Abstract

Anthocyanins, they key pigments present in wine, are polyphenol molecules naturally found in many plants. In this part of the project we turn to nature to search for enzymes able to degrade these pigments, and we take it a step further by enhancing their stain removal activity. We aim to harness the power of nature and make the changes that would direct it to our goal. First, we searched the literature for enzymes that had potential to degrade Anthocyanins. Then, making use of the Fabric Binding Domains found in this project, we directed their activity to our stains.
Here we show that our enzymes are correctly expressed and maintain their activity even when fused with the Fabric Binding Domains. We also show that our Fabric Binding Domains have specificity to different fabrics, opening a new door for enzymatic detergents for stain removal. SPEAK ABOUT HOW WE DEGRADE ANTHOCYANINS!!!

Motivation and Background

Nature, from inspiration to a canvas

Throughout human history, people have always taken inspiration on nature for their daily problems. We find numerous examples of turning to nature in many fields. The most famous and successful example of this is probably velcro, which was inspired in the way the burrs’ structure allows them to attach to furry bodies, such as dogs.
Over the years, however, with the increasing control that humans have over nature, we have been allowed to go one step further. We no longer only take inspiration on nature, but also control it and change it, “enhancing” it for our specific purposes.
It is with this mindset that we approached this part of our project. It is clear that in the past we have turned to nature to find enzymes that can act as specific detergents in our laundry. We wanted to do the same, to find specific enzymes for getting rid of wine stains, but we wanted to go a step further. We wanted to enhance the stain removal capacity of these enzymes by focusing their activity on our stains.

Polyphenol degrading enzymes

Throughout this project we tested several polyphenol enzymes on their ability to degrade wine stains. We decided to focus on polyphenol degrading enzymes because Anthocyanins, red wine pigments, belong to this group of proteins.
In order to choose the enzymes with which to work with, we analysed the literature and applied two different criteria: first, we chose enzymes that had been expressed in E. coli before, and second, enzymes whose activity could be tested using spectrography. This two criteria were chosen to make our project realistic, both given the time we had for the project and the facilities we had at our disposal.
First, we decided to work with the Polyphenol Oxidase from Camellia sinensis (PPO). We chose this enzyme because Anthocyanins had been already described as natural substrates for it (Jaiswal 2009). In a study where the authors expressed the enzyme in E. coli, a structure-activity analysis was performed, and it was determined that the optimal way of expressing this enzyme in E. coli was to eliminate the signal peptide region that guided the protein to cell wall in plant cells (Wu 2010). We therefore cloned a codon optimised version of the gene, without the signal peptide, called PPO2.
We decided to work with Bacillus pumilus’ BpuI laccase as well, because laccases have been shown to degrade Anthocyanins (Rodriguez Couto 2006). B. subtilis’ laccase already existed in the Biobrick registry for iGEM (BBa_K863000), so we took the already existing sequence and codon optimised it for E. coli.
Next, we decided to work with two Catechol-dioxygenases, XylE from Pseudonomas putida and CatA from Acinetobacter pittii. These two enzymes have Catechol as substrate, which shares structural similarities with Anthocyanins. We thought that these enzymes would be strong candidates for removing red-wine stains, specially XylE, given that it had been described as being a promiscuous enzyme (Cerdan 1995, Kobayasi 1995 and Lin 2015).
The last enzyme we decided to work with was Lactobacillus plantarum’s tannin acyl hydrolase TanLpI. Tannins are Polyphenol molecules that are important for wine colour. Although the red colour associated with red wine comes from Anthocyanins, Tannins are important molecules that need to form covalent bonds with Anthocyanins in order for them to be stable over time. We thought that an alternative way of getting rid of wine stains would be to attack the Tannin molecules, hence making the Anthocyanin ones unstable. We chose to work with this specific tannin acyl hydrolase because it fit the criteria we exposed before (Curiel 2009).

Design of our expression system

The first step of this project was to design a system that allowed us to test all our proteins in an easy way. We aimed to create a method that would allow us to use the same primers for all the versions of the proteins, as well as a system that would make it easy for then transit our proteins to the Phytobricks.
The first step was to obtain the codon optimised sequences of the five proteins we decided to work with. We obtained gBlocks of all the proteins, with a histidine tail on the C-terminus. We designed the synthetic fragment so that it would allow us test the proteins both with and without the histidine tag. Two primers common to all proteins were designed for inserting the protein sequences with the his-tag on our backbone. A third primer, individual to each of the proteins, was designed as well, to allow for cloning of the protein sequence alone, just in case the his-tag inhibited the protein activity. All primers incluses BsaI restriction sites.

In order to have a plasmid that allowed for overexpression of our proteins, we adapted the pCOLA plasmid from the pDUET system to make it compatible with Golden Gate assembly. We deleted all BsaI restriction sites and synthesised a plasmid with only one T7 promoter. We placed a BsaI restriction site on the desired position so as to be able to insert our genes under the T7 promoter.
In the end, we had a simple workflow for expressing our proteins under the T7 promoter. We constructed all the plasmids with Golden Gate, replicated them in E. coli DH5alfa and transformed them in E. coli BL21(DE3) for protein expression.

Fabric Binding Domain fusion proteins - enhancing of wine stain degrading properties

An important part of our project was to enhance the ability of the chosen enzymes to degrade wine stains. Our aim was to enhance the stain removal capacity of our enzymes by directing their activity to the fabric. We believed that by increasing the time of contact between our enzymes and the stain, we would get an enhancement on stain removal.
We looked for ways of enhancing enzymatic activity in the literature, and one usual method we found was by creating fusion proteins. There are several examples of this methodology. For example, by fusing either a transcriptional repressor or an transcriptional activator to dCas9, enhanced repression and enhanced activation of genes can be accomplished. This happens because dCas9 guides the repressor or the activator to the desired place of action, and therefore the process is more efficient.
Inspired by this examples, we wanted to find something that would guide our enzymes to the fabric in which the stains were found. We found peptides with affinity for specific fabrics using phage display. More information on the FBD. These peptides were thoroughly tested to see whether they were able to bind to specific type of fabrics.
Nonetheless we wanted to know whether the binding of these Fabric Binding Domains (FBD) would affect the activity of our proteins. We tested them first with GFP and then with some of our enzymes. Testing them with GFP allowed us to know in a straightforward way whether it was possible to express functional FBD-fusion proteins.

Results

Outline of the project

Text about the outline

Confirming expression of our proteins

Text about the SDS-PAGE gels

Testing our enzymes in their natural substrate

Text about the tests with Catechol.

Testing the effect of fusing FBD with proteins

Text about the tests with the GFP-FBD. Also about the binding of the FBD to our proteins!

Testing our enzymes on their ability to degrade Anthocyanins

Text about the tests with Anthocyanins.

Testing our enzymes on their ability to degrade wine stains

Text about the tests with wine on fabric.

Methods

Codon optimisation of plant and bacterial genes of interest

One important step when expressing heterologously genes in E. coli, specially those of eukaryotic origin, is to perform Codon Optimisation. Different organisms have different codon preferences, which means that each organism has different abundances of the several codons that translate to a same amino acid.
We performed Codon Optimisation using the IDT Codon Optimisation tool.

Golden Gate and Gibson Assembly for Biobrick Construction

We built our plasmids using either Golden Gate or Gibson Assembly.
In the case of Golden Gate, we used NEB’s Golden Gate Assembly mix. 40fmol of backbone and 40fmol of insert were mixed in a 10uL total reaction, following the instructions provided by NEB. The mixure was incubated with the following cycle: 37ºC for 5 min, (37ºC for 2 min, 16ºC for 5min)x50, 37ºC for 5min, 50ºC for 10min and 80ºC for 10min.
In the case of Gibson Assembly, 200ng of DNA (backbone and insert with a ratio of 1:2) were mixed in a 15uL total reaction, using NEB’s 2X Hifi DNA Assembly MasterMix. The mixure was incubated 1h at 37ºC.

Induced protein expression by the use of expression systems

We used E. coli BL21(DE3) as our expression strain. Cells transformed with our constructs were incubated at appropriate temperature until they reached an OD(600nm) of 0.4-0.6. They were then induced with 0.5mM IPTG.

Preparation of cell extract for protein activity testing

We performed our cell extract preparation using ThermoScientific’s B-PER Bacterial Protein Extraction Reagent with Enzymes.

His-tag purification of proteins

We performed the purification of our proteins using ThermoScientific’s His-Pur Ni-NTA Resin and all buffers as indicated by the manufacturer.

SDS-PAGE analysis

We analysed our proteins using pre-made, 10% SDS-PAGE gels from BioRad.

Assaying the binding strength of the Fabric Binding Domains More information on the FBD

We assayed the strength or the Fabric Binding Domains using the assay developped by our team, carrying cotton, wool, silk and linnen untreated, unbleached fabrics. Cell extracts of BL21(DE3) strains carrying GFP-FBD fusion proteins were performed and 1:10 dilutions were made. The diluted cell extracts were incubated in the 96-well-plates-based assay with the fabrics overnight. The several measurements were performed in a Infinite M200-Pro Tecan plate reader.

Assaying the enhanced stain removal activity of fusion proteins

We assayed the stain removal activity of our fusion proteins in our assay, with the different fabrics stained with wine. The different measurements wera carried out in the Infinite M200-Pro Tecan plate reader.

Attributions

This project was done by Alicia and Sebastián. BpuI data was obtained by Mislav. A special thanks to our advisor Nadine for all the insight on Golden Gate and for the GFP sequences. A special thanks to our advisor Jake for all the help with the figures and the pDUET system. Thank you to Afonso Bravo for the help with the his-tag purification. Thank you to IDT for the synthesis of our genes, and thanks to NEB for the enzymatic kits.

References

• Cerdan P, Rekik M and Harayama S (1995). Substrate specificity differences between two catechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from Pseudomonas putida. European Journal of Biochemistry. 229, 113-118
• Curiel JA, Rodriguez H, Acebron I, Mancheño JM, de las Rivas B and Muñoz R (2009). Production and physicochemical properties of recombinant Lactobacillus plantarum tannase. Journal of Agricultural Food Chemistry. 57(14), 6224-6230
• Jaiswal V, DerManderosian A, and Porter JR. (2009). Anthocyanins and polyphenol oxidase from dried arils of pomegranate (Punica granatum L.). Food chemistry. 118(1), 11-16
• Kobayashi T, Ishida T, Horiike K, Takara Y, Numao N, Nakazawa A, Nakazawa T and Nozaki M (1995). Overexpression of Pseudomonas putida Catechol 2,3-Dioxygenase with High Specific Activity by Genetically Engineered Escherichia coli. Journal of Biochemistry. 117, 614-622
• Lin J and Milase RN (2015) Purification and characterization of catechol 1,2-dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli transformants. Protein Journal. 34(6), 421-433
• Rodriguez Couto S, Toca Herrera JL, (2006). Industrial and biotechnological applications of laccases: a review. Biotechnology Advances. 24/5), 500-513
• Wu YL, Pan LP, Yu SL and Li HH (2010). Cloning, microbial expression and structure-activity relationship of polyphenol oxidases from Camellia sinensis. Journal of Biotechnology. 145(1), 66-72