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.

The goals of the recently finished research project P29745-B11 were as follows: i. Identification and sequencing of the surface (S-)layer gene spaA and the slg (S-layer glycosylation) gene cluster of Paenibacillus alvei CCM 2051. ii. Proof of concept study of heterologous glycoprotein production by exchange of the endogeneous slg gene cluster by the slg gene cluster of Geobacillus stearothermophilus NRS 2004/3a. iii. Identification and characterization of the gene cluster of P. alvei CCM 2051 encoding the biosynthesis of the secondary cell wall polymer (SCWP) anchor. iv. Knock-out of essential genes from the SCWP gene cluster to abolish the predicted interaction between SCWP and S-layer protein, leading to the secretion of the S-layer glycoprotein into the surrounding medium.At the beginning of this project best understood was the S-layer glycosylation system of the thermophilic bacterium G. stearothermophilus NRS 2004/3a. However, a drawback of this organism is its resistance to take up foreign DNA. Based on successful transformation experiments, the mesophilic strain P. alvei CCM 2051 was used to set-up a system for genetic manipulation. As the adaptor saccharide backbones of the S-layer glycans of P. alvei CCM 2051 and G. stearothermophilus NRS 2004/3a are identical, it was conceivable that an enzyme homologous to the lipid carrier transferase WsaP would initiate S-layer glycosylation in P. alvei. Thus, wsfP served as a first target for the gene knockout system to be developed in P. alvei. This target was chosen, because loss of function would be easily screenable, resulting in an S-layer glycosylation deficient mutant. For proof of concept, we specifically dealt with the reconstitution of enzyme activity of WsfP by its predicted functional homologue WsaP.The structural gene encoding the S-layer protein SpaA of P. alvei CCM 2051 was sequenced and used for development of an in vivo surface co-display system by using this protein as a cell wall anchor. This was exemplified by the presentation of a heterologous peptide epitope and a functional fluorescence protein, respectively, in addition to the native S-layer glycan chain. Immunoblot analyses indicated the suitability of this system for future in vivo cell surface co-display of interesting engineered epitopes.Besides presentation of functionalized S-layer protein on the cell envelope, also secretion into the medium can be a desired option, avoiding time- and cost-consuming purification steps. In this context, the anchoring mechanism of the S-layer was investigated, starting with the observation of three SLH domains located at the N-terminus of SpaA. These domains are known to play a key role in mediating binding of the S-layer to the cell wall and may have great importance in future biotech applications.