Metabolic engineering of Pichia pastoris GS115 for enhanced pentose phosphate pathway (PPP) flux toward recombinant human interferon gamma (hIFN-γ) production

In the present study, the effects of individual as well as multiple genes of pentose phosphate pathway (PPP) on human interferon gamma (hIFN-gamma) production were analyzed. With overexpression of 6-phosphogluconate dehydrogenase (GND2), 1.9-fold increase in hIFN-gamma was achieved, while synergetic effect of 6-phosphogluconolactonase (SOL3) and d-ribulose-5-phosphate 3-epimerase (RPE1) resulted in 2.56-fold increase in hIFN-gamma as compared to control. Fed batch fermentation using mixed feeding of gluconate and methanol (carbon source) was carried out, resulting in 80 and 123 mg L-1 of hIFN-gamma enhancement in recombinant Pichia GS115 strain encoding codon optimized hIFN-gamma (GS115/hIFN-gamma) and Pichia GS115 strain encoding codon optimized hIFN-gamma with co-expressed 6-phosphogluconolactonase(SOL3) and d-ribulose-5-phosphate 3-epimerase (RPE1) (GS115/hIFN-gamma/SR) respectively. To get more insight of the flux distribution towards hIFN-gamma, studies were carried out by applying flux balance analysis during methanol fed batch phase for both strains. In both strains (GS115/hIFN-gamma and GS115/hIFN-gamma/SR) more than 95% of formaldehyde flux is directed towards assimilatory pathway. The analysis revealed that with the overexpression of SOL3 and RPE1 the flux towards PPP triggering the alleviation in hIFN-gamma production.