Development of Agaricus Bisporus as a Platform for Heterologous Expression of Biopharmaceuticals

Open Access
Author:
Woolston, Benjamin Michael
Area of Honors:
Chemical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Wayne Roger Curtis, Thesis Supervisor
  • Wayne Roger Curtis, Honors Advisor
  • Charles Peter Romaine, Thesis Supervisor
  • Andrew Zydney, Faculty Reader
Keywords:
  • Pharming
  • Protein Transport
  • Biopharmaceuticals
  • GUS Expression System
Abstract:
Agaricus bisporus (the button mushroom) is examined as a viable platform for commercial-scale therapeutic protein expression in light of newly developed genetic manipulation techniques, coupled with established methods for rapid and economical biomass production. To this end, studies were undertaken to optimize protein expression using the β-glucuronidase (GUS) reporter system. In the course of these experiments, it was discovered that by manipulating the commercial bi-layered growth system, high levels of heterologous protein could be accumulated in the mushroom despite the absence of the cognate transgene. Further investigation into this unexpected result, including RT-PCR experiments and analysis of expression in basidiospores and compost mycelium, led to a hypothesis that protein expressed in the lower compost layer is shuttled upward during development of the fruiting body, which forms from the upper casing layer. This work set the stage for investigation into the mechanism of this phenomenon, prompting studies with an epitope-tagged native glyceraldehyde-3-phosphate dehydrogenase (GPD-HA) gene to explore whether protein transport was due solely to the presence of a heterologous gene, or whether native proteins could also be translocated. In a separate line of experiments, designed to boost heterologous expression levels, genetic constructs were assembled with GUS flanked by the untranslated regions from RNA-2 of the Cowpea Mosaic Virus (CPMV), sequences which have been shown to increase translation rate in transgenic plants and could potentially have the same effect in the mushroom.