Paenibacillus Glycoengineering & Nanobiotechnology

Today nanobiotechnology is an emerging scientific discipline with great application potential for the life and non-life sciences. One of the most relevant areas of nanobiotechnological research concerns technological utilization of self-assembly systems, wherein molecules spontaneously associate into reproducible supramolecular structures. In this context, glycosylated crystalline bacterial cell surface layers (S-layer glycoproteins) represent a unique self-assembly system that can be used in a bottom-up process as a patterning element for a biomolecular construction kit. Since cell surface glycoconjugates play critical roles in biological processes - from bacteria to mankind - tailor-made S-layer glycoproteins are not only promising means for the development of functionalized nanoscaled materials but will also decisively alter our capacity to influence and control complex biological systems. Detailed knowledge of glycoprotein biosynthesis pathways and cloning of the key enzymes involved are prerequisite for the exploitation of glycobiology within the fields of nanobiotechnology. P. alvei was selected for this endeavor, because according to a recent transformation screen, it is the only S-layer glycoprotein-carrying strain that can be readily transformed with foreign DNA. To accomplish our long-term research goal, that is the rational modification of “nonsense” S-layer glycoproteins by “carbohydrate engineering” to create “intelligent” S-layer neoglycoproteins, we will take advantage of the S-layer protein glycosylation system of Paenibacillus alvei CCM 2051T and its elucidated glycan structure to establish this Gram-positive organism (i) as an endotoxin-free host for in vivo surface display of rational glycans via the S-layer protein anchor, and (ii) as an efficient S-layer neoglycoprotein secretion system. In a “proof of concept” study, in vivo glycan surface display will be accomplished by replacing the P. alvei slg gene cluster by that of Geobacillus stearothermophilus NRS 2004/3a. The establishment of the S-layer neoglycoprotein secretion system is based on the finding that non-covalent attachment of the P. alvei S-layer protein to the bacterial cell wall is mediated by a negatively charged polysaccharide of known structure. Knock-out of essential genes from the corresponding polysaccharide gene cluster will abolish the interaction between the polysaccharide and the S-layer protein, leading to the secretion of S-layer (neo)glycoprotein into the medium. The planned experiments may be beneficial for the production of high yields of recombinant (neo)glycoproteins that can be easily purified from the culture broth. The outlined project will lead to the establishment of P. alvei as a host for surface display as well as for the secretion of natural or rationally designed S-layer (neo)glycoproteins with potential applications in the fields of nanobiotechnology, biomedicine, and nutrition.