Enhanced protein production by sorbitol co-feeding with methanol in recombinant Pichia pastoris strains

Abstract

Pichia pastoris strains carrying 1, 6, 12, and 18 copies of the porcine insulin precursor (PIP) gene, were employed to investigate the effects of sorbitol co-feeding with methanol on the physiology of the strains. Multicopy clones of the methylotrophic yeast were generated to vary the PIP gene dosage and recombinant proteins. Elevated gene dosage increased levels of the recombinant PIP protein when methanol served as the sole carbon and energy source i.e., an increase of 1.9% for a strain carrying 1 copy, 42.6% for a strain carrying 6 copies, 34.7% for a strain carrying 12 copies and 80.9% for a strain carrying 18 copies, respectively (using sorbitol co-feeding with methanol during the induction phase). However, it had no significant influence on a lower gene dosage strain (1 copy), but this approach affirmed enhancement in cell growth and PIP production for higher gene dosage strain (6, 12, and 18 copies) via using sorbitol co-feeding with methanol. Additionally, the co-feeding strategy could hold vital importance for recombinant protein production by a multi-copy P. pastoris system.

PAOX1 expression in mixed-substrate continuous cultures of Komagataella phaffii (Pichia pastoris) is completely determined by methanol consumption regardless of the secondary carbon source

“…Repressing carbon sources, such as glycerol, abolish P AOX1 expression, whereas non-repressing carbon sources, such as sorbitol, permit P AOX1 expression. The same conclusion has been reached from studies of mixed-substrate growth in fed-batch cultures ( Brierley et al, 1990 ; Thorpe et al, 1999 ; Xie et al, 2005 ; Çelik et al, 2009 ; Wang et al, 2010 ; Gao et al, 2012 ; Niu et al, 2013 ; Carly et al, 2016 ; Azadi et al, 2017 ; Chen et al, 2017 ) and continuous cultures ( Jungo et al, 2006 ; Jungo et al, 2007a ; Jungo et al, 2007b ; Canales et al, 2015 ; Berrios et al, 2017 ). Indeed, even though glycerol is commonly used as the secondary carbon source, the use of sorbitol has been almost unanimously recommended on the grounds that glycerol represses P AOX1 expression.…”

Burden Imposed by Heterologous Protein Production in Two Major Industrial Yeast Cell Factories: Identifying Sources and Mitigation Strategies

“…In another study, a 10-copy strain of K. phaffii producing 1.2 g/L Thermomyces lanuginosus xylanase A performed better than 6- and 18-copy strains producing ~0.85 and 0.25 g/L, respectively (Mellitzer et al, 2012 ). In a third study, K. phaffii production of the porcine insulin precursor reached a maximum of 0.9 g/L at 6 copies, showing approximately the same production titers for 6, 12, and 18 copies (Chen et al, 2017 ). These findings indicate that cells possess mechanisms for downregulating heterologous genes when reaching a certain gene dosage plateau, a Goldilocks range of maximum expression, above which burden is triggered (Mellitzer et al, 2014 ).…”

“…Insulin precursor Human, porcine Sc, Kp Hormone used for treatment of diabetes Zhu et al, 2009;Kazemi Seresht et al, 2013;Vanz et al, 2014;Chen et al, 2017;Wright et al, 2020;Shen et al, 2021 Immunoglobulin G (IgG) amounts. S. cerevisiae can tolerate harsh growth conditions, such as low pH, and finally, it has gained the generally recognized as safe (GRAS) status allowing for easier approval processes (U.S. Food Drug Administration, 2018).…”

Detection of recombinant Pichia pastoris producing glargine

“…Pichia pastoris can also use methanol as its only carbon and energy source. When grown on methanol, yeast vigorously activates two alcohol oxidases (Aox1 and Aox2), which are required in the initial phase of the methanol consumption pathway [11,[17][18][19][20][21][22][23][24].…”