Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

Cós, Oriol; Ramon, Ramon; Montesinos, José Luis González; Valero, Francisco

doi:10.1186/1475-2859-5-17

Cited by 287 publications

(138 citation statements)

References 95 publications

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“…In the GAP promoter expression system, the cloned heterologous protein will be expressed along with cell growth if the protein is not toxic for the cell [25]. A similar result was observed in this study.…”

Section: Discussionsupporting

confidence: 87%

Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis

Yang

Huang

Chen

et al. 2009

J Ind Microbiol Biotechnol

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A gene encoding the thermostable raw starch digesting alpha-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into Pichia pastoris X-33 host strain using the vector pGAPZalphaA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 510 U/l in the Hinton flask culture broth. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-beta-N-acetylglycosaminidase H, and this agrees with the predicted size based on the nucleotide sequence. About 75% of the original activity remained after heat treatment at 60 degrees C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60 degrees C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 48-h treatment, the DPw of raw sago starch obviously decreased from 830,945 to 378,732. The surface of starch granules was rough, and some granules displayed deep cavities.

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Section: Discussionsupporting

confidence: 87%

Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis

Yang

Huang

Chen

et al. 2009

J Ind Microbiol Biotechnol

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“…In this context, the level of protein expression in P. pastoris depends critically on the growth conditions, and the attainment of high cell densities has been shown to improve protein yields substantially (Stratton et al, 1998). Although production of recombinant proteins under such culture conditions is typically induced by methanol, which activates the aox-1 promoter controlling the heterologous gene, feeding mixtures of glycerol (or other multicarbon sources) to the culture has also been successfully used as a means for improving process productivities (for a review see Cos et al, 2006). In view of the outstanding role of P. pastoris for biotechnology research, this organism represents an obvious target for studies of its metabolism and physiology.…”

Section: Introductionmentioning

confidence: 99%

Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates

et al. 2007

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The metabolic pathways associated with the tricarboxylic acid cycle intermediates of Pichia pastoris were studied using biosynthetically directed fractional 13 C labelling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (1.39610 "5 s "1 and 4.44610) with varying mixtures of glycerol and methanol as sole carbon sources. The results show that, with co-assimilation of methanol, the common amino acids are synthesized as in P. pastoris cells grown on glycerol only. During growth at the lower dilution rate, when both substrates are entirely consumed, the incorporation of methanol into the biomass increases as the methanol fraction in the feed is increased. Moreover, the co-assimilation of methanol impacts on how key intermediates of the pentose phosphate pathway (PPP) are synthesized. In contrast, such an impact on the PPP is not observed at the higher dilution rate, where methanol is only partially consumed. This finding possibly indicates that the distribution of methanol carbon into assimilatory and dissimilatory (direct oxidation to CO 2 ) pathways are different at the two dilution rates. Remarkably, distinct flux ratios were registered at each of the two growth rates, while the dependency of the flux ratios on the varying fraction of methanol in the medium was much less pronounced. This study brings new insights into the complex regulation of P. pastoris methanol metabolism in the presence of a second carbon source, revealing important implications for biotechnological applications.

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“…In the batch phase the initial concentration of glycerol was 10 g/l in order to obtain a significant level of biomass prior to triggering the expression of the recombinant gene. It is known that the maximum specific growth rate of P. pastoris on methanol is, generally, lower when the yeast is producing a heterologous protein because of the negative effect that the metabolic burden of heterologous protein production exerts on the cells [17], as it proved to be in our case (data not shown). The production of chimeras was triggered by the daily addition of methanol and growth was monitored for 4 days.…”

Section: Resultsmentioning

confidence: 50%

Optimization of construct design and fermentation strategy for the production of bioactive ATF-SAP, a saporin based anti-tumoral uPAR-targeted chimera

et al. 2016

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BackgroundThe big challenge in any anti-tumor therapeutic approach is represented by the development of drugs selectively acting on the target with limited side effects, that exploit the unique characteristics of malignant cells. The urokinase (urokinase-type plasminogen activator, uPA) and its receptor uPAR have been identified as preferential target candidates since they play a key role in the evolution of neoplasms and are associated with neoplasm aggressiveness and poor clinical outcome in several different tumor types.ResultsTo selectively target uPAR over-expressing cancer cells, we prepared a set of chimeric proteins (ATF-SAP) formed by the human amino terminal fragments (ATF) of uPA and the plant ribosome inactivating protein saporin (SAP). Codon-usage optimization was used to increase the expression levels of the chimera in the methylotrophic yeast Pichia pastoris. We then moved the bioprocess to bioreactors and demonstrated that the fed-batch production of the recombinant protein can be successfully achieved, obtaining homogeneous discrete batches of the desired constructs. We also determined the cytotoxic activity of the obtained batch of ATF-SAP which was specifically cytotoxic for U937 leukemia cells, while another construct containing a catalytically inactive mutant form of SAP showed no activity.ConclusionOur results demonstrate that the uPAR-targeted, saporin-based recombinant fusion ATF-SAP can be produced in a fed-batch fermentation with full retention of the molecules selective cytotoxicity and hence therapeutic potential.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0589-1) contains supplementary material, which is available to authorized users.

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Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

Cited by 287 publications

References 95 publications

Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis

Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis

Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates

Optimization of construct design and fermentation strategy for the production of bioactive ATF-SAP, a saporin based anti-tumoral uPAR-targeted chimera

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