Team:NRP-UEA-Norwich/Approach
“An ounce of prevention is worth more than a million pounds of cure”
- David Agus, New York Times bestselling author.
Approaches
House of Carbs is taking two approaches to increase the levels of butyrate in the colon
1. Butrylated Starches
The first approach aims for the production of butrylated starches that, when consumed, will release butyrate into the digestive system. Butrylated starch produced by chemical modification of purified maize starch have previously been shown to confer physiological changes associated with colon cancer resistance in rats1. The alteration of carbohydrate molecules in vivo would also be of great benefit, as modified starches are used in many industries. These starches are usually produced by chemical modification of purified starches and expensive processes using large quantities of environmentally damaging chemicals. For example, acetylated starch is commonly used in food products such as frozen food, sauces and puddings due to its viscous, cohesive, gel-forming and freezing-stability properties2.
Figure 1: Schematic representation of a starch molecule with butyrate groups attached to it. We plan to do this enzymatically.
1. We have developed an experimental system to screen putative acyltransferases for their ability to modify alpha-1,4 linked carbohydrates. We are initially testing putative acyltransferases to determine if they can make acylated carbohydrates using acyl-CoA and butryl-CoA as substrates. Acyl-CoA is available in the cells of our chassis organisms but to provide butryl-CoA, we are also expressing two enzymes (butyrate kinase + phophotransbutrylase) from Coprococcus sp. L2-50 (DSM 16842), a colonic bacteria.
2. As the modification of plants is time-consuming, our initial experiments are the modification of the alpha-1,4 linked carbohydrate known as glycogen produced in laboratory strains of Escherichia coli. We also progressed to testing the enzymes for their ability to modify the starches produced in the plastids of plants. In the bacterial system, we also modified the structure of the glycogen using branching and de-branching enzymes in order to increase the number of free ends available to the putative acyltransferases whilst maintaining the solubility of the glycogen to aid downstream analysis.
3. To help us understand how carbohydrate structure affects the number of free ends we have modelled the growth of glycogen molecules. As we do not know which carbon positions the acyltransferases will utilise or if they will compete with carbohydrate synthases, we have also modelled the effects on carbohydrate structure and yield of the modification of different carbon positions during growth. Finally, we have made a kinetic model of glycogen production from glucose.
2. A Butyrate-producing Probiotic
The second approach aims to produce a culturable bacterial strain that produces butyrate. This would be particularly useful for individuals whose colonic microbiomes are lacking in butyrate-producing bacteria.
1. We have cloned six coding sequences that encode the enzymes that catalyse the biosynthesis of butyrate from acetate from the colonic bacterium Coprococcus sp. L2-50 (DSM 16842)3.
2. We have assembled these enzymes into a BioBrick that we are initially testing for butyrate production in a laboratory strain of Escherichia coli . We are also aiming to engineer bacterial species such as Escherichia coli "Nissle" (Mutaflor®) that are already used in probiotics.
3. Our final test will be to test the performance of our bacteria in realistic digestive conditions. We will therefore introduce our bacteria into an artificial gut system consisting of faecal microbiota.
Figure 2: The butyrate biosynthetic pathway.
References
1. Bajka B.H., Clarke J.M., Topping D.L., Cobiac L., Abeywardena M.Y., and Patten G.S., 2010, Butyrylated starch increases large bowel butyrate levels and lowers colonic smooth muscle contractility in rats , Nutr. Res., 30(6), p. 427–34
2. Thomas D., and Atwell A., 1999, Matching Starches to Applications , In. Starches, p. 49–55.
3. Louis P., Petra L., Pauline Y., Grietje H., and Flint H.J., 2010, Diversity of human colonic butyrate-producing bacteria revealed by analysis of the butyryl-CoA:acetate CoA-transferase gene , Environmental Microbiology, 12(2) p. 304–14.
