Development of a platform for immobilization of proteins based on Bacillus subtilis spores

The increasing demand for development of sustainable production processes has extended the application repertoire of biotechnology. Microorganisms and enzymes are the protagonists in the race towards sustainability. To this end, surface display of polypeptides on biological carriers has evolved as a rather valuable tool for the cost efficient production of biocatalysts. Among a variety of available bio-carriers, spores from Bacillus subtilis have several advantages such as genetic amenability, robust structure that endures harsh conditions, cost and time efficient methods for production and purification. The most common strategy for spore display is the fusion protein technology. Proteins or peptides of interest are fused to coat proteins thus facilitating immobilization on the spore surface. The main objective of the present dissertation was to utilize spores as bioparticles for immobilization of enzymes. To this end, the current study focused on the improvement of specific properties of the B. subtilis spores and established state of the art methods for their qualitative and quantitative analysis. Conceptually this study is separated in two research projects. Chapter 3 comprises the genetic engineering of B. subtilis as well as the development of a flow cytometric method for quantification and analysis of spores. Within this framework B. subtilis germination deficient strains were developed. In addition a mutant strain with higher sporulation efficiency was constructed. Following, the investigation was focused on the development of a rapid method for quantification of spores. To this end, flow cytometry combined with nucleic acid fluorescence staining were employed. The developed method allowed the resolution of up to three cell populations formed during sporulation including spores, cells and cells containing forespores. Chapter 4 covers the application of recombinant spores in biocatalytic processes. Initially a small size library of components for spore display was designed and tested by utilizing fluorescent proteins. In view of applying spore display for biocatalysis, a recently discovered light inducible fatty acid decarboxylase from Chlorella variabilis (CvFAP) was presented on the spore surface. The displayed enzyme was extensively characterized using palmitic acid as substrate. Aiming at the production of hydrocarbons from natural oils CvFAP displaying spores were combined with lipase in one pot one step bienzymatic cascade. From the bioconversion, hydrocarbon titers up to 64 mg/L were obtained. Finally the applicability of spore display was tested for the production of glycosides from the low cost substrate, sucrose. For this purpose, two different variants of sucrose phosphorylase were displayed, one deriving from Leuconostoc mesenteroides (LmSucP) and the other one deriving from Bifidobacterium adolescentis (BaSucP). Both enzymes were evaluated for their efficiency to perform phosphorolysis of sucrose to alpha-D-glucose-1-phosphat (G1P) and fructose. Only trace amounts of G1P were detected for the reaction catalyzed by LmSucP while BaSucP could convert up to 66 % of the initial substrate

Quantification and isolation of Bacillus subtilis spores using cell sorting and automated gating

The Gram-positive bacterium Bacillus subtilis is able to form endospores which have a variety of biotechnological applications. Due to this ability, B. subtilis is as well a model organism for cellular differentiation processes. Sporulating cultures of B. subtilis form sub-populations which include vegetative cells, sporulating cells and spores. In order to readily and rapidly quantify spore formation we employed flow cytometric and fluorescence activated cell sorting techniques in combination with nucleic acid fluorescent staining in order to investigate the distribution of sporulating cultures on a single cell level. Automated gating procedures using Gaussian mixture modeling (GMM) were employed to avoid subjective gating and allow for the simultaneous measurement of controls. We utilized the presented method for monitoring sporulation over time in germination deficient strains harboring different genome modifications. A decrease in the sporulation efficiency of strain Bs02018, utilized for the display of sfGFP on the spores surface was observed. On the contrary, a double knock-out mutant of the phosphatase gene encoding Spo0E and of the spore killing factor SkfA (Bs02025) exhibited the highest sporulation efficiency, as within 24 h of cultivation in sporulation medium, cultures of BS02025 already consisted of 80% spores as opposed to 18% for the control strain. We confirmed the identity of the different subpopulations formed during sporulation by employing sorting and microscopy

Observation of WWW Production in pp Collisions at √s = 13 TeV with the ATLAS Detector

This Letter reports the observation of W W W production and a measurement of its cross section using 139     fb − 1 of proton-proton collision data recorded at a center-of-mass energy of 13 TeV by the ATLAS detector at the Large Hadron Collider. Events with two same-sign leptons (electrons or muons) and at least two jets, as well as events with three charged leptons, are selected. A multivariate technique is then used to discriminate between signal and background events. Events from W W W production are observed with a significance of 8.0 standard deviations, where the expectation is 5.4 standard deviations. The inclusive W W W production cross section is measured to be 820 ± 100   ( stat ) ± 80   ( syst )     fb , approximately 2.6 standard deviations from the predicted cross section of 511 ± 18     fb calculated at next-to-leading-order QCD and leading-order electroweak accuracy