Expression of multidrug transporter P‐glycoprotein in Pichia pastoris affects the host's methanol metabolism

Abstract

Summary Pichia pastoris KM71H (MutS) is an efficient producer of hard‐to‐express proteins such as the membrane protein P‐glycoprotein (Pgp), an ATP‐powered efflux pump which is expressed properly, but at very low concentration, using the conventional induction strategy. Evaluation of different induction strategies indicated that it was possible to increase Pgp expression by inducing the culture with 20% media containing 2.5% methanol. By quantifying methanol, formaldehyde, hydrogen peroxide and formate, and by measuring alcohol oxidase, catalase, formaldehyde dehydrogenase, formate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase and α‐ketoglutarate dehydrogenases, it was possible to correlate Pgp expression to the induction strategy. Inducing the culture by adding methanol with fresh media was associated with decreases in formaldehyde and hydrogen peroxide, and increases in formaldehyde dehydrogenase, formate dehydrogenase, isocitrate dehydrogenase and α‐ketoglutarate dehydrogenases. At these conditions, Pgp expression was 1400‐fold higher, an indication that Pgp expression is affected by increases in formaldehyde and hydrogen peroxide. It is possible that Pgp is responsible for this behaviour, since the increased metabolite concentrations and decreased enzymatic activities were not observed when parental Pichia was subjected to the same growth conditions. This report adds information on methanol metabolism during expression of Pgp from P. pastoris MutS strain and suggests an expression procedure for hard‐to‐express proteins from P. pastoris.

Development of a New High-Cell Density Fermentation Strategy for Enhanced Production of a Fungus β-Glucosidase in Pichia pastoris

“…As previously reported methods ( Liu et al, 2019b ), methanol in the fermentation broth was measured with an GM8 Micro-stat Analyzer (Analox Instruments Ltd., Hammersmith, London, United Kingdom). Using a formaldehyde assay kit (Sigma-Aldrich, St. Louis, MO, United States, MAK131) as per manufacturer’s instruction, formaldehyde (CH 2 O) was measured by derivatization with acetoacetanilide in the presence of ammonia.…”

“…SDS-PAGE was performed based on previously reported methods with some modifications (Liu et al, 2016(Liu et al, , 2019b. Briefly, 0.5 g freeze-dried yeast cells were suspended in 5 mL 50 mM phosphate buffer containing 1 mM PSMF (pH 7.5) and subjected to 25 cycles of glass beads assisted cell disruption at 4 • C for 25 min.…”

“…Other calculations including specific cell growth rate (µ), specific methanol consumption rate (g/g/h), volumetric FBG1 production (mg/L), specific FBG1 production (mg/g), and specific production rate (mg/g/h) were determined using described methods (Liu et al, 2016(Liu et al, , 2019b. Experiments were done in triplicates.…”

Development of a New Bioprocess for Clean Diosgenin Production through Submerged Fermentation of an Endophytic Fungus

“…Agitation and aeration may often cause excessive foam formation and thus influence cell growth and biotransformation efficiency. In our previous study, a certain quantity of antifoam reagent was added into the fermentation medium and reaction broth for the SmF of yeast and recombinant enzyme catalysis, respectively. − The antifoam reagent has been used commonly in SmF, and the mechanisms of action were also summarized, such as bridging-stretching, spreading fluid entrainment, bridging-dewetting, etc . However, there is very little knowledge on the effect of antifoam reagent addition on the growth of Fusarium strains.…”

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…The applications of systems biology in P. pastoris range from an increased understanding of cell physiology to improving recombinant protein expression have been described (Zahrl et al, 2017). A study by Liu W. et al (2019a) provided information on methanol metabolism during the expression of P-glycoprotein from the P. pastoris MutS strain and suggested an expression procedure for hard-to-express proteins from P. pastoris.…”