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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.