Free fatty acids reduce metabolic stress and favor a stable production of heterologous proteins in Pichia pastoris

Abstract

The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an inhibitor of the heterologous protein production pathway; oxygen concentration can generate aerobic or anaerobic environments and affects the fermentation rate; and temperature affects the central carbon metabolism and stress response protein folding. The main goal of this study was determine the implication of free fatty acids on the production of heterologous proteins in different culture conditions in cultures of Pichia pastoris. We evaluated cell viability using propidium iodide by flow cytometry and thiobarbituric acid reactive substances to measure cell membrane damage. The results indicate that the use of low temperatures and low methanol concentrations favors the decrease in lipid peroxidation in the transition phase from glycerol to methanol. In addition, a temperature of 14 °C + 1%M provided the most stable viability. By contrast, the temperature of 18 °C + 1.5%M favored the production of a higher antibody fragment concentration. In summary, these results demonstrate that the decrease in lipid peroxidation is related to an increased production of free fatty acids.

Current advances of Pichia pastoris as cell factories for production of recombinant proteins

“…It was primarily used for measuring the fluorescence intensity produced by fluorescent-labeled antibodies which served for detecting proteins or ligands that bind to various molecules related to cells. Specially, it was used for quantifying the physiological state regarding P. pastoris in heterologous protein production process in cultures with high cell density ( Zepeda et al, 2018 ). Thousands of individual cells were measured simultaneously by the use of fluorescent probes and several parameters ( Cossarizza et al, 2019 ).…”

Controlling Specific Growth Rate for Recombinant Protein Production by Pichia pastoris Under Oxidation Stress in Fed-batch Fermentation

“…Cells were collected at the end of fermentation from each run, washed with PBS buffer and suspended in the same buffer. Malondialdehyde (MDA) was quanti ed by measuring thiobarbituric acid reactive substances as described previously [25]. The cell suspension treated with snailase and digestion buffer (Sangon Biotech, shanghai, China) were centrifuged at 10,000 × g for 15 min at 4 °C.…”

Controlling methanol feeding for recombinant protein production by Pichia pastoris under oxidation stress in fed-batch fermentation

“…Cells were collected at the time point corresponding to the maximum enzyme activities, washed with PBS buffer and suspended in the same buffer. MDA was quanti ed by measuring thiobarbituric acid reactive substances as described previously [34]. The cell suspension treated with snailase and digestion buffer (Sangon Biotech, shanghai, China) were centrifuged at 10,000 × g for 15 min at 4 °C.…”