scholarly journals The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

2004 ◽  
Vol 3 (3) ◽  
pp. 589-597 ◽  
Author(s):  
Michele Saliola ◽  
Paola Chiara Bartoccioni ◽  
Ilaria De Maria ◽  
Tiziana Lodi ◽  
Claudio Falcone
Keyword(s):  
Succinate Dehydrogenase ◽  
Glyoxylate Cycle ◽  
Alternative Pathway ◽  
Kluyveromyces Lactis ◽  
Carbon Sources ◽  
Pyruvate Decarboxylase ◽  
Northern Analysis ◽  
Gene Encoding ◽  
Dehydrogenase Gene ◽  
High Sequence Conservation

ABSTRACT We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose.

scholarly journals Deletion of the Glucose-6-Phosphate Dehydrogenase Gene KlZWF1 Affects both Fermentative and Respiratory Metabolism in Kluyveromyces lactis

2006 ◽  
Vol 6 (1) ◽  
pp. 19-27 ◽  
Author(s):  
Michele Saliola ◽  
Gina Scappucci ◽  
Ilaria De Maria ◽  
Tiziana Lodi ◽  
Patrizia Mancini ◽  
...  
Keyword(s):  
Biomass Yield ◽  
Kluyveromyces Lactis ◽  
Carbon Sources ◽  
Pentose Phosphate ◽  
Respiratory Metabolism ◽  
Wild Type ◽  
Dehydrogenase Gene ◽  
Increased Sensitivity ◽  
Dimeric Form ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACT In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1Δ strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield.

scholarly journals Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis

2022 ◽  
Vol 23 (2) ◽  
pp. 772
Author(s):  
Rosaura Rodicio ◽  
Hans-Peter Schmitz ◽  
Jürgen J. Heinisch
Keyword(s):  
Kluyveromyces Lactis ◽  
Regulation Of Gene Expression ◽  
Carbon Sources ◽  
Pentose Phosphate ◽  
Gene Encoding ◽  
Physiological Characterization ◽  
Genes Encoding ◽  
Fructose 1,6 Bisphosphate

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

scholarly journals Auxins Upregulate Expression of the Indole-3-Pyruvate Decarboxylase Gene in Azospirillum brasilense

1999 ◽  
Vol 181 (4) ◽  
pp. 1338-1342 ◽  
Author(s):  
Ann Vande Broek ◽  
Mark Lambrecht ◽  
Kristel Eggermont ◽  
Jos Vanderleyden
Keyword(s):  
Acetic Acid ◽  
Azospirillum Brasilense ◽  
Pyruvate Decarboxylase ◽  
Northern Analysis ◽  
Naphthaleneacetic Acid ◽  
Gene Encoding ◽  
Bacterial Gene ◽  
Synthetic Auxins ◽  
Expression Studies ◽  
Indole 3 Acetic Acid

ABSTRACT Transcription of the Azospirillum brasilense ipdC gene, encoding an indole-3-pyruvate decarboxylase involved in the biosynthesis of indole-3-acetic acid (IAA), is induced by IAA as determined by ipdC-gusA expression studies and Northern analysis. Besides IAA, exogenously added synthetic auxins such as 1-naphthaleneacetic acid, 2,4-dichlorophenoxypropionic acid, andp-chlorophenoxyacetic acid were also found to upregulateipdC expression. No upregulation was observed with tryptophan, acetic acid, or propionic acid or with the IAA conjugates IAA ethyl ester and IAA-l-phenylalanine, indicating structural specificity is required for ipdC induction. This is the first report describing the induction of a bacterial gene by auxin.

scholarly journals Two homologs of the Cat8 transcription factor are involved in the regulation of ethanol utilization in Komagataella phaffii

2021 ◽  
Author(s):  
Diane Barbay ◽  
Monika Mačáková ◽  
Leander Sützl ◽  
Sonakshi De ◽  
Diethard Mattanovich ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Sequence Similarity ◽  
Glyoxylate Cycle ◽  
Kluyveromyces Lactis ◽  
Carbon Sources ◽  
Methylotrophic Yeast ◽  
Growth Conditions ◽  
Komagataella Phaffii ◽  
Binding Domains

AbstractThe transcription factors Cat8 and Sip4 were described in Saccharomyces cerevisiae and Kluyveromyces lactis to have very similar DNA binding domains and to be necessary for derepression of a variety of genes under non-fermentative growth conditions via binding to the carbon source responsive elements (CSREs). The methylotrophic yeast Komagataella phaffii (syn Pichia pastoris) has two transcription factors (TFs), which are putative homologs of Cat8 based on sequence similarity, termed Cat8-1 and Cat8-2. It is yet unclear in which cellular processes they are involved and if one of them is actually the homolog of Sip4. To study the roles of the Cat8 homologs in K. phaffii, overexpression or deletion strains were generated for the two TFs. The ability of these mutant strains to grow on different carbon sources was tested, and transcript levels of selected genes from the carbon metabolism were quantified. Our experiments showed that the TFs are required for the growth of K. phaffii on C2 carbon sources, but not on glucose, glycerol or methanol. K. phaffii deleted for Cat8-1 showed impaired growth on acetate, while both Cat8-1 and Cat8-2 are involved in the growth of K. phaffii on ethanol. Correspondingly, both TFs are participating in the activation of ADH2, ALD4 and ACS1, three genes encoding enzymes important for the assimilation of ethanol. Different from S. cerevisiae and K. lactis, Cat8-1 is not regulating the transcription of the putative Sip4-family member Cat8-2 in K. phaffii. Furthermore, Cat8-1 is necessary for the activation of genes from the glyoxylate cycle, whereas Cat8-2 is necessary for the activation of genes from the carnitine shuttle. Neither Cat8-1 nor Cat8-2 are required for the activation of gluconeogenesis genes. Finally, the CAT8-2 gene is repressed by the Mig1-2 transcription factor on glucose and autorepressed by the Cat8-2 protein on all tested carbon sources. Our study identified the involvement of K. phaffii Cat8-1 and Cat8-2 in C2-metabolism, and highlighted similarities and differences to their homologs in other yeast species.

scholarly journals Production of Xylitol from d-Xylose by a Xylitol Dehydrogenase Gene-Disrupted Mutant of Candida tropicalis

2006 ◽  
Vol 72 (6) ◽  
pp. 4207-4213 ◽  
Author(s):  
Byoung Sam Ko ◽  
Jinmi Kim ◽  
Jung Hoe Kim
Keyword(s):  
Candida Tropicalis ◽  
Carbon Sources ◽  
Fermentation Medium ◽  
Xylitol Dehydrogenase ◽  
Specific Productivity ◽  
Xylitol Production ◽  
Xylose Metabolism ◽  
Gene Encoding ◽  
Dehydrogenase Gene ◽  
Xylitol Fermentation

ABSTRACT Xylitol dehydrogenase (XDH) is one of the key enzymes in d-xylose metabolism, catalyzing the oxidation of xylitol to d-xylulose. Two copies of the XYL2 gene encoding XDH in the diploid yeast Candida tropicalis were sequentially disrupted using the Ura-blasting method. The XYL2-disrupted mutant, BSXDH-3, did not grow on a minimal medium containing d-xylose as a sole carbon source. An enzyme assay experiment indicated that BSXDH-3 lost apparently all XDH activity. Xylitol production by BSXDH-3 was evaluated using a xylitol fermentation medium with glucose as a cosubstrate. As glucose was found to be an insufficient cosubstrate, various carbon sources were screened for efficient cofactor regeneration, and glycerol was found to be the best cosubstrate. BSXDH-3 produced xylitol with a volumetric productivity of 3.23 g liter−1 h−1, a specific productivity of 0.76 g g−1 h−1, and a xylitol yield of 98%. This is the first report of gene disruption of C. tropicalis for enhancing the efficiency of xylitol production.

scholarly journals Changes in the enzyme activities of Saccharomyces cerevisiae during aerobic growth on different carbon sources

1965 ◽  
Vol 97 (1) ◽  
pp. 284-297 ◽  
Author(s):  
ES Polakis ◽  
W Bartley
Keyword(s):  
Citric Acid ◽  
Citric Acid Cycle ◽  
Glyoxylate Cycle ◽  
Carbon Sources ◽  
Pyruvate Decarboxylase ◽  
Regulatory Control ◽  
Growth Media ◽  
Aerobic Growth ◽  
Acid Cycle ◽  
Citric Acid Cycle Enzymes

1. The activities of the enzymes of the citric acid cycle, the glyoxylate by-pass and some other enzymes acting on the substrates of these cycles have been measured at the pH of the yeast cell during the aerobic growth of yeast on different carbon sources and in different growth media. 2. Sugars induced an anaerobic type of metabolism as measured by ethanol production. Glucose was much more effective in inducing the anaerobic pathways than was galactose. The production of ethanol by cells grown on pyruvate was very small. 3. Glucose was also a more effective repressor than was galactose of the citric acid-cycle enzymes but both were equally effective in repressing almost completely the enzymes of the glyoxylate by-pass. 4. Disappearance of the sugars from the growth medium resulted in an increase in the activities of the enzymes of the citric acid cycle and in the appearance of substantial activities of the enzymes of the glyoxylate cycle. By contrast, the activities of purely biosynthetic enzymes (glutamate-oxaloacetate transaminase, NADP(+)-linked glutamate dehydrogenase) and of pyruvate decarboxylase were decreased. 5. The 2-oxoglutarate-oxidase system was found to be the least active enzyme of the citric acid cycle. 6. The regulatory control at the levels of pyruvate and acetaldehyde and the control of the citric acid cycle are discussed.

scholarly journals Genetic Analysis of Mutations Affecting pckA Regulation in Rhizobium (Sinorhizobium) meliloti

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1521-1531 ◽  
Author(s):  
Magne Østerås ◽  
Shelley A P O'Brien ◽  
Turlough M Finan
Keyword(s):  
Sinorhizobium Meliloti ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Root Nodules ◽  
Genetic Regulation ◽  
Carbon Sources ◽  
Dicarboxylic Acids ◽  
Phenotypic Analysis ◽  
Gene Encoding ◽  
Type Locus ◽  
Rapid Induction

Abstract The enzyme phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step in the gluconeogenic pathway in most organisms. We are examining the genetic regulation of the gene encoding Pck, pckA, in Rhizobium (now Sinorhizobium) meliloti. This bacterium forms N2-fixing root nodules on alfalfa, and the major energy sources supplied to the bacteria within these nodules are C4-dicarboxylic acids such as malate and succinate. R. meliloti cells growing in glucose minimal medium show very low pckA expression whereas addition of succinate to this medium results in a rapid induction of pckA transcription. We identified spontaneous mutations (rpk) that alter the regulation of pckA expression such that pckA is expressed in media containing the non-inducing carbon sources lactose and glucose. Genetic and phenotypic analysis allowed us to differentiate at least four rpk mutant classes that map to different locations on the R. meliloti chromosome. The wild-type locus corresponding to one of these rpk loci was cloned by complementation, and two Tn5 insertions within the insert DNA that no longer complemented the rpk mutation were identified. The nucleotide sequence of this region revealed that both Tn5 insertions lay within a gene encoding a protein homologous to the Ga1R/LacI family of transcriptional regulators that are involved in metabolism.

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

1996 ◽  
Vol 20 (4) ◽  
pp. 765-772 ◽  
Author(s):  
C. Prior ◽  
L. Tizzani ◽  
H. Fukuhara ◽  
M. Wésolowski-Louvel
Keyword(s):  
Transcriptional Regulation ◽  
Kluyveromyces Lactis ◽  
Pyruvate Decarboxylase

scholarly journals Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

2004 ◽  
Vol 70 (5) ◽  
pp. 2632-2638 ◽  
Author(s):  
Claudia Donnini ◽  
Francesca Farina ◽  
Barbara Neglia ◽  
Maria Concetta Compagno ◽  
Daniela Uccelletti ◽  
...  
Keyword(s):  
Glucose Repression ◽  
Kluyveromyces Lactis ◽  
Kinase Domain ◽  
Arxula Adeninivorans ◽  
Nucleotide Position ◽  
Heterologous Proteins ◽  
Reporter Protein ◽  
Gene Encoding ◽  
Defective Mutant ◽  
Highly Sensitive

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.

Control of mRNA turnover as a mechanism of glucose repression in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (7) ◽  
pp. 2941-2948
Author(s):  
A Lombardo ◽  
G P Cereghino ◽  
I E Scheffler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Succinate Dehydrogenase ◽  
Half Life ◽  
Glucose Repression ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Mrna Levels ◽  
Protein Subunit ◽  
Gene Encoding ◽  
Regulatory Complex

We have examined the expression of the gene encoding the iron-protein subunit (Ip) of succinate dehydrogenase in Saccharomyces cerevisiae. The gene had been cloned by us and shown to be subject to glucose regulation (A. Lombardo, K. Carine, and I. E. Scheffler, J. Biol. Chem. 265:10419-10423, 1990). We discovered that a significant part of the regulation of the Ip mRNA levels by glucose involves the regulation of the turnover rate of this mRNA. In the presence of glucose, the half-life appears to be less than 5 min, while in glycerol medium, the half-life is greater than 60 min. The gene is also regulated transcriptionally by glucose. The upstream promoter sequence appeared to have four regulatory elements with consensus sequences shown to be responsible for the interaction with the HAP2/3/4 regulatory complex. A deletion analysis has shown that the two distal elements are redundant. These measurements were carried out by Northern (RNA) analyses of Ip mRNA transcripts as well as by assays of beta-galactosidase activity in cells carrying constructs of the Ip promoter linked to the lacZ coding sequence. These observations on the regulation of mRNA stability were also extended to the mRNA of the flavoprotein subunit of succinate dehydrogenase and in some experiments of iso-1-cytochrome c.

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