Isocitrate lyase of the yeast Kluyveromyces lactis is subject to glucose repression but not to catabolite inactivation

ABSTRACT The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1β compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.

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Expression of the transcriptional activator LAC9 (KlGAL4) in Kluyveromyces lactis is controlled by autoregulation

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.3058-3066.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 3058-3066

Author(s):

W Zachariae ◽

K D Breunig

Keyword(s):

Carbon Source ◽

Binding Site ◽

Glucose Repression ◽

Kluyveromyces Lactis ◽

Transcriptional Activator ◽

Optimal Growth ◽

Source Control ◽

Poor Growth ◽

Protein Protein Interaction ◽

Metabolic Genes

The concentration of the transcriptional activator LAC9 (KlGAL4) of Kluyveromyces lactis is moderately regulated by the carbon source as is the case for GAL4, its homolog in Saccharomyces cerevisiae. Expression of the LAC9 gene is induced about twofold in galactose. This induction is due to autoregulation. The LAC9 gene product binds to a low-affinity binding site in the LAC9 promoter and moderately activates transcription in response to galactose above a basal level. As for the LAC9-controlled metabolic genes, induction of LAC9 is inhibited in the presence of glucose. This inhibition of induction is a prerequisite for glucose repression of the lactose-galactose metabolic pathway. On the other hand, induced LAC9 levels are required for optimal growth on galactose, since mutating the LAC9 binding site in the LAC9 promoter resulted in poor growth and reduced expression of LAC9-controlled genes. Thus, in addition to the GAL80-dependent regulation by protein-protein interaction, the regulation of LAC9 gene expression is an important parameter in determining carbon source control of the LAC-GAL regulon. Although the mode of control is different, the pattern of LAC9 gene regulation resembles that of the S. cerevisiae GAL4 gene, being lower in glucose and glucose-galactose than in galactose.

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Conservation of Histone Binding and Transcriptional Repressor Functions in a Schizosaccharomyces pombeTup1p Homolog

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8461 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8461-8468 ◽

Cited By ~ 34

Author(s):

Yukio Mukai ◽

Eri Matsuo ◽

Sharon Y. Roth ◽

Satoshi Harashima

Keyword(s):

Glucose Repression ◽

Kluyveromyces Lactis ◽

Chimeric Protein ◽

Evolutionary Conservation ◽

Histone Binding ◽

Amino Terminal ◽

Corepressor Complex ◽

Related Proteins ◽

Repressor Activity

ABSTRACT The Ssn6p-Tup1p corepressor complex is important to the regulation of several diverse genes in Saccharomyces cerevisiae and serves as a model for corepressor functions. To investigate the evolutionary conservation of these functions, sequences homologous to the S. cerevisiae TUP1 gene were cloned fromKluyveromyces lactis (TUP1) andSchizosaccharomyces pombe (tup11 +). Interestingly, while the K. lactis TUP1 gene complemented an S. cerevisiae tup1 null mutation, the S. pombe tup11 + gene did not, even when expressed under the control of the S. cerevisiae TUP1 promoter. However, anS. pombe Tup11p-LexA fusion protein repressed transcription of a corresponding reporter gene, indicating that this Tup1p homolog has intrinsic repressor activity. Moreover, a chimeric protein containing the amino-terminal Ssn6p-binding domain of S. cerevisiae Tup1p and 544 amino acids from the C-terminal region of S. pombe Tup11p complemented the S. cerevisiae tup1 mutation. The failure of native S. pombe Tup11p to complement loss of Tup1p functions in S. cerevisiaecorresponds to an inability to bind to S. cerevisiae Ssn6p in vitro. Disruption of tup11 + in combination with a disruption of tup12 +, anotherTUP1 homolog gene in S. pombe, causes a defect in glucose repression of fbp1 +, suggesting thatS. pombe Tup1p homologs function as repressors in S. pombe. Furthermore, Tup11p binds specifically to histones H3 and H4 in vitro, indicating that both the repression and histone binding functions of Tup1p-related proteins are conserved across species.

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Isolation and characterization of dominant mutations resistant to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.1.2.83-93.1981 ◽

1981 ◽

Vol 1 (2) ◽

pp. 83-93

Author(s):

K Matsumoto ◽

A Toh-e ◽

Y Oshima

Keyword(s):

Partial Resistance ◽

Carbon Catabolite Repression ◽

Glucose Repression ◽

Isocitrate Lyase ◽

Isolation And Characterization ◽

Enhanced Resistance ◽

Leloir Pathway ◽

Resistant Mutants ◽

A Site

Seven dominant mutations showing greatly enhanced resistance to the glucose repression of galactokinase synthesis have been isolated from GAL81 mutants, which have the constitutive phenotype but are still strongly repressible by glucose for the synthesis of the Leloir enzymes. These glucose-resistant mutants were due to semidominant mutations at either of two loci, GAL82 and GAL83. Both loci are unlinked to the GAL81- gal4, gal80, or gal7 X gal10 X gal1 locus or to each other. The GAL83 locus was mapped on chromosome V at a site between arg9 and cho1. The GAL82 and GAL83 mutations produced partial resistance of galactokinase to glucose repression only when one or both of these mutations were combined with a GAL81 or a gal80 mutation. The GAL82 and GAL83 mutations are probably specific for expression of the Leloir pathway and related enzymes, because they do not affect the synthesis of alpha-D-glucosidase, invertase, or isocitrate lyase.

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The Evolutionary Rewiring of Ubiquitination Targets Has Reprogrammed the Regulation of Carbon Assimilation in the Pathogenic Yeast Candida albicans

mBio ◽

10.1128/mbio.00495-12 ◽

2012 ◽

Vol 3 (6) ◽

Cited By ~ 54

Author(s):

Doblin Sandai ◽

Zhikang Yin ◽

Laura Selway ◽

David Stead ◽

Janet Walker ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Fatty Acids ◽

Candida Albicans ◽

Isocitrate Lyase ◽

Carbon Sources ◽

Carbon Assimilation ◽

Pathogenic Yeast ◽

Content Type ◽

Catabolite Inactivation ◽

Accelerated Degradation

ABSTRACTMicrobes must assimilate carbon to grow and colonize their niches. Transcript profiling has suggested thatCandida albicans, a major pathogen of humans, regulates its carbon assimilation in an analogous fashion to the model yeastSaccharomyces cerevisiae, repressing metabolic pathways required for the use of alterative nonpreferred carbon sources when sugars are available. However, we show that there is significant dislocation between the proteome and transcriptome inC. albicans. Glucose triggers the degradation of theICL1andPCK1transcripts inC. albicans, yet isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are stable and are retained. Indeed, numerous enzymes required for the assimilation of carboxylic and fatty acids are not degraded in response to glucose. However, when expressed inC. albicans,S. cerevisiaeIcl1 (ScIcl1) is subjected to glucose-accelerated degradation, indicating that likeS. cerevisiae, this pathogen has the molecular apparatus required to execute ubiquitin-dependent catabolite inactivation.C. albicansIcl1 (CaIcl1) lacks analogous ubiquitination sites and is stable under these conditions, but the addition of a ubiquitination site programs glucose-accelerated degradation of CaIcl1. Also, catabolite inactivation is slowed inC. albicans ubi4cells. Ubiquitination sites are present in gluconeogenic and glyoxylate cycle enzymes fromS. cerevisiaebut absent from theirC. albicanshomologues. We conclude that evolutionary rewiring of ubiquitination targets has meant that following glucose exposure,C. albicansretains key metabolic functions, allowing it to continue to assimilate alternative carbon sources. This metabolic flexibility may be critical during infection, facilitating the rapid colonization of dynamic host niches containing complex arrays of nutrients.IMPORTANCEPathogenic microbes must assimilate a range of carbon sources to grow and colonize their hosts. Current views about carbon assimilation in the pathogenic yeastCandida albicansare strongly influenced by theSaccharomyces cerevisiaeparadigm in which cells faced with choices of nutrients first use energetically favorable sugars, degrading enzymes required for the assimilation of less favorable alternative carbon sources. We show that this is not the case inC. albicansbecause there has been significant evolutionary rewiring of the molecular signals that promote enzyme degradation in response to glucose. As a result, this major pathogen of humans retains enzymes required for the utilization of physiologically relevant carbon sources such as lactic acid and fatty acids, allowing it to continue to use these host nutrients even when glucose is available. This phenomenon probably enhances efficient colonization of host niches where sugars are only transiently available.

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Characterization of a positive regulatory gene, LAC9, that controls induction of the lactose-galactose regulon of Kluyveromyces lactis: structural and functional relationships to GAL4 of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.1111 ◽

1987 ◽

Vol 7 (3) ◽

pp. 1111-1121 ◽

Cited By ~ 74

Author(s):

L V Wray ◽

M M Witte ◽

R C Dickson ◽

M I Riley

Keyword(s):

Saccharomyces Cerevisiae ◽

Metal Binding ◽

Glucose Repression ◽

Kluyveromyces Lactis ◽

Regulatory Gene ◽

Regulatory Protein ◽

Constitutive Expression ◽

Functional Relationships ◽

Functional Implications

Lactose or galactose induces the expression of the lactose-galactose regulon in Kluyveromyces lactis. We show here that the regulon is not induced in strains defective in LAC9. We demonstrate that this gene codes for a regulatory protein that acts in a positive manner to induce transcription. The LAC9 gene was isolated by complementation of a lac9 defective strain. DNA sequence analysis of the gene gave a deduced protein of 865 amino acids. Comparison of this sequence with that of the GAL4 protein of Saccharomyces cerevisiae revealed three regions of homology. One region of about 90 amino acid occurs at the amino terminus, which is known to mediate binding of GAL4 protein to upstream activator sequences. We speculate that a portion of this region, adjacent to the "metal-binding finger," specifies DNA binding. We discuss possible functions of the two other regions of homology. The functional implications of these structural similarities were examined. When LAC9 was introduced into a gal4 defective strain of S. cerevisiae it complemented the mutation and activated the galactose-melibiose regulon. However, LAC9 did not simply mimic GAL4. Unlike normal S. cerevisiae carrying GAL4, the strain carrying LAC9 gave constitutive expression of GAL1 and MEL1, two genes in the regulon. The strain did show glucose repression of the regulon, but repression was less severe with LAC9 than with GAL4. We discuss the implications of these results and how they may facilitate our understanding of the LAC9 and GAL4 regulatory proteins.

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Yarrowia lipolytica Mutants Devoid of Pyruvate Carboxylase Activity Show an Unusual Growth Phenotype

Eukaryotic Cell ◽

10.1128/ec.4.2.356-364.2005 ◽

2005 ◽

Vol 4 (2) ◽

pp. 356-364 ◽

Cited By ~ 19

Author(s):

Carmen-Lisset Flores ◽

Carlos Gancedo

Keyword(s):

Yarrowia Lipolytica ◽

Pyruvate Carboxylase ◽

Glucose Repression ◽

Isocitrate Lyase ◽

Glyoxylate Cycle ◽

Growth Defect ◽

Carboxylase Activity ◽

Spliceosomal Introns ◽

Eukaryotic Microorganisms ◽

Growth Phenotype

ABSTRACT We have cloned and characterized the gene PYC1, encoding the unique pyruvate carboxylase in the dimorphic yeast Yarrowia lipolytica. The protein putatively encoded by the cDNA has a length of 1,192 amino acids and shows around 70% identity with pyruvate carboxylases from other organisms. The corresponding genomic DNA possesses an intron of 269 bp located 133 bp downstream of the starting ATG. In the branch motif of the intron, the sequence CCCTAAC, not previously found at this place in spliceosomal introns of Y. lipolytica, was uncovered. Disruption of the PYC1 gene from Y. lipolytica did not abolish growth in glucose-ammonium medium, as is the case in other eukaryotic microorganisms. This unusual growth phenotype was due to an incomplete glucose repression of the function of the glyoxylate cycle, as shown by the lack of growth in that medium of double pyc1 icl1 mutants lacking both pyruvate carboxylase and isocitrate lyase activity. These mutants grew when glutamate, aspartate, or Casamino Acids were added to the glucose-ammonium medium. The cDNA from the Y. lipolytica PYC1 gene complemented the growth defect of a Saccharomyces cerevisiae pyc1 pyc2 mutant, but introduction of either the S. cerevisiae PYC1 or PYC2 gene into Y. lipolytica did not result in detectable pyruvate carboxylase activity or in growth on glucose-ammonium of a Y. lipolytica pyc1 icl1 double mutant.

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Gal80 proteins of Kluyveromyces lactis and Saccharomyces cerevisiae are highly conserved but contribute differently to glucose repression of the galactose regulon

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7566-7576.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7566-7576

Author(s):

F T Zenke ◽

W Zachariae ◽

A Lunkes ◽

K D Breunig

Keyword(s):

Saccharomyces Cerevisiae ◽

Binding Sites ◽

Glucose Repression ◽

Kluyveromyces Lactis ◽

Constitutive Expression ◽

Indirect Evidence ◽

Transcriptional Activator ◽

Negative Regulator ◽

Gene Encoding ◽

Fold Induction

We cloned the GAL80 gene encoding the negative regulator of the transcriptional activator Gal4 (Lac9) from the yeast Kluyveromyces lactis. The deduced amino acid sequence of K. lactis GAL80 revealed a strong structural conservation between K. lactis Gal80 and the homologous Saccharomyces cerevisiae protein, with an overall identity of 60% and two conserved blocks with over 80% identical residues. K. lactis gal80 disruption mutants show constitutive expression of the lactose/galactose metabolic genes, confirming that K. lactis Gal80 functions in essentially in the same way as does S. cerevisiae Gal80, blocking activation by the transcriptional activator Lac9 (K. lactis Gal4) in the absence of an inducing sugar. However, in contrast to S. cerevisiae, in which Gal4-dependent activation is strongly inhibited by glucose even in a gal80 mutant, glucose repressibility is almost completely lost in gal80 mutants of K. lactis. Indirect evidence suggests that this difference in phenotype is due to a higher activator concentration in K. lactis which is able to overcome glucose repression. Expression of the K. lactis GAL80 gene is controlled by Lac9. Two high-affinity binding sites in the GAL80 promoter mediate a 70-fold induction by galactose and hence negative autoregulation by Gal80. Gal80 in turn not only controls Lac9 activity but also has a moderate influence on its rate of synthesis. Thus, a feedback control mechanism exists between the positive and negative regulators. By mutating the Lac9 binding sites of the GAL80 promoter, we could show that induction of GAL80 is required to prevent activation of the lactose/galactose regulon in glycerol or glucose plus galactose, whereas the noninduced level of Gal80 is sufficient to completely block Lac9 function in glucose.

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