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

Molecular Biology Reports
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 towa...Continue Reading

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Citations

Jan 3, 2019·Critical Reviews in Biotechnology·Wan-Cang LiuPing Zhu
Feb 2, 2019·Preparative Biochemistry & Biotechnology·Ahmad BeirotiDariush Norouzian
Aug 30, 2020·International Journal of Molecular Sciences·Patricia Guerrero-OchoaAlberto Anel
Dec 22, 2020·Preparative Biochemistry & Biotechnology·Yagmur UnverSeyda Yildiz

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Methods Mentioned

BETA
PCR
ELISA

Software Mentioned

MATLAB
FBA
ExPASy
Fluxomic
ProtParam

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