K. Lactis (k + lacti)

Distribution by Scientific Domains

Selected Abstracts

Experimental and steady-state analysis of the GAL regulatory system in Kluyveromyces lactis

FEBS JOURNAL, Issue 14 2010
Venkat R. Pannala
The galactose uptake mechanism in yeast is a well-studied regulatory network. The regulatory players in the galactose regulatory mechanism (GAL system) are conserved in Saccharomyces cerevisiae and Kluyveromyces lactis, but the molecular mechanisms that occur as a result of the molecular interactions between them are different. The key differences in the GAL system of K. lactis relative to that of S. cerevisiae are: (a) the autoregulation of KlGAL4; (b) the dual role of KlGal1p as a metabolizing enzyme as well as a galactose-sensing protein; (c) the shuttling of KlGal1p between nucleus and cytoplasm; and (d) the nuclear confinement of KlGal80p. A steady-state model was used to elucidate the roles of these molecular mechanisms in the transcriptional response of the GAL system. The steady-state results were validated experimentally using measurements of ,-galactosidase to represent the expression for genes having two binding sites. The results showed that the autoregulation of the synthesis of activator KlGal4p is responsible for the leaky expression of GAL genes, even at high glucose concentrations. Furthermore, GAL gene expression in K. lactis shows low expression levels because of the limiting function of the bifunctional protein KlGal1p towards the induction process in order to cope with the need for the metabolism of lactose/galactose. The steady-state model of the GAL system of K. lactis provides an opportunity to compare with the design prevailing in S. cerevisiae. The comparison indicates that the existence of a protein, Gal3p, dedicated to the sensing of galactose in S. cerevisiae as a result of genome duplication has resulted in a system which metabolizes galactose efficiently. [source]

A tool kit for molecular genetics of Kluyveromyces lactis comprising a congenic strain series and a set of versatile vectors

Jürgen J. Heinisch
Abstract A set of different marker deletions starting with a ura3 derivative of the Kluyveromyces lactis type strain CBS2359 was constructed. After a first cross to obtain a strain with the opposite mating type that also carried a leu2 allele, continuous back-crosses were used to obtain a congenic strain series with different marker combinations, including deletions in KlHIS3, KlADE2 and KlLAC4. Enzymes involved in carbohydrate metabolism were shown to behave very similarly to the original type strain and other K. lactis strains investigated previously. Moreover, a vector series of Saccharomyces cerevisiae genes flanked by loxP sites was constructed to be used as heterologous deletion cassettes in K. lactis, together with two plasmids for expression of Cre-recombinase for marker regeneration. To increase the frequency of homologous recombination, the Klku80 deletion was also introduced into the congenic strain series. A PCR-based method for determination of mating type is provided. [source]

Significance of the KlLAC1 gene in glucosylceramide production by Kluyveromyces lactis

Naoya Takakuwa
Abstract Each of the 12 genes involved in the synthesis of glucosylceramide was overexpressed in cells of Kluyveromyces lactis to construct a strain accumulating a high quantity of glucosylceramide. Glucosylceramide was doubled by the KlLAC1 gene, which encodes ceramide synthase, and not by 11 other genes, including the KlLAG1 gene, a homologue of KlLAC1. Disruption of the KlLAC1 gene reduced the content below the detection level. Heterologous expression of the KlLAC1 gene in the cells of Saccharomyces cerevisiae caused the accumulation of ceramide, composed of C18 fatty acid. The KlLAC1 protein preferred long-chain (C18) fatty acids to very-long-chain (C26) fatty acids for condensation with sphingoid bases and seemed to supply a ceramide moiety as the substrate for the formation of glucosylceramide. When the amino acid sequences of ceramide synthase derived from eight yeast species were compared, LAC1 proteins from five species producing glucosylceramide were clearly discriminated from those of the other three species and all LAG1 proteins. The LAC1 protein of K. lactis is the enzyme that plays a crucial role in the synthesis of glucosylceramide. [source]

Current status of Kluyveromyces systematics

Marc-André Lachance
Abstract A brief outline of the current taxonomic status of the genus Kluyveromyces is presented. Noteworthy are the transfer of several former Kluyveromyces species to other genera, the retention of the name Kluyveromyces for K. lactis, K. marxianus, and four related species, and some recent attempts to clarify the variety status of strains assigned to K. lactis. [source]

A respiratory-deficient mutation associated with high salt sensitivity in Kluyveromyces lactis

Paola Goffrini
Abstract A salt-sensitive mutant of Kluyveromyces lactis was isolated that was unable to grow in high-salt media. This mutant was also respiratory-deficient and temperature-sensitive for growth. The mutation mapped in a single nuclear gene that is the ortholog of BCS1 of Saccharomyces cerevisiae. The BCS1 product is a mitochondrial protein required for the assembly of respiratory complex III. The bcs1 mutation of S. cerevisiae leads to a loss of respiration, but, unlike in K. lactis, it is not accompanied by salt sensitivity. All the respiratory-deficient K. lactis mutants tested were found to be salt-sensitive compared to their isogenic wild-type strains. In the presence of the respiratory inhibitor antimycin A, the wild-type strain also became salt-sensitive. By contrast, none of the S. cerevisiae respiratory-deficient mutants tested showed increased salt sensitivity. The salt sensitivity of the Klbcs1 mutant, but not its respiratory deficiency, was suppressed by the multicopy KlVMA13 gene, a homolog of the S. cerevisiae VMA13 gene encoding a subunit of the vacuolar H+ -ATPase. These results suggest that cellular salt homeostasis in K. lactis is strongly dependent on mitochondrial respiratory activity, and/or that the ion homeostasis of mitochondria themselves could be a primary target of salt stress. [source]

Why does Kluyveromyces lactis not grow under anaerobic conditions?

Comparison of essential anaerobic genes of Saccharomyces cerevisiae with the Kluyveromyces lactis genome
Abstract Although some yeast species, e.g. Saccharomyces cerevisiae, can grow under anaerobic conditions, Kluyveromyces lactis cannot. In a systematic study, we have determined which S. cerevisiae genes are required for growth without oxygen. This has been done by using the yeast deletion library. Both aerobically essential and nonessential genes have been tested for their necessity for anaerobic growth. Upon comparison of the K. lactis genome with the genes found to be anaerobically important in S. cerevisiae, which yielded 20 genes that are missing in K. lactis, we hypothesize that lack of import of sterols might be one of the more important reasons that K. lactis cannot grow in the absence of oxygen. [source]

Production of Low-Lactose Milk by Means of Nonisothermal Bioreactors

Valentina Grano
The effect of the immobilization time on the activity of immobilized ,-galactosidase from K. lactis was investigated. Six biocatalytic membranes, different only for the time of the enzyme immobilization, were obtained by using nylon membranes grafted with glycidyl methacrylate (GMA) and activated by hexamethylenediamine (HMDA) and glutaraldehyde (Glu), used as spacer and coupling agent, respectively. Comparison between the isothermal and nonisothermal yield of these biocatalytic membranes was carried out in the process of lactose hydrolysis in milk. All of the results, reported as a function of the immobilization time, have evidenced the influence of our variable parameter on the activity of the catalytic membranes. The membrane giving highest yield under isothermal and nonisothermal conditions was that obtained with 2 h of immobilization time. The industrial application of these membranes has been discussed in terms of percentage reduction of the production times. [source]