Optimization of five environmental factors to increase beta‐propeller phytase production in <i>Pichia pastoris</i> and impact on the physiological response of the host

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.

mentioning

confidence: 86%

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

Valenzuela

Altamirano

Berríos

2015

“…Compared to other yeast expression systems, Pichia expression system has several advantages for recombinant protein production such as high cell density, genetic stability, low endogenous protein secretion, posttranslational modification (glycosylation and disulfide bond formation), and high secretory capacity . Thus, P. pastoris has been widely used for recombinant protein production along with bioprocess optimization that is specific to a protein of interest …”

Section: Introductionmentioning

confidence: 99%

Maximizing recombinant human serum albumin production in a Mut^sPichia pastoris strain

Mallem

Warburton

et al. 2014

Human serum albumin (HSA) is a cysteine rich molecule that is most abundant in human blood plasma. To remain viable in the market due to lower marketing costs for HSA, it is important to produce a large quantity in an economical manner by recombinant technology. The objective of this study was to maximize recombinant HSA (rHSA) production using a Mut(s) Pichia pastoris strain by fermentation process optimization. We evaluated the impact of process parameters on the production of rHSA, including induction cell density (wet cell weight, g/L) and the control of specific growth rate at induction. In this study, we demonstrated that induction cell density is a critical factor for high level production of rHSA under controlled specific growth rate. We observed higher specific productivities at higher induction cell densities (285 g/L) and at lower specific growth rates (0.0022-0.0024/h) during methanol induction phase, and achieved the broth titer of rHSA up to 10 g/L. The temperature shift from 24 to 28(o) C was effective to control the specific growth rate at low level (≤0.0024/h) during methanol induction phase while maintaining high specific productivity [0.0908 mgrHSA /(gwcw h)].

“…There are reports in the literature about strategies that have proven to be effective in minimizing the proteolytic degradation of recombinant proteins, namely: addition of amino‐acid rich supplements, such as peptone or casamino acids; adjust the pH of the culture medium to one that is not optimal for the problem protease and reduction of induction temperature; and the use of a protease‐deficient P. pastoris host strain (e.g., SMD1163, SMD1156, and SMD1168) . However, in an industrial scale, the use of specific protease inhibitors has been proven to be cost‐prohibitive .…”

Section: Resultsmentioning

confidence: 99%

Enhanced heterologous protein production in Pichia pastoris under increased air pressure

Lopes

Oliveira

Domingues

et al. 2014

Pichia pastoris is a widely used host for the production of heterologous proteins. In this case, high cell densities are needed and oxygen is a major limiting factor. The increased air pressure could be used to improve the oxygen solubility in the medium and to reach the high oxygen demand of methanol metabolism. In this study, two P. pastoris strains producing two different recombinant proteins, one intracellular (β-galactosidase) and other extracellular (frutalin), were used to investigate the effect of increased air pressure on yeast growth in glycerol and heterologous protein production, using the methanol AOX1-inducible system. Experiments were carried out in a stainless steel bioreactor under total air pressure of 1 bar and 5 bar. The use of an air pressure raise of up to 5 bar proved to be applicable for P. pastoris cultivation. Moreover, no effects on the kinetic growth parameters and methanol utilization (Mut) phenotype of strains were found, while an increase in recombinant β-galactosidase-specific activity (ninefold) and recombinant frutalin production was observed. Furthermore, the air pressure raise led to a reduction in the secreted protease specific activity. This work shows for the first time that the application of an air pressure of 5 bar may be used as a strategy to decrease protease secretion and improve recombinant protein production in P. pastoris.