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.
  

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Some Background

• Present in immense numbers of in the gut1 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.

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The Question Posed Is

• How can we engineer bacteria as to make a
  superior gut microbe?

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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.

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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

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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.

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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.
• Lycopene cyclase, 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.

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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)

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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.

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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)

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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.

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References

• Controlling the metabolic flux through the
  carotenoid pathway using directed mRNA processing
  and stabilization. C D Smolke, V J Martin, and J
  D Keasling Metab Eng. 2001 October 3(4)
  313321.
• Microbial ecology Human gut microbes associated
  with obesity Ruth E. Ley, Peter J. Turnbaugh
  Nature 444, 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
  Hardcastle Gut. 1988 August 29(8) 10351041.
• Intestinal immunity of Escherichia coli NISSLE
  1917 a safe carrier for therapeutic molecules
  Astrid M. Westendorf, Florian Gunzer, Stefanie
  Deppenmeier FEMS Immunol Med Microbiol 2005 Mar
  1 43(3) 373-84.