Engineering agarolytic enzymes into Bacteroides thetaiotaomicron for the development of a drug delivery system with algal polysaccharide-derived capsules

• Author / Creator

  Monteith, Stephanie M.

• The microbiota of the distal gastrointestinal tract (GIT) of monogastric animals plays a vital role in maintaining host health, such as releasing energy and nutrients from dietary polysaccharides that are otherwise indigestible by human enzymes. These dietary polysaccharides can be coupled to the metabolism and proliferation of defined bacteria in order to achieve an improved health outcome, which is known as synbiotics. Agarose is a marine polysaccharide that is well suited to act as a selective nutrient in a designer synbiotic system because it is resistant to digestion by the vast majority of microorganisms residing in the distal GIT of terrestrial animals. Agarose can be completely saccharified into its monosaccharide substituents by three agarases: GH16, GH117, and GH2, found in a polysaccharide utilization locus from a terrestrial bacterium Bacteroides uniformis NP1. The first objective of this research project aimed to engineer an agarolytic strain of the commensal gut bacteria, B. thetaiotaomicron, by introducing intrachromasomal copies of the three agarases with extracellular-directed signal peptides on the N-terminal domain. The transgenic agarases will be expressed to the outside of the cell in order to access the agarose substrates and import the released D-galactose into the cell to use as a carbon source. The second area of my research focused on developing an assay to measure release of cargo from algal-polysaccharide-derived capsules after digestion from the engineered strain. The agarase genes were successfully introduced into the B. thetaiotaomicron genome through homologous recombination, and all three agarases were produced by the bacterium in detectable amounts on western blots. Notably, enzymatic products were observed using thin layer chromatography following incubation with the engineered strains indicating the transgenic agarases are functional and able to hydrolyse agarose substrates. Only the GH16 enzyme was confirmed to be active on the outer membrane of the cell. Supplementation of the GH117 and GH2 into the medium on the GH16 producing strain was able to confer growth on agarose. The second objective was completed by measuring the oxidation activity of released HRP from porphyran- and carrageenan-derived capsules. The capsules were leaky and optimization of the integrity of the capsules as well as the cargo used would be important to confirm the polysaccharides efficacy of capsules to release therapeutic molecules within the distal GIT. Importantly, oligosaccharides observed from capsules digested with purified GH16 enzymes as well as GH16 enzymes produced from both B. uniformis NP1 and engineered B. thetaiotaomicron support the development of a drug delivery system using algal polysaccharide-derived capsules and engineered agarolytic bacteria to deliver therapeutic molecules within the distal GIT.

• Subjects / Keywords

  • genetic engineering
  • agarase
  • algal polysaccharides
  • dysbiosis
  • carbohydrate-active enzymes

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-9enq-7613

• License

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