ACR1, a gene encoding a protein related to mitochondrial carriers, is essential for acetyl-CoA synthetase activity in Saccharomyces cerevisiae

The utilization of ethanol via acetate by the yeast Saccharomyces cerevisiae requires the presence of the enzyme acetyl-coenzyme A synthetase (acetyl-CoA synthetase), which catalyzes the activation of acetate to acetyl-coenzyme A (acetyl-CoA). We have isolated a mutant, termed acr1, defective for this activity by screening for mutants unable to utilize ethanol as a sole carbon source. Genetic and biochemical characterization show that, in this mutant, the structural gene for acetyl-CoA synthetase is not affected. Cloning and sequencing demonstrated that the ACR1 gene encodes a protein of 321 amino acids with a molecular mass of 35 370 Da. Computer analysis suggested that the ACR1 gene product (ACR1) is an integral membrane protein related to the family of mitochondrial carriers. The expression of the gene is induced by growing yeast cells in media containing ethanol or acetate as sole carbon sources and is repressed by glucose. ACR1 is essential for the utilization of ethanol and acetate since a mutant carrying a disruption in this gene is unable to grow on these compounds.     

ACR1, a gene encoding a protein related to mitochondrial carriers,is essential for acetyl-CoA synthetase activity in Saccharomyces cerevisiae

The utilization of ethanol via acetate by the yeast Saccharomyces cerevisiae requires the presence of the enzyme acetyl-coenzyme A synthetase (acetyl-CoA synthetase), which catalyzes the activation of acetate to acetyl-coenzyme A (acetyl-CoA). We have isolated a mutant, termed acr1, defective for this activity by screening for mutants unable to utilize ethanol as a sole carbon source. Genetic and biochemical characterization show that, in this mutant, the structural gene for acetyl-CoA synthetase is not affected. Cloning and sequencing demonstrated that the ACR1 gene encodes a protein of 321 amino acids with a molecular mass of 35 370 Da. Computer analysis suggested that the ACR1 gene product (ACR1) is an integral membrane protein related to the family of mitochondrial carriers. The expression of the gene is induced by growing yeast cells in media containing ethanol or acetate as sole carbon sources and is repressed by glucose. ACR1 is essential for the utilization of ethanol and acetate since a mutant carrying a disruption in this gene is unable to grow on these compounds.     

The Neurospora crassa cfp promoter drives a carbon source-dependent expression of transgenes in filamentous fungi

AIMS: The objective of the present study was to determine the potential of promoter sequences from the cfp gene of Neurospora crassa to drive the expression of transgenes in filamentous fungi. METHODS AND RESULTS: Northern blot analyses showed that the mRNA levels of cfp were rapidly modified in response to either inducing or repressing culture conditions. The hygromycin phosphotransferase (hph) and S-adenosylmethionine synthetase (eth-1) genes were fused to a minimal cfp promoter fragment (Pcfp) and used as reporter genes. These constructs were highly expressed in transformant N. crassa strains grown in media containing glucose or sucrose and repressed in media containing ethanol or ethanol plus glucose. A gene fusion of the cfp promoter to the beta-glucuronidase gene (cfp-uidA) showed identical patterns of expression in the heterologous filamentous fungus Aspergillus nidulans. CONCLUSIONS: Our results show that the levels of expression of the native cfp gene, as well as reporter genes driven by cfp promoter sequences, can be rapidly modified in response to different carbon sources. These modified levels of expression are maintained by continuous growth in the presence of the corresponding carbon source. SIGNIFICANCE AND IMPACT OF THE STUDY: We propose that the cfp promoter can be used to control the expression of transgenes in filamentous fungi in a carbon source-dependent fashion.     

Ethanol-inducible gene expression using gld1 + promoter in the fission yeast Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, the gld1 ⁺ gene encoding glycerol dehydrogenase is repressed by glucose and induced by ethanol and 1-propanol. The promoter region of gld1 ⁺ was cloned into a multicopy vector designated as pEG1 for evaluation as an ethanol-inducible expression vector using EGFP as a model heterologous protein. Expression of EGFP was repressed in the presence of high glucose and induced in the presence of ethanol, low-glucose, and 1-propanol in the absence of glucose. Addition of ethanol to cells harboring pEG1–EGFP was found to be the most effective means for inducing EGFP production. Protein yields were found to increase in proportion to ethanol concentration. As a further test of effectiveness, secreted recombinant human growth hormone was produced using the pEG1 expression vector in medium containing glycerol and ethanol. The pEG1 gene expression system is an effective tool for the production of heterologous proteins under glucose-limiting conditions, including medium containing glycerol as a carbon source.     

Promoter analysis of the barley Pht1;1 phosphate transporter gene identifies regions controlling root expression and responsiveness to phosphate deprivation

Previous studies have shown that the promoter from the barley (Hordeum vulgare) phosphate transporter gene, HvPht1;1, activates high levels of expression in rice (Oryza sativa) roots and that the expression level was induced by up to 4-fold in response to phosphorus (P) deprivation. To identify promoter regions controlling gene regulation specificities, successive promoter truncations were made and attached to reporter genes. Promoters of between 856 and 1,400 nucleotides activated gene expression in a number of cell types but with maximal expression in trichoblast (root hair) cells. For shorter promoters the trichoblast specificity was lost, but in other tissues the distribution pattern was unchanged. The low P induction response was unaffected by promoter length. Domain exchange experiments subsequently identified that the region between -856 and -547 nucleotides (relative to the translational start) is required for epidermal cell expression. A second region located between 0 and -195 nucleotides controls root-tip expression. The HvPht1;1 promoter contains one PHO-like motif and three motifs similar to the dicot P1BS element. Analysis of promoters from which the PHO-like element was eliminated (by truncation) showed no change in the gene induction response to P deficiency. In contrast, mutation of the P1BS elements eliminated any induction of gene expression in response to low P. An internal HvPht1;1 promoter fragment, incorporating a single P1BS element, had an increased response to P deprivation in comparison with the unmodified promoter (containing three elements). Together these findings further our understanding of the regulation of the HvPht1;1 gene and provide direct evidence for a functional role of the P1BS element in the expression of P-regulated genes.