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

• Published in: Molecular biology reports Vol. 45; no. 5; pp. 961 - 972
• Main Authors: Prabhu, Ashish A., Veeranki, Venkata Dasu
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01-10-2018; Springer Nature B.V
• Subjects: 6-Phosphogluconolactonase; Animal Anatomy; Animal Biochemistry; Batch Cell Culture Techniques; Biomedical and Life Sciences; Carbohydrate Epimerases - genetics; Carbon sources; Carboxylic Ester Hydrolases - genetics; Epimerase; Fermentation; Histology; Humans; Interferon-gamma - genetics; Interferon-gamma - metabolism; Life Sciences; Metabolic engineering; Metabolic Engineering - methods; Methanol; Morphology; Original Article; Pentose Phosphate Pathway; Phosphogluconate dehydrogenase (decarboxylating); Phosphogluconate Dehydrogenase - genetics; Pichia; Pichia - genetics; Pichia - growth & development; Recombinant Proteins - metabolism; Human interferon gamma (hIFN-γ); Fed batch studies; Pentose phosphate pathway gene; Flux balance analysis(FBA); Pichia pastoris
• ISSN: 0301-4851; 1573-4978
• DOI: 10.1007/s11033-018-4244-2

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.