RESUMO
The objective of this work is to demonstrate if the hexaprenyl pyrophosphate synthetase Coq1p might be involved in monoterpenes synthesis in Saccharomyces cerevisiae, although its currently known function in yeast is to catalyze the first step in ubiquinone biosynthesis. However, in a BY4743 laboratory strain, the presence of an empty plasmid in a chemically defined grape juice medium results in a statistically significant increase of linalool, (E)-nerolidol and (E,E)-farnesol. When COQ1 is overexpressed from a plasmid, the levels of the volatile isoprenoids are further increased. Furthermore, overexpression of COQ1 in the same genetic context but with a mutated farnesyl pyrophosphate synthetase (erg20 mutation K197E), results in statistically significant higher levels of linalool (above 750⯵g/L), geraniol, α-terpineol, and the sesquiterpenes, farnesol and nerolidol (total concentration of volatile isoprenoids surpasses 1300⯵g/L). We show that the levels of monoterpenes and sesquiterpenes that S. cerevisiae can produce, in the absence of plant genes, depend on the composition of the medium and the genetic context. To the best of our knowledge, this is the highest level of linalool produced by S. cerevisiae up to now. Further research will be needed for understanding how COQ1 and the medium composition might interact to increase flavor complexity of fermented beverages.
Assuntos
Saccharomyces cerevisiae/metabolismo , Terpenos/metabolismo , Vitis/microbiologia , Monoterpenos Acíclicos , Geraniltranstransferase/genética , Geraniltranstransferase/metabolismo , Monoterpenos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Both prokaryotes and eukaryotes respond to a decrease in temperature with the expression of a specific subset of proteins. Although a large body of information concerning cold shock-induced genes has been gathered, studies on temperature regulation have not clearly identified the key regulatory factor(s) responsible for thermosensing and signal transduction at low temperatures. Here we identified a two-component signal transduction system composed of a sensor kinase, DesK, and a response regulator, DesR, responsible for cold induction of the des gene coding for the Delta5-lipid desaturase from Bacillus subtilis. We found that DesR binds to a DNA sequence extending from position -28 to -77 relative to the start site of the temperature-regulated des gene. We show further that unsaturated fatty acids (UFAs), the products of the Delta5-desaturase, act as negative signalling molecules of des transcription. Thus, a regulatory loop composed of the DesK-DesR two-component signal transduction system and UFAs provides a novel mechanism for the control of gene expression at low temperatures.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Ligação a DNA/fisiologia , Ácidos Graxos Dessaturases/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Proteínas de Neoplasias , Proteínas Nucleares/fisiologia , Proteínas Quinases/genética , Transdução de Sinais/fisiologia , Fusão Gênica Artificial , Bacillus subtilis/genética , Bacillus subtilis/fisiologia , Sequência de Bases , Temperatura Baixa , DNA Bacteriano/metabolismo , Ácidos Graxos Insaturados/metabolismo , Genes Bacterianos , Histidina Quinase , Óperon Lac , Dados de Sequência Molecular , Mutagênese , Óperon , Regiões Promotoras Genéticas , Proteínas Quinases/metabolismo , RNA Bacteriano/biossíntese , RNA Mensageiro/biossíntese , Proteínas Repressoras , Fatores de Transcrição , Ativação Transcricional , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The Bacillus subtilis des gene encodes the cold-inducible Delta5 lipid desaturase involved in the formation of unsaturated fatty acids from saturated phospholipid precursors. Here, we describe the expression pattern of the des gene in response to a temperature downshift from 37 to 20 degrees C. We found that the synthesis of des mRNA is undetectable at 37 degrees C but dramatically induced upon the temperature downshift. Decay characteristics of the des transcript as well as the in vivo decay of B. subtilis bulk mRNA were investigated. The results showed that the stability of the des transcript as well as of bulk mRNA lasted substantially longer at 20 degrees C than at 37 degrees C. Functional expression of des at 37 degrees C was achieved by exchanging its promoter with the non-cold shock spac promoter. These data provide the first direct evidence that temperature-mediated control of transcription is the major mechanism regulating the mRNA levels of the B. subtilis desaturase. The present results also demonstrate that the only component of the desaturation system regulated by temperature is the desaturase enzyme.
Assuntos
Bacillus subtilis/genética , Temperatura Baixa , Ácidos Graxos Dessaturases/genética , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Bacillus subtilis/enzimologia , Dessaturase de Ácido Graxo Delta-5 , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/análise , Plasmídeos , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
Bacillus subtilis grown at 37 degrees C synthesizes saturated fatty acids with only traces of unsaturated fatty acids (UFAs). However, when cultures growing at 37 degrees C are transferred to 20 degrees C, UFA synthesis is induced. We report the identification and characterization of the gene encoding the fatty acid desaturase of B. subtilis. This gene, called des, was isolated by complementation of Escherichia coli strains with mutations in either of two different genes of UFA synthesis. The des gene encodes a polypeptide of 352 amino acid residues containing the three conserved histidine cluster motifs and two putative membrane-spanning domains characteristic of the membrane-bound desaturases of plants and cyanobacteria. Expression of the des gene in E. coli resulted in desaturation of palmitic acid moieties of the membrane phospholipids to give the novel mono-UFA cis-5-hexadecenoic acid, indicating that the B. subtilis des gene product is a delta5 acyl-lipid desaturase. The des gene was disrupted, and the resulting null mutant strains were unable to synthesize UFAs upon a shift to low growth temperatures. The des null mutant strain grew as well as its congenic parent at 20 or 37 degrees C but showed severely reduced survival during stationary phase. Analysis of operon fusions in which the des promoter directed the synthesis of a lacZ reporter gene showed that des expression is repressed at 37 degrees C, but a shift of cultures from 37 to 20 degrees C resulted in a 10- to 15-fold increase in transcription. This is the first report of a membrane phospholipid desaturase in a nonphotosynthetic organism and the first direct evidence for cold induction of a desaturase.