Abstract

Anthocyanins, the key pigments found in red wine, are abundant in grapes, berries, flowers and many plants. Like all naturally ocurring metabolites, they eventually biodegrade and re-enter the carbon cycle. In this project, we search nature for enzymatic pathways that can break down anthocyanins into simpler, unpigmented molecules. We reasoned that microbes living in the soil near vineyards were likely to catabolize and consume anthocyanin. Therefore we collected soil samples from XX vinyards around France, Europe and the world with the help of our fellow iGEM teams. In total we isolated 182 strains through selective and non-selective plating on different media. All of the strains were identified by 16s rRNA sequencing, then characterized for their ability to degrade quercitin, a compound structurally similar to anthocyanin. By phylogenetic analysis, we were able to connect quercitin degradation to specific bacterial phyla including Micrococcus, Pseudomonas, Lysinibacillus and Oerskovia. The most effective strains were further characterized by whole genome sequencing to identify enzymes linked to natural quercitin degradation. By bioprospecting with the help of the worldwide iGEM community, we were able to find the best stain fighting enzymes that nature has to offer.

Motivation and Background

Bioprospecting and Bioremediation

Bioprospecting is the process of searching nature for genetic information that can be adapted in useful or profitable ways. In recent years, bioprospecting efforts have focused on the search for small molecule pharmaceuticals and other bioactive compounds (Müller, 2016). Bioremediation is the use of living organisms to remove environmental toxins from contaminated areas. Microbes in particular are well known for their ability to degrade organic pollutants like petroleum, pesticides and phenolic compounds. Bioremediation has always been a popular topic in iGEM, producing many notable projects with diverse organisms and applications.

Anthocyanin is not harmful, but in the context of a stain it is unwanted and so could be considered a target for bioremediation. Therefore a mechanism to degrade anthcyanin could be revealed with a classic bioremediation strategy. 1) Select organisms from a contaminated environment, where enzymatic decontamination may have naturally evolved. 2) Isolate pure strains and measure their activity. 3) Connect the activity to specific pathways using molecular genetics.

Anthocyanidin and Quercitin

In these experiments, we use quercitin as a chemical proxy for anthocyanins. Naturally occurring anthocyanidins are chemically diverse derivatives of a a core flayvlium cation. Plant sources of anthocyanidin carry a range of anthocyanidin pigments substituted at any of up to seven positions, with the relative concentrations contributing to a characteristic color. Pure anthocyanidins, like malvidin, are expensive (120 EUR for 1 mg) and do not necessarily represent the full chemical diversity of a natrual wine stain. Therefore, in this work we use quercitin, a flavonol, as a structural proxy. Quercitn is cheap (40 EUR for 10 g), stable, and can be quantified by absorbance at 375 nm. In follow up experiments, microbes are tested on real wine and on bulk anthocyanins that we extract directly from grape skins.

Results

Selection of quercetin as an anthocyanin substitute

Direct testing of anthocyanin presented a challenge for the team, as anthocyanin can be difficult to isolate and purchasing large quantities is prohibitively expensive. We surveyed the literature to find an inexpensive substitute with a highly similar structure to anthocyanin. All flavonoids are structured as two phenyl rings and a heterocyclic ring. Anthocyanin itself is structured as a chromane ring with an aromatic ring on C2 (figure). Cyanindin and malvidin (most commonly found in wine) comprise 90% of the anthocyanins found in nature. These chemicals differ only in their cyclic B groups, and the chromane ring is well conserved in most flavonoids. Therefore, we theorized that the chromane ring itself presented an ideal target for degradation.

Based on these criteria, we chose the flavanol quercetin as our anthocyanin substitute. This molecule differs from anthocyanins only in the presence of a carbonyl group (in4?). Additionally, quercetin is present in wine, and contributes to its color. Thus, even in the case where enzymes are isolated that break down Quercetin and not anthocyanin, the possibility exists of reducing the color or intensity of wine stains. Finally, co-pigmentation chemical interactions occur between anthocyanin and quercetin, leading to the possibility that quercetin degradation could impact anthocyanin stability (need more background here?).

Concurrent to the quercetin degradation screen, we optimized an anthocyanin isolation protocol from grapes in order to similarly test our samples on anthocyanin.

Soil sample collection map

Our sample collection included soil and grapes from XX locations in France, XX in Europe including X, and X locations in Australia (more?). We tested soil resuspensions, individual isolated microbes, and whole cell extracts for their ability to degrade quercetin and anthocyanin. Samples from locations in XX were capable of degrading q or a (X for q, Y for a), and samples from YY could degrade both (figure).

Figure X: Quercitin degradation by 189 microbes collected from global soil samples Single colonies were inoculated in M9 minimal medium and grown for 6 days. Quercitin was measured by absorbance.

Figure X: Quercitin degradation detail The top-performing strains included isolated from both selective and nonselective media. Strains marked with an asterisk were selected for further investigation.

Methods

Anthocyanin isolation protocol

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Preparation of fabric samples for panning

Fabrics were washed with x prior to panning to remove coatings, preservatives or other treatments that may have been applied in the factory.

Detailed protocol for phage display

10 µl of phage was mixed with 1 gram of fabric...

Sequence similarity was calculated as BLOSUM. Trees were made using Geneious with nearest neighbor joining.

Sequence clustering analysis

Binding quantification with ELISA

Attributions

This project was done mostly by Antoine Villa Antoine Poirot and Sébastien Gaultier. Put here everyone who helped including other iGEM teams

References

• Sweetlove, L. J., & Fernie, A. R. (2013). The Spatial Organization of Metabolism Within the Plant Cell. Annual Review of Plant Biology, 64(1), 723–746.
• Lee, H., DeLoache, W. C., & Dueber, J. E. (2012). Spatial organization of enzymes for metabolic engineering. Metabolic Engineering, 14(3), 242–251.
• Pröschel, M., Detsch, R., Boccaccini, A. R., & Sonnewald, U. (2015). Engineering of Metabolic Pathways by Artificial Enzyme Channels. Frontiers in Bioengineering and Biotechnology, 3(Pt 5), 123–13.