Biology subjects

Weber, T.

Publications and source records attributed to Weber, T..

48 records · Page 3Linked to original sources

bioRxiv2021

Identification of the 216kbp gene cluster and structure elucidation of gargantulides B and C, new complex 52-membered macrolides from Amycolatopsis sp.

Gargantulides B and C, two new and highly complex 52-membered glycosylated macrolactones, were isolated from Amycolatopsis sp. strain CA-230715 during an antibacterial screening campaign. The structures of these giant macrolides were elucidated by 2D NMR spectroscopy and shown to be related to gargantulide A, although containing additional {beta}-glucopyranose and/or -arabinofuranose monosaccharides separately attached to their backbones. Genome sequencing allowed the identification of a strikingly large 216 kbp biosynthetic gene cluster, among the largest type I PKS clusters described so far, and the proposal of a biosynthetic pathway for gargantulides A-C. Additionally, genes putatively responsible for the biosynthesis of the amino sugar {beta}-3,6-deoxy-3-methylamino glucose, reported exclusively in gargantulide macrolides, were also found in the cluster and described in this work. The absolute configurations of gargantulides B and C were assigned based on a combination of NMR and bioinformatics analysis of ketoreductase and enoylreductase domains within the multimodular type I PKS. Furthermore, the absolute stereochemistry of the related macrolide gargantulide A has now been revised and completed. Gargantulides B and C display potent antibacterial activity against a set of drug-resistant Gram-positive bacteria and moderate activity against the clinically relevant Gram-negative pathogen Acinetobacter baumannii.

microbiology↗

bioRxiv2020

Characterization and Engineering of Streptomyces griseofuscus DSM 40191 as a Potential Host for Heterologous Expression of Biosynthetic Gene Clusters

Streptomyces griseofuscus DSM 40191 is a fast growing Streptomyces strain that remains largely underexplored as a heterologous host. Here, we report the genome mining of S. griseofuscus, followed by the detailed exploration of its phenotype, including production of native secondary metabolites and ability to utilise carbon, nitrogen, sulphur and phosphorus sources. Furthermore, several routes for genetic engineering of S. griseofuscus were explored, including use of GusA-based vectors, CRISPR-Cas9 and CRISPR-cBEST-mediated knockouts. Using CRISPR-BEST technology, core genes of 4 biosynthetic gene clusters (BGCs) that are situated on the chromosome arms were inactivated and the outcomes of the inactivations were tested. Two out of the three native plasmids were cured using CRISPR-Cas9 technology, leading to the generation of strain S. griseofuscus DEL1. DEL1 was further modified by full deletion of a pentamycin BGC and an unknown NRPS BGC, leading to the generation of strain DEL2, lacking approx. 500 kbp of the genome, which corresponds to a 5,19% genome reduction. Sequencing confirmed that DEL2 does not bear any crucial off-target effects or rearrangements in its genome. It can be characterized by faster growth and inability to produce three main native metabolites of S. griseofuscus: lankacidin, lankamycin, pentamycin and their derivatives. To test the ability of DEL2 to heterologously produce secondary metabolites, the actinorhodin BGC was used. We were able to confirm the production of actinorhodin by both S. griseofuscus wild type and DEL2. We believe that this strain will serve as a good chassis for heterologous expression of BGCs. ImportanceThe rise of antibacterial resistance calls on the development of the next generation of antibiotics, majority of which are derived from natural compounds, produced by actinomycetes. The manipulation, refactoring and expression of BGCs coding for such natural products is a promising approach in secondary metabolite discovery. Thus, the development of a versatile panel of heterologous hosts for the expression of BGCs is essential. We believe that first-to-date systematic, detailed characterisation of S. griseofuscus, a highly promising chassis strain, will not only facilitate the further development of this particular strain, but also will set a blueprint for characterisation of other potential hosts.

microbiology↗

bioRxiv2020

Phylogenetic distribution of secondary metabolites in the Bacillus subtilis species complex

Microbes produce a plethora of secondary metabolites that although not essential for primary metabolism benefit them to survive in the environment, communicate, and influence differentiation. Biosynthetic gene clusters (BGCs) responsible for the production of these secondary metabolites are readily identifiable on the genome sequence of bacteria. Understanding the phylogeny and distribution of BGCs helps us to predict natural product synthesis ability of new isolates. Here, we examined the inter- and intraspecies patterns of absence/presence for all BGCs identified with antiSMASH 5.0 in 310 genomes from the B. subtilis group and assigned them to defined gene cluster families (GCFs). This allowed us to establish patterns in distribution for both known and unknown products. Further, we analyzed variations in the BGC structure of particular families encoding for natural products such as plipastatin, fengycin, iturin, mycosubtilin and bacillomycin. Our detailed analysis revealed multiple GCFs that are species or clade specific and few others that are scattered within or between species, which will guide exploration of the chemodiversity within the B. subtilis group. Uniquely, we discovered that partial deletion of BGCs and frameshift mutations in selected biosynthetic genes are conserved within phylogenetically related isolates, although isolated from around the globe. Our results highlight the importance of detailed analysis of BGCs and the remarkable phylogenetically conserved errodation of secondary metabolite biosynthetic potential in the B. subtilis group. IMPORTANCEMembers of the B. subtilis species complex are commonly recognized producers of secondary metabolites, among those the production of antifungals makes them promising biocontrol strains. However, while there are studies examining the distribution of well-known B. subtilis metabolites, this has not yet been systematically reported for the group. Here, we report the complete biosynthetic potential within the Bacillus subtilis group species to explore the distribution of the biosynthetic gene clusters and to provide an exhaustive phylogenetic conservation of secondary metabolite production supporting the chemodiversity of Bacilli. We identify that certain gene clusters acquired deletions of genes and particular frame-shift mutations rendering them inactive for secondary metabolite biosynthesis, a conserved genetic trait within phylogenetically conserved clades of certain species. The overview presented will superbly guide assigning the secondary metabolite production potential of newly isolated strains based on genome sequence and phylogenetic relatedness.

bioinformatics↗

bioRxiv2020

CRISPR-nRAGE, a Cas9 nickase-reverse transcriptase assisted versatile genetic engineering toolkit for E. coli

In most prokaryotes, missing and poorly active non-homologous end joining (NHEJ) DNA repair pathways heavily restrict the direct application of CRISPR-Cas for DNA double-strand break (DSB)-based genome engineering without providing editing templates. CRISPR base editors, on the other hand, can be directly used for genome engineering in a number of bacteria, including E. coli, showing advantages over CRISPR-Cas9, since they do not require DSBs. However, as the current CRISPR base editors can only engineer DNA by A to G or C to T/G/A substitutions, they are incapable of mediating deletions, insertions, and combinations of deletions, insertions and substitutions. To address these challenges, we developed a Cas9 nickase (Cas9n)-reverse transcriptase (Moloney Murine Leukemia Virus, M-MLV) mediated, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes, termed CRISPR-nRAGE (CRISPR-Cas9n Reverse transcriptase Assisted Genome Engineering) system. CRISPR-nRAGE can be used to introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli. Notably, small sized-deletion shows better editing efficiency compared to other kinds of DNA engineering. CRISPR-nRAGE has been used to delete and insert DNA fragments up to 97 bp and 33 bp, respectively. Efficiencies, however, drop sharply with the increase of the fragment size. It is not only a useful addition to the genome engineering arsenal for E. coli, but also may be the basis for the development of similar toolkits for other organisms.

bioengineering↗

bioRxiv2020

Genomic and chemical diversity of Bacillus subtilis secondary metabolites against plant pathogenic fungi

Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant-growth-promoting and biocontrol abilities. These secondary metabolites include non-ribosomal peptides (NRPs) with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates and therefore, a comparative overview of B. subtilis secondary metabolites is missing. In this study, we have isolated 23 B. subtilis strains from eleven sampling sites, compared the fungal inhibition profiles of wild types and their NRPs mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that non-ribosomal peptide production varied among B. subtilis strains co-isolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium sp., a combination of plipastatin and surfactin is required to hinder the growth of Botrytis cinerea. Detailed genomic analysis revealed that altered NRP production profiles in certain isolates is due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical NRPs detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis. IMPORTANCESecondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which non-ribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack production of certain secondary metabolites.

microbiology↗

bioRxiv2020

Distribution of Epsilon-Polylysine Synthetases in Coryneform Bacteria Isolated from Cheese and Human Skin

Epsilon-polylysine ({varepsilon}-PL) is an antimicrobial commercially produced by Streptomyces fermentation and widely used in Asian countries for food preservation. Here we discovered a gene from cheese bacterium Corynebacterium variabile that showed high similarity to the {varepsilon}-PL synthetase from Streptomyces in terms of enzymatic domain architecture and gene context. By cloning it into Streptomyces coelicolor with a Streptomyces albulus {varepsilon}-PL synthetase promoter, we confirmed that its product is indeed {varepsilon}-PL. A comprehensive sequence analysis suggests that {varepsilon}-PL synthetases are widely spread among coryneform bacteria isolated from cheese and human skin; 14 out of 15 Brevibacterium isolates and 10 out of 12 Corynebacterium isolates contain Pls gene. This discovery raises the possibility that {varepsilon}-PL as a bioactive secondary metabolite might be produced and play a role in the cheese and skin ecosystems. IMPORTANCEEvery year, microbial contamination causes billions of tons of food wasted and millions of cases of foodborne illness. {varepsilon}-PL is an excellent food preservative as it is potent, wide spectrum and is heat stable and biodegradable. It has not been accepted by all countries (e.g those in the EU) partially because it was not a natural composition of food but rather originated from the soil bacteria Streptomyces, a famous producer of various antibiotic drugs and toxins. The unexpected finding of {varepsilon}-PL synthetases in cheese and skin bacteria suggests that {varepsilon}-PL may naturally exist in cheese and on our skin.

microbiology↗

bioRxiv2020

Longitudinal isolation of potent near-germline SARS-CoV-2-neutralizing antibodies from COVID-19 patients

The SARS-CoV-2 pandemic has unprecedented implications for public health, social life, and world economy. Since approved drugs and vaccines are not available, new options for COVID-19 treatment and prevention are highly demanded. To identify SARS-CoV-2 neutralizing antibodies, we analysed the antibody response of 12 COVID-19 patients from 8 to 69 days post diagnosis. By screening 4,313 SARS-CoV-2-reactive B cells, we isolated 255 antibodies from different time points as early as 8 days post diagnosis. Among these, 28 potently neutralized authentic SARS-CoV-2 (IC100 as low as 0.04 g/ml), showing a broad spectrum of V genes and low levels of somatic mutations. Interestingly, potential precursors were identified in naive B cell repertoires from 48 healthy individuals that were sampled before the COVID-19 pandemic. Our results demonstrate that SARS-CoV-2 neutralizing antibodies are readily generated from a diverse pool of precursors, fostering the hope of rapid induction of a protective immune response upon vaccination.

immunology↗

bioRxiv2019

Highly parallelized microfluidic droplet cultivation and prioritization on antibiotic producers from complex natural microbial communities

To investigate the overwhelming part of the bacterial diversity still evading standard cultivation for its potential use in antibiotic synthesis, we have compiled a microscale-cultivation and screening system. We devised a strategy based on droplet-microfluidics taking advantage of the inherent miniaturization and high throughput. Single cells of natural samples were confined in 9 x 106 aqueous droplets and subjected to long-term incubation under controlled conditions. Subsequent a high-throughput screening for antimicrobial natural products was implemented, employing a whole cell reporting system using the viability of reporter strains as a probe for antimicrobial activity. Due to the described microscale cultivation a novel subset of bacterial strains was made available for the following screening for antimicrobials. We demonstrate the merits of the in-droplet cultivation by comparing the cultivation outcome in microfluidic droplets and on conventional agar plates for a bacterial community derived from soil by 16S rRNA gene amplicon sequencing. In-droplet cultivation resulted in a significantly higher bacterial diversity without the common overrepresentation of Firmicutes. Natural strains able to inhibit either a Gram-positive or a Gram-negative reporter strain were isolated from the microscale system and further cultivated. Thereby a variety of rare isolates was obtained. The natural products with antimicrobial activity were elucidated for the most promising candidate. Our method combines a new cultivation approach with a high-throughput search for antibiotic producers to increase the chances of finding new lead substances.

microbiology↗

bioRxiv2019

Pangenome Analysis of Enterobacteria Reveals Richness of Secondary Metabolite Gene Clusters and their Associated Gene Sets

The growing number of sequenced genomes enables the study of secondary metabolite biosynthetic gene clusters (BGC) in phyla beyond well-studied soil bacteria. We mined 2627 enterobacterial genomes to detect 8604 BGCs, including nonribosomal peptide synthetases, siderophores, polyketide-nonribosomal peptide hybrids, and 60 other BGC types, with an average of around 3.3 BGCs per genome. These BGCs represented 212 distinct BGC families, of which only 20 have associated products in the MIBiG standard database with functions such as siderophores, antibiotics, and genotoxins. Pangenome analysis identified genes associated with a specific BGC encoding for colon cancer-related colibactin. In one example, we associated genes involved in the type VI secretion system with the presence of a colibactin BGC in Escherichia. This richness of BGCs in enterobacteria opens up the possibility to discover novel secondary metabolites, their physiological roles and provides a guide to identify and understand PKS associated gene sets.

systems biology↗

bioRxiv2019

Versatile polyketide biosynthesis platform for production of aromatic compounds in yeast

To accelerate the shift to bio-based production and overcome complicated functional implementation of natural and artificial biosynthetic pathways to industry relevant organisms, development of new, versatile, bio-based production platforms is required. Here we present a novel yeast platform for biosynthesis of bacterial aromatic polyketides. The platform is based on a synthetic polyketide synthase system enabling a first demonstration of bacterial aromatic polyketide biosynthesis in a eukaryotic host.

synthetic biology↗

bioRxiv2019

CRISPR-BEST: a highly efficient DSB-free base editor for filamentous actinomycetes

Filamentous actinomycetes serve as major producers of various natural products including antimicrobial compounds. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSB) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem). Specifically targeted by an sgRNA, the cytidine deaminase component of CRISPR-BEST efficiently converts C:G to T:A within a window of approximately seven-nucleotides. The system was validated and successfully used in different Streptomyces species.

synthetic biology↗

bioRxiv2019

Depiction of secondary metabolites and antifungal activity of Bacillus velezensis DTU001

For a safe and sustainable environment, effective microbes as biocontrol agents are in high demand. We have isolated a new Bacillus velezensis strain DTU001, investigated its antifungal spectrum, sequenced its genome, and uncovered the production of lipopeptides in HPLC-HRMS analysis. To test the antifungal efficacy, extracts of B. velezensis DTU001 was tested against a range of twenty human or plant pathogenic fungi. We demonstrate that inhibitory potential of B. velezensis DTU001 against selected fungi is superior in comparison to single lipopeptide, either iturin or fengycin. The isolate showed analogous biofilm formation to other closely related Bacilli. To further support the biocontrol properties of the isolate, coculture with Candida albicans demonstrated that B. velezensis DTU001 exhibited excellent antiproliferation effect against C. albicans. In summary, the described isolate is a potential antifungal agent with a broad antifungal spectrum that might assist our aims to avoid hazardous pathogenic fungi and provide alternative to toxicity caused by chemicals.

microbiology↗