Abstract

We want to support the natural functions of our gut and extend its metabolic capacity. This is mediated by a modifiable cell free biofilm matrix which can be modulated according to the need of the user. The system is based on csgA nano fibers and the SpyTag/SpyCatcher system.

Aim

With our food, we take up toxic and unnecessary substances which harm our bodies. In order to prevent these substances to be resorbed by the intestinal mucosa, we wanted to develop a system which converts, reduces or detoxifies these components. For these three modes of action we chose
a) the lactase for converting lactose into glucose and galactose,
b) the D-galactose/D-glucose binding protein to reduce the natural glucose resorption and
c) the alcohol dehydrogenase for detoxification.
To combine these functions, we chose a natural occurring biofilm matrix on which we immobilize the corresponding proteins. This platform consists of modified curli fibers which have the capacity to bind proteins via the SypTag/SpyCatcher system. This enables us to design personalized food additives.

Background

Curli fibers

Some bacterial strains are producing an extracellular matrix called biofilm, which is protecting them from environmental impacts. This matrix is composed of proteins, polysaccharides, lipids and nucleic acids. One of the main structural components in Escherichia coli biofilms are curli fibers, with a diameter of 4-7 nanometer that can made up to 10-40% of the whole biofilm.[1] These fibers are amyloid structures, which are anchored on the bacterial cell surface and are assembled of 13 kDa CsgA proteins. For the production of these fibers the curli-system consists of two operons, containing seven genes: csgBAC and csgDEFG. The self-assembly and nucleation of CsgA on the cell surface is mediated by CsgB. CsgC/G are responsible for the secretion and CsgE/F for producing of CsgA. CsgD is the transcriptional regulator of this system. The following figure shows the Curli-producing process.

Lactase

Alcoholdehydrogenase

D-Galactose-/D-Glucose binding protein

SpyTag/SpyCatcher system

The SpyTag/SpyCatcher tagging system consists of two parts. The first part is a small, 13 amino acid long peptide chain called SpyTag (AHIVMVDAYKPTK) which can be fused either at the N-terminus, C-terminus or at an internal position of any protein. Whereas the counterpart is a 116 amino acid larger protein called SpyCatcher.[3] Due to the reaction between Asp7 of the SpyTag peptide and Lys31 of the SpyCatcher an intramoleculare, irreversible isopeptide bond is formed. [4] The resulting complex forms a compact β-sandwich structure, which is stable to boiling in SDS and to thousands of piconewtons.[3]