Engineering Gut Microbiota

1. Engineering Gut Microbiota Fei Chen 5/11/08

2. The Big Picture • Gut microbiota provide a dynamic and very beneficial symbiotic relationship with human hosts. • Gut microbiota perform key functions in metabolism • They influence drug response, and the development of many diseases. • Genetically engineered gut bacteria can have far-ranging impact on health and quality of life.

3. Some Background • Present in immense numbers of in the gut:~1 x 1014 bacteria. • 30-40 species compose of 99% of the population. • Mutualistic/symbiotic relationship with hosts. In humans, they serve several key roles: • Metabolism and fermenting unused energy substrates. • Training the immune system • Production of vitamins. • And competitive inhibition of harmful species. • Very significant for health: • Obesity • Disease • Aging • ‘Gut’ refers to the general digestive tract, in terms of microflora, we are interested in the large intestine, especially the Cecum, where a large population of bacteria is present.

4. The Question Posed Is: • How can we engineer bacteria as to make a superior gut microbe?

5. Is It Viable? Some Considerations… • Gut flora maintains a dynamic relationship with the host immune-system. The introduced engineered bacteria must not promote a immune-response from the host. • Research has already shown that a specific strain of E. Coli, NISSLE 1917, can be engineered and introduced to the mice gut without provoking an immune response. • Gut flora is very sensitive to environmental changes. Small changes in pH or population can cause drastic changes to population make-up. • We have to consider the interaction between our genetically engineered flora and the local microbes.

6. Possible Ideas • There are many possible applications in engineering gut microbes. Five projects based on these ideas are: • Gut pH management • Vitamin Production • Lactase Production/Digestion • Pathogenic Defense • Varied expression through Slipped Strand Mismatching

7. Gut pH Maintenance • The flora in the gut is very sensitive to pH. • The body maintains a healthy bacterial ecosystem using pH. Our bacteria could do the same. • The pH of an healthy adult Cecum is around 6.4. (std. dev. 0.4) • Design a system which maintains the pH in this range. • System Requirements: • Sense and respond to external pH. • Create byproducts which buffers external pH. • Reducing pH would be easy, acids are byproducts of metabolism. • Increasing pH is harder.

8. Vitamin Production • Bacteria are responsible for the production of several vitamins required for the body. Example: E. Coli produces vitamin K. • Engineer bacteria which produce other Vitamins and nutritional benefits. • One example: Beta-Carotene. • Lycopenecyclase, an enzyme separated from cyanobacteria which produces Beta-Carotene from lycopene. • Lycopene is commonly found in human diet, a source is tomatoes. • Design Goal: Engineer bacteria which can produce Beta-Carotene/ Vitamins.

9. Lactase Production and Dietary Regulation • Lactose is a sugar predominantly found in milk. Lactose intolerance is the inability to metabolize glucose, and is found in a large percentage of Asians and Africans. It is estimated that 70% of adults are lactose intolerant. • Lactase is a glycoside hydrolase which breaks lactose disaccharides into galactose and glucose monomers. • This gene can be incorporated into our engineered bacteria to metabolize lactose in those who have lactose intolerance. • Further ideas in this subsystem include dietary regulation. • Bacteria has been shown to have a significant link with obesity. The cause of this is due to the balance between various subpopulations inhabiting the gut. (How to exploit this is unclear)

10. Fend Off Pathogens • Example Pathogen: Salmonella (S. Typhimurium ) • One of the major causes of food poisoning. Found in inadequately cooked eggs, pets. • Release of lysogenic phage held in our engineered bacteria to infect and lyse salmonella. • P22 is an well documented phage which infects Salmonella, and has lysogenic/lytic life-cyles. Can enter lytic life cycle after UV-exposure/DNA damage.

11. Overall System Construction • Use of Slipped-Strand-Mismatching to generate different subpopulations with varied expression. • Possible Ways to Utilize the Idea: • Combinations of Slip-Strand Mismatching devices to create many different phenotypes. We have multiple functions needed by the cell. • Set one phenotype to be more predominant than others. (Example: Set Vitamin production to be the primary phenotype)

12. Pros and Cons • Many different subsystems which can be constructed because gut microbiota do so much. • Bacteria already inhabit the gut, including as our favorite, E. Coli. • Many of the subsystems are very interesting by themselves and could be very useful for future teams: • Bacterial Phage Production • Slipped-Strand Mismatching for multiple subpopulations. • External pH buffering • The flora of the gut is very complicated and very sensitive. • A lot of work required for each subsection.

13. References: • Controlling the metabolic flux through the carotenoid pathway using directed mRNA processing and stabilization. C D Smolke, V J Martin, and J D KeaslingMetab Eng. 2001 October; 3(4): 313–321. • Microbial ecology: Human gut microbes associated with obesity Ruth E. Ley, Peter J. TurnbaughNature444, 1022-1023. • Measurement of gastrointestinal pH profiles in normal ambulant human subjects. D F Evans, G Pye, R Bramley, A G Clark, T J Dyson, and J D HardcastleGut. 1988 August; 29(8): 1035–1041. • Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules Astrid M.Westendorf, FlorianGunzer, Stefanie Deppenmeier FEMS Immunol Med Microbiol 2005 Mar 1; 43(3) 373-84.