct clade within the genus Amycolatopsis . The 16S rRNA gene sequence similarity analysis showed that both strains were mostly related to Amycolatopsis rhabdoformis NCIMB 14900 T with 99.0 and 99.4% sequence similarity, respectively. The genome-based comparison indicated that strain 2-2 T shared the highest digital DNA-DNA hybridization value of 35.6% and average nucleotide identity value of 86.9% with Amycolatopsis pretoriensis DSM 44654 T , and strain 2-15 T shared the corresponding values of 36.5 and 87.9% with A. rhabdoformis NCIMB 14900 T , all of which being well below the thresholds for species delineation. The chemotaxonomic properties of both strains were typical of the genus Amycolatopsis . In silico prediction of chemotaxonomic markers was also carried out, and the results were consistent with the chemotaxonomic profiles of the genus. Genome mining for secondary metabolite production in strains 2-2 T and 2-15 T revealed the presence of 29 and 24 biosynthetic gene clusters involved in the production of polyketide synthase, non-ribosomal peptide synthetase, ribosomally synthesized and post-translationally modified peptides, lanthipeptide, terpenes, siderophore, and a number of other unknown type compounds. Both strains showed broad antifungal activity against several filamentous fungi and also antibacterial activity against methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii . The phenotypic, biochemical, and chemotaxonomic properties indicated that both strains could be clearly distinguished from other species of Amycolatopsis , and thus the names Amycolatopsis nalaikhensis sp. nov. (type strain, 2-2 T =KCTC 29695 T =JCM 30462 T ) and Amycolatopsis carbonis (type strain, 2-15 T =KCTC 39525 T =JCM 30563 T ) are proposed accordingly.

Background and aims: The application of chemical fungicides is the first strategy to control plant fungal diseases. This approach is highly polluting for the environment and affects human health. The artificial introduction of beneficial rhizobacteria into the soil can be an economical and practical way to control phytopathogenic fungi in commercial greenhouses. Here, we recount the travel of a rare Actinomycete (Amycolatopsis strain 1119) from a maize field to a commercial cucumber greenhouse. Methods and results: Culturable bacteria from rhizosphere and bulk soils of dicot and monocot crops were isolated and screened. About 20% of the representative colonies showed Actinobacteria appearance. 106 Actinobacteria that had antagonistic activity against Phytophthora capsici and were able to produce IAA-like molecules were selected for further analysis. Two Streptomyces strains (432 and 615) and 2 Amycolatopsis strains (3513 and 1119) that showed a positive effect on plant growth in greenhouse conditions were selected to evaluate the biocontrol potential. Strains 432, 3513, 615 and 1119 controlled incidence of the damping-off by 65%, 42%, 83% and 100% respectively. Application of strain 1119 under commercial greenhouse conditions resulted in an increase in fruit yield (20%) and a decrease in fruit nitrate content (70%). Increased antioxidant enzymes activity and increased LOX and APX transcription and also, increased expression of two genes PR1-1a and GLU (SAR genes) showed that strain 1119 could induce both ISR and SAR in cucumber without pathogen exposure. Conclusion: Our results demonstrate that the Amycolatopsis strain 1119 has a great potential to be used as an active principle for bio-inoculant development because of the ability to improve cucumber fruit yield and induce plant defense responses in a commercial greenhouse. [ABSTRACT FROM AUTHOR]

Benzo(b)fluoranthene (BbF) is a polycyclic aromatic hydrocarbon (PAH) with five fused benzene rings; it is a highly recalcitrant compound and a priority environmental pollutant due to its detrimental effects on human health and the survival of wild animals. Biodegradation of BbF by microorganisms is an attractive alternative, and few studies have been focused on this issue. In this work, bacteria with the ability to degrade BbF were isolated and selected. The capability of the isolates to tolerate concentrations of 50 and 75 mg l−1 of BbF in liquid medium was evaluated. The selected isolates were identified by the 16S rRNA gene sequencing as belonging to Bacillus, Gordonia, Pseudomonas, Rhodococcus, Ochrobactrum, and Amycolatopsis. All isolates were tolerant and grew at the BbF concentrations tested, some isolates were more competitive than others, and the most prominent was Amycolatopsis sp. Ver12, which removed 47% of BbF, furthermore, with the addition of yeast extract, removed 59% of the compound. In summary, the report shows that Amycolatopsis sp. Ver12 can degrade BbF efficiently and could be considered for bioremediation of BbF‐contaminated environments. Significance and Impact of the Study: Benzo(b)fluoranthene (BbF) is a polycyclic aromatic hydrocarbon (PAH) with five fused benzene rings and is highly recalcitrant. The BbF can generate mutations that result in cancer. Due to the risk that BbF represents to human health, its elimination from the environment is a priority. An alternative for the removal of BbF is by biodegradation. There are few bacteria capable of degrading BbF. In this work, Amycolatopsis sp. Ver12 was isolated and identified from an oil‐contaminated soil, as a potential BbF degrader, which could be used in the future to remediate BbF‐contaminated soils. [ABSTRACT FROM AUTHOR]

Actinobacteria provide a rich spectrum of bioactive natural products and therefore display an invaluable source towards commercially valuable pharmaceuticals and agrochemicals. Here, we studied the use of inorganic talc microparticles (hydrous magnesium silicate, 3MgO·4SiO2·H2O, 10 µm) as a general supplement to enhance natural product formation in this important class of bacteria. Added to cultures of recombinant Streptomyces lividans, talc enhanced production of the macrocyclic peptide antibiotic bottromycin A2 and its methylated derivative Met‐bottromycin A2 up to 109 mg L−1, the highest titer reported so far. Hereby, the microparticles fundamentally affected metabolism. With 10 g L−1 talc, S. lividans grew to 40% smaller pellets and, using RNA sequencing, revealed accelerated morphogenesis and aging, indicated by early upregulation of developmental regulator genes such as ssgA, ssgB, wblA, sigN, and bldN. Furthermore, the microparticles re‐balanced the expression of individual bottromycin cluster genes, resulting in a higher macrocyclization efficiency at the level of BotAH and correspondingly lower levels of non‐cyclized shunt by‐products, driving the production of mature bottromycin. Testing a variety of Streptomyces species, talc addition resulted in up to 13‐fold higher titers for the RiPPs bottromycin and cinnamycin, the alkaloid undecylprodigiosin, the polyketide pamamycin, the tetracycline‐type oxytetracycline, and the anthramycin‐analogs usabamycins. Moreover, talc addition boosted production in other actinobacteria, outside of the genus of Streptomyces: vancomycin (Amycolatopsis japonicum DSM 44213), teicoplanin (Actinoplanes teichomyceticus ATCC 31121), and the angucyclinone‐type antibiotic simocyclinone (Kitasatospora sp.). For teicoplanin, the microparticles were even crucial to activate production. Taken together, the use of talc was beneficial in 75% of all tested cases and optimized natural and heterologous hosts forming the substance of interest with clusters under native and synthetic control. Given its simplicity and broad benefits, microparticle‐supplementation appears as an enabling technology in natural product research of these most important microbes. [ABSTRACT FROM AUTHOR]

Abstract Background Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify more integrating vectors, using phage int/attP loci, that would efficiently integrate site-specifically in the rare Actinomycete, Amycolatopsis marina DSM45569. Results Analysis of the genome of A. marina DSM45569 indicated the presence of attB-like sequences for TG1 and R4 integrases. The TG1 and R4 attBs were active in in vitro recombination assays with their cognate purified integrases and attP loci. Integrating vectors containing either the TG1 or R4 int/attP loci yielded exconjugants in conjugation assays from Escherichia coli to A. marina DSM45569. Site-specific recombination of the plasmids into the host TG1 or R4 attB sites was confirmed by sequencing. Conclusions The homologous TG1 and R4 attB sites within the genus Amycolatopsis have been identified. The results indicate that vectors based on TG1 and R4 integrases could be widely applicable in this genus.

Actinobacteria of the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as bioconversion or bioremediation. Despite their importance, tools and methods for their genetic manipulation are less developed than in other actinobacteria such as Streptomyces. We report here the use of the pSAM2 site-specific recombination system to delete antibiotic resistance cassettes used in gene replacement experiments or to create large genomic deletions. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the integrase and the excisionase from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the attL and attR sites on each side of a large region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei by obtaining with high efficiency a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster. These robust and simple tools enrich the toolbox for genome engineering in Amycolatopsis.

Abstract Background Genome mining tools have enabled us to predict biosynthetic gene clusters that might encode compounds with valuable functions for industrial and medical applications. With the continuously increasing number of genomes sequenced, we are confronted with an overwhelming number of predicted clusters. In order to guide the effective prioritization of biosynthetic gene clusters towards finding the most promising compounds, knowledge about diversity, phylogenetic relationships and distribution patterns of biosynthetic gene clusters is necessary. Results Here, we provide a comprehensive analysis of the model actinobacterial genus Amycolatopsis and its potential for the production of secondary metabolites. A phylogenetic characterization, together with a pan-genome analysis showed that within this highly diverse genus, four major lineages could be distinguished which differed in their potential to produce secondary metabolites. Furthermore, we were able to distinguish gene cluster families whose distribution correlated with phylogeny, indicating that vertical gene transfer plays a major role in the evolution of secondary metabolite gene clusters. Still, the vast majority of the diverse biosynthetic gene clusters were derived from clusters unique to the genus, and also unique in comparison to a database of known compounds. Our study on the locations of biosynthetic gene clusters in the genomes of Amycolatopsis’ strains showed that clusters acquired by horizontal gene transfer tend to be incorporated into non-conserved regions of the genome thereby allowing us to distinguish core and hypervariable regions in Amycolatopsis genomes. Conclusions Using a comparative genomics approach, it was possible to determine the potential of the genus Amycolatopsis to produce a huge diversity of secondary metabolites. Furthermore, the analysis demonstrates that horizontal and vertical gene transfer play an important role in the acquisition and maintenance of valuable secondary metabolites. Our results cast light on the interconnections between secondary metabolite gene clusters and provide a way to prioritize biosynthetic pathways in the search and discovery of novel compounds.

Ever since the isolation of Amycolatopsis mediterranei in 1957, this strain has been the focus of research worldwide. In the last 60 years or more, our understanding of the taxonomy, development of cloning vectors and conjugation system, physiology, genetics, genomics, and biosynthetic pathway of rifamycin B production in A . mediterranei has substantially increased. In particular, the development of cloning vectors, transformation system, characterization of the rifamycin biosynthetic gene cluster, and the regulation of rifamycin B production by the pioneering work of Heinz Floss have made the rifamycin polyketide biosynthetic gene cluster (PKS) an attractive target for extensive genetic manipulations to produce rifamycin B analogues which could be effective against multi-drug-resistant tuberculosis. Additionally, a better understanding of the regulation of rifamycin B production and the application of newer genomics tools, including CRISPR-assisted genome editing systems, might prove useful to overcome the limitations associated with low production of rifamycin analogues.

A novel filamentous actinobacterium designated strain 4-36 T showing broad-spectrum antifungal activity was isolated from a coal mining site in Mongolia, and its taxonomic position was determined using polyphasic approach. Optimum growth occurred at 30 °C, pH 7.5 and in the absence of NaCl. Aerial and substrate mycelia were abundantly formed on agar media. The colour of aerial mycelium was white and diffusible pigments were not formed. Phylogenetic analyses based on 16S rRNA gene sequence showed that strain 4-36 T formed a distinct clade within the genus Amycolatopsis . The 16S rRNA gene sequence similarity showed that the strain was mostly related to Amycolatopsis lexingtonensis DSM 44544 T and Amycolatopsis rifamycinica DSM 46095 T with 99.3 % sequence similarity. However, the highest digital DNA-DNA hybridization value to closest species was 44.1 %, and the highest average nucleotide identity value was 90.2 %, both of which were well below the species delineation thresholds. Chemotaxonomic properties were typical of the genus Amycolatopsis , as the major fatty acids were C 15 : 0 , iso-C 16 : 0 and C 16 : 0 , the cell-wall diamino acid was meso- diaminopimelic acid, the quinone was MK-9(H 4 ), and the main polar lipids were diphosphatidylglycerol, phosphatidylmethanolamine and phosphatidylethanolamine. The in silico prediction of chemotaxonomic markers was also carried out by phylogenetic analysis. The genome mining for biosynthetic gene clusters of secondary metabolites in strain 4-36 T revealed the presence of 34 gene clusters involved in the production of polyketide synthase, nonribosomal peptide synthetase, ribosomally synthesized and post-translationally modified peptide, lanthipeptide, terpenes, siderophore and many other unknown clusters. Strain 4-36 T showed broad antifungal activity against several filamentous fungi. The phenotypic, biochemical and chemotaxonomic properties indicated that the strain could be clearly distinguished from other species of Amycolatopsis , and thus the name Amycolatopsis mongoliensis sp. nov. is proposed accordingly (type strain, 4-36 T =KCTC 39526 T =JCM 30565 T ).

The pollution of aquatic environments by drugs is a problem for which scarce research has been conducted in regards of their removal. Amycolatopsis sp. Poz 14 presents the ability to biotransformation naphthalene at high efficiency, therefore, in this work this bacterium was proposed as an assimilator of naproxen and carbamazepine. Growth curves at different concentrations of naproxen and carbamazepine showed that Amycolatopsis sp. Poz 14 is able to utilize these drugs at a concentration of 50 mg L−1 as a source of carbon and energy. At higher concentrations, the bacterial growth was inhibited. The transformation kinetics of naproxen showed the total elimination of the compound in 18 days, but carbamazepine was only eliminated in 19.9%. The supplementation with cometabolites such as yeast extract and naphthalene (structure similar to naproxen) at 50 mg L−1, showed that the yeast extract shortened the naproxen elimination to 6 days and reached a higher global consumption rate compared to the naphthalene cometabolite. The biotransformation of carbamazepine was not improved by the addition of cometabolites. The partial sequencing of the genome of Amycolatopsis sp. Poz 14 detected genes encoding putative enzymes for the degradation of cyclic aromatic compounds and the activities of aromatic monooxygenase, catechol 1,2-dioxygenase and gentisate 1,2-dioxygenase exhibited their involving in the naproxen biodegradation. The HPLC–MS analysis detected the 5-methoxysalicylic acid at the end of the biotransformation kinetics. This work demonstrates that Amycolatopsis sp. Poz 14 utilizes naproxen and transforms it to 5-methoxysalicylic acid which is the initial compound for the catechol and gentisic acid metabolic pathway. [ABSTRACT FROM AUTHOR]

Abstract In the present study the polycyclic aromatic hydrocarbon removal and metabolic adaptation of Amycolatopsis tucumanensis DSM 45259 were investigated. Analysis of one-dimensional gel electrophoresis of crude cell extracts revealed differential synthesis of proteins which were identified by MALDI-TOF. To elucidate the phenanthrene metabolic pathway in A. tucumanensis DSM45259, two-dimensional electrophoresis and detection of phenanthrene degradation intermediates by GS-MS were performed. The presence of aromatic substrates resulted in changes in the abundance of proteins involved in the metabolism of aromatic compounds, oxidative stress response, energy production and protein synthesis. The obtained results allowed us to clarify the phenanthrene catabolic pathway, by confirming the roles of several proteins involved in the degradation process and comprehensive adaptation. This may clear the way for more efficient engineering of bacteria in the direction of more effective bioremediation applications. Graphical abstract fx1 Highlights • PAHs induce differential proteomic responses in DSM 45259. • 22 over-synthesis proteins induced by PHE were detected. • Mono and dihydroxylation are proposed for PHE upper catabolic pathway in DSM 45259. • Phthalate and catechol are proposed for PHE lower catabolic pathways in DSM 45259. • Novel PHE degradation pathway is proposed for strain DSM 45259. [ABSTRACT FROM AUTHOR]

Background: Microbial metabolites are of great importance to the pharmaceutical industries. There is an urgent need of novel microbial metabolites in the present scenario to combat antimicrobial resistance. Selection and screening of potent microbial strains for production of antimicrobial metabolites as well as optimization of their culture conditions is of utmost importance in drug discovery. Therefore, the study was carried out to evaluate the effect of nutritional and cultural conditions on the production of bioactive metabolites by a rare tea garden actinobacterial strain Amycolatopsis sp. ST-28. Materials and methods: Submerged fermentation of the actinobacterial isolate was carried out on different culture media and different culture conditions such as carbon and nitrogen sources, inoculum volume, pH, fermentation period and agitation speed. The culture filtrate was assayed against Staphylococcus aureus. Agar well diffusion method was employed to determine the maximum diameter of zone of inhibition (mm). The dried mycelial weight (mg) in a fixed volume of culture media was used for the determination of the total biomass produced. Results: Maximum bioactive metabolite and biomass production was observed when submerged fermentation was carried out with mannose and peptone respectively as a sole carbon and nitrogen source. Maintaining other environmental parameters viz. inoculum 11% (v/v), pH of 6.5, temperature of 32°C and incubation period of 11 days at 150 rpm were found optimum for maximum antimicrobial activity. Conclusion: This study demonstrated optimized cultural conditions for improved production of antimicrobial compound by Amycolatopsis sp. ST-28. Contexte: Les métabolites microbiens revêtent une grande importance pour les industries pharmaceutiques. Il existe un besoin urgent de nouveaux métabolites microbiens dans le scénario actuel pour lutter contre la résistance aux antimicrobiens. La sélection et le criblage de souches microbiennes puissantes pour la production de métabolites antimicrobiens, ainsi que l'optimisation de leurs conditions de culture, revêtent une importance capitale pour la découverte de médicaments. Par conséquent, l'étude a été réalisée pour évaluer l'effet des conditions nutritionnelles et culturelles sur la production de métabolites bioactifs par une rare souche d'actinobactéries de jardin de thé, Amycolatopsis sp. ST-28. Matériels et méthodes: La fermentation immergée de l'isolat actinobactérien a été réalisée sur différents milieux de culture et différentes conditions de culture tels que les sources de carbone et d'azote, le volume d'inoculum, le pH, la période de fermentation et la vitesse d'agitation. Le filtrat de culture a été testé contre Staphylococcus aureus. La méthode de diffusion sur puits d'agar a été utilisée pour déterminer le diamètre maximum de la zone d'inhibition (mm). Le poids du mycélium séché (mg) dans un volume fixe de milieu de culture a été utilisé pour la détermination de la biomasse totale produite. Résultats: Une production maximale de métabolites bioactifs et de biomasse a été observée lors de la fermentation en immersion avec du mannose et de la peptone, respectivement, comme seule source de carbone et d'azote. Maintenir d'autres paramètres environnementaux à savoir. inoculum 11% (v / v), pH de 6,5, température de 32 ° C et période d'incubation de 11 jours à 150 tr / min ont été jugés optimaux pour une activité antimicrobienne maximale. Conclusion: Cette étude a démontré des conditions de culture optimisées pour une production améliorée de composé antimicrobien par Amycolatopsis sp. ST-28. [ABSTRACT FROM AUTHOR]

A new polycyclic antibiotic, pradimicin-IRD, was isolated from actinobacteria Amycolatopsis sp. IRD-009 recovered from soil of Brazilian rainforest undergoing restoration area. This molecule is the major compound produced in solid culture media. The new compound was detected by a focused method of precursor ion (high-performance liquid chromatography coupled to tandem mass spectrometer) developed previously to identify unusual aminoglycosyl sugar moieties. The compound was isolated and its structure was, therefore, elucidated by high-resolution mass spectrometry, and 1D and 2D nuclear magnetic resonance experiments. Pradimicin-IRD displayed potential antimicrobial activity against Streptococcus agalactiae (MIC 3.1 μg/mL), Pseudomonas aeruginosa (MIC 3.1 μg/mL) and Staphylococcus aureus (MIC 3.1 μg/mL), and also cytotoxicity against tumour and non-tumour cell lines with IC50 values ranging from 0.8 μM in HCT-116 colon carcinoma cells to 2.7 μM in MM 200 melanoma cells. Particularly, these biological properties are described for the first time for this chemical class. [ABSTRACT FROM AUTHOR]

A new phthalazinone derivative, named amycophthalazinone A (1), and a new isoflavonoid glycoside, 7- O -methyl-5- O -α-L-rhamnopyranosylgenestein (2), along with an isoflavonoid glycoside, 7- O -α-D-arabinofuranosyl daidzein (3) firstly found from natural sources, and eight known compounds (4–11), were isolated from the culture broth of the lichen-associated Amycolatopsis sp. YIM 130642. The structures of new compounds were elucidated on the basis of spectroscopic analysis. Compound 1 was the first example of naturally occurring phthalazinone derivative. The antimicrobial activities of all compounds towards five pathogenic strains were evaluated by a broth microdilution assay. Compound 1 exhibited the most potent inhibitory activity against Staphylococcus aureus , Salmonella typhi , and Candida albicans with MIC values of 32, 32, and 64 μg/mL, respectively. Unlabelled Image [ABSTRACT FROM AUTHOR]

Actinomycetes are regarded as important sources for the generation of various bioactive secondary metabolites with rich chemical and bioactive diversities. Amycolatopsis falls under the rare actinomycete genus with the potential to produce antibiotics. In this review, all literatures were searched in the Web of Science, Google Scholar and PubMed up to March 2021. The keywords used in the search strategy were “Amycolatopsis”, “secondary metabolite”, “new or novel compound”, “bioactivity”, “biosynthetic pathway” and “derivatives”. The objective in this review is to summarize the chemical structures and biological activities of secondary metabolites from the genus Amycolatopsis. A total of 159 compounds derived from 8 known and 18 unidentified species are summarized in this paper. These secondary metabolites are mainly categorized into polyphenols, linear polyketides, macrolides, macrolactams, thiazolyl peptides, cyclic peptides, glycopeptides, amide and amino derivatives, glycoside derivatives, enediyne derivatives and sesquiterpenes. Meanwhile, they mainly showed unique antimicrobial, anti-cancer, antioxidant, anti-hyperglycemic, and enzyme inhibition activities. In addition, the biosynthetic pathways of several potent bioactive compounds and derivatives are included and the prospect of the chemical substances obtained from Amycolatopsis is also discussed to provide ideas for their implementation in the field of therapeutics and drug discovery.

l -Asparaginase (l -ASNase) is a well known chemotherapeutic enzyme, to treat different neoplasms, especially in treating Acute Lymphoblastic Leukaemia (ALL) of children and adults. However, production of glutaminase free l -ASNase is the central area of research to produce cytotoxicity free L-ASNases in the cancer therapeutics. The present study explored the potential glutaminase free l -ASNase production from rare actinobacterium, Amycolatopsis thermoflava SFMA-103 isolated from agricultural field soils. The attention of the enzyme production was sequentially optimized using various methods starting from One-factor-at-a-time (OFAT) to Taguchi orthogonal array (OA) experimental design (DOE). Four critical factors, pH, temperature, arabinose and yeast extract were identified initially, hence, three levels with a layout of L9 (34) were used and further processed with Qualitek-4 software. Upon validation an enhanced enzyme production of 68.55% was observed (from 10 IU −1 min−1 to 31.8 IU −1 min−1). As far as our knowledge extends, glutaminase-free l -ASNase production is the first time report from Amycolatopsis thermoflava SFMA-103. Further, scale-up studies were performed using bench scale bioreactor (5L capacity) with the statistically optimized conditions (aeration of 0.5 VVM and agitation of 150 rpm at 40 °C) resulted in an improved enzyme production to the level of 132.69% (61.6 IU −1 min−1) with an incubation period of 66 h. Taguchi methods for scale-up studies of l -ASNase proves useful in bioprocess optimization with minor experimental set up, saving cost and time consumption is low. [Display omitted] • Isolated Amycolatopsis thermoflava SFMA-103, a rare actinobacterium having potential to produce glutaminase free l -Asparaginase (ASNase). • ASNase production optimized using Taguchi orthogonal array experimental design. • pH, temperature, arabinose and yeast extract were noticed as significant factors and at optimized condition production yield was improved >68%. • Bioreactor based scale up studies enhanced the enzyme production to the tune of 6 fold upon step up optimization. [ABSTRACT FROM AUTHOR]

Actinomycetes constitute a large group of Gram-positive bacteria present in different habitats. One of these habitats involves the association of these bacteria with insects. In this work, we have studied twenty-four actinomycetes strains isolated from the intestinal tract and feces from larvae of the xylophagous coleopteran Cerambyx welensii and have shown that seventeen strains present hydrolytic activity of some of the following substrates: cellulose, hemicellulose, starch and proteins. Fourteen of the isolates produce antimicrobial molecules against the Gram-positive bacteria Micrococcus luteus. Analysis of seven strains led us to identify the production of a wide number of compounds including streptanoate, alpiniamide A, alteramides A and B, coproporphyrin III, deferoxamine, demethylenenocardamine, dihydropicromycin, nocardamine, picromycin, surugamides A, B, C, D and E, tirandamycins A and B, and valinomycin. A significant number of other compounds, whose molecular formulae are not included in the Dictionary of Natural Products (DNP), were also present in the extracts analyzed, which opens up the possibility of identifying new active antibiotics. Molecular identification of ten of the isolated bacteria determined that six of them belong to the genus Streptomyces, two of them are included in the genus Amycolatopsis and two in the genus Nocardiopsis.

Strains belonging to the genus Amycolatopsis are well known for the production of a number of important antimicrobials and other bioactive molecules. In this study, we have sequenced the genomes of five Amycolatopsis strains including Amycolatopsis circi DSM 45561T, Amycolatopsis palatopharyngis DSM 44832T and Amycolatopsis thermalba NRRL B-24845T. The genome sequences were analyzed with 52 other publically available Amycolatopsis genomes, representing 34 species, and 12 representatives from related genera including Saccharomonospora, Saccharopolyspora, Saccharothrix, Pseudonocardia and Thermobispora. Based on the core genome phylogeny, Amycolatopsis strains were subdivided into four major clades and several singletons. The genus Amycolatopsis is homogeneous with only three strains noted to group with other genera. Amycolatopsis halophila YIM93223T is quite distinct from other Amycolatopsis strains, both phylogenetically and taxonomically, and belongs to a distinct genus. In addition, Amycolatopsis palatopharyngis DSM 44832T and Amycolatopsis marina CGMCC4 3568T grouped in a clade with Saccharomonospora strains and showed similar taxogenomic differences to this genus as well as other Amycolatopsis strains. The study found a number of strains, particularly those identified as Amycolatopsis orientalis, whose incorrect identification could be resolved by taxogenomic analyses. Similarly, some unclassified strains could be assigned with species designations. The genome sequences of some strains that were independently sequenced by different laboratories were almost identical (99–100% average nucleotide and amino acid identities) consistent with them being the same strain, and confirming the reproducibility and robustness of genomic data. These analyses further demonstrate that whole genome sequencing can reliably resolve intra- and, inter-generic structures and should be incorporated into prokaryotic systematics.

Actinomycetes have been extensively exploited as one of the most prolific secondary metabolite-producer sources and continue to be in the focus of interest in the constant search of novel bioactive compounds. The availability of less expensive next generation genome sequencing techniques has not only confirmed the extraordinary richness and broad distribution of silent natural product biosynthetic gene clusters among these bacterial genomes, but also has allowed the incorporation of genomics in bacterial taxonomy and systematics. As part of our efforts to isolate novel strains from unique environments, we explored lichen-associated microbial communities as unique assemblages to be studied as potential sources of novel bioactive natural products with application in biotechnology and drug discovery. In this work, we have studied the whole genome sequences of two new Amycolatopsis strains (CA-126428 and CA-128772) isolated from tropical lichens, and performed a comparative genomic analysis with 41 publicly available Amycolatopsis genomes. This work has not only permitted to infer and discuss their taxonomic position on the basis of the different phylogenetic approaches used, but has also allowed to assess the richness and uniqueness of the biosynthetic pathways associated to primary and secondary metabolism, and to provide a first insight on the potential role of these bacteria in the lichen-associated microbial community.

Here, we report the draft genome sequence of Amycolatopsis camponoti RTGN1, a bacterial endophyte of Alnus glutinosa root nodules, collected from Saltwell Park, United Kingdom. The genome is 11.9 Mbp in size, composed of 147 contigs, with an N 50 of 179,211 bp and presenting a GC content of 70.9%., Competing Interests: The authors declare no conflict of interest.

Cancer cells including colorectal cancer cells are resistant to anoikis, an anchorage-independent programmed death, which enables metastasis and subsequent survival in a new tumor microenvironment. In this study, we identified a new anoikis inducer, amoxetamide A (1) with a β-lactone moiety, that was produced by combined-culture of Amycolatopsis sp. 26-4 and mycolic acid-containing bacteria (MACB) Tsukamurella pulmonis TP-B0596. The structure of 1 including the stereochemistry of C8 was determined by MS and NMR spectroscopy and modified Mosher's method, and the absolute configurations of C11 and C12 were suggested as 11R and 12S, respectively, by GIAO NMR calculations. Amoxetamide A (1) exhibited anoikis-inducing activity in human colorectal cancer HT-29 cells in anchorage-independent culture conditions., (© 2023. The Author(s), under exclusive licence to the Japan Antibiotics Research Association.)

Strains belonging to the genus Amycolatopsis are well known for the production of a number of important antimicrobials and other bioactive molecules. In this study, we have sequenced the genomes of five Amycolatopsis strains including Amycolatopsis circi DSM 45561T, Amycolatopsis palatopharyngis DSM 44832T and Amycolatopsis thermalba NRRL B-24845T. The genome sequences were analyzed with 52 other publically available Amycolatopsis genomes, representing 34 species, and 12 representatives from related genera including Saccharomonospora, Saccharopolyspora, Saccharothrix, Pseudonocardia and Thermobispora. Based on the core genome phylogeny, Amycolatopsis strains were subdivided into four major clades and several singletons. The genus Amycolatopsis is homogeneous with only three strains noted to group with other genera. Amycolatopsis halophila YIM93223T is quite distinct from other Amycolatopsis strains, both phylogenetically and taxonomically, and belongs to a distinct genus. In addition, Amycolatopsis palatopharyngis DSM 44832T and Amycolatopsis marina CGMCC4 3568T grouped in a clade with Saccharomonospora strains and showed similar taxogenomic differences to this genus as well as other Amycolatopsis strains. The study found a number of strains, particularly those identified as Amycolatopsis orientalis, whose incorrect identification could be resolved by taxogenomic analyses. Similarly, some unclassified strains could be assigned with species designations. The genome sequences of some strains that were independently sequenced by different laboratories were almost identical (99-100% average nucleotide and amino acid identities) consistent with them being the same strain, and confirming the reproducibility and robustness of genomic data. These analyses further demonstrate that whole genome sequencing can reliably resolve intraand, inter-generic structures and should be incorporated into prokaryotic systematics. [ABSTRACT FROM AUTHOR]

The pseudonocardiate Amycolatopsis sp. ATCC 39116 is used for the biotechnical production of natural vanillin from ferulic acid. Our laboratory has performed genetic modifications of this strain previously, but there are still many gaps in our knowledge regarding its vanillin tolerance and the general metabolism. We performed cultivations with this bacterium and compared the proteomes of stationary phase cells before ferulic acid feeding with those during ferulic acid feeding. Thereby, we identified 143 differently expressed proteins. Deletion mutants were constructed and characterized to analyze the function of nine corresponding genes. Using these mutants, we identified an active ferulic acid β-oxidation pathway and the enzymes which constitute this pathway. A combined deletion mutant in which the β-oxidation as well as non-β-oxidation pathways of ferulic acid degradation were deleted was unable to grow on ferulic acid as the sole source of carbon and energy. This mutant differs from the single deletion mutants and was unable to grow on ferulic acid. Furthermore, we showed that the non-β-oxidation pathway is involved in caffeic acid degradation; however, its deletion is complemented even in the double deletion mutant. This shows that both pathways can complement each other. The β-oxidation deletion mutant produced significantly reduced amounts of vanillic acid (0.12 instead of 0.35 g/l). Therefore, the resulting mutant could be used as an improved production strain. The quinone oxidoreductase deletion mutant (ΔytfG) degraded ferulic acid slower at first but produced comparable amounts of vanillin and significantly less vanillyl alcohol when compared to the parent strain. [ABSTRACT FROM AUTHOR]