Abstract
The use of methylotrophic yeast Pichia pastoris for the production of heterologous proteins under control of promoter PAOX1 is widespread. However, the physiological effects controlling PAOX1 have not been thoroughly investigated. A series of experiments were performed investigating either the effect of oxygen limitation or mixed glycerol/methanol feeding regimes on heterologous protein production. Oxygen limitation revealed that increases in Human serum albumin (HSA) mRNA did not reflect the actual level of HSA protein produced. Mixed glycerol/methanol feeding showed the negative effect of glycerol on HSA production. There was not a statistically significant change in the level of HSA mRNA dependent on the ratio of glycerol:methanol used. This was believed to be due to the effects of mixed feeding overriding the inhibitory/stimulatory effects of glycerol and methanol respectively. A stoichiometric bioreaction network for P. pastoris consisting of 85 reactions was constructed, and used to determine the distribution of metabolic fluxes under the different physiological conditions analyzed. Metabolic flux analysis of data from both the oxygen limitation and mixed feed experiments showed amino acid biosynthesis, oxygen and methanol uptake reactions were positively correlated to HSA excretion. TCA cycle reactions and amino acid synthesis reactions competing with HSA production were negatively correlated to HSA excretion. Glycerol uptake rate was negatively correlated with fluxes in the Pentose Phosphate pathway. Metabolic flux analysis also revealed that energy and biomass producing reactions "compete" with HAS producing reactions for the flux of metabolites. Using the data from this work it should be possible to engineer strain of P. pastoris that can produce increased quantities of heterologous protein.