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Currently most fermentation products are produced from first-generation biomass by well characterized and genetically modifiable model strains, often referred to as cell-factories. To efficiently valorise second-generation biomass using fermentation, strains are needed that can grow on the diversity of sugars and tolerate the inhibitory compounds present in this substrate, traits that are often challenging to engineer in model organisms. In this project we have isolated bacteria from soil samples that are tolerant to hemicellulosic biomass hydrolysate from beech wood. This has yielded several novel environmental strains that are able to grow on and tolerate this generally toxic substrate. The most interesting isolates are a novel species of Pseudomonas and a novel strain of the Pantoea genus. Both strains show relatively high tolerance to the inhibitors present and can grow on a wide range of different substrates. The genome sequences of these strains have been determined using nanopore sequencing and basic physiological features have been characterized. The Pseudomonas strain has been shown to grow in a wide pH range; from pH 3 up to a pH of 9, which could be an advantage depending on the substrate and product. Both strains showed great robustness and tolerance to high density fermentation in a bioreactor, indicating a large potential for industrial applications. In order to use these novel strains to produce valuable compounds from lignocellulosic biomass they need to be modifiable and domesticated. To do this, a wide range of molecular tools must be identified and developed. We first established efficient transformation protocols for both strains. We have characterized a number of different constitutive and inducible promoters, a prerequisite for further development of efficient engineering tools. We are currently in the process of developing molecular tools for gene deletions and integrations to i

The lack of financial incentive from the pharmaceutical industry has led to a stagnation in the discovery of new antimicrobial compounds, creating a severe threat to human health from multiresistant bacterial pathogens. Advances in cost-efficient sequencing equipment and bioinformatics tools enable the identification of biosynthetic gene clusters with desirable properties, leading to the discovery of new secondary metabolite compounds and producer strains from Actinomyces bacteria. In order to investigate the biosynthetic potential in Actinomycetes, we have developed a comprehensive and efficient pipeline for generating a dataset of more than 1000 Actinomycete genomes sequenced using Nanopore and Illumina sequencing platforms. The pipeline spans the entire process from sampling to benchmarking of the assembled genome. By predicting the domains required for synthesizing natural products using antiSMASH (antibiotics & Secondary Metabolite Analysis Shell) and comparing them to known biosynthetic pathways we will be able to expand our knowledge of the properties, underlying mechanisms and in turn discover currently unknown features of sustainable natural products.

Here, we report the complete, circular genome sequence of a potential novel species from the underexplored Alphaproteobacterial genus Bosea. Bosea sp. NBC_00550 was isolated from a soil sample collected in Lyngby, Denmark. We explore the biosynthetic potential of Bosea sp. NBC_00550 and compare it with that of other Bosea species.

Actinomycetota (Actinobacteria) is an ecologically and industrially important phylum which is challenging to extract pure high-molecular-weight (HMW) DNA from. This protocol provides a parallelized, cost-effective, and straightforward approach for consistently extracting pure HMW DNA using modified non-toxic commercial kits suitable for higher throughput applications. We further provide a workflow for sequencing and assembly of complete genomes using an optimized Oxford Nanopore rapid barcoding protocol and Illumina data error correction.

Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the Initiative for the Critical Assessment of Metagenome Interpretation (CAMI). The CAMI II challenge engaged the community to assess methods on realistic and complex datasets with long- and short-read sequences, created computationally from around 1,700 new and known genomes, as well as 600 new plasmids and viruses. Here we analyze 5,002 results by 76 program versions. Substantial improvements were seen in assembly, some due to long-read data. Related strains still were challenging for assembly and genome recovery through binning, as was assembly quality for the latter. Profilers markedly matured, with taxon profilers and binners excelling at higher bacterial ranks, but underperforming for viruses and Archaea. Clinical pathogen detection results revealed a need to improve reproducibility. Runtime and memory usage analyses identified efficient programs, including top performers with other metrics. The results identify challenges and guide researchers in selecting methods for analyses.

The actinomycete Streptomyces sp. strain Gö40/10 has the potential to produce a range of secondary metabolites, one of which is collinolactone, a compound with neuroprotective properties and potential for pharmaceutical applications. The genome was sequenced with Oxford Nanopore Technologies MinION and Illumina MiSeq systems and consists of a single 9,635,564-nucleotide linear chromosome.

ε-poly-L-lysine is a potent antimicrobial produced through fermentation of Streptomyces and used in many Asian countries as a food preservative. It is synthesized and excreted by a special NRPS-like enzyme called Pls. Here we discovered a gene from cheese bacterium Corynebacterium variabile that showed high similarity to the Pls from Streptomyces in terms of domain architecture and gene context. By cloning it into Streptomyces coelicolor with a Streptomyces albulus Pls promoter, we confirmed that its product is indeed ε-poly-L-lysine. A comprehensive sequence analysis suggests that Pls genes are widely spread among coryneform actinobacteria isolated from cheese and human skin; 14 out of 15 Brevibacterium isolates and 10 out of 12 Corynebacterium isolates contain it in their genomes. This finding raises the possibility that ε-poly-L-lysine as a bioactive secondary metabolite might be produced and play a role in the cheese and skin ecosystems. IMPORTANCE Every year, microbial contamination causes billions of tons of food wasted and millions of cases of illness. ε-poly-L-lysine has potent, wide spectrum of inhibitory activity and is heat stable and biodegradable. It has been approved for food preservation by an increasing number of countries. ε-poly-L-lysine is produced from the soil bacteria Streptomyces, also a producer of various antibiotic drugs and toxins, and not considered to be a naturally-occurring food component. The frequent finding of pls in cheese and skin bacteria suggests that ε-poly-L-lysine may naturally exist in cheese and on our skin, and ε-poly-L-lysine producers are not limited to filamentous actinobacteria.

Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant growth-promoting and biocontrol abilities. These secondary metabolites include nonribosomal peptides 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 secondary metabolites from various environmental B. subtilis strains is missing. In this study, we isolated 23 B. subtilis strains from 11 sampling sites, compared the fungal inhibition profiles of wild types and their nonribosomal peptide mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that nonribosomal peptide production varied among B. subtilis strains coisolated 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 spp., a combination of plipastatin and surfactin is required to hinder growth of Botrytis cinerea. Detailed genomic analysis revealed that altered nonribosomal peptide production profiles in specific isolates are due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical nonribosomal peptide 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. IMPORTANCE Secondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus

Here, we report the sequencing, assembly, and annotation of the genome of Streptomyces griseofuscus DSM 40191. The genome of S. griseofuscus was sequenced using PacBio and Illumina technologies. It consists of a linear 8,721,740-bp chromosome and three plasmids, pSGRIFU1 (220 kb), pSGRIFU2 (88 kb), and pSGRIFU3 (86 kb).

Isolation, sequencing, and analysis of circular genetic elements bring new insights to mobile genetic elements related to microbial ecology. One method used to study circular plasmids, viruses, and other elements is called the mobilome method. The mobilome method presented here is an unamplified mobilome approach allowing fast isolation of circular DNA elements from a variety of samples followed by directly building unamplified Illumina-compatible sequencing libraries using enzymatic tagging and fragmentation. Several methods for bioinformatic analysis of mobilome data are also suggested.

Bacillus subtilis is a plant-benefiting soil-dwelling Gram-positive bacterium with secondary metabolite production potential. Here, we report the complete genome sequences of 13 B. subtilis strains isolated from different soil samples in Germany and Denmark.