Although CRISPR/Cas9 genome editing has provided numerous opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome. Here we interrogate the molecular consequences of CRISPR/Cas9-mediated deletions at 17 sites in four loci of the mouse genome. We sequence targeted sites in 632 founder mice and analyse 54 established lines. While the median deletion size using single sgRNAs is 9 bp, we also obtain large deletions of up to 600 bp. Furthermore, we show unreported asymmetric deletions and large insertions of middle repetitive sequences. Simultaneous targeting of distant loci results in the removal of the intervening sequences. Reliable deletion of juxtaposed sites is only achieved through two-step targeting. Our findings also demonstrate that an extended analysis of F1 genotypes is required to obtain conclusive information on the exact molecular consequences of targeting events.

Biosurfactants increasingly gain attention due to the manifold of possible applications and production on the basis of renewable resources. Owing to its various characteristics, Surfactin is one of the most studied biosurfactants. Since its discovery, several Surfactin producers have been identified, but their capacity to produce Surfactin has not been evaluated in a comparison. Six different Bacillus strains were analyzed regarding their ability to produce Surfactin in model fermentations with integrated foam fractionation, for in situ product enrichment and removal. Three of the investigated strains are commonly used in Surfactin production (ATCC 21332, DSM 3256, DSM 3258), whereas two Bacillus strains are described for the first time (DSM 1090, LM43a50°C) as Surfactin producers. Additionally, the Bacillus subtilis type strain DSM 10(T) was included in the evaluation. Interestingly, all strains, except DSM 3256, featured high values for Surfactin recovered from foam in comparison to other studies, ranging between 0.4 and 1.05 g. The fermentation process was characterized by calculating procedural parameters like substrate yield Y X/S, product yield Y P/X, specific growth rate μ, specific productivity q Surfactin, volumetric productivity q Surfactin, Surfactin and bacterial enrichment as well as Surfactin recovery. The strains differ most in specific and volumetric productivity; nevertheless, it is evident that it is not possible to name a Bacillus strain that is the most appropriate for the production of Surfactin under these conditions. In contrast, it becomes apparent that the choice of a specific strain should depend on the applied fermentation conditions.

BackgroundThe overreliance on dwindling fossil fuel reserves and the negative climatic effects of using such fuels are driving the development of new clean energy sources. One such alternative source is hydrogen (H2), which can be generated from renewable sources. Parageobacillus thermoglucosidasius is a facultative anaerobic thermophilic bacterium which is frequently isolated from high temperature environments including hot springs and compost.ResultsComparative genomics performed in the present study showed that P. thermoglucosidasius encodes two evolutionary distinct H2-uptake [Ni-Fe]-hydrogenases and one H2-evolving hydrogenases. In addition, genes encoding an anaerobic CO dehydrogenase (CODH) are co-localized with genes encoding a putative H2-evolving hydrogenase. The co-localized of CODH and uptake hydrogenase form an enzyme complex that might potentially be involved in catalyzing the water-gas shift reaction (CO + H2O → CO2 + H2) in P. thermoglucosidasius. Cultivation of P. thermoglucosidasius DSM 2542T with an initial gas atmosphere of 50% CO and 50% air showed it to be capable of growth at elevated CO concentrations (50%). Furthermore, GC analyses showed that it was capable of producing hydrogen at an equimolar conversion with a final yield of 1.08 H2/CO.ConclusionsThis study highlights the potential of the facultative anaerobic P. thermoglucosidasius DSM 2542T for developing new strategies for the biohydrogen production.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0954-3) contains supplementary material, which is available to authorized users.

Surfactin is one of the most promising biosurfactants due to its extraordinary surface activity. Commonly, the well-established Cooper medium, a glucose-based mineral salt medium, is utilized for the microbial production of Surfactin. The current study investigated the enhancement of Surfactin yields by analyzing the effects of different glucose concentrations, next to the introduction of an alternative chelating agent and nitrogen source. The utilization of 8 g/L glucose, 0.008 mM Na3citrate and 50 mM (NH4)2SO4 increased Surfactin yields from 0.7 to 1.1 g/L during shake flask experiments applying Bacillus subtilis DSM10T. Consequentially conducted shake flask experiments, employing five other Surfactin producer strains during cultivation in the former and enhanced version of the Cooper medium, suggest a general enhancement of Surfactin yields during application of the enhanced version of the Cooper medium. The enhancement of the medium composition is therefore most likely independent from the employed producer strain. The following utilization of the enhanced medium composition during fed-batch fermentation with integrated foam fractionation yielded 30 % more Surfactin in comparison to batch fermentations with integrated foam fractionation employing the former version of the Cooper medium.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-015-0145-0) contains supplementary material, which is available to authorized users.