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

Molecular biology reports (2018-07-19)
Ashish A Prabhu, Venkata Dasu Veeranki
ABSTRACT

In the present study, the effects of individual as well as multiple genes of pentose phosphate pathway (PPP) on human interferon gamma (hIFN-γ) production were analyzed. With overexpression of 6-phosphogluconate dehydrogenase (GND2), 1.9-fold increase in hIFN-γ 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-γ 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-γ enhancement in recombinant Pichia GS115 strain encoding codon optimized hIFN-γ (GS115/hIFN-γ) and Pichia GS115 strain encoding codon optimized hIFN-γ with co-expressed 6-phosphogluconolactonase(SOL3) and D-ribulose-5-phosphate 3-epimerase (RPE1) (GS115/hIFN-γ/SR) respectively. To get more insight of the flux distribution towards hIFN-γ, studies were carried out by applying flux balance analysis during methanol fed batch phase for both strains. In both strains (GS115/hIFN-γ and GS115/hIFN-γ/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-γ production.

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Sigma-Aldrich
Picropodophyllotoxin, ≥96% (HPLC), powder