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.