Mutants partially deficient in alcohol dehydrogenase in Schizosaccharomyces pombe

Megnet, Roland

doi:10.1016/0003-9861(67)90024-0

Cited by 48 publications

(20 citation statements)

References 2 publications

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“…Colonies growing on this medium do not oxidize allyl alcohol as the product, acreolein, is toxic for the yeast and prevents the wildtype strain to grow (Megnet, 1967). With glucose as substrate adc-mutants are obtained and under derepressed conditions in the presence of glycerol it is possible to isolate adr-mutants.…”

Section: Resultsmentioning

confidence: 98%

Enhanced production of glycerol in an alcohol dehydrogenase (ADH I) deficient mutant of Saccharomyces cerevisiae

Johansson¹,

Sjöström²

1984

Biotechnol Lett

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Section: Resultsmentioning

confidence: 98%

Enhanced production of glycerol in an alcohol dehydrogenase (ADH I) deficient mutant of Saccharomyces cerevisiae

Johansson¹,

Sjöström²

1984

Biotechnol Lett

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“…ADH-deficient mutants were obtained by positive selection on YPG agar medium supplemented with allyl alcohol (0.002%). Allyl aldehyde formed from allyl alcohol by oxidation by ADH was lethal to the cells (14). Allyl alcohol-resistant colonies were randomly selected, and the enzyme activities of the mutants were examined.…”

Section: Methodsmentioning

confidence: 99%

Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus

1995

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The membrane-bound alcohol dehydrogenase (ADH) of Acetobacter pasteurianus NCI1452 consists of three different subunits, a 78-kDa dehydrogenase subunit, a 48-kDa cytochrome c subunit, and a 20-kDa subunit of unknown function. For elucidation of the function of the smallest subunit, this gene was cloned from this strain by the oligonucleotide-probing method, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequence and the NH 2 -terminal sequence determined for the purified protein indicated that the smallest subunit contained a typical signal peptide of 28 amino acids, as did the larger two subunits. This gene complemented the ADH activity of a mutant strain which had lost the smallest subunit. Disruption of this gene on the chromosome resulted in loss of ADH activity in Acetobacter aceti, indicating that the smallest subunit was essential for ADH activity. Immunoblot analyses of cell lysates prepared from various ADH mutants suggested that the smallest subunit was concerned with the stability of the 78-kDa subunit and functioned as a molecular coupler of the 78-kDa subunit to the 48-kDa subunit on the cytoplasmic membrane.Acetic acid fermentation, in which ethanol is oxidized to acetic acid by acetic acid bacteria, is the most characteristic process in vinegar production (10). This ethanol oxidation is catalyzed by two membrane-bound enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) (1-3, 6, 11, 27). The ADHs of Acetobacter spp., a genus of acetic acid bacteria (10), are classified into two types, two component and three component. The former in Acetobacter polyoxogenes consists of a 72-to 78-kDa dehydrogenase subunit (subunit I) and a 44-to 48-kDa cytochrome c subunit (subunit II), and the latter type in Acetobacter aceti and Acetobacter pasteurianus has one more subunit, a 20-kDa subunit of unknown function (subunit III). We recently purified both types of ADHs and cloned the genes encoding the larger two subunits of both types of ADHs. These studies revealed that both subunit I and subunit II were essential for ADH activity (25-27). The genes encoding subunits I and II were clustered (25, 26), but the gene encoding subunit III was not present close to the cluster (25). It had thus been postulated that only the two larger components were required for ADH activity.In subsequent investigations, we found that some spontaneous mutants of A. pasteurianus NCI1452 which lost subunit III showed an ADH activity-deficient phenotype. We therefore began to study how subunit III of the three-component ADH participates in the enzymatic activity and to clarify its function. In the present study, we cloned the gene encoding subunit III from A. pasteurianus NCI1452 and analyzed various ethanol oxidation-defective mutants derived from the parental strain genetically and immunologically. This paper provides evidence that this subunit is indispensable for ADH activity. The function of subunit III in the three-component-type ADH is also discussed. MATERIALS AND METHODSBacteria...

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“…ADHs are usually readily stained in gels (Megnet, 1967). We isoelectrofocused cell-free extracts of the wildtype M. extorqzlens AM1, two representatives of a d b A mutants, ADH70 and ADH76, and ADH70 carrying pLC31O.SS (the 13.5 kb Hind111 fragment cloned into pRK31O), in which the a d h A product should be overexpressed.…”

Section: Search For Adh Activitymentioning

confidence: 99%

Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate in the isocitrate-lyase-negative methylotroph Methylobacterium extorquens AM1

Chistoserdova

Lidstrom

1996

Microbiology

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A region on the Methylobacterium extorquens AM1 chromosome previously shown to complement a chemically induced mutant (PCT48) unable to convert acetyl-CoA into glyoxylate was characterized in detail in order to identify the gene(s) involved in the unknown pathway for acetyl-CoA oxidation. Six complete and two partial ORFs were identified by sequencing. Sequence comparisons suggested these might code for, respectively, a dehydrogenase of unknown specificity, a polypeptide of at least 15 kDa with unknown function, a coenzyme-B,,-linked mutase, a catalase, an alcohol dehydrogenase (ADH) of unknown function, a polypeptide of 28 kDa, a ketol-acid reductoisomerase and a propionyl-CoA carboxylase (PCC), Insertion mutations were introduced into each ORF in order to determine their involvement in C, and C, metabolism. Mutations in three genes, encoding the mutase, ADH and PCC, resulted in a phenotype characteristic of mutants unable to oxidize acetyl-CoA, i.e. they were C, -and C,-negative and their growth on these compounds was restored by the addition of glycolate or glyoxylate. Mutants in the genes thought to encode catalase and PCC were found to be deficient in the corresponding enzyme activity, confirming the identity of these genes, while physiological substrates for the mutase and ADH remain unidentified. This work, in which three new genes necessary for conversion of acetyl-CoA into glyoxylate were identified, is an intermediary step on the way to the solution of the unknown pathway for acetyl-CoA oxidation in isocitrate-lyase-negative methylotrophs.

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Mutants partially deficient in alcohol dehydrogenase in Schizosaccharomyces pombe

Cited by 48 publications

References 2 publications

Enhanced production of glycerol in an alcohol dehydrogenase (ADH I) deficient mutant of Saccharomyces cerevisiae

Enhanced production of glycerol in an alcohol dehydrogenase (ADH I) deficient mutant of Saccharomyces cerevisiae

Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus

Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate in the isocitrate-lyase-negative methylotroph Methylobacterium extorquens AM1

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