Discover natural enzymes for anthocyanin degradation

Highlights

Goals

• • To screen soil samples from around the world to find bacterial and/or fungal strains that naturally degrade anthocyanin, either as a metabolic substrate or a dedicated carbon source.

Methods

• Microbiome cultivation
• Anthocyanin extraction
• Anthocyanin/Quercitin degradation screen
• DNA extraction
• 16S/18S PCR amplification and sequencing
• Whole genome sequencing
• Phylogenetic analysis
• Creation of microbial library
• Quantitative ELISA

Results

• Sample origin
• Species isolated
• How well samples/species degraded quercitin
• How well samples/species degraded anthocyanin
• Phylogenetic tree of the different species of bacteria and fungus
• Common candidate genes

BioBricks

• BioBrick 1
• BioBrick 2
• BioBrick 3

Abstract

In this part of the project, we screened bacteria previously isolated from vineyards around the world to look for strains able to degrade flavonoids such as Quercitin and anthocyanin. This was done to find non-toxic alternatives to PERC, a toxic chemical widely used in the dry cleaning industry. Through our screen from XX locations around the world, we isolated X species capable of degrading these compounds, which were identified through 16S sequencing and subjected to phylogenetic analysis. We also chose a few promising, unknown strains of [species XX] for whole-genome sequencing to look for common enzymes. This allowed us to construct a microbial library that could be passed on to the assay team to directly test microbial enzyme activity on fabric samples.

In another hand, we made a selection process of microbes on a single carbon source media composed by Quercetin and soil sample. The idea was to get at the end some microbes able to use Quercetin as a carbon source. After 6 days of experiments, we had about 9 different fungi able to degrade Quercetin in a very efficient way.

Motivation and Background

Wine stains are notoriously difficult to remove from clothing. This is true for ordinary consumers as well as for professional cleaning services, a fact that our human practices team confirmed through a widespread survey of Parisian dry cleaners. Perchloroetylene (PERC) is a common solvent used by dry cleaners to remove stains; however, it is toxic both for human health and the environment, and will be phased out of use in France by 2022. The difficulty in stain removal is due to the complex chemical composition of the wine itself, which includes phenolic compounds such as flavonoids.

Flavonoids, specifically anthocyanins, are abundant in grapes and are the main contributors to red wine pigmentation (Kennedy 2005). In order to find a more sustainable, non-toxic alternative to PERC, we screened bacteria for enzymes that break down anthocyanins, either as a metabolic substrate or as a carbon source. Microbes living in vineyard soil and on the grapes themselves have been suggested to play a role in wine quality itself (Bokulich 2016). As it seemed likely that microbes growing in vineyards would be capable of anthocyanin degradation, we focused on sample collection from a diverse range of vineyard locations. In the course of our screen, we gathered grape and soil samples from Australia, Croatia, Namibia, Spain, and France, primarily through collaboration with other iGEM teams, and tested them for flavonoid degradation.

We tested anthocyanins from multiple sources: one the one hand, we purchased the anthocyanin malvidin in order to make a standard curve from (Sigma?). On the other, we also isolated anthocyanin from vineyard grapes in order to have a more “natural” chemical representation sample; this had the added benefit of obtaining anthocyanins in a more cost effective manner. We also tested for degradation of the flavonolic compound quercetin, which we could purchase much more cheaply than anthocyanin.

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).

Methods

Anthocyanin isolation protocol

blabla ###.

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.