Development of Pichia pastoris as a ruminal escape vehicle
Strauss, Colin Earl
University of Lethbridge. Faculty of Arts and Science
Lethbridge, Alta. : University of Lethbridge, Faculty of Arts and Science, 2000
The yeast expression system Pichia pastoris was investigated as an encapsulation technology capable of serving as a rumen escape vehicle. Cellularly encapsulated protein is protected from the ruminal environment so long as the cell membrane, which surrounds and isolates the intracellular protein is physically intact. Intracellular expression of Green Fluorescent Protein (GFP) allows for the monitoring of cellular integrity as necessary for the protection of encapsulated protein from ruminal proteases. Upon cellular lysis GFP is exposed to extracellular proteases which result in both the proteolytic degradation of the protein-based GFP chromophore and its associated fluorescence. Visualization of rumen fluid under epifluorescent microscopy revealed a high level of background autofluorescence owing to the fluorescent plant particles, microbes, and fluorescent compounds therein. Visualization of GFP in rumen fluid can be optimized through GFP variant selection, filter set design, and light source selection based on bulb emission spectra. Incubation of intracellular GFP expressing P. pastoris in batch culture ruminal in vitro simulations demonstrated that 93%, 97%, and 25% of the P. pastoris inoculum maintained cellular integrity in clarified rumen fluid, bacterial fraction of rumen fluid, and whole rumen fluid, respectively, when incubated over 36 to 48 h. Continuous fermentation in vitro rumen simulations (Rusitec) demonstrated a P. pastoris escape rate of 19% when added daily to fully adapted Rusitec vessels having a dilution rate of 0.75d-1. Abomasal in vitro simulations demonstrated that 84% of the P. pastoris inoculum was lysed within 12 h, as necessary for the release of encapsulated protein. P.pastoris may be an effective post-fuminal delivery vehicle, provided that similar results are obtained in vivo.
xiv, 120 leaves : ill. ; 28 cm.
Pichia pastoris , Yeast fungi -- Biotechnology , Dissertations, Academic , Rumen -- Microbiology