Inducible overexpression of the FUM1 gene in saccharomyces cerevisiae: Localization of fumarase and efficient fumaric acid bioconversion to L-Malic acid

Peleg, Y; Rokem, J S; Goldberg, I; Pines, O

Title: Inducible overexpression of the FUM1 gene in saccharomyces cerevisiae: Localization of fumarase and efficient fumaric acid bioconversion to L-Malic acid

Full Record
Other Related Research

Abstract

Cloning of the Saccharomyces cerevisiae FUM1 gene downstream of the strong GAL10 promoter resulted in inducible overexpression of fumarase in the yeast. The overproducing strain exhibited efficient bioconversion of fumaric acid to L-malic acid with an apparent conversion value of 88% and a conversion rate of 80.4 mmol of fumaric acid/h per g of cell wet weight, both of which are much higher than parameters known for industrial bacterial strains. The only product of the conversion reaction was L-malic acid, which was essentially free of the unwanted by-product succinic acid. The GAL10 promoter situated upstream of a promoterless FUM1 gene led to production and correct distribution of the two fumarase isoenzyme activities between cytosolic and mitochondrial subcellular fractions. The amino-terminal sequence of fumarase contains the mitochondrial signal sequence since (i) 92 of 463 amino acid residues from the amino terminus of fumarase are sufficient to localize fumarase-lacZ fusions to mitochondria and (ii) fumarase and fumarase-lacZ fusions lacking the amino-terminal sequence are localized exclusively in the cytosol. The possibility that both mitochondrial and cytosolic fumarases are derived from the same initial translation product is discussed.

Authors:

Peleg, Y; Rokem, J S; Goldberg, I; Pines, O ^[1]

The Hebrew Univ., Jerusalem (Israel)

Publication Date:: Sat Sep 01 00:00:00 EDT 1990

OSTI Identifier:: 6273188

Resource Type:: Journal Article

Journal Name:: Applied and Environmental Microbiology; (USA)

Additional Journal Information:: Journal Volume: 56:9; Journal ID: ISSN 0099-2240

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; HYDRO-LYASES; DNA-CLONING; MALIC ACID; BIOSYNTHESIS; BIOCONVERSION; FUMARIC ACID; MOLECULAR BIOLOGY; SACCHAROMYCES CEREVISIAE; CARBON-OXYGEN LYASES; CARBOXYLIC ACIDS; CLONING; DICARBOXYLIC ACIDS; DNA HYBRIDIZATION; ENZYMES; EUMYCOTA; FUNGI; HYBRIDIZATION; HYDROXY ACIDS; LYASES; MICROORGANISMS; ORGANIC ACIDS; ORGANIC COMPOUNDS; PLANTS; SACCHAROMYCES; SYNTHESIS; YEASTS; 550200* - Biochemistry

Citation Formats


                    Peleg, Y, Rokem, J S, Goldberg, I, and Pines, O. Inducible overexpression of the FUM1 gene in saccharomyces cerevisiae: Localization of fumarase and efficient fumaric acid bioconversion to L-Malic acid.  United States: N. p., 1990. 
        Web.

Copy to clipboard


                    Peleg, Y, Rokem, J S, Goldberg, I, & Pines, O. Inducible overexpression of the FUM1 gene in saccharomyces cerevisiae: Localization of fumarase and efficient fumaric acid bioconversion to L-Malic acid.  United States.

Copy to clipboard


                    Peleg, Y, Rokem, J S, Goldberg, I, and Pines, O. 1990.  
        "Inducible overexpression of the FUM1 gene in saccharomyces cerevisiae: Localization of fumarase and efficient fumaric acid bioconversion to L-Malic acid".  United States.

Copy to clipboard


                    
@article{osti_6273188,

  title        = {Inducible overexpression of the FUM1 gene in saccharomyces cerevisiae: Localization of fumarase and efficient fumaric acid bioconversion to L-Malic acid},

  author       = {Peleg, Y and Rokem, J S and Goldberg, I and Pines, O},

  abstractNote = {Cloning of the Saccharomyces cerevisiae FUM1 gene downstream of the strong GAL10 promoter resulted in inducible overexpression of fumarase in the yeast. The overproducing strain exhibited efficient bioconversion of fumaric acid to L-malic acid with an apparent conversion value of 88% and a conversion rate of 80.4 mmol of fumaric acid/h per g of cell wet weight, both of which are much higher than parameters known for industrial bacterial strains. The only product of the conversion reaction was L-malic acid, which was essentially free of the unwanted by-product succinic acid. The GAL10 promoter situated upstream of a promoterless FUM1 gene led to production and correct distribution of the two fumarase isoenzyme activities between cytosolic and mitochondrial subcellular fractions. The amino-terminal sequence of fumarase contains the mitochondrial signal sequence since (i) 92 of 463 amino acid residues from the amino terminus of fumarase are sufficient to localize fumarase-lacZ fusions to mitochondria and (ii) fumarase and fumarase-lacZ fusions lacking the amino-terminal sequence are localized exclusively in the cytosol. The possibility that both mitochondrial and cytosolic fumarases are derived from the same initial translation product is discussed.},

  doi          = {},

  url          = {https://www.osti.gov/biblio/6273188},
  journal      = {Applied and Environmental Microbiology; (USA)},

  issn         = {0099-2240},

  number       = ,

  volume       = 56:9,

  place        = {United States},

  year         = {Sat Sep 01 00:00:00 EDT 1990},

  month        = {Sat Sep 01 00:00:00 EDT 1990}

}

Copy to clipboard

Journal Article:

Other availability

Find in Google Scholar

Search WorldCat to find libraries that may hold this journal

Save / Share:

Export Metadata

Save to My Library

Similar records in OSTI.GOV collections:

Fatal mitochondrial encephalopathy caused by fumarase deficiency: A molecular-genetic study

Journal Article Gellera, C; Cavadini, P; Baratta, S - American Journal of Human Genetics

Fumarase deficiency is a rare autosomal recessive disorder of the citric acid cycle resulting in severe organic aciduria and encephalopathy. Mammalian cells contain two fumarase isoenzymes, one mitochondrial and one cytosolic. In rat, the two proteins are encoded by the same gene and are synthesized by alternative initiation of translation at two in-phase AUG codons. One single fumarase gene locus has been identified on human chromosome 1. In most of the patients so far described, the activities of both isozymes are severely affected, suggesting that mutations within a single gene may underlie the disease. Here, we report the molecular studymore »« less
Mitochondrial import of human and yeast fumarase in live mammalian cells: Retrograde translocation of the yeast enzyme is mainly caused by its poor targeting sequence

Journal Article Singh, Bhag; Gupta, Radhey - Biochemical and Biophysical Research Communications

Studies on yeast fumarase provide the main evidence for dual localization of a protein in mitochondria and cytosol by means of retrograde translocation. We have examined the subcellular targeting of yeast and human fumarase in live cells to identify factors responsible for this. The cDNAs for mature yeast or human fumarase were fused to the gene for enhanced green fluorescent protein (eGFP) and they contained, at their N-terminus, a mitochondrial targeting sequence (MTS) derived from either yeast fumarase, human fumarase, or cytochrome c oxidase subunit VIII (COX) protein. Two nuclear localization sequences (2x NLS) were also added to these constructsmore »« less
https://doi.org/10.1016/j.bbrc.2006.05.188
Absolute configuration of a chiral CHD group via neutron diffraction: confirmation of the absolute stereochemistry of the enzymatic formation of malic acid

Journal Article Bau, R; Brewer, I; Chiang, M; ... - Biochem. Biophys. Res. Commun.; (United States)

Neutron diffraction has been used to monitor the absolute stereochemistry of an enzymatic reaction. (-)(2S)malic-3-d acid was prepared by the action of fumarase on fumaric acid in D/sub 2/O. After a large number of cations were screened, it was found that (+)(R)..cap alpha..-phenylethylamine forms the large crystals necessary for a neutron diffraction analysis. The subsequent structure determination showed that (+)(R)..cap alpha..-phenylethylammonium (-)(2S)malate-3-d has an absolute configuration of R at the CHD site. This result confirms the absolute stereochemistry of fumarate-to-malate transformation as catalyzed by the enzyme fumarase.
https://doi.org/10.1016/S0006-291X(83)80041-2
Production of succinic acid through overexpression of NAD{sup +}-dependent malic enzyme in an Escherichia coli mutant

Journal Article Stols, L; Donnelly, M - Applied and Environmental Microbiology

NAD{sup +}-dependent malic enzyme was cloned from the Escherichia coli genome by PCR based on the published partial sequence of the gene. The enzyme was overexpressed and purified to near homogeneity in two chromatographic steps and was analyzed kinetically in the forward and reverse directions. The K{sub m} values determined in the presence of saturating cofactor and manganese ion were 0.26 mM for malate (physiological direction) and 16 mM for pyruvate (reverse direction). When malic enzyme was induced under appropriate culture conditions in a strain of E. coli that was unable to ferment glucose and accumulated pyruvate, fermentative metabolism ofmore »« less
Isolation, structure, and characterization of the RAD3 gene of the yeast

Thesis/Dissertation Higgins, D

In Saccharomyces cerevisiae excision of UV-induced pyrimidine dimers from the DNA involves at least 10 genes. In this work, the RAD3 gene has been cloned, its nucleotide sequence determined, and some structural and functional features examined. The RAD3 gene codes for an mRNA of 2.5 kb. The RAD3 open reading frame is 2334 nucleotides long with an encoded protein of 89,779 daltons. Genomic deletions of the RAD3 gene are recessive lethal, demonstrating that it is an essential gene and suggesting that the RAD3 gene product plays a vital role in the cell in addition to its role in excision repair.more »« less

Similar Records