Modeling of Phytase Production by Cultivation of Pichia pastoris Under the Control of the GAP Promoter

Cell growth and recombinant phytase production under the control of the GAP promoter were studied in continuous cultivation experiments of Pichia pastoris. Based on these studies, a simple kinetic model was established to predict biomass and enzyme production. Although parameters were mainly estimated using the results of continuous cultivation, the proposed model was able to successfully predict cell growth and phytase production in three different fed-batch cultivations of P. pastoris using limited glucose feeding.

Download Full-text

Medium Optimization for the Production of Phytase by Recombinant Pichia pastoris Grown on Glycerol

International Journal of Chemical Reactor Engineering ◽

10.2202/1542-6580.2709 ◽

2011 ◽

Vol 9 (1) ◽

Author(s):

Min Liu ◽

Gabriel Potvin ◽

Yiru Gan ◽

Zhanbin Huang ◽

Zisheng Zhang

Keyword(s):

Pichia Pastoris ◽

Phytase Production ◽

Medium Components ◽

Gap Promoter ◽

Recombinant Pichia Pastoris ◽

Burman Design ◽

Statistical Designs ◽

Plackett Burman Design ◽

Central Composite ◽

Three Components

Based on statistical designs, minimal salts medium, commonly used for yeast cultivation, was optimized to maximize GAP promoter-mediated phytase production by recombinant Pichia pastoris grown on glycerol. A Plackett-Burman design was followed to screen medium components to determine those that significantly affected phytase production. Of the 8 components studied, the concentrations of K2SO4, CaSO4•2H2O and MgSO4•7H2O were identified as having a significant effect. These three components were subsequently optimized by response surface methodology using a central composite design. The optimal concentrations of the three components, leading to a maximal extracellular phytase activity of 161.64 U/ml, were K2SO4 13.25g/l, CaSO4•2H2O 1.03g/l and MgSO4•7H2O 17.94g/l. The activity measured in cultures using optimized growth medium is significantly higher than the 73.31 U/ml measured in cultures using standard minimal salts media. The theoretical phytase yields predicted by the developed model were very close to experimentally obtained values.

Download Full-text

Statistical Medium Optimization for the Increased Production of Recombinant Phytase in the Fed-Batch Cultivation of Pichia pastoris

International Journal of Chemical Reactor Engineering ◽

10.1515/ijcre-2015-0055 ◽

2015 ◽

Vol 13 (3) ◽

pp. 427-435 ◽

Cited By ~ 3

Author(s):

Gabriel Potvin ◽

Zhi Li ◽

Zisheng Zhang

Keyword(s):

Pichia Pastoris ◽

Inductively Coupled Plasma ◽

Batch Cultivation ◽

Medium Composition ◽

Fed Batch ◽

Inductively Coupled ◽

Recombinant Product ◽

Residual Elements ◽

Recombinant Phytase ◽

Central Composite

Abstract Using a 20-run central composite design, standard Basal Salt Media (BSM) was optimized for the production of recombinant phytase by Pichia pastoris in bioreactor-based fed-batch cultivations using glucose as the carbon source. The phytase activity in the supernatant of the cultures at the end of 48-hour runs was modeled as a function of the medium composition, and this model was successfully validated. Using Inductively Coupled Plasma spectroscopy (ICP), residual elements were quantified in the cultivation broth at the end of each run to confirm that differences in final cell density and enzyme activity were not due to nutrient depletion, but indeed associated with the variations in medium composition. The optimized media contains significantly lower concentration of the potassium and magnesium sources (28.7 g/l KH2PO4 and 4.8 g/l MgSO4·7H2O respectively), as well as a reduced concentration of trace salts, and based on experimental results, significantly outperforms standard media in terms of recombinant product yields under the same conditions.

Download Full-text

Engineering the expression system for Komagataella phaffii (Pichia pastoris): an attempt to develop a methanol-free expression system

FEMS Yeast Research ◽

10.1093/femsyr/foz059 ◽

2019 ◽

Vol 19 (6) ◽

Cited By ~ 1

Author(s):

Shinobu Takagi ◽

Noriko Tsutsumi ◽

Yuji Terui ◽

XiangYu Kong ◽

Hiroya Yurimoto ◽

...

Keyword(s):

Pichia Pastoris ◽

Expression System ◽

Constitutive Expression ◽

Free Expression ◽

Aox1 Promoter ◽

Phytase Production ◽

Model Case ◽

Komagataella Phaffii ◽

Gap Promoter ◽

Important Region

ABSTRACT The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1—a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another important region to enhance the effect of KpTrm1p under a methanol-free condition was identified in the DAS1 promoter, and was termed ESPDAS1. Nevertheless, methanol-free phytase production using an engineered DAS1 promoter outperformed phytase production with the GAP promoter by 25%. Difference in regulation by known transcription factors on the AOX1 promoter and the DAS1 promoter was also illustrated.

Download Full-text

Fed-batch operational strategies for recombinant Fab production with Pichia pastoris using the constitutive GAP promoter

Biochemical Engineering Journal ◽

10.1016/j.bej.2013.07.013 ◽

2013 ◽

Vol 79 ◽

pp. 172-181 ◽

Cited By ~ 34

Author(s):

Xavier Garcia-Ortega ◽

Pau Ferrer ◽

José Luis Montesinos ◽

Francisco Valero

Keyword(s):

Pichia Pastoris ◽

Fed Batch ◽

Operational Strategies ◽

Gap Promoter

Download Full-text

Integration of GC-MS in identification of possible final metabolites from phytase production in Pichia Pastoris based on sorbitol induction optimization

Archives of Biotechnology and Biomedicine ◽

10.29328/journal.abb.1001024 ◽

2021 ◽

Vol 5 (1) ◽

pp. 020-025

Author(s):

Jean Bernard Ndayambaje ◽

Gratien Habarurema ◽

Janvier Habinshuti ◽

Angelique Ingabire ◽

Sabine Ingabire Ange ◽

...

Keyword(s):

Pichia Pastoris ◽

Large Scale ◽

Culture Media ◽

Induction Phase ◽

Scale Production ◽

Significant Activity ◽

Fed Batch ◽

Phytase Production ◽

Methanol Induction ◽

Induction Technique

The isolation of phytase using Pichia Pastoris under methanol/sorbitol co-feeding induction technique was investigated. The biological activity of extracellular phytase after optimization with co-substrates induction in 4 liters fermentor (NBS) increased to 13250 U/ml. This led to a 509 fold increases in comparison to the other type of phytase. This effect was studied via induction with sorbitol/methanol in fermentation by Pichia Pastoris GS115 (Mut+) at 20 °C. The interference of by products; methylal, hexamine and (S)-(+)-1,2-propanediol with release of phytase in Pichia Pastoris under methanol induction were detected and cannot be repressed by methanol induction alone. The TLC was used for glycerin analysis under methanol/sorbitol induction and the results were lesser compare to that obtained during phytase production under methanol induction alone. This work showed the higher expression of heterologous proteins and by fed batch fermentation; the expression identified an advantage of producing a significant activity of phytase. Practical applications Plant derived products including sorbitol have been used as alternative medicines for the therapeutic treatment of various diseases, food supplements and could be used in many manufacturing processes. It serves as a culture media for bacteria, and helps to distinguish the pathogenic E. coli O157:H7 from its most other strains. Cells growing on methanol require high oxygen consumption. Sorbitol was used as an alternative cheap co-feeding for the production of proteins and is a non-repressing carbon source for AOX1 promoter with no effect on the level of r-protein at its induction phase. This report describes the isolation of phytase using Pichia Pastoris under methanol/sorbitol co-feeding induction techniques, and sorbitol showed to be a promising co-substrate, as it could enhance both cell growth and targeted protein productivity. This co-feeding and fed-batch induction technique was used for recombinant phytase production in a small and large scale production and the metabolites were analyzed.

Download Full-text

Screening of Alternative Carbon Sources for Recombinant Protein Production in Pichia pastoris

International Journal of Chemical Reactor Engineering ◽

10.1515/ijcre-2015-0092 ◽

2016 ◽

Vol 14 (1) ◽

pp. 251-257 ◽

Cited By ~ 3

Author(s):

Gabriel Potvin ◽

Zisheng Zhang ◽

Amanda Defela ◽

Howard Lam

Keyword(s):

Pichia Pastoris ◽

Enzyme Production ◽

Recombinant Protein Production ◽

Protein Production ◽

Carbon Sources ◽

Extracellular Enzyme ◽

Fed Batch ◽

Fermentation Time ◽

Batch Cultures ◽

Batch Bioreactor

AbstractSeventeen carbon sources were screened to identify those with the potential to support pGAP-regulated recombinant enzyme production by Pichia pastoris, using phytase as a model product. Of these, four, namely glucose, glycerol, fructose and ethanol, supported cell growth and enzyme production, and the performance of the latter two was analyzed. Ranges of acceptable residual carbon source concentrations, i.e. those at which no substrate-related growth inhibition occurred, were determined and used to design fed-batch bioreactor-based processes. In fed-batch cultures, fructose supported higher biomass concentrations and equivalent extracellular enzyme activities than glucose. The same metrics for the cultures grown on ethanol were comparable to those of the cultures grown on glucose, but with a greater required fermentation time.

Download Full-text