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 nanofiber matrix which is able to pick up disruptive elements. To accomplish this goal, we used a Curli fiber network 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 intracellularly, whereas they assemble extracellularly into amyloid fibers. Further, we decided to use W3110 as our work strain because of its native Curli production. As described before, we used the SpyTag/SpyCatcher system (SpySystem) to modify these Curli fibers posttranslationally. For this reason, we fused the small SpyTag peptide to the CsgA monomers. SpyTag is the smaller component of the SpySystem which does not interfere with the fusion protein’s secretion. 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 immobilizing 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 in different manners, we decided to fuse several, functional domains to the SpyCatcher. First, we designed a SpyCatcher-glucose binding protein (GGPB) hybrid, in regard of decreasing the glucose concentration in the gut. Another created fusion protein contains the alcohol dehydrogenase instead which catalyzes the oxidation of alcohol. Additionally, we combined the SpyCatcher with lactase enabling people suffering from lactose intolerance to metabolize lactose sufficiently.

Results

During our time in the lab, we achieved the establishment of a novel carrier matrix based on Curli fibers, the protein compound of bacterial biofilms. Taking advantage of their high stability and their fast self-assembly, we designed CsgA monomers which enable the modification with functional domains and properties on demand. This was facilitated by introducing the SpySystem to our Curli matrix. Here we show both the successful expression and detection of our modified construct as well as its accurate functionality.

PickUp monomers

We designed a plasmid construct for the production of the SpyTagged CsgA monomers (pPickUp). Therefore, we used a T5 promoter sequence in front of the CsgA-SpyTag gene fusion, which encodes for the modified Curli Fiber monomer, with RBS in between.

An additional mCherry gene was meant to indicate the correct gene transcription. The translation is stopped by two terminators which are are coded on the plasmid backbone (pSB1C3) with chloramphenicol resistance. The native csgA gene contains a pstI restriction site which we deleted by mutagenesis for submission as a biobrick (part BBa_K1650047). We also transformed this construct into another backbone for selection on kanamycin (pPickUp2). To investigate the proper CsgA production, we assayed various strains by applying crystal violet and congo red staining, enabling the detection and relative quantification of biofilms. Crystal violet colors the cells inside of the biofilm population, whereas congo red interacts with the amyloid structures like Curli fibers.

As illustrated in Fig. x, W3310D+pPickUp shows an increase in absorbance after applying both staining procedures, in comparison to W3110 and W3110D. This result indicates a higher biofilm production in our modified strain. The background absorption from the deficient mutant can be affiliated to other amyloid structures like flagella’s. The production of the CsgA fibers is verified via studies of the biofilm morphology on congo red agar plates, shown in figure X.

The pictures shown above, point out that the CsgA-deficient mutant does not produce amyloid structures in comparison to the same mutant carrying pPickUp. These experiments indicate the expression and secretion of CsgA units as well as the self-assembly to curli fibers. Additionally, electron microscopy images confirm our hypothesis.

The SpyCatcher-GFP-His construct

To prove the accurate interaction between the the Spy-tagged CsgA and its counterpart, the SpyCatcher, we designed a plasmid encoding for a SpyCatcher-GFP fusion product with an additional, C-terminal His-Tag (pGlow). The small peptide tag allows an efficient purification via IMAC.

The successful production of the SpyCatcher-GFP construct was determined by western blot analysis using a primary anti-His-Antibody. Whole cell lysates were generated derived from an overnight culture and purified utilizing Ni+-affinity chromatography. The immunoblot shows a protein whose molecular weight corresponds to the estimated size of 41 kDa. Besides, the strong detection signal underlines the efficient accumulation of the SpyCatcher-GFP construct as our protein of interest.

SpyTag/SpyCatcher systemt

To assay the interaction between both components of the SpySystem, we co-transformed DH5alpha with pPickUp2 and pGlow. The selection was performed using chloramphenicol-kanamycin plates. As described before, whole cell lysates where purified applying IMAC.

Detection of the anti-His-signal exposes a single band of an approximately 54 kDa protein revealed the intracellular, covalent fusion of CsgA-SpyTag and SpyCatcher-GFP-His. A smaller band at approximately 37 kDa could be a degradation product scince neither CsgA-SpyTag nor SpyCatcher-GFP behave like that.

Extracellular binding to the modified curlis

In order to investigate wether the system is still functional when the CsgA are assembled outside of the cell, both W3310 transformed with pGlow and W3310d CsgA as negative control were grown on a microscopyplate. After two days of growth, the cells were incubated with crude cell lysate containing SpyCatcher-GFP-His which was meant to induce covalent fusion of the interaction partners. The cells were microscoped after repetitive washing to remove unbound protein.

As illustrated in Figure, W3110 pGlow treated with SpyCatcher-GFP containing cell lysate show a clear membrane-associated GFP signal indicating the accurate functionality of the two-component system and the proper self-assembly of secreted CsgA monomers. In contrast to that, the GFP signal of treated W331 dCsgA shows no distinct localization stressing the specificity of the desired reaction.

Alcohol dehydrogenase

To obtain a detoxification protein, we fused the alcohol dehydrogenase II to the SpyCatcher. Therefore, we designed a plasmid encoding for a SpyCatcher-ADHII fusion product (pDeTox).

For investigation the proper translation, we did a SDS-Page with the crude cell lysate and the natural strain as control. The desired product was obtained, with a molecular weight of 52 kDa.

Hier noch das WT bild

For further characterization of the remaining catalytic activity of the ADHII after fusion to the SpyCatcher, an enzymatic kinetic assay has been proceed. The natural property of the ADHII is to oxidase ethanol to acetaldehyde under consumption the cofactor NAD+. The reduction product, NADH, is measured at a wavelength of 365 nm. For this measurement, a crude cell extract was used.
An enzymatic kinetic assay has shown, that the catalytic activity of the ADHII still remains after fusing it to the SpyCatcher. The ADHII catalyze the reaction of ethanol to acetaldehyde, while reducing the cofactor NAD+ to NADH. The absorption of the formed NADH is measured at a wavelength of 365 nm. For this measurement, a crude cell extract was used.

As shown in figure XX, the conversion of NAD to NADH, caused by the others enzymes in the lysate, has a constant increase of the gradient (m = 0.1). After adding the ethanol, the gradient of the curve raises, which indicates an increasing amount of NADH (m = 0.183). After a short time, the gradient falls back to the initial state (m = 0.1). After adding another amount of ethanol, the gradient raises again from 0.1 to 0.15.

As a control we used the DH5alpha strain, which got no alcohol dehydrogenase and therefore can’t catalyze this oxidation reaction. We can that the gradient constantly increases. After adding the ethanol, no change in the gradient could be observed. Hence we can conclude, that NAD+ hasn’t undergone the conversion to NADH.

Bild negativ probe

Glucose binding protein

We constructed a plasmid, which encodes a fusion protein of the SpyCatcher with the glucose binding protein in order to lower the glucose concentration in the gut (pSweet).

To ensure a correct production of the fusion protein, we performed a SDS-PAGE with crude cell lysate and the natural strain as control. The fusion product has a molecular weight of 51 kDa, which can be observed on the SDS-PAGE.

Hier noch das WT bild

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.(Nguyen et.al) 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.(Zakeri et. al) Due to the reaction between Asp7 of the SpyTag peptide and Lys31 of the SpyCatcher an intramoleculare, irreversible isopeptide bond is formed. (Li et. al) The resulting complex forms a compact β-sandwich structure, which is stable to boiling in SDS and to thousands of piconewtons.(Zakeri et. al)

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. 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. 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

b-Galactosidase is an enzyme wich naturally converts Lactose. It catalyses the hydrolytic cleavage of Lactose into Glucose and Galactose while keeping the stereochemistry oft he product. There are also two other katalytical activities oft he lactase: The transformation of lactose into allolactose and the cleavage of allolactose into Glucose and Galactose. (Juers et al.)
b-Galactosidase is a homotetramer (464 kDa), build up of four identical peptidchains with 1023 amino acids. These are linked via non-covalent bounds. The four active sites oft he enzyme are each build up oft wo Monomers and Sodium and magnesia kations. However the whole enzyme only active in ist tetrameric form. (Juers et. al)