Effect of dilution rate and methanol‐glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastorisMut⁺ phenotype in continuous culture

Abstract

The induction using substrate mixtures is an operational strategy for improving the productivity of heterologous protein production with Pichia pastoris. Glycerol as a cosubstrate allows for growth at a higher specific growth rate, but also has been reported to be repressor of the expression from the AOX1 promoter. Thus, further insights about the effects of glycerol are required for designing the induction stage with mixed substrates. The production of Rhizopus oryzae lipase (ROL) was used as a model system to investigate the application of methanol-glycerol feeding mixtures in fast metabolizing methanol phenotype. Cultures were performed in a simple chemostat system and the response surface methodology was used for the evaluation of both dilution rate and methanol-glycerol feeding composition as experimental factors. Our results indicate that productivity and yield of ROL are strongly affected by dilution rate, with no interaction effect between the involved factors. Productivity showed the highest value around 0.04-0.06 h(-1) , while ROL yield decreased along the whole dilution rate range evaluated (0.03-0.1 h(-1) ). Compared to production level achieved with methanol-only feeding, the highest specific productivity was similar in mixed feeding (0.9 UA g-biomass(-1) h(-1) ), but volumetric productivity was 70% higher. Kinetic analysis showed that these results are explained by the effects of dilution rate on specific methanol uptake rate, instead of a repressor effect caused by glycerol feeding. It is concluded that despite the effect of dilution rate on ROL yield, mixed feeding strategy is a proper process option to be applied to P. pastoris Mut(+) phenotype for heterologous protein production.

Continuous Cultivation as a Tool Toward the Rational Bioprocess Development With Pichia Pastoris Cell Factory

“…With methanol fractions higher than 0.6 C-mol C-mol −1 , q P was like cultures performed with methanol as sole carbon source. Canales et al (2015) used the production of Rhizopus oryzae lipase (Rol) as a model system to investigate the application of methanol-glycerol feeding mixtures in the P. pastoris Mut + producer clone. Cultures were grown in a simple chemostat system and response surface methodology was used to evaluate the effects of D and the methanol/glycerol ratio in the feed as experimental variables.…”

Truncated Prosequence of Rhizopus oryzae Lipase: Key Factor for Production Improvement and Biocatalyst Stability

“…Using this yeast provides a number of advantages, including little endogenous protein secretion, the absence of endogenous lipases and esterases, the presence of a powerful, tightly regulated methanol-inducible alcohol oxidase 1 promoter (P AOX1 ), and the ability to grow at high cell densities on defined media [30]. The high suitability of P. pastoris for ROL production is confirmed by the vast amount of literature on its cloning and expression [31][32][33][34][35]. Previous studies have examined the effects of cloning and expressing the whole ROL prosequence (97 amino acids) with the mature sequence in P. pastoris [36,37].…”

Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris (Komagataella phaffii) PAOX1-driven recombinant producer strains with different target gene dosage

“…These results led them to devise an optimized bioprocess strategy based on a stepwise decrease in µ during their fed-batch experiments. Canales et al [34] studied the effect of glycerol:methanol mixtures in the chemostat feeding stream and the specific growth rate on Rol production under P AOX1 promoter. They found µ to be much more influential on q p than was the methanol fraction in the feeding.…”