Metabolic and Process Engineering of Pichia Pastoris for the Production of Value-added Products

Abstract

Motivated by the surging demand of recombinant proteins and biofuels derived from renewable substrates, increasing attention has been paid to the development of novel strains via metabolic engineering strategies. Pichia pastoris is a eukaryotic platform suitable for protein expression and potentially for biofuel production due to its advantageous traits over Escherichia coli or Saccharomyces cerevisiae. In this thesis, we constructed a xylanase-producing P. pastoris strain. The fungal xylanase Xyn11A was successfully overexpressed under the constitutive GAP promoter. Biochemical characterization of the xylanase revealed that Xyn11A is optimally active at 70 °C and pH 7.4. This xylanase was stable over a wide range of pH ranging from pH 2 to pH 11. Excellent thermal stability was observed at temperature 60 °C. Enhanced production of Xyn11A was achieved by investigating the effect of carbon source and feeding strategies. The highest xylanase activity was detected at 15000 U/mL using high cell density cultivation. Production of optically pure (2R, 3R)-2, 3-BD was achieved by engineering P. pastoris with a heterologous pathway. The pathway genes consisting of Bacillus subtilis alsS, alsD and S. cerevisiae BDH1 were assembled and transformed into P. pastoris. Cultivation conditions were optimized and the highest titer of 2, 3-BD obtained using YPD media was 45 g/L in fed-batch cultivation. To enhance the economic viability of 2, 3-BD production in P. pastoris, statistical medium optimization was performed. It was found that 75 g/L of 2, 3-BD was produced using optimized media in fed-batch cultivation.