Hoynes, Gemma (2021) Investigating the metabolism of tartrazine by the human gut microbiome. (MSc(R) thesis), Kingston University, .
Abstract
Azo dyes are a class of food dyes that are widely used in a variety of commercial industries. These synthetic colours are aromatic in nature and are characterised by one or more functional –N N– groups within its molecular structure. One example of an azo dye is tartrazine. It’s lemon-yellow shade lends a bright, appealing colour to food products and pharmaceuticals, and is one of the most popular artificial dyes in the food trade. Tartrazine has also long been associated with negative health effects such as ADHD like symptoms, allergic responses and tissue damage. Posing a risk to human health has led to the prohibition and removal of many azo dyes from consumer products. At present, despite a long history of investigation, the toxicity of tartrazine has not yet been established and is still authorised for use in many countries. Azoreductases are enzymes present in mammals, bacteria and yeast that are recognised for their ability to reduce azo-dyes which in turn generates metabolites such as aromatic amines, often with carcinogenic and toxic properties. It is well known that the human gut microbiota play an integral part in the reduction, activation and detoxification of xenobiotics, however the majority of these metabolic pathways remain uncharacterised. This study aims to help advance these efforts, by selecting a common gut bacterial strain that has already demonstrated azoreductase capabilities, yet the enzymes responsible remain uncharacterised. To identify the genes that code for an azoreductase enzyme, the full genomic DNA of Odoribacter splanchnicus, a bacterial strain common to the human gut, was selected and a blastp search was performed, using a library of already characterised azoreductase protein sequences. This generated one protein sequence result which bore 51% sequence similarity to AzoC, an azoreductase from the anaerobic bacteria Clostridium perfringens. To amplify this gene, PCR conditions were optimised by applying a range of annealing temperatures. The temperature at which gene amplification was highest allowed for the gene of interest to be cloned into a choice of two pET TOPO® vectors which was then transformed into E. coli BL21 Star™ (DE3) cells. Varying combinations of environmental conditions were applied during protein expression trials to optimise the expression of soluble protein. Time constraints prevented further experiments which would aid in the identification and subsequent characterisation of a putative azoreductase enzyme from the genome of O. splanchnicus. However, this study highlights the importance in uncovering the enzymatic properties of our gut microbiome, particularly in species that have not yet been identified.
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