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 cell free 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.

Project Design

With our project we want to support the human gut to raise its efficiency. Therefore we constructed a modifiable cell free biofilm matrix, which is able to pick up disruptive elements.
To accomplish this goal, we used a Curli-biofilm as a scaffold matrix. We introduced a plasmid containing the encoding for the CsgA-monomer, which is the major part of the Curli-fibers. The CsgA-monomers are produced intracellular whereas these monomers self-assemble extracellular to fibers.
As described we used the SpyTag/SpyCatcher-System (SpySystem) to modify these Curli fibers. Therefore we fused the small SpyTag peptide to the CsgA monomers. SpyTag is the smaller component of the SpySystem, so that the fusion protein is still able to pass the membrane-pore.
To link specific proteins (or enzymes) to our matrix, we fused the corresponding protein to the SpyCatcher. This catcher is able to bind to its counterpart, the SpyTag. With this SpySystem it is possible to modify the matrix with any desired functions by linking the particular proteins. To prove the viability of this System we first created a fusion protein, consisting of the SpyCatcher and GFP.
To support the gut we decided to fuse several proteins to the SpyCatcher. First, we fused a glucose binding protein (GGPB) to the SpyCatcher, in regard of decreasing the glucose concentration in the gut. Another created fusion protein contains the alcohol dehydrogenase, which is able to reduce the alcohol level. We chose lactase as further protein, linked to the SpyCatcher. This enables people, which are suffering from lactose intolerance, to metabolize lactose.

Results

Constructs

We designed a plasmid construct for the production of biofilm-template with curli proteins. Therefore we used a T5 promoter, controlled by a lacI-system. As inducer we used IPTG. The Promoter is followed by an RBS and csgA gene, which encodes for Curli. As control for expression we added another RBS and the mCherry gene. Inbetween csgA and the RBS of mCherry are restriction sites for Nhe I and Bam HI to enable the addition of further genes or the fusion of csgA with other proteins. The construct is surrounded by the prefix (Eco RI, Not I and Xba I) and the suffix (Spe I, Not I and Pst I). The natural csgA gene contains a pstI restriction site, which we deleted via mutation for submission as a biobrick. This construct we used to create a fusion protein out of csgA and a SpyTag . This is enabling the matrix to catch other proteins which are fused to the SpyCatcher. Therefore we used the described restriction sites that surrounds the csgA gene.

For labeling the extracellular curli fibers, we designed a plasmid for the production of a marker protein which can bind to the SpyTag. This plasmid contains a lacI controlled T5 promoter, followed by a RBS, the SpyCatcher gene and the green fluorescent protein (GFP) gene. The construct is surrounded with the restriction sites (Eco RI, XbaI NcoI, XhoI and SmaI as suffix and SpeI, NotI PStI as prefix) as well. Between the two genes are the NcoI and the NdeI restriction sites (Fig. XX).

For reducing the glucose concentration in the gut, we fused the SpyCatcher to the Galactose/Glucose binding protein (GGBP). This construct includes the restriction sites, the lacI controlled T5 promoter, the SpyCatcher gene and the mglB gene, which encodes the GGBP (Fig. XX).

To support the human gut and intestine in detoxication processes, we fused the SpyCatcher to the alcohol dehydrogenase II (ADH II), which is able to oxidize alcohols (especially ethanol) to acetaldehyde. The construct is surrounded by several restriction sites.

Protein detection

Crystal violet staining

Congo red staining

The congo red stain is used for the visual detection of amyloid fibers like cellulose and curli. The congo red plates should give us a hint for expressed amyloid fibers and the stain should show, in which backbone the expression of csgA is favored. The CR plates and the samples were prepared as described in the protocol section.

SDS-Page

EM?

Functional SpySystem

Real world application

Outlook

In conclusion one of the biggest advantages, besides the opportunity to obtain a cell free system is, that the matrix can be customized for every user individually. In the next few years will be a significant upturn in the field of personalized nutrition and personalized health care (personalized medicine). In this project we showed the first prove of principal, of functionalizing the curli matrix. More development and research is needed, to reach the full potential of this functionalized protein matrix. This would be the first major step in the field of personalized food supplementary and medicine.
As we could show in our results, it is possible to link proteins to the extracellular curli matrix via the SpyCatcher. One of the next important steps to a cell free system would be, to separate the matrix from the bacteria. The first step would be, to kill the bacteria to stop further production. This could be achieved by implanting a killswitch, which got activated after producing a certain amount of biofilm for example. Another method would be blue light to kill the bacteria. The dead bacteria could then be separated from the matrix to minimize the immune response.
One of the biggest advantages, besides the cell free system is, that the remaining, purified matrix can be customized for every user individually. For this, there are no limits in customizable, as long as the SpyCatcher is still able to bind to the matrix. This represents the opportunity to create an “all in one” food and medicine supplementary. An example would be, that next to the glucose binding protein could be a fructose binding protein, for people who are intolerant against it. That would kill two birds with one stone.
Another option is, to optimize the existing parts, like adding an aldehyde dehydrogenase besides the ADH II. Because the ADH II is oxidizing the ethanol to acetaldehyde, which is toxic to the human body, it would be an ideal addition to the ADH II to prevent users of the toxic effect.
A third option is to use the modularity of the biofilm not only for food and health support, but also for other applications. Like already mentioned, it is already possible to link every protein with a functional SpyCatcher to the matrix. One application could be, to build a filter system with different functions, like absorbing pollutions or mikroplastic from seawater.

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.

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]

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.

Lactase