Search Results (9)

Background: Myxol, a monocyclic carotenoid with β- and ψ-end groups, has been identified in only a limited number of bacteria, such as flavobacteria and cyanobacteria. Despite its biological significance, the biosynthetic pathway of myxol is not well understood, and studies on its physiological functions and biological activities are limited because of its rarity. Methods: BLAST homology searches for carotenoid biosynthesis genes in the genome of Nonlabens were performed. The carotenogenesis-related genes in the genome of the marine flavobacteria Nonlabens spongiae were individually cloned and functionally characterized using a heterologous Escherichia coli expression system. Carotenoids from N. spongiae were identified using an LC-MS analysis. Results: We identified a gene cluster involved in carotenoid biosynthesis in the genome of N. spongiae. This cluster includes genes encoding phytoene synthase (CrtB), phytoene desaturase (CrtI), lycopene cyclase (CrtY), carotenoid 1,2-hydratase (CruF), carotenoid 3,4-desaturase (ψ-end group) (CrtD), carotenoid 2-hydroxylase (ψ-end group) (CrtA-OH), and carotene hydro-xylase (CrtZ). Based on the characteristics of these enzymes, the primary products were predicted to be myxol and/or zeaxanthin. A spectroscopic analysis confirmed that myxol was the primary carotenoid. Furthermore, a plasmid containing a reconstructed gene cluster and geranylgeranyl pyrophosphate synthase (CrtE) located outside the cluster was introduced into E. coli. This system predominantly accumulated myxol, indicating that the reconstructed gene cluster enabled efficient myxol production in E. coli. Conclusions: This study highlighted the potential biotechnological applications of the carotenoid biosynthesis gene clusters for myxol production. Full article

Bacteria from the genus Sulfitobacter are distributed across various marine habitats and play a significant role in sulfur cycling. However, the metabolic features of Sulfitobacter inhabiting marine biofilms are still not well understood. Here, complete genomes and paired metatranscriptomes of eight Sulfitobacter strains, isolated from biofilms on subtidal stones, have been analyzed to explore their central energy metabolism and potential of secondary metabolite biosynthesis. Based on average nucleotide identity and phylogenetic analysis, the eight strains were classified into six novel species and two novel strains. The reconstruction of the metabolic pathways indicated that all strains had a complete Entner–Doudoroff pathway, pentose phosphate pathway, and diverse pathways for amino acid metabolism, suggesting the presence of an optimized central carbon metabolism. Pangenome analysis further revealed the differences between the gene cluster distribution patterns among the eight strains, suggesting significant functional variation. Moreover, a total of 47 biosynthetic gene clusters were discovered, which were further classified into 37 gene cluster families that showed low similarity with previously documented clusters. Furthermore, metatranscriptomic analysis revealed the expressions of key functional genes involved in the biosynthesis of ribosomal peptides in in situ marine biofilms. Overall, this study sheds new light on the metabolic features, adaptive strategies, and value of genome mining in this group of biofilm-associated Sulfitobacter bacteria. Full article

The pseudotetrasaccharide acarbose, produced by Actinoplanes sp. SE50/110, is a relevant secondary metabolite used in diabetes type II medication. Although maltose plays a crucial role in acarbose biosynthesis, the understanding of the maltose/maltodextrin metabolism and its involvement in acarbose production is at an early stage. Here, we reconstructed the predicted maltose–maltodextrin pathway that involves four enzymes AmlE, MalZ, MalP, and MalQ. An investigation of enzyme activities was conducted through in vitro assays, leading to an expansion of previously postulated substrate spectra. The maltose-induced α-glucosidase AmlE is noteworthy for its high hydrolysis rate of linear α-1,4-glucans, and its capability to hydrolyze various glycosidic bonds. The predicted maltodextrin glucosidase MalZ showed slow hydrolysis activity on linear α-glucans, but it was resistant to acarbose and capable of releasing glucose from acarbose. AmlE compensates for the low activity of MalZ to ensure glucose supply. We determined the enzyme activity of MalP and its dual function as maltodextrin and glycogen phosphorylase. The 4-α-glucanotransferase MalQ plays a central role in the maltose/maltodextrin metabolism, alongside MalP. This study confirmed the simultaneous degradation and synthesis of long-chain α-glucans. The product distribution showed that with an increasing number of glycosidic bonds, less glucose is formed. We found that MalQ, like its sequence homolog AcbQ from the acarbose biosynthetic gene cluster, is involved in the formation of elongated acarviosyl metabolites. However, MalQ does not participate in the elongation of acarbose 7-phosphate, which is likely the more readily available acceptor molecule in vivo. Accordingly, MalQ is not involved in the formation of acarviosyl impurities in Actinoplanes sp. SE50/110. Full article

Flavonoids are a large family of polyphenolic compounds with important agro-industrial, nutraceutical, and pharmaceutical applications. Among the structural diversity found in the flavonoid family, methylated flavonoids show interesting characteristics such as greater stability and improved oral bioavailability. This work is focused on the reconstruction of the entire biosynthetic pathway of the methylated flavones diosmetin and chrysoeriol in Streptomyces albidoflavus. A total of eight different genes (TAL, 4CL, CHS, CHI, FNS1, F3′H/CPR, 3′-OMT, 4′-OMT) are necessary for the heterologous biosynthesis of these two flavonoids, and all of them have been integrated along the chromosome of the bacterial host. The biosynthesis of diosmetin and chrysoeriol has been achieved, reaching titers of 2.44 mg/L and 2.34 mg/L, respectively. Furthermore, an additional compound, putatively identified as luteolin 3′,4′-dimethyl ether, was produced in both diosmetin and chrysoeriol-producing strains. With the purpose of increasing flavonoid titers, a 3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase (DAHP synthase) from an antibiotic biosynthetic gene cluster (BGC) from Amycolatopsis balhimycina was heterologously expressed in S. albidoflavus, enhancing diosmetin and chrysoeriol production titers of 4.03 mg/L and 3.13 mg/L, which is an increase of 65% and 34%, respectively. To the best of our knowledge, this is the first report on the de novo biosynthesis of diosmetin and chrysoeriol in a heterologous host. Full article

Advances in the computational annotation of genomes and the predictive potential of current metabolic models, based on more than thousands of experimental phenotypes, allow them to be applied to identify the diversity of metabolic pathways at the level of ecophysiology differentiation within taxa and to predict phenotypes, secondary metabolites, host-associated interactions, survivability, and biochemical productivity under proposed environmental conditions. The significantly distinctive phenotypes of members of the marine bacterial species Pseudoalteromonas distincta and an inability to use common molecular markers make their identification within the genus Pseudoalteromonas and prediction of their biotechnology potential impossible without genome-scale analysis and metabolic reconstruction. A new strain, KMM 6257, of a carotenoid-like phenotype, isolated from a deep-habituating starfish, emended the description of P. distincta, particularly in the temperature growth range from 4 to 37 °C. The taxonomic status of all available closely related species was elucidated by phylogenomics. P. distincta possesses putative methylerythritol phosphate pathway II and 4,4′-diapolycopenedioate biosynthesis, related to C30 carotenoids, and their functional analogues, aryl polyene biosynthetic gene clusters (BGC). However, the yellow-orange pigmentation phenotypes in some strains coincide with the presence of a hybrid BGC encoding for aryl polyene esterified with resorcinol. The alginate degradation and glycosylated immunosuppressant production, similar to brasilicardin, streptorubin, and nucleocidines, are the common predicted features. Starch, agar, carrageenan, xylose, lignin-derived compound degradation, polysaccharide, folate, and cobalamin biosynthesis are all strain-specific. Full article

National Aeronautics and Space Administration’s (NASA) spacecraft assembly facilities are monitored for the presence of any bacteria or fungi that might conceivably survive a transfer to an extraterrestrial environment. Fungi present a broad and diverse range of phenotypic and functional traits to adapt to extreme conditions, hence the detection of fungi and subsequent eradication of them are needed to prevent forward contamination for future NASA missions. During the construction and assembly for the Mars 2020 mission, three fungal strains with unique morphological and phylogenetic properties were isolated from spacecraft assembly facilities. The reconstruction of phylogenetic trees based on several gene loci (ITS, LSU, SSU, RPB, TUB, TEF1) using multi-locus sequence typing (MLST) and whole genome sequencing (WGS) analyses supported the hypothesis that these were novel species. Here we report the genus or species-level classification of these three novel strains via a polyphasic approach using phylogenetic analysis, colony and cell morphology, and comparative analysis of WGS. The strain FJI-L9-BK-P1 isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) exhibited a putative phylogenetic relationship with the strain Aaosphaeria arxii CBS175.79 but showed distinct morphology and microscopic features. Another JPL-SAF strain, FJII-L3-CM-DR1, was phylogenetically distinct from members of the family Trichomeriaceae and exhibited morphologically different features from the genera Lithohypha and Strelitziana. The strain FKI-L1-BK-DR1 isolated from the Kennedy Space Center facility was identified as a member of Dothideomycetes incertae sedis and is closely related to the family Kirschsteiniotheliaceae according to a phylogenetic analysis. The polyphasic taxonomic approach supported the recommendation for establishing two novel genera and one novel species. The names Aaosphaeria pasadenensis (FJI-L9-BK-P1 = NRRL 64424 = DSM 114621), Pasadenomyces melaninifex (FJII-L3-CM-DR1 = NRRL 64433 = DSM 114623), and Floridaphiala radiotolerans (FKI-L1-BK-DR1 = NRRL 64434 = DSM 114624) are proposed as type species. Furthermore, resistance to ultraviolet-C and presence of specific biosynthetic gene cluster(s) coding for metabolically active compounds are unique to these strains. Full article

The discovery of novel secondary metabolites is actively being pursued in new ecosystems. Sponge-associated bacteria have been in the limelight in recent years on account of their ability to produce bioactive compounds. In this study, heterotrophic bacteria associated with four sponge species were isolated, taxonomically identified, and subjected to screening for the production of bioactive entities against a panel of nine microorganisms, including Gram-positive and negative bacteria, as well as yeast and fungi. Of the 105 isolated strains, 66% were represented by Proteobacteria, 16% by Bacteriodetes, 7% by Actinobacteria, and 11% by Firmicutes. Bioactivity screening revealed that 40% of the total isolated strains showed antimicrobial activity against one or more of the target microorganisms tested. Further, active extracts from selective species were narrowed down by bioassay-guided fractionation and subsequently identified by HR-ESI-MS analyses to locate the active peaks. Presumably responsible compounds for the observed bioactivities were identified as pentadecenoic acid, oleic acid, and palmitoleic acid. One isolate, Qipengyuania pacifica NZ-96^T, based on 16S rRNA novelty, was subjected to comparative metabolic reconstruction analysis with its closest phylogenetic neighbors, revealing 79 unique functional roles in the novel isolate. In addition, genome mining of Qipengyuania pacifica NZ-96^T revealed three biosynthetic gene clusters responsible for the biosynthesis of terpene, beta lactone, lasso peptide, and hserlactone secondary metabolites. Our results demonstrate the ability to target the sponge microbiome as a potential source of novel microbial life with biotechnological potential. Full article

Monacolin J (MJ), a key precursor of Lovastatin, could synthesize important statin drug simvastatin by hydrolyzing lovastatin and adding different side chains. In this study, to reduce the cumbersome hydrolysis of lovastatin to produce MJ in the native strain Aspergillus terreus, the MJ biosynthetic pathway genes (lovB, lovC, lovG, and lovA) were heterologously integrated into the genome of Aspergillus. niger CBS513.88 with strong promoters and suitable integration sites, via yeast 2μ homologous recombination to construct expression cassettes of long-length genes and CRISPR/Cas9 homology-directed recombination (CRISPR-HDR) to integrate MJ genes in the genome of A. niger. RT-PCR results proved that pathway synthesis-related genes could be heterologously expressed in A. niger. Finally, we constructed an engineered strain that could produce monacolin J, detected by LC-HR-ESIMS (MJ, 339.22 [M-H]⁺). The yield of MJ reached 92.90 mg/L after 7-day cultivation. By optimizing the cultivation conditions and adding precursor, the final titer of MJ was 142.61 mg/L on the fourth day of fed-batch cultivation, which was increased by 53.5% compared to the original growth conditions. Due to the wide application of A. niger in industrial fermentation for food and medicine, the following work will be dedicated to optimizing the metabolic network to improve the MJ production in the engineered strain. Full article

The spread of antimicrobial resistance (AMR) creates a challenge for global health security, rendering many previously successful classes of antibiotics useless. Unfortunately, this also includes glycopeptide antibiotics (GPAs), such as vancomycin and teicoplanin, which are currently being considered last-resort drugs. Emerging resistance towards GPAs risks limiting the clinical use of this class of antibiotics—our ultimate line of defense against multidrug-resistant (MDR) Gram-positive pathogens. But where does this resistance come from? It is widely recognized that the GPA resistance determinants—van genes—might have originated from GPA producers, such as soil-dwelling Gram-positive actinobacteria, that use them for self-protection. In the current work, we present a comprehensive bioinformatics study on the distribution and phylogeny of GPA resistance determinants within the Actinobacteria phylum. Interestingly, van-like genes (vlgs) were found distributed in different arrangements not only among GPA-producing actinobacteria but also in the non-producers: more than 10% of the screened actinobacterial genomes contained one or multiple vlgs, while less than 1% encoded for a biosynthetic gene cluster (BGC). By phylogenetic reconstructions, our results highlight the co-evolution of the different vlgs, indicating that the most diffused are the ones coding for putative VanY carboxypeptidases, which can be found alone in the genomes or associated with a vanS/R regulatory pair. Full article