scholarly journals Mining for novel cyclomaltodextrin glucanotransferases unravels the carbohydrate metabolism pathway via cyclodextrins in Thermoanaerobacterales

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Sara Centeno-Leija ◽  
Laura Espinosa-Barrera ◽  
Beatriz Velazquez-Cruz ◽  
Yair Cárdenas-Conejo ◽  
Raúl Virgen-Ortíz ◽  
...  

AbstractCarbohydrate metabolism via cyclodextrins (CM-CD) is an uncommon starch-converting pathway that thoroughly depends on extracellular cyclomaltodextrin glucanotransferases (CGTases) to transform the surrounding starch substrate to α-(1,4)-linked oligosaccharides and cyclodextrins (CDs). The CM-CD pathway has emerged as a convenient microbial adaptation to thrive under extreme temperatures, as CDs are functional amphipathic toroids with higher heat-resistant values than linear dextrins. Nevertheless, although the CM-CD pathway has been described in a few mesophilic bacteria and archaea, it remains obscure in extremely thermophilic prokaryotes (Topt ≥ 70 °C). Here, a new monophyletic group of CGTases with an exceptional three-domain ABC architecture was detected by (meta)genome mining of extremely thermophilic Thermoanaerobacterales living in a wide variety of hot starch-poor environments on Earth. Functional studies of a representative member, CldA, showed a maximum activity in a thermoacidophilic range (pH 4.0 and 80 °C) with remarkable product diversification that yielded a mixture of α:β:γ-CDs (34:62:4) from soluble starch, as well as G3–G7 linear dextrins and fermentable sugars as the primary products. Together, comparative genomics and predictive functional analysis, combined with data of the functionally characterized key proteins of the gene clusters encoding CGTases, revealed the CM-CD pathway in Thermoanaerobacterales and showed that it is involved in the synthesis, transportation, degradation, and metabolic assimilation of CDs.

Author(s):  
Patrick Videau ◽  
Kaitlyn Wells ◽  
Arun Singh ◽  
Jessie Eiting ◽  
Philip Proteau ◽  
...  

Cyanobacteria are prolific producers of natural products and genome mining has shown that many orphan biosynthetic gene clusters can be found in sequenced cyanobacterial genomes. New tools and methodologies are required to investigate these biosynthetic gene clusters and here we present the use of <i>Anabaena </i>sp. strain PCC 7120 as a host for combinatorial biosynthesis of natural products using the indolactam natural products (lyngbyatoxin A, pendolmycin, and teleocidin B-4) as a test case. We were able to successfully produce all three compounds using codon optimized genes from Actinobacteria. We also introduce a new plasmid backbone based on the native <i>Anabaena</i>7120 plasmid pCC7120ζ and show that production of teleocidin B-4 can be accomplished using a two-plasmid system, which can be introduced by co-conjugation.


2021 ◽  
Author(s):  
Valentin Waschulin ◽  
Chiara Borsetto ◽  
Robert James ◽  
Kevin K. Newsham ◽  
Stefano Donadio ◽  
...  

AbstractThe growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes.


2016 ◽  
Vol 4 (2) ◽  
Author(s):  
Fabian Horn ◽  
Jörg Linde ◽  
Derek J. Mattern ◽  
Grit Walther ◽  
Reinhard Guthke ◽  
...  

Here, we report the draft genome sequence of Aspergillus calidoustus (strain SF006504) . The functional annotation of A. calidoustus predicts a relatively large number of secondary metabolite gene clusters. The presented genome sequence builds the basis for further genome mining.


2014 ◽  
Vol 58 (10) ◽  
pp. 6185-6196 ◽  
Author(s):  
Marius Spohn ◽  
Norbert Kirchner ◽  
Andreas Kulik ◽  
Angelika Jochim ◽  
Felix Wolf ◽  
...  

ABSTRACTThe emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products isAmycolatopsis. However,Amycolatopsis japonicumdoes not produce an antibiotic under standard laboratory conditions. In contrast to mostAmycolatopsisstrains,A. japonicumis genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, thebbrgene fromAmycolatopsis balhimycina(bbrAba), intoA. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing ofA. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed thein silicoprediction that the recombinantA. japonicum/pRM4-bbrAbasynthesizes ristomycin A.


2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Domonique A. Carson ◽  
Herman W. Barkema ◽  
Sohail Naushad ◽  
Jeroen De Buck

ABSTRACT Non-aureus staphylococci (NAS), the bacteria most commonly isolated from the bovine udder, potentially protect the udder against infection by major mastitis pathogens due to bacteriocin production. In this study, we determined the inhibitory capability of 441 bovine NAS isolates (comprising 26 species) against bovine Staphylococcus aureus. Furthermore, inhibiting isolates were tested against a human methicillin-resistant S. aureus (MRSA) isolate using a cross-streaking method. We determined the presence of bacteriocin clusters in NAS whole genomes using genome mining tools, BLAST, and comparison of genomes of closely related inhibiting and noninhibiting isolates and determined the genetic organization of any identified bacteriocin biosynthetic gene clusters. Forty isolates from 9 species (S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. saprophyticus, S. sciuri, S. simulans, S. warneri, and S. xylosus) inhibited growth of S. aureus in vitro, 23 isolates of which, from S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. simulans, and S. xylosus, also inhibited MRSA. One hundred five putative bacteriocin gene clusters encompassing 6 different classes (lanthipeptides, sactipeptides, lasso peptides, class IIa, class IIc, and class IId) in 95 whole genomes from 16 species were identified. A total of 25 novel bacteriocin precursors were described. In conclusion, NAS from bovine mammary glands are a source of potential bacteriocins, with >21% being possible producers, representing potential for future characterization and prospective clinical applications. IMPORTANCE Mastitis (particularly infections caused by Staphylococcus aureus) costs Canadian dairy producers $400 million/year and is the leading cause of antibiotic use on dairy farms. With increasing antibiotic resistance and regulations regarding use, there is impetus to explore bacteriocins (bacterially produced antimicrobial peptides) for treatment and prevention of bacterial infections. We examined the ability of 441 NAS bacteria from Canadian bovine milk samples to inhibit growth of S. aureus in the laboratory. Overall, 9% inhibited growth of S. aureus and 58% of those also inhibited MRSA. In NAS whole-genome sequences, we identified >21% of NAS as having bacteriocin genes. Our study represents a foundation to further explore NAS bacteriocins for clinical use.


mSystems ◽  
2018 ◽  
Vol 3 (2) ◽  
Author(s):  
Daniela B. B. Trivella ◽  
Rafael de Felicio

ABSTRACT Natural products are the richest source of chemical compounds for drug discovery. Particularly, bacterial secondary metabolites are in the spotlight due to advances in genome sequencing and mining, as well as for the potential of biosynthetic pathway manipulation to awake silent (cryptic) gene clusters under laboratory cultivation. Further progress in compound detection, such as the development of the tandem mass spectrometry (MS/MS) molecular networking approach, has contributed to the discovery of novel bacterial natural products. The latter can be applied directly to bacterial crude extracts for identifying and dereplicating known compounds, therefore assisting the prioritization of extracts containing novel natural products, for example. In our opinion, these three approaches—genome mining, silent pathway induction, and MS-based molecular networking—compose the tripod for modern bacterial natural product discovery and will be discussed in this perspective.


2020 ◽  
Vol 74 (5) ◽  
pp. 382-390 ◽  
Author(s):  
Fabienne Arn ◽  
David Frasson ◽  
Ivana Kroslakova ◽  
Fabio Rezzonico ◽  
Joël F. Pothier ◽  
...  

Actinomycetes strains isolated from different habitats in Switzerland were investigated for production of antibacterial and antitumoral compounds. Based on partial 16S rRNA gene sequences, the isolated strains were identified to genus level. Streptomyces as the largest genus of Actinobacteriawas isolated the most frequently. A screening assay using the OmniLog instrument was established to facilitate the detection of active compounds from actinomycetes. Extracts prepared from the cultivated strains able to inhibit Staphylococcus aureusand Escherichia coliwere further analysed by HPLC and MALDI-TOF MS to identify the produced antibiotics. In this study, the bioactive compound echinomycin was identified from two isolated Streptomycesstrains. Natural compounds similar to TPU-0037-C, azalomycin F4a 2-ethylpentyl ester, a derivative of bafilomycin A1, milbemycin-α8 and dihydropicromycin were detected from different isolated Streptomyces strains. Milbemycin-α8 showed cytotoxic activity against HT-29 colon cancer cells. The rare actinomycete,Micromonospora sp. Stup16_C148 produced a compound that matches with the antibiotic bottromycin A2. The draft genome sequence from Actinokineospora strain B136.1 was determined using Illumina and nanopore-based technologies. The isolated strain was not able to produce antibacterial compounds under standard cultivation conditions. The antiSMASH bioinformatics analyses of the genome from strain B136.1 identified biosynthetic gene clusters with identity values between 4% to 90% to known gene clusters encoding antibiotics. The combinations of cultivation conditions, screening assays, analytical methods and genome mining are important tools to characterize strains of actinomycetes for the identification of their potential to produce natural compounds with antimicrobial activity.


Biology ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 137
Author(s):  
Maedeh Kamali ◽  
Dianjing Guo ◽  
Shahram Naeimi ◽  
Jafar Ahmadi

Tomato Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (Fol), is a destructive disease that threatens the agricultural production of tomatoes. In the present study, the biocontrol potential of strain KR2-7 against Fol was investigated through integrated genome mining and chemical analysis. Strain KR2-7 was identified as B. inaquosorum based on phylogenetic analysis. Through the genome mining of strain KR2-7, we identified nine antifungal and antibacterial compound biosynthetic gene clusters (BGCs) including fengycin, surfactin and Bacillomycin F, bacillaene, macrolactin, sporulation killing factor (skf), subtilosin A, bacilysin, and bacillibactin. The corresponding compounds were confirmed through MALDI-TOF-MS chemical analysis. The gene/gene clusters involved in plant colonization, plant growth promotion, and induced systemic resistance were also identified in the KR2-7 genome, and their related secondary metabolites were detected. In light of these results, the biocontrol potential of strain KR2-7 against tomato Fusarium wilt was identified. This study highlights the potential to use strain KR2-7 as a plant-growth promotion agent.


Marine Drugs ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. 458
Author(s):  
Hui-Min Gao ◽  
Peng-Fei Xie ◽  
Xiao-Ling Zhang ◽  
Qiao Yang

A new versatile actinobacterium designated as strain NJES-13 was isolated from the feces of the Antarctic emperor penguin. This new isolate was found to produce two active gephyromycin analogues and bioflocculanting exopolysaccharides (EPS) metabolites. Phylogenetic analysis based on pairwise comparison of 16S rRNA gene sequences showed that strain NJES-13 was closely related to Mobilicoccus pelagius Aji5-31T with a gene similarity of 95.9%, which was lower than the threshold value (98.65%) for novel species delineation. Additional phylogenomic calculations of the average nucleotide identity (ANI, 75.9–79.1%), average amino acid identity (AAI, 52.4–66.9%) and digital DNA–DNA hybridization (dDDH, 18.6–21.9%), along with the constructed phylogenomic tree based on the up-to-date bacterial core gene (UBCG) set from the bacterial genomes, unequivocally separated strain NJES-13 from its close relatives within the family Dermatophilaceae. Hence, it clearly indicated that strain NJES-13 represented a putative new actinobacterial species isolated from the gut microbiota of mammals inhabiting the Antarctic. The obtained complete genome of strain NJES-13 consisted of a circular 3.45 Mb chromosome with a DNA G+C content of 67.0 mol%. Furthering genome mining of strain NJES-13 showed the presence of five biosynthetic gene clusters (BGCs) including one type III PKS responsible for the biosynthesis of the core of gephyromycins, and a series of genes encoding for bacterial EPS biosynthesis. Thus, based on the combined phylogenetic and active metabolites characterization presented in this study, we confidently conclude that strain NJES-13 is a novel, fresh actinobacterial candidate to produce active gephyromycins and microbial bioflocculanting EPS, with potential pharmaceutical, environmental and biotechnological implications.


2021 ◽  
Author(s):  
Dharmesh Harwani ◽  
Jyotsna Begani ◽  
Jyoti Lakhani

AbstractWe sequenced the genome of Streptomyces sp. JB150, isolated from a unique site of the Thar desert in India. Genome mining of the JB150 genome revealed the presence of many interesting secondary metabolic biosynthetic gene clusters (BGCs). The encoded parvome of JB150 includes non-ribosomal peptides, polyketides including β-lactone, butyrolactone, ectoine, lantipeptides, lasso peptides, melanin, resorcinol, siderophores, terpenoids, thiopeptides, and other types of hybrid compounds. Among them, ~30% BGCs displayed a high degree of novelty. The genome of JB150 was enriched for a large assortment of specialized genes coding for the production of many interesting biomolecules comprising compatible solutes, multiple stress-response regulators, transport proteins, protein secretion systems, signaling molecules, chaperones and storage reserves, etc. The presence of diverse members of CAZymes enzyme families, high numbers of riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), trehalose and aromatic compounds synthesis genes, putative orthologues to several of the classical fatty acid synthesis components, prototrophy for many essential amino acids exhibit metabolic versatility of JB150 to inhabit in the extreme desert environment. Besides, the genome of JB150 was observed to specifically encode thiazole-oxazole-modified thiazolemicrocin (TOMM) and ectoine. The comparison of the complete genomes of Streptomyces sp. JB150 and seven other actinomycete strains belonging to different desert ecosystems unveiled the presence of many previously undetected, distinctive, biological, and genomic signatures. We propose that these genetic traits endowed by these strains are essential for their adaptation in the highly underprivileged, extreme ecosystem of the Thar desert to cope with multiple abiotic stressors, oligotrophic nutrient conditions and to produce a huge repertoire of diverse secondary metabolites.


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