Glycans are critical components in many biological processes. Cells use them to communicate with each other,
bacteria and viruses use them to infect their hosts, and cancer cells use them to chart new territory in the body.
Frequently, carbohydrate-mediated phenomena fall in the categories of biostimulation and biotargeting, with the
carbohydrates in many cases being coupled to a protein. Thus, glycosylation engineering to obtain customized
protein glycosylation will decisively increase our capabilities in influencing and controlling complex biological
systems. Bacteria are regarded promising candidates for this endeavor, because they are easily tractable, have
favorable process economics to produce glycoproteins, and, most importantly, according to recent data, there are
effective ways to make homogeneously glycosylated proteins in bioengineered bacteria.
Despite of the power of bacterial cell surface display for protein display in basic and applied research, this strategy
has not yet been exploited for customized carbohydrates. In the present project, we propose to undertake a
biomimetic approach, in which bacterial protein glycosylation engineering shall be combined with multivalent
surface display of customized glycans by using the naturally glycosylated bacterial cell surface layer protein of the
Gram-positive bacterium Paenibacillus alvei CCM 2051T as a 2D crystalline, nanometer-scale display matrix. This
matrix is generated through self-assembly, with the attached glycan chains being naturally orientated towards the
We hypothesize that due to the similarities between the S layer protein glycosylation pathway and other bacterial
polysaccharide biosynthesis systems, engineering of customized S layer glycoprotein glycans will be possible. As a
way to analyze the basis for S layer glycosylation engineering and to test our hypothesis, our research goals are: A)
Investigation of the native tyrosine O glycosylation sites on the S layer protein; B) Unraveling mechanisms for
glycan chain length regulation, export and oligosaccharide:protein transfer; C) Engineering S layer protein SpaA
glycosylation in selected glycosylation mutants of Pa with diverse heterologous saccharides to demonstrate the
convergence of the S-layer glycosylation machinery with foreign bacterial glycosylation systems; D) Extension of
the glycosylation potential of the S layer protein SpaA by exchanging the native O glycosylation site(s) by bacterial
N glycosylation site(s) that are recognized by the cognate glycosylation machineries.
This system shall allow ultimate control over the position, quantity and type of carbohydrate structure present on
the bacterium. It provides a unique framework for studying the biostimulation and biotargeting behavior of various
carbohydrates of interest in a setting that mirrors the natural dynamic behavior of a cell and may help to enhance
the affinity and specificity of interaction between carbohydrates and their ligands through multivalent presentation.