The <i>ICL1</i> gene of <i>Pichia pastoris</i>, transcriptional regulation and use of its promoter

Menéndez, Javier; Valdés, Iris; Cabrera, Nelson

doi:10.1002/yea.1028

Cited by 56 publications

(24 citation statements)

References 35 publications

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“…Recently, the ICL1 (isocitrate lyase) gene of P. pastoris was investigated as an alternative promoter to the commonly used AOX1 and GAP promoters [72]. A plasmid (pPICLDEX) containing a single copy of the gene encoding dextranase from Penicillium minioluteum was transformed into P. pastoris.…”

Section: Icl1 Promotermentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,095

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: Icl1 Promotermentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,095

633

View full text Add to dashboard Cite

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“…ICL1 promoter sequences from several yeasts such as S. cerevisiae , P. pastoris , Yarrowia lipolytica or Candida tropicalis are well established and frequently applied to protein expression [77,78,114]. In K. lactis , ICL1 is assumed to be regulated without a Mig1 repressor, even if the derepression and induction are mediated by the Snf1/Snf4 complex [115]; it is still unclear if other repressor proteins are involved with URS regulation of ICL1 .…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, the volumetric activity of an expressed enzyme does not necessarily correlate with the strength of transcription. This might also be a reason why in Pichia pastoris the native P ICL1 was praised as a good alternative for methanol free protein production [77], while on the other hand, according to a recent review, the transcription levels of this promoter in Pichia pastoris appear to be lower than with the classic P AOX1 or P GAP [117]. …”

Section: Introductionmentioning

confidence: 99%

Carbon source dependent promoters in yeasts

et al. 2014

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Budding yeasts are important expression hosts for the production of recombinant proteins.The choice of the right promoter is a crucial point for efficient gene expression, as most regulations take place at the transcriptional level. A wide and constantly increasing range of inducible, derepressed and constitutive promoters have been applied for gene expression in yeasts in the past; their different behaviours were a reflection of the different needs of individual processes.Within this review we summarize the majority of the large available set of carbon source dependent promoters for protein expression in yeasts, either induced or derepressed by the particular carbon source provided. We examined the most common derepressed promoters for Saccharomyces cerevisiae and other yeasts, and described carbon source inducible promoters and promoters induced by non-sugar carbon sources. A special focus is given to promoters that are activated as soon as glucose is depleted, since such promoters can be very effective and offer an uncomplicated and scalable cultivation procedure.

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“…As such, the P. pastoris-derived GAP (46), ICL1 (26), FLD1 (35), PEX8 (20), TEF1 (1), PGK1 (7), and YPT1 (34) promoters are available. P FLD1 is an attractive alternative to P AOX1 for the expression of foreign genes in P. pastoris, but besides methanol, it needs methylamine for induction, which is also toxic and flammable.…”

mentioning

confidence: 99%

Catabolite Repression of Aox inPichia pastorisIs Dependent on Hexose Transporter PpHxt1 and Pexophagy

Zhang

Zhou

et al. 2010

Appl Environ Microbiol

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In this work, the identification and characterization of two hexose transporter homologs in the methylotrophic yeast Pichia pastoris, P. pastoris Hxt1 (PpHxt1) and PpHxt2, are described. When expressed in a Saccharomyces cerevisiae hxt-null mutant strain that is unable to take up monosaccharides, either protein restored growth on glucose or fructose. Both PpHXT genes are transcriptionally regulated by glucose. Transcript levels of PpHXT1 are induced by high levels of glucose, whereas transcript levels of PpHXT2 are relatively lower and are fully induced by low levels of glucose. In addition, PpHxt2 plays an important role in glycolysis-dependent fermentative growth, since PpHxt2 is essential for growth on glucose or fructose when respiration is inhibited. Notably, we firstly found that the deletion of PpHXT1, but not PpHXT2, leads to the induced expression of the alcohol oxidase I gene (AOX1) in response to glucose or fructose. We also elucidated that a sharp dropping of the sugar-induced expression level of Aox at a later growth phase is caused mainly by pexophagy, a degradation pathway in methylotrophic yeast. The sugar-inducible AOX1 promoter in an ⌬hxt1 strain may be promising as a host for the expression of heterologous proteins. The functional analysis of these two hexose transporters is the first step in elucidating the mechanisms of sugar metabolism and catabolite repression in P. pastoris.Pichia pastoris, a methylotrophic yeast, has been developed as a successful expression platform for heterologous proteins. The increasing popularity of this particular expression system can be attributed to several reasons (3, 6): (i) the easy techniques needed for the molecular genetic manipulation and the simple growth medium or culture conditions required for growth compared with those for mammalian cells; (ii) high levels of protein expression at the intra-or extracellular level; (iii) the ability to perform higher-eukaryotic protein modifications, such as glycosylation, disulfide bond formation, and proteolytic processing; and (iv) the availability of the expression system as a commercially available kit.In the P. pastoris system, the expression of foreign genes is usually driven by the outstanding promoter of the alcohol oxidase I gene (AOX1), which encodes the first enzyme in the methanol utilization pathway. The expression of Aox can be regulated with both repression and derepression mechanisms responding to different carbon sources. The AOX1 promoter (P AOX1 ) is induced only in response to methanol and repressed by other carbon sources, such as glucose or ethanol (3, 5, 6). However, the P AOX1 -based platform is not free of drawbacks (12, 23). The strength of induction of P AOX1 is strictly dependent on methanol as the carbon source. Methanol is derived mainly from petrochemical sources, which is unsuitable for use in the production of certain food products and additives. Methanol metabolism in methylotrophic yeast produces a toxic byproduct, hydrogen peroxide (H 2 O 2 ), which causes oxidative stress and eli...

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The ICL1 gene of Pichia pastoris, transcriptional regulation and use of its promoter

Cited by 56 publications

References 35 publications

Heterologous protein production using the Pichia pastoris expression system

Heterologous protein production using the Pichia pastoris expression system

Carbon source dependent promoters in yeasts

Catabolite Repression of Aox inPichia pastorisIs Dependent on Hexose Transporter PpHxt1 and Pexophagy

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