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

Alcohol dehydrogenase

The alcohol dehydrogenase (ADH) plays a functional role in fermentation in Saccharomyces cerevisiae. It has five different alcohol dehydrogenases (ADH I-V). Four of these enzymes, ADH I, ADH III, ADH IV and ADH V, reduce acetaldehyde to ethanol during glucose fermentation, while the NAD+ dependent ADH II catalyzes the reverse reaction of oxidizing ethanol to acetaldehyde.[5] When glucose becomes depleted from the environment, ADH II is responsible for catalyzing the initial step in the utilization of ethanol as a carbon source. While ADH I has a methionine residue at position 294, ADH II has a leucine residue, their gene products differ in metabolic directionality due to their differences in substrate affinity; ADH II has a ten-fold lower Km for ethanol than all the other alcohol dehydrogenases.[5] In natural occurring systems the presence of glucose leads to a repression of the ADH II expression by several hundred fold.

D-Galactose-/D-Glucose binding protein

The D-Galactose/D-Glucose binding protein (GGPB) belongs to the periplasmic binding proteins and is involved in chemotaxis, transport and quorum sensing for D-Galactose and D-Glucose. The GGBP is constructed of two globular domains, which are arranged in the so called “Venus flytrap“-structure. The connecting hinge region is built by 3 strands which are responsible for the binding of D-Galactose-/D-glucose. The glucose binding site is placed in the hinge region of the protein where ten residues form a „shell“ around the sugar molecule. Upon binding of glucose to the protein, the conformation changes from the open to closed state. This two different structures can be recognized by membrane components for chemotaxis. The affinity of the protein towards glucose is very high, in micromolar regions (0.2 μM). The following video shows the binding of Glucose to the closed form of GGBP.

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]