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 10 vinyards around France, Europe and the world, notably 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 and genus 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 natural 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 315 nm. In follow up experiments, microbes are tested on real wine and on bulk anthocyanins that we extract directly from grape skins.

Figure X: Structure and absorbance of malvidin, an anthocyanidin, and quercetin, a flavonol 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. Cyanindin and malvidin 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, increasing wine color stability, mainly through π-π stacking between their phenolic cycles. Thus, it leads to the possibility that quercetin degradation could also impact anthocyanin stability.

Results

Collection of the soil samples

Soil samples were collected from France, Spain, Croatia, Namibia and Australia. Samples from the Paris region were collected by members of our team. Other samples were sent by friends, family members, and collaborating iGEM teams. Soil samples were declared to French customs authorities with a Facture Proforma, printed out by the sample donor and included in the shipment.
Upon arrival, samples were washed gently with phosphate-buffered saline (PBS) solution, then left to stand, allowing large particles to settle. The resulting eluate was diluted further with PBS then used directly as a source of soil microbes.

Country	Location	Collector
France	Clos Monmartre Vineyard, Paris	Our Team
France	Cochin Port Royal, Paris	Our team
France	Vaucluse region’s vineyard	INSA-Lyon iGEM team
Spain	Barcelona	UPF-CRG Barcelona iGEM team
Spain	Utiel Requena	UPV Valencia iGEM team
Australia	Hunter Valley	UNSW
Australia		Macquarie 2016 iGEM team
Namibia	Etosha National Park	Our team
Algeria	Alger	Our team
Croatia	Kricke	Our team
Israel	Jerusalem	Our team

Preparation of the microbe library

For safety, microbial isolation was performed in a fume hood in a BSL 2 facility. More safety information in provided on our safety page.
Microbes were isolated from soil samples using either selective or nonselective plating. In nonselective plating, soil eluate was serially diluted and plated on rich media (Trypic Soy Agar, TSA) or minimal media (M9 Glucose). In selective plating, soil eluate was used to innoculate a 20 mL culture of M9 quercitin media, in which quercitin was the only supplied carbon source. The resulting culture was incubated at 37 C for 48 hours, then serially diluted and plated on TSA. The purpose of selective plating was to enrich for microbes with the ability to metabolize quercitin.
In each case, single colonies were isolated then re-streaked to eliminate potential contamination. To maximize the library diversity, we preferentially chose colonies with unique morphology and we took no more than 5 clones from a single soil sample. After isolation, we performed colony PCR with universal 16s rRNA primers (see methods). Sequencing the resulting PCR products allowed us to identify the strains and position them within the greater bacterial taxonomy.

Quanitification of quercitin degradation

Single colonies from the selective and nonselective microbe libraries were innoculated in M9 Glucose media containing X g of quercitin. Samples were cultured at 37 C for six days. Following incubaction, samples were centrifuged to remove cells and contaminants. Quercitin was measured by absorbance at 375 nm. Of 182 strains tested, 50 produced quercitin levels significantly lower than controls (Figure X). 20 strain produced more than 50% quercitin degradation and 2 strains degraded quercitin more than 80%.
Both selective an nonselective plating methods were able to produce quercitin-degrading strains. 4 of the 5 strains showing the most quercitin degradation were obtained by selective plating. However, 40 of the 50 strains showing significant degradation were obtained by nonselective plating.

Phylogenetic analysis of quercitin degradation

Testing microbes with real wine and real fabrics

Mining genomes for quercitin-degrading enzymes

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.

Figure X: Phylogenetic Tree.

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.