Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter

Resina, David

doi:10.1016/j.jbiotec.2003.10.029

Cited by 96 publications

(68 citation statements)

References 26 publications

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“…The advantage of this is that when using methylamine for induction of foreign protein expression, glucose or glycerol can be used as the carbon source instead of methanol, or methanol itself can be used as the sole carbon source and also for induction [89]. This, coupled with the AOX1-comparable tight regulation and transcriptional efficiency, makes the FLD1 promoter an attractive alternative for methanol-independent expression of recombinant Heterologous protein production using Pichia pastoris expression system 253 Figure 1.…”

Section: Fld1 Promotermentioning

confidence: 99%

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Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,097

633

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The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

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Section: Fld1 Promotermentioning

confidence: 99%

“…Resina et al [89] found that induction levels of the FLD1 promoter by methanol were dependent on the nitrogen source used, with co-induction using methanol and methylamine resulting in higher expression levels (19 U/ml) of a Rhizopus oryzae lipase than induction of the FLD1 promoter with methanol and ammonium (12 U/ml).…”

Section: Fld1 Promotermentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,097

633

View full text Add to dashboard Cite

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“…Lipolytic activity of the supernatant was determined by a modified method based on a colorimetric commercial kit assay (LIP kit from Roche Diagnostics, Mannheim, Germany), as described elsewhere (Resina et al, 2004). One unit of lipolytic activity was defined as the amount of enzyme required to release 1 mmol of fatty acid per minute under assay conditions.…”

Section: Lipase Activity Assaymentioning

confidence: 99%

A simple model‐based control for Pichia pastoris allows a more efficient heterologous protein production bioprocess

Cós

Ramon

Montesinos

et al. 2006

Biotech & Bioengineering

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A predictive control algorithm coupled with a PI feedback controller has been satisfactorily implemented in the heterologous Rhizopus oryzae lipase production by Pichia pastoris methanol utilization slow (Mut(s)) phenotype. This control algorithm has allowed the study of the effect of methanol concentration, ranging from 0.5 to 1.75 g/L, on heterologous protein production. The maximal lipolytic activity (490 UA/mL), specific yield (11,236 UA/g(biomass)), productivity (4,901 UA/L . h), and specific productivity (112 UA/g(biomass)h were reached for a methanol concentration of 1 g/L. These parameters are almost double than those obtained with a manual control at a similar methanol set-point. The study of the specific growth, consumption, and production rates showed different patterns for these rates depending on the methanol concentration set-point. Results obtained have shown the need of implementing a robust control scheme when reproducible quality and productivity are sought. It has been demonstrated that the model-based control proposed here is a very efficient, robust, and easy-to-implement strategy from an industrial application point of view.

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“…Therefore, a promoter that does not require methanol is attractive for expression of foreign genes in P. pastoris. Potential promoters that are alternatives to P AOX1 include the glutathione-dependent formaldehyde dehydrogenase promoter (P FLD1 ) (26), for which either methanol or methylamine can act as an inducer, and the glyceraldehyde-3-phosphate dehydrogenase promoter (P GAP ) (33), which is a strong constitutive promoter in various microorganisms. Recently, we also developed the translation elongation factor 1␣ promoter (P TEF1 ), with more highly growth-associated expression characteristics, as an alternative to P GAP (2).…”

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confidence: 99%

Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter

Ahn

Hong

Park

et al. 2009

Appl Environ Microbiol

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To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Na ؉ )-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (P PHO89 ) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. P PHO89 was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1␣ and the glyceraldehyde-3-phosphate dehydrogenase promoter, P PHO89 exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple P PHO89 -based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of P PHO89 for controlled production of recombinant proteins in P. pastoris.Pichia pastoris is a methylotrophic yeast species that is increasingly used as a host system for heterologous protein expression for both academic and industrial purposes. To date, more than 500 proteins have been cloned and expressed using this system (19; http://faculty.kgi.edu/cregg/index htm), and it has provided the protein production platform for several structural genomics programs (25,35).In this system, most recombinant proteins have been produced using the alcohol oxidase I promoter (P AOX1 ), which is completely repressed when cells are grown on glucose and is induced in the presence of methanol (31). However, there are circumstances in which the promoter may not be suitable; the principal problem is that the highly volatile and inflammable compound methanol is required for transcription (29). Use of methanol for the induction of gene expression is often not permitted in the production of food products since methanol is a petroleum by-product (13, 33). Also, P AOX1 -based fermentation requires relatively long times and sophisticated feeding strategies for maintenance of the induction phase. These properties hamper the industrial applicability of this approach. Therefore, a promoter that does not require methanol is attractive for expression of foreign genes in P. pastoris. Potential promoters that are alternatives to P AOX1 include the glutathione-dependent formaldehyde dehydrogenase promoter (P FLD1 ) (26), for which either methanol or methylamine can act as an inducer, and the glyceraldehyde-3-phosphate dehydrogenase promoter (P GAP ) (33), which is a strong constitutive promoter in various microorganisms. Recently, we also developed the translation elongation factor 1␣ promoter (P TEF1 ), with more highly growth-associated expression characteristics, as an alternative to P GAP (2). The current study describes the use of a phosphate-responsive promoter as an alternative to constitutive and inducible promoters to drive expres...

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Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter

Cited by 96 publications

References 26 publications

Heterologous protein production using the Pichia pastoris expression system

Heterologous protein production using the Pichia pastoris expression system

A simple model‐based control for Pichia pastoris allows a more efficient heterologous protein production bioprocess

Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter

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