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Researchers have achieved a high yield of lactic acid per gram of glucose consumed by Kluyveromyces lactis that is devoid of pyruvate dehydrogenase and pyruvate decarboxylase activity but has high levels of lactate dehydrogenase activity. In addition, this technique produces few additional metabolites, which is one of the goals of industrial fermentation.

Research is conducted on the construction of the yeast Kluyveromyces lactis strain that contains a chromosomal recombinase A gene copy. Results demonstrate that copy number amplification increases the production of heterologous protein genes.

The development of a homolactic Kluyveromyces lactis yeast strain, which displays high concentration and high productivity of lactic acid at a pH level below 5 to 5.5, the typical minimum pH range for lactate production by conventional microbial cells, is reported. Results represent the first example of a total replacement of a fermentation product mediated by metabolic engineering methods in yeast genera.

An analysis of the recombination of the circular 2micrometer-like plasmid pKD1 in Kluyveromyces lactis was reported. The results revealed that unlike the 2micrometer plasmid of Saccharomyces cerevisiae, the partitioning genes B and C are directly involved in plasmid recombination. It was suggested that B and C products are required by the plasmid recombinase, encoded by gene A, to promote site-specific recombination.

In unicellular organisms like yeasts, which do not have specialized tissues for protection against environmental challenges, the presence of cellular mechanisms to respond and adapt to stress conditions is fundamental. In this work, we aimed to investigate the response to environmental light in Kluyveromyces lactis. Yeast lacks specialized light-sensing proteins; however, Saccharomyces cerevisiae has been reported to respond to light by increasing hydrogen peroxide level and triggering nuclear translocation of Msn2. This is a stress-sensitive transcription factor also present in K. lactis. To investigate light response in this yeast, we analyzed the different phenotypes generated by the deletion of the hypoxia responsive and lipid biosynthesis transcription factor Kl Mga2. Alterations in growth rate, mitochondrial functioning, ROS metabolism, and fatty acid biosynthesis provide evidence that light was a source of stress in K. lactis and that Kl Mga2 had a role in the light-stress response. The involvement of Kl Msn2 and Kl Crz1 in light stress was also explored, but the latter showed no function in this response. [ABSTRACT FROM AUTHOR]

The possibility of a practical application of the hypoxic regulation of the K1PDC1 promoter is investigated by cloning the whole promoter sequence upstream to several heterologous genes coding for proteins widely heterogeneous in their characteristics is presented. Based on the results it is proposed that the Zygosaccharomyces bailii yeast could be used as K1PDC1 promoter-based expression vectors to achieve heterologous production in a simple and cheap procedure.

In the yeast Kluyveromyces lactis, the inactivation of structural or regulatory glycolytic and fermentative genes generates obligate respiratory mutants which can be characterized by sensitivity to the mitochondrial drug antimycin A on glucose medium (Rag- phenotype). Rag- mutations can occasionally be generated by the inactivation of genes not evidently related to glycolysis or fermentation. One such gene is the hypoxic regulatory gene KlMGA2. In this work, we report a study of the many defects, in addition to the Rag- phenotype, generated by KlMGA2 deletion. We analyzed the fermentative and respiratory metabolism, mitochondrial functioning and morphology in the Klmga2Δ strain. We also examined alterations in the regulation of the expression of lipid biosynthetic genes, in particular fatty acids, ergosterol and cardiolipin, under hypoxic and cold stress and the phenotypic suppression by unsaturated fatty acids of the deleted strain. Results indicate that, despite the fact that the deleted mutant strain had a typical glycolytic/fermentative phenotype and KlMGA2 is a hypoxic regulatory gene, the deletion of this gene generated defects linked to mitochondrial functions suggesting new roles of this protein in the general regulation and cellular fitness of K. lactis. supplementation of unsaturated fatty acids suppressed or modified these defects suggesting that KlMga2 modulates membrane functioning or membrane-associated functions, both cytoplasmic and mitochondrial. [ABSTRACT FROM AUTHOR]

Laccases are multicopper oxidases of wide specificity that catalyze the oxidation of phenolic and related compounds using molecular oxygen as the electron acceptor. Here, we report the production of the Lcc1 laccase of the fungus Trametes trogii in strains of the yeast Kluyveromyces lactis, using the pyruvate decarboxylase promoter ( KlPDC1) as an expression system. We assayed laccase production in various strains, with replicative and integrative transformants and with different cultivation parameters. A comparison with Lcc1 enzymes from other yeasts and from the original organism was also performed. The best production conditions were obtained with integrative transformants of an individual strain, whereas cultivation conditions were less stringent than the use of the regulated KlPDC1 promoter could anticipate. The secreted recombinant laccase showed better enzyme properties than the native enzyme or recombinant enzyme from other yeasts. We conclude that selected K. lactis strains, with opportune physiological properties and transcription regulation of the heterologous gene, could be optimal hosts for laccase isoenzyme production. [ABSTRACT FROM AUTHOR]

In yeast, many environmental stimuli are sensed and signaled by the MAP kinases pathways. In a previous work, we showed that cesium chloride activates the HOG pathway and modulates the transcription of several genes, especially those involved in cell wall biosynthesis and organization. The response to cesium was largely overlapping with the response to salt and osmotic stress. However, when low cesium chloride concentrations were used, a specific response was eventually elicited. The cesium-specific response involved the Yaf9 protein and its activity of chromatin remodeling and transcription regulation. In this paper we show that the osmotic activity of cesium salt is detected and signaled by the two branches downstream of the Sln1 and Sho1 sensors of the HOG pathway, that seem to possess different but exchangeables functions in cesium signaling. However, the cesium-specific response mediated by Yaf9, that counteracts the efficiency of the HOG pathway, is not routed by these sensors. In addition, the cesium response also involves the cell wall integrity (CWI) pathway, which is activated by low concentration of cesium chloride. Mutations blocking the CWI pathway show sensitivity to this salt. [ABSTRACT FROM AUTHOR]

We analyzed the global transcriptional response of Saccharomyces cerevisiae cells exposed to different concentrations of CsCl in the growth medium and at different times after addition. Early responsive genes were mainly involved in cell wall structure and biosynthesis. About half of the induced genes were previously shown to respond to other alkali metal cations in a Hog1-dependent fashion. Western blot analysis confirmed that cesium concentrations as low as 100 mM activate Hog1 phosphorylation. Another important fraction of the cesium-modulated genes requires Yaf9p for full responsiveness as shown by the transcriptome of a yaf9-deleted strain in the presence of cesium. We showed that a cell wall-restructuring process promptly occurs in response to cesium addition, which is dependent on the presence of both Hog1 and Yaf9 proteins. Moreover, the sensitivity to low concentration of cesium of the yaf9-deleted strain is not observed in a strain carrying the hog1/yaf9 double deletion. We conclude that the observed early transcriptional modulation of cell wall genes has a crucial role in S. cerevisiae adaptation to cesium. [ABSTRACT FROM AUTHOR]

In Kluyveromyces lactis, Rag3 regulates both fermentative metabolism and thiamine biosynthesis. Regulation of fermentation is exerted at the level of transcription of KlPDC1. We have isolated and identified a mutation of the transcription factor KlGCR1, Klgcr1-1, which suppressed the fermentative-deficient phenotype associated with the RAG3 deletion. In the mutant, the transcription of KlPDC1 was restored. However, we found that the suppression was not specific to the RAG3 mutation, as the Klgcr1-1 mutation could also suppress the fermentative defect associated with mutation of Sck1, another regulator of glycolysis. [ABSTRACT FROM AUTHOR]