Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris

Abstract

Pichia pastoris is methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). Three yeast cultures were performed: 3X = 3% methanol-10 °C, 4X = 3% methanol-30 °C, and 5X = 1% methanol-10°C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. Ours results show that at 3% methanol −30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol −10 °C and 1% and at methanol −10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris.

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

“…This observation agrees with the results obtained in recombinant P. pastoris at 10 °C. 8 The decrease in the formation of lipid peroxides is induced due to a drop in temperature. This may be because the temperature induces a modulation in the formation of membrane lipids.…”

“…For the reactivation step, 1 mL of frozen material was inoculated in 500 mL Erlenmeyer flasks containing 100 mL of the growth medium BMGY (buffered glycerol complex medium) (1% (w/v) yeast extract, 2% (w/v) casein peptone, 0.34% (w/v) yeast nitrogen base, 1% (w/v) ammonium sulfate, 100 mM buffer potassium phosphate pH 6.0, 4 × 10 −5 % (w/v) biotin, 1% (v/v) glycerol, 2% (w/v) casamino acids with deionized water) and incubated at 30 °C and 250 rpm for 19 h in a shaking incubator (Model SI-300, Jeio Tech, Seoul, Korea). 8 The reactivation step should be realized once because freeze–thaw stress affects the viability and activity of yeast cells, beside it has been reported that several cellular factors affect freeze–thaw stress tolerance. 36 , 37 …”

“…It has also been reported that proteolytic degradation can be minimized through the manipulation of pH and temperature, 6 with cell viability enhanced for over 24 h in batch fermentations at very low (10 °C) and very high temperatures (37 °C). 7 Conversely, growth using high concentrations of methanol as the sole carbon source at high temperatures induces strong oxidative stress 8 due to autoxidation of polyunsaturated fatty acids (PUFAs) or lipid peroxidation that reduces biomass production. 9 PUFA residues of membrane phospholipids are very sensitive to oxidation and the action of ROS, 34 wherein PUFAs easily undergo non-enzymatic oxidation and fragmentation during lipid peroxidation and form numerous toxic products.…”

Cultivation of Pichia pastoris carrying the scFv anti LDL (−) antibody fragment. Effect of preculture carbon source

“…Thus, the use of glycerol as a cosubstrate could be an interesting, alternative strategy,13, 16 provided that it does not repress AOX1 promoter at relatively low level 14 . Based on this information, a typical rAb production by P. pastoris consists of three distinct phases: (a) glycerol batch phase to stimulate growth, (b) glycerol fed-batch phase for AOX1 derepression, and (c) methanol induction phase to express the recombinant protein 17, 18, 19. In the last phase methanol is typically pulsed repeatedly after complete depletion of substrate 20 …”

Produção biotecnológica de L-asparaginase <i>(ASP1)</i> de <i>Saccharomyces cerevisiae</i> em sistema de expressão heterólogo <i>Pichia pastoris</i>

“…Verificou-se que em temperaturas acima de 32 °C ou muito baixas decai a expressão de proteínas heterólogas (COS et al, 2006;ZEPEDA et al,2014).…”