Optimization of Archael Enzyme Expression in Yeast

A few decades ago life was discovered that thrived in extremes of temperature (4 and 100oC), pH (2 and 10), salinity (5 M NaCl), and pressure (250 atm). These organisms, termed Archaea, were found to be a form of life distinct from both bacteria and eukaryotes. Archael enzymes are of great interest for industrial applications in the pulp and paper industry, hydraulic fracturing of gas and oil wells, and specialty chemicals, because of their inherent stability under extremes of pH, temperature, and salinity. The native archael hosts are often difficult to grow in large culture, because of lower biomass yields and the growth dependence on elemental sulfur and subsequent release of hydrogen sulfide as a byproduct.

One goal of this project is to create a high-yield archael enzyme expression system in the yeast, Saccharomyces cerevisiae. We chose the Pyrococcus furiousus b-glucosidase, a homotetrameric enzyme, for initial studies. Through optimization of gene copy number, we have achieved secretion levels of 10 mg/L in batch culture. Using sucrose gradient cell fractionation and confocal microscopy, we have identified an intracellular bottleneck in the first compartment in secretion, the endoplasmic reticulum (ER). 50% of retained protein is inactive, and likely forms aggregates during routine expression. Overexpression of ER chaperones enables a two-fold increase in secretion levels. Increasing the culture growth temperature to 40°C lowers the fraction of inactive protein, and increases the secretion yields by over 400%. We are currently engineering yeast strains to further improve archael protein expression levels. To identify improved strains, we have developed a multi-well enzyme assay to screen expression from 96 yeast colonies in parallel.