Influence of Mutations in Hexose-Transporter Genes on Glucose Repression in Kluyveromyces Lactis

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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Identification of Hexose Transporter-Like Sensor HXS1 and Functional Hexose Transporter HXT1 in the Methylotrophic Yeast Hansenula polymorpha

Eukaryotic Cell ◽

10.1128/ec.00028-08 ◽

2008 ◽

Vol 7 (4) ◽

pp. 735-746 ◽

Cited By ~ 27

Author(s):

Olena G. Stasyk ◽

Mykola M. Maidan ◽

Oleh V. Stasyk ◽

Patrick Van Dijck ◽

Johan M. Thevelein ◽

...

Keyword(s):

Transcriptional Repression ◽

Glucose Transporter ◽

Glucose Repression ◽

Sequence Similarity ◽

Hansenula Polymorpha ◽

Methylotrophic Yeast ◽

Hexose Transporter ◽

Autophagic Degradation ◽

Hexose Carrier ◽

Hexose Uptake

ABSTRACT We identified in the methylotrophic yeast Hansenula polymorpha (syn. Pichia angusta) a novel hexose transporter homologue gene, HXS1 (hexose sensor), involved in transcriptional regulation in response to hexoses, and a regular hexose carrier gene, HXT1 (hexose transporter). The Hxs1 protein exhibits the highest degree of primary sequence similarity to the Saccharomyces cerevisiae transporter-like glucose sensors, Snf3 and Rgt2. When heterologously overexpressed in an S. cerevisiae hexose transporter-less mutant, Hxt1, but not Hxs1, restores growth on glucose or fructose, suggesting that Hxs1 is nonfunctional as a carrier. In its native host, HXS1 is expressed at moderately low level and is required for glucose induction of the H. polymorpha functional low-affinity glucose transporter Hxt1. Similarly to other yeast sensors, one conserved amino acid substitution in the Hxs1 sequence (R203K) converts the protein into a constitutively signaling form and the C-terminal region of Hxs1 is essential for its function in hexose sensing. Hxs1 is not required for glucose repression or catabolite inactivation that involves autophagic degradation of peroxisomes. However, HXS1 deficiency leads to significantly impaired transient transcriptional repression in response to fructose, probably due to the stronger defect in transport of this hexose in the hxs1Δ deletion strain. Our combined results suggest that in the Crabtree-negative yeast H. polymorpha, the single transporter-like sensor Hxs1 mediates signaling in the hexose induction pathway, whereas the rate of hexose uptake affects the strength of catabolite repression.

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

Current Genetics ◽

10.1007/s00294-004-0489-5 ◽

2004 ◽

Vol 45 (4) ◽

pp. 256-256

Author(s):

M. Luz L�pez ◽

Bego�a Redruello ◽

Eva Vald�s ◽

Fernando Moreno ◽

J�rgen J. Heinisch ◽

...

Keyword(s):

Glucose Repression ◽

Isocitrate Lyase ◽

Kluyveromyces Lactis ◽

Catabolite Inactivation

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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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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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The hexokinase gene is required for transcriptional regulation of the glucose transporter gene RAG1 in Kluyveromyces lactis

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3882-3889.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3882-3889

Author(s):

C Prior ◽

P Mamessier ◽

H Fukuhara ◽

X J Chen ◽

M Wesolowski-Louvel

Keyword(s):

Glucose Transporter ◽

Kinase Activity ◽

Glucose Repression ◽

Kluyveromyces Lactis ◽

Transport Systems ◽

Transporter Gene ◽

Rag1 Gene ◽

Sugar Kinase

The RAG1 gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAG5, and RAG8 genes are trans-acting genes controlling the expression of the RAG1 gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAG5 gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. rag5 mutants showed no detectable hexokinase or glucokinase activity, suggesting that the sugar kinase activity encoded by this gene is the only hexokinase in K. lactis. Both high- and low-affinity transport systems of glucose were affected in rag5 mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG1 gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the HXK2 gene of S. cerevisiae and complementation of hxk1 hxk2 mutations of S. cerevisiae by the RAG5 gene showed that RAG5 and HXK2 were equivalent for sugar-phosphorylating activity but that RAG5 could not restore glucose repression in the S. cerevisiae hexokinase mutants.

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Extension of Chronological Lifespan by Hexokinase Mutation inKluyveromyces lactisInvolves Increased Level of the Mitochondrial Chaperonin Hsp60

Journal of Aging Research ◽

10.1155/2012/946586 ◽

2012 ◽

Vol 2012 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Lisa Rizzetto ◽

Elena Zanni ◽

Daniela Uccelletti ◽

Ileana Ferrero ◽

Paola Goffrini

Keyword(s):

Glucose Repression ◽

Kluyveromyces Lactis ◽

Metabolic Adaptation ◽

Molecular Processes ◽

Respiratory Enzymes ◽

Telomere Shortening ◽

Chronological Lifespan ◽

Important Player ◽

Complex Regulation ◽

Glucose Reduction

Oxidative damage, mitochondrial dysfunction, genomic instability, and telomere shortening represent all molecular processes proposed as causal factors in aging. Lifespan can be increased by metabolism through an influence on such processes. Glucose reduction extends chronological lifespan (CLS) ofSaccharomyces cerevisiaethrough metabolic adaptation to respiration. To answer the question if the reduced CLS could be ascribed to glucoseper seor to glucose repression of respiratory enzymes, we used theKluyveromyces lactisyeast, where glucose repression does not affect the respiratory function. We identified the unique hexokinase, encoded byRAG5gene, as an important player in influencing yeast lifespan by modulating mitochondrial functionality and the level of the mitochondrial chaperonin Hsp60. In this context, this hexokinase might have a regulatory role in the influence of CLS, shedding new light on the complex regulation played by hexokinases.

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Transcriptomic Analysis of Extensive Changes in Metabolic Regulation in Kluyveromyces lactis Strains

Eukaryotic Cell ◽

10.1128/ec.00087-06 ◽

2006 ◽

Vol 5 (8) ◽

pp. 1360-1370 ◽

Cited By ~ 14

Author(s):

Audrey Suleau ◽

Pierre Gourdon ◽

Joëlle Reitz-Ausseur ◽

Serge Casaregola

Keyword(s):

Gene Expression ◽

Metabolic Regulation ◽

Glucose Repression ◽

Kluyveromyces Lactis ◽

Sugar Transport ◽

Point Mutations ◽

Growth Control ◽

Ethanol Metabolism ◽

Regulation Mechanism ◽

Reference Laboratory

ABSTRACT Genome-wide analysis of transcriptional regulation is generally carried out on well-characterized reference laboratory strains; hence, the characteristics of industrial isolates are therefore overlooked. In a previous study on the major cheese yeast Kluyveromyces lactis, we have shown that the reference strain and an industrial strain used in cheese making display a differential gene expression when grown on a single carbon source. Here, we have used more controlled conditions, i.e., growth in a fermentor with pH and oxygen maintained constant, to study how these two isolates grown in glucose reacted to an addition of lactose. The observed differences between sugar consumption and the production of various metabolites, ethanol, acetate, and glycerol, correlated with the response were monitored by the analysis of the expression of 482 genes. Extensive differences in gene expression between the strains were revealed in sugar transport, glucose repression, ethanol metabolism, and amino acid import. These differences were partly due to repression by glucose and another, yet-unknown regulation mechanism. Our results bring to light a new type of K. lactis strain with respect to hexose transport gene content and repression by glucose. We found that a combination of point mutations and variation in gene regulation generates a biodiversity within the K. lactis species that was not anticipated. In contrast to S. cerevisiae, in which there is a massive increase in the number of sugar transporter and fermentation genes, in K. lactis, interstrain diversity in adaptation to a changing environment is based on small changes at the level of key genes and cell growth control.

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