Conversion of Starch to Ethanol in a Recombinant Saccharomyces cerevisiae Strain Expressing Rice α-Amylase from a Novel Pichia pastoris Alcohol Oxidase Promoter

Kumagai, Monto H.; Sverlow, Genadie G.; della-Cioppa, Guy; Grill, Laurence K.

doi:10.1038/nbt0593-606

Cited by 13 publications

(11 citation statements)

References 40 publications

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“…Of the two MREs, MRE1 exhibited strong response to methanol compared to MRE2. To date, several consensus sequences have been reported to be conserved in promoter regions of methanol-metabolizing enzyme genes from P. pastoris, H. polymorpha, and C. boidinii (7,12). Recently, the sequence responsible for methanol or both methanol and formate in C. boidnii P FDH1 were identified (6).…”

Section: Discussionmentioning

confidence: 99%

Trm1p, a Zn(II) ₂ Cys ₆ -Type Transcription Factor, Is a Master Regulator of Methanol-Specific Gene Activation in the Methylotrophic Yeast Candida boidinii

et al. 2008

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The methylotrophic yeasts are commonly used as hosts for heterologous gene expression. In this study, we describe a novel gene, TRM1, in Candida boidinii, responsible for the transcriptional activation of several methanol-inducible promoters. The encoded protein, Trm1p, is a Zn(II) 2 Cys 6 -type zinc cluster protein. Deletion of TRM1 completely inhibits growth on methanol but causes no growth defect on glucose or other nonfermentative carbon sources, glycerol, ethanol, or oleate. Trm1p is responsible for transcriptional activation of five methanol-inducible promoters tested, but not for peroxisome assembly or peroxisomal protein transport. Expression of the TRM1 gene was constitutive, and Trm1p localizes to the nuclei regardless of the carbon source. Two cis-acting methanol response elements (MREs), MRE1 and MRE2 are present in the promoter of the dihydroxyacetone synthase gene. Trm1p is shown to be required for MRE1-dependent methanol-inducible gene expression. Chromatin immunoprecipitation assays reveal that Trm1p binds to five methanol-inducible promoters upon methanol induction but does not bind in glucose-grown cells. Thus, the TRM1 gene encodes a master transcriptional regulator responsible for methanol-specific gene activation in the methylotrophic yeasts.Methylotrophic yeasts, which can utilize methanol as the sole carbon and energy source, have been used as the hosts for production of a wide variety of heterologous proteins and also as a model organism for studies of peroxisome biogenesis and degradation (4,14,19). To date, heterologous expression systems have been established in Candida boidinii, Hansenula polymorpha (Pichia angusta), Pichia methanolica, and Pichia pastoris (2,4,15,22). With these expression systems, a large number of useful proteins have been produced, e.g., enzymes, antibodies, cytokines, plasma proteins, and hormones (4). The unique feature of these systems is that heterologous gene expression can be driven by methanol-inducible promoters, allowing production of the target protein to be tightly regulated; this is particularly important when the desired protein is toxic to the host cell. The molecular mechanism of methanol-inducible gene expression, however, has yet to be elucidated.When methylotrophic yeasts grow on methanol as the sole carbon source, peroxisomes massively proliferate and can occupy up to 80% of the intracellular volume. Methanol-induced peroxisomes contain methanol-metabolizing enzymes, alcohol oxidase (AOD), dihydroxyacetone synthase (DAS), and catalase. AOD catalyzes the oxidation of methanol to form formaldehyde and hydrogen peroxide. Formaldehyde is situated at the branching point of the assimilation and dissimilation pathways. DAS catalyzes the first reaction of the assimilation pathway by fixing formaldehyde with xylulose-5-phosphate. Alternatively, formaldehyde can be oxidized to carbon dioxide by the glutathione-dependent formaldehyde oxidation pathway, catalyzed by formaldehyde dehydrogenase, S-formylglutathione hydrolase, and formate dehydrogenase (FDH) i...

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Section: Discussionmentioning

confidence: 99%

Trm1p, a Zn(II) ₂ Cys ₆ -Type Transcription Factor, Is a Master Regulator of Methanol-Specific Gene Activation in the Methylotrophic Yeast Candida boidinii

et al. 2008

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“…For differential centrifugation experiments, Western blots, and enzyme assays, protein concentrations were determined using the Pierce (Rockford, IL) bicinchoninic acid protein assay kit with bovine serum albumin as a standard. Cytosolic and pellet fractions containing 25 g of protein from differential centrifugations as well as total cellular protein from extracts were loaded onto stacked sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels for electrophoresis (29). Proteins were then transferred onto nitrocellulose using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad, Hercules, CA) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…Relative specific activities were indicated as a percentage of the specific activity of the wild-type (WT) strain harboring the pHWG0 vector on methanol induction medium. AOX2, region in P. pastoris alcohol oxidase II promoter hypothesized by Ohi et al (35) to be a methanol-regulatory region; K, sequence identified by Kumagai et al (29) in several promoters that is hypothesized to be a methanol-regulatory region.…”

Section: Fig 2 Mxr1 Is Necessary For Normal Accumulation Of Peroxinmentioning

confidence: 99%

“…6A) that were identical except that each contained an AOX1 upstream fragment with a different 5Ј deletion end point. Deletion end points were selected based on homology between AOX1 sequences and defined cis-acting sequences in yeast genes that are modulated by a carbon source (29,35). Each AOX1 upstream fragment was inserted into the vector and positioned in front of the bacterial bla gene so that the transcriptional activity could be evaluated by measuring the level of ␤-lactamase activity present.…”

Section: Fig 2 Mxr1 Is Necessary For Normal Accumulation Of Peroxinmentioning

confidence: 99%

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Mxr1p, a Key Regulator of the Methanol Utilization Pathway and Peroxisomal Genes in Pichia pastoris

Lin-Cereghino

Godfrey

Cruz

et al. 2006

Molecular and Cellular Biology

141

192

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Growth of the yeast Pichia pastoris on methanol induces the expression of genes whose products are required for its metabolism. Three of the methanol pathway enzymes are located in an organelle called the peroxisome. As a result, both methanol pathway enzymes and proteins involved in peroxisome biogenesis (PEX proteins) are induced in response to this substrate. The most highly regulated of these genes is AOX1, which encodes alcohol oxidase, the first enzyme of the methanol pathway, and a peroxisomal enzyme. To elucidate the molecular mechanisms responsible for methanol regulation, we identify genes required for the expression of AOX1. Mutations in one gene, named MXR1 (methanol expression regulator 1), result in strains that are unable to (i) grow on the peroxisomal substrates methanol and oleic acid, (ii) induce the transcription of AOX1 and other methanol pathway and PEX genes, and (iii) form normal-appearing peroxisomes in response to methanol. MXR1 encodes a large protein with a zinc finger DNA-binding domain near its N terminus that has similarity to Saccharomyces cerevisiae Adr1p. In addition, Mxr1p is localized to the nucleus in cells grown on methanol or other gluconeogenic substrates. Finally, Mxr1p specifically binds to sequences upstream of AOX1. We conclude that Mxr1p is a transcription factor that is necessary for the activation of many genes in response to methanol. We propose that MXR1 is the P. pastoris homologue of S. cerevisiae ADR1 but that it has gained new functions and lost others through evolution as a result of changes in the spectrum of genes that it controls.The ability to utilize methanol as a carbon and energy source is limited in eukaryotes to a few yeast species (1,34,57). The metabolic pathway is nearly identical in each species and begins with the oxidation of methanol to formaldehyde, which is catalyzed by the peroxisomal matrix enzyme alcohol oxidase (Aox). A by-product of this reaction is hydrogen peroxide, which is subsequently degraded to water and oxygen by a second peroxisomal enzyme catalase (Cat). The formaldehyde generated by Aox follows one of two paths. A portion leaves the peroxisome and is further oxidized by two cytoplasmic enzymes, formaldehyde dehydrogenase (Fld) and formate dehydrogenase (Fdh), to generate energy for the cell. The remaining formaldehyde is condensed with xylulose-5-phosphate by a third peroxisomal enzyme, dihydroxyacetone synthase (Dhas), to generate two three-carbon molecules that leave the peroxisome and enter a cyclic pathway that regenerates xylulose-5-phosphate and produces one net molecule of glyceraldehyde-3-phosphate for every three turns of this cycle (1, 57).Because three of the methanol pathway enzymes (Aox, Cat, and Dhas) are peroxisomal, the function of this organelle is also essential for methanol growth (21,26,33). This observation has made Pichia pastoris a major model system for the elucidation of peroxisome biogenesis and function (2,40,49). One advantage of P. pastoris for peroxisome studies is that in addition to methanol uti...

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“…[4][5][6] This system contains 5?-AOX, a 942-bp fragment containing the AOX 1 promoter that allows methanol-inducible and high-level expression in P. pastoris. In addition, P. pastoris utilizes most of the post-translational modiˆcation pathway typically associated with higher eukaryotes.…”

mentioning

confidence: 99%

Functional Properties of Glycosylated Lysozyme Secreted inPichia pastoris

Saito¹,

Sako²,

Usui³

et al. 2003

Bioscience, Biotechnology, and Biochemistry

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Conversion of Starch to Ethanol in a Recombinant Saccharomyces cerevisiae Strain Expressing Rice α-Amylase from a Novel Pichia pastoris Alcohol Oxidase Promoter

Cited by 13 publications

References 40 publications

Trm1p, a Zn(II) ₂ Cys ₆ -Type Transcription Factor, Is a Master Regulator of Methanol-Specific Gene Activation in the Methylotrophic Yeast Candida boidinii

Trm1p, a Zn(II) ₂ Cys ₆ -Type Transcription Factor, Is a Master Regulator of Methanol-Specific Gene Activation in the Methylotrophic Yeast Candida boidinii

Mxr1p, a Key Regulator of the Methanol Utilization Pathway and Peroxisomal Genes in Pichia pastoris

Functional Properties of Glycosylated Lysozyme Secreted inPichia pastoris

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Conversion of Starch to Ethanol in a Recombinant Saccharomyces cerevisiae Strain Expressing Rice α-Amylase from a Novel Pichia pastoris Alcohol Oxidase Promoter

Cited by 13 publications

References 40 publications

Trm1p, a Zn(II) 2 Cys 6 -Type Transcription Factor, Is a Master Regulator of Methanol-Specific Gene Activation in the Methylotrophic Yeast Candida boidinii

Trm1p, a Zn(II) 2 Cys 6 -Type Transcription Factor, Is a Master Regulator of Methanol-Specific Gene Activation in the Methylotrophic Yeast Candida boidinii

Mxr1p, a Key Regulator of the Methanol Utilization Pathway and Peroxisomal Genes in Pichia pastoris

Functional Properties of Glycosylated Lysozyme Secreted inPichia pastoris

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Trm1p, a Zn(II) ₂ Cys ₆ -Type Transcription Factor, Is a Master Regulator of Methanol-Specific Gene Activation in the Methylotrophic Yeast Candida boidinii

Trm1p, a Zn(II) ₂ Cys ₆ -Type Transcription Factor, Is a Master Regulator of Methanol-Specific Gene Activation in the Methylotrophic Yeast Candida boidinii