(269a) Systematic Optimization of Protein Secretory Pathways in Saccharomyces Cerevisiae to Increase Production of Hepatitis B Vaccine

Systematic
Optimization of Protein Secretory Pathways in Saccharomyces cerevisiae to
Increase Production of Hepatitis B Vaccine

Hunter
Flick, Jiayuan Sheng, Xueyang Feng*

Department
of Biological Systems Engineering, Virginia Polytechnic Institute and State
University, Blacksburg, VA 24060

Hepatitis B is a major disease that threats
over 40% of the population in the world, especially in developing countries. Currently,
one of the effective vaccines to treat Hepatitis B is the Hepatitis B Small
Antigen (HBsAg), which is produced by the recombinant
yeast Saccharomyces cerevisiae.  However, the price for the Hepatitis B vaccine
is still too high (~$20/dose) for people in developing countries to afford. In
this study, we aim to improve the production of HBsAg
by systematically engineering the protein secretory pathways. In general, we
screened 194 S. cerevisiae strains with single gene knocked out in four
major steps of the secretory pathway: ER
degradation (47 genes), protein folding (67 genes), unfolded protein response
(39 genes), and translocation and exocytosis (40 genes). From these screenings,
we found that the deletion of YPT32 (YGL210W), SBH1 (YER087C-A), and HSP42 (YDR171W)
led to the most dramatic increase of HBsAg production,
with 1.92, 1.66, and 1.62-fold increases over the wild type S. cerevisiae
strain, respectively. During the screening, we also found that the deletion of IRE1
(YHR079C) led to dramatic decrease of HBsAg production. We next
overexpressed IRE1 and found that the HBsAg
production was increased by 1.29-fold. We then tested the combination of two
strategies: gene knockout and gene overexpression and found that deletion
of YPT32 with the overexpression of IRE1 led to a 2.12-fold increase in HBsAg
production over the wild type which indicates potential synergistic
effect among different genes in protein secretory pathways.
In summary, our discoveries promoted the bio-manufacturing of a broad scope
vaccines in a cost-effective ways, allowing the vaccine to be more accessible
throughout the world.