RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis

Flores

2016

Microbiol Mol Biol Rev

SUMMARYMoonlighting proteins are multifunctional proteins that participate in unrelated biological processes and that are not the result of gene fusion. A certain number of these proteins have been characterized in yeasts, and the easy genetic manipulation of these microorganisms has been useful for a thorough analysis of some cases of moonlighting. As the awareness of the moonlighting phenomenon has increased, a growing number of these proteins are being uncovered. In this review, we present a crop of newly identified moonlighting proteins from yeasts and discuss the experimental evidence that qualifies them to be classified as such. The variety of moonlighting functions encompassed by the proteins considered extends from control of transcription to DNA repair or binding to plasminogen. We also discuss several questions pertaining to the moonlighting condition in general. The cases presented show that yeasts are important organisms to be used as tools to understand different aspects of moonlighting proteins.

Section: Pyruvate Decarboxylase Regulates the Transcription Of Its Enmentioning

confidence: 93%

The Expanding Landscape of Moonlighting Proteins in Yeasts

Flores

2016

Microbiol Mol Biol Rev

“…The well-documented influence of several positive effectors upon KlPDC1 expression (glucose metabolism [2,17,24], hypoxia [5], and the presence of several putative consensus sequences for transcription factors [5]) suggests that the promoter activity could be the result of different stimuli which might not necessarily be synergic. In order to get rid of interfering regulations and to identify a promoter sequence specifically responding to hypoxia, we decided to assay a panel of promoter sequences, generated by enzymatic digestions and/or PCR amplification of the KlPDC1 promoter sequence, as hypoxic mediators of lacZ gene expression.…”

Section: Production Of Recombinant C-33 Protein From Hepatitis C Virumentioning

confidence: 99%

Induction by Hypoxia of Heterologous-Protein Production with the Kl PDC1 Promoter in Yeasts

Camattari

Bianchi

Branduardi

et al. 2007

Appl Environ Microbiol

The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the KlPDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1␤ was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The lacZ gene from Escherichia coli was cloned downstream of the KlPDC1 promoter in order to get ␤-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii, and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii; thus, this promotor constitutes a possible inducible system for this new nonconventional host.

“…In contrast, in the Crabtree-negative yeast Kluyveromyces lactis both respiratory and fermentative pathways co-exist during growth on glucose (De Deken, 1966;Breunig et al, 2000), although respiration appears to be dispensable since antimycin A (AA) does not inhibit growth on glucose (Rag + phenotype) (Goffrini et al, 1989; Wésolowski-Louvel et al, 1992a). Sensitivity to AA on glucose (Rag -) is associated with mutations in genes encoding glycolytic enzymes, glucose transporters and sensing components, as well as their regulatory factors (Goffrini et al, 1991; Wésolowski-Louvel et al, 1992b;Prior et al, 1993; Bianchi et al, 1996;Prior et al, 1996;Blaisonneau et al, 1997;Betina et al, 2001; Lemaire & Wésolowski-Louvel, 2004; Hnatova et al, 2008).During the characterization of the putative cell cycle gene KlHSL1 in K. lactis, we found that its deletion led to a Rag -phenotype, suggesting involvement in glucose metabolism. To identify the relationship between RAG and KlHSL1, rag and Klhsl1D mutant strains were further analysed.…”

mentioning

confidence: 99%

KlHsl1 is a component of glycerol response pathways in the milk yeast Kluyveromyces lactis

et al. 2011

In Saccharomyces cerevisiae, HSL1 (NIK1) encodes a serine-threonine protein kinase involved in cell cycle control and morphogenesis. Deletion of its putative orthologue in Kluyveromyces lactis, KlHSL1, gives rise to sensitivity to the respiratory inhibitor antimycin A (AA). Resistance to AA on glucose (Rag + phenotype) is associated with genes (RAG) required for glucose metabolism/ glycolysis. To understand the relationship between RAG and KlHSL1, rag and Klhsl1D mutant strains were investigated. The analysis showed that all the mutants contained a phosphorylated form of Hog1 and displayed an inability to synthesize/accumulate glycerol as a compatible solute. In addition, rag mutants also showed alterations in both cell wall and membrane fatty acids. The pleiotropic defects of these strains indicate that a common pathway regulates glucose utilization and stress response mechanisms, suggesting impaired adaptation of the plasma membrane/cell wall during the respiratory-fermentative transition. KlHsl1 could be the link between these adaptive pathways and the morphogenetic checkpoint. INTRODUCTIONIn Saccharomyces cerevisiae, the cell cycle is regulated by the Cdc28 kinase and its regulatory subunits (cyclins). Swe1 kinase negatively regulates entry into mitosis through inhibitory phosphorylation of Cdc28 (Booher et al., 1993). Swe1 accumulates through the late G 1 and S phases, and is then degraded in G 2 by hyperphosphorylation (Sia et al., 1998). HSL1, also called NIK1, encodes a protein kinase, which is a negative regulator of Swe1 (Tanaka & Nojima, 1996;Ma et al., 1996). Hsl1 is a component of the septin morphogenetic checkpoint that promotes bud development and the recruitment of Swe1 at the septin ring where it is phosphorylated/degraded (McMillan et al., 1999;Lew, 2003).S. cerevisiae is a Crabtree-positive yeast in that respiration is completely repressed by glucose even under aerobic conditions, growth being supported solely by fermentation (Gancedo, 1998). In contrast, in the Crabtree-negative yeast Kluyveromyces lactis both respiratory and fermentative pathways co-exist during growth on glucose (De Deken, 1966;Breunig et al., 2000), although respiration appears to be dispensable since antimycin A (AA) does not inhibit growth on glucose (Rag + phenotype) (Goffrini et al., 1989; Wésolowski-Louvel et al., 1992a). Sensitivity to AA on glucose (Rag -) is associated with mutations in genes encoding glycolytic enzymes, glucose transporters and sensing components, as well as their regulatory factors (Goffrini et al., 1991; Wésolowski-Louvel et al., 1992b;Prior et al., 1993; Bianchi et al., 1996;Prior et al., 1996;Blaisonneau et al., 1997;Betina et al., 2001; Lemaire & Wésolowski-Louvel, 2004; Hnatova et al., 2008).During the characterization of the putative cell cycle gene KlHSL1 in K. lactis, we found that its deletion led to a Rag -phenotype, suggesting involvement in glucose metabolism. To identify the relationship between RAG and KlHSL1, rag and Klhsl1D mutant strains were further analysed. All rag Abbreviati...