S-layer glycosylation enzymes for nanobiotechnology

Glycoproteins are ubiquitous biomolecules that are involved in many crucial biological processes, with the associated glycan structures frequently being the key to biological function. Thus, the emerging technology of glycoengineering, that allows engineering of tailor-made glycoproteins, will decisively change our capabilities in influencing and controlling complex biological systems. Due to the complexity of these essential biosynthetic pathways in eukaryotic cells, glycoproteins have escaped biotechnological applications so far. However, the recent successful functional transfer of protein glycosylation pathways into the experimental model organism Escherichia coli opens new avenues for glycoengineering. In the present project, we propose to exploit the S-layer protein O-glycosylation pathway of the Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a to perform glyco-engineering in an E. coli background. This approach combines, for the first time, a natural S-layer glycoprotein self-assembly system with prokaryotic glycoengineering to enable high-density surface display of "intelligent glycosylation motifs" in a nanometer- scaled, periodic set-up. This will open up new possibilities in the fields of nanobiotechnology and biomedicine, such as surface functionalization or design of novel, glycan-based vaccines. For this endeavor, the investigation of key modules of the S-layer protein glycosylation process - that is the initiation enzyme and the oligosaccharyl:protein transferase - is a prerequisite. S-layer protein O glycosylation is encoded by a chromosomal gene cluster, and it is evident that this glycosylation pathway utilizes distinct modules from the lipopolysaccharide assembly route in Gram-negative bacteria. Due to the similarities between S-layer glycoprotein and lipopolysaccharide biosyntheses, and based on a detailed understanding of key modules of the S layer protein glycosylation process, which shall be generated within this project, the heterologous transfer of glycans onto the known S layer protein glycosylation sites should be feasible. As a way to characterize these modules of S layer glycan biosynthesis and to test our hypothesis, our research goals include: A) Characterization of the G. stearothermophilus NRS 2004/3a initiation enzyme of S layer glycoprotein glycan biosynthesis. This is especially important in the light of recent data that indicate dual specificity of this enzyme. B) Heterologous production of an S layer glycoprotein carrying an authentic S layer glycan in E. coli to prove the concerted action of the S layer glycosylation machinery in the host background. C) Proof of principle by O glycosylation of the S layer protein with distinct lipopolysaccharide O antigens in glycosylation-competent E. coli cells. D) Addressing the question of oligosaccharyl:protein transferase specificity by testing the capability of the enzyme to transfer different lipid-linked oligosaccharides.

Bacterial glycosylation systems have come under enhanced scrutiny because of their potential for exploitation in recombinant glycosylation engineering to produce biologically active glycoproteins in an easily tractable, bacterial system. Glycoproteins have escaped biotechnological applications so far due to their complex biosynthesis pathways. Bacterial cell surface (S-) layer glycoproteins are present as outermost cell envelope structures of many bacteria. They possess the intrinsic capability to self-assemble into 2D crystalline arrays, which makes them ideal candidates for being exploited as display matrix in the fields of (nano)biotechnology and biomedicine providing nanometer-scaled accuracy and control over glycan display. The project goal was to perform glycosylation engineering based on the S-layer protein O-glycosylation pathway of the bacterium Geobacillus stearothermophilus NRS 2004/3a. We hypothesized that based on the understanding of key modules of S-layer protein glycosylation and on the proposed similarities between this O-glycosylation pathway and other bacterial polysaccharide pathways, exchange of glycosylation modules should allow the transfer of designed glycans onto the S-layer protein in glycosylation competent cell factories. The major achievements of this project are: (i) Elucidation of the biosynthesis pathway of the model bacteria Geobacillus stearothermophilus NRS 2004/3a und Paenibacillus alvei CCM 2051. X-ray crystallography and molecular dynamic simulation allowed first insights into the structure-function relationship of selected glycosylation enzymes (ii) A picture of how S-layer glycosylation modules can be utilized for glycosylation engineering is evolving. Individual modules can be combined with other bacterial polysaccharide biosynthesis modules and the S-layer protein matrix can be engineered to become an acceptor for heterologous oligosaccharyltransferases to act on. First nanopatterned biomaterials were constructed based on the acquired knowledge, such as S-layer proteins carrying either a heptasaccharide from Camplyobacter jejuni or the E. coli O7 antigen. (iii) An S-layer based liposome-type biocatalyst was designed with a carbohydrate-active model enzyme. Due to the improved properties of this composite in comparison to the sole enzyme, it is expected that nanopatterned biohybrids can lead to new functional concepts for biocatalytic applications. (iv) A strategy for in vivo cell surface co-display of designed peptide and glycan epitopes was developed in the model organism P. alvei. Tailor-made nanopatterned, self-assembly proteins with designed glycosylation may open up new strategies for influencing and controlling complex biological systems. This might be of great value for the fields of receptor mimics, vaccine development, or drug delivery. In the future, it will be challenging to fully exploit the S-layer glycome for glycosylation engineering purposes and to link it to the bacterial interactome.