Kluyveromyces marxianus (K. marxianus) is a facultatively fermentative, non-conventional yeast (Dijken et al., 1993). K. marxianus has been isolated from many dairy products such as yoghurt, fermented milk and cheeses. This is due to the fact that this yeast has the ability to assimilate lactose and use it as a carbon source.
Traditionally, the most studied yeast has been that of Saccharomyces cerevisiae (S. Cerevisiae), which is used extensively in the biotechnological industry. This is a conventional yeast, and is studied due to its application in the production of fermented beers. Not as much information is known about K. marxianus compared to that of S. cerevisiae, with most of the findings about K. marxianus spanning many different strains. 20% of the K. marxianus strain CBS 712 has been randomly sequenced by Llorente et al (Llorente et al., 2000). However, no complete genome sequence currently exists for any K. marxianus strain.
K.marxianus is of great interest in the biotechnological industry due to many attractive features, such as thermotolerance, secretion of lytic enzymes, and it's extremely high growth rates, in fact one of the fastest growth rates of any eukaryotic microbe. Due to this, interest and study in this yeast species for biotechnological applications has increased rapidly over the last few years. The typical generation time of K. marxianus is approximately 70 minutes. Due to thermotolerance, this yeast has the ability to grow at up to 52 degrees(Fonseca et al., 2008).
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Carbon Catabolite Repression
The ability of yeast to adapt to the nutrients in their medium is very important for their growth and metabolism. Although S.cerevisiae and K.marxianus can grow on a variety of different carbon sources, the preferred carbon source for these yeast strains and others is generally glucose. Levels of glucose present can have an impact on aspects of the yeasts development, such as its growth rates and stress resistance. Depending on the different carbon sources which are present in the media, different enzymes and metabolic pathways are active. If glucose is present in the media in which the yeast is growing, the production of enzymes associated with other carbon sources is downregulated, or ceases entirely. This is known as carbon catabolite repression, and as the carbon source is usually glucose, it is often commonly referred to as glucose repression. Carbon catabolite repression in yeast is a very complex system.
When being grown on a nonfermentable carbon source, energy is provided through the respiratory chain. When glucose is present in the medium, the metabolic conditions are changed, as glycolysis begins and glucose is converted into ethanol and carbon dioxide. As glucose repression takes place, genes involved in respiration, gluconeogenesis and glyoxylate cycle are repressed, as well genes which are involved in the uptake and metabolism of other carbon sources. Such examples include GAL, SUC and MAL genes, which are involved in the metabolism of galactose, sucrose and maltose, respectively. Genes which are involved in the utilization of lactate, ethanol and glycerol are also repressed. Gluconeogenesis becomes inhibited. An increase in ribosomal RNA (rRNA) and protein synthesis leads to an over all increase in growth rates in this glucose rich medium.
Carbon Catabolite Repression in S.cerevisiae
Saccharomyces serevisiae (S. Cerevisiae), has the ability to recognise a change in the nutrients present in the media in which it is growing. S.cerevisiae is a glucose sensitive yeast. It has many sensing and signalling mechanisms which are in place to ensure that the glucose which is present is used optimally. By having the ability to recognise a change in glucose levels in the media in which it is growing, S.cerevisiae is able to create intracellulalar signals. These intracellular signals in turn create a cellular response which deals with the level of glucose present.
Glucose repression inside the cell is controlled by (hexokinase 2) Hxk2 and Mig1. Both these proteins are necessary in order for the glucose repression signal to be transmitted. Hxk2 was one of the first proteins found to be associated with glucose repression. Hxk2 is a glucose phosphorylating enzyme. In early experiments where the Hkx2 protein was deleted in S.cerevisiae strains, it was shown that glucose repression was severely disrupted. It was therefore presumed that Hxk2 was the main gene involved in glucose repression. Further experimentation by Rose et al. and De Winde et al .,however, demonstrated that deletion of the Hxk1 gene also had the ability to maintain catabolite repression. From the research carried out by Rolland et al., it was shown that catabolite repression in S.cerevisiae consists of an initial phase in which a rapid response is generated by a kinase which has the ability to phosphorylate glucose. For long term repression by glucose, Hxk2 is required. In the presence of high levels of glucose, Hxk2-dependent Mig1-repression mechanism is required for repression to be carried out.
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Mig1 is a zinc finger that binds to DNA. The sub-cellular location of Mig1 is important in glucose repression. When there is a large volume of glucose present, Mig1 moves from the cytoplasm to the nucleus. Once in the nucleus, it binds to the promoters of glucose-repressed genes, and also ceases transcription of e.g. GAL4 and MAL63, which are gene encoding activators. When the volume of glucose present decreases, Mig1 returns to the cytoplasm. When glucose is in excess, Snf1 kinase activity is repressed. When glucose is limited, Snf1 kinase activity is stimulated. The Snf1-protein kinase complex is important for the transcription of glucose repressible genes. It is believed that in the absence of glucose, Snf1 phosphorylates, therefore leading to the translocation of Mig1.
Carbon Catabolite Repression in non-conventional yeasts
Some genera of non-conventional yeasts include, Candida, Pichia, Yarrowia, as well as Kluyveromyces. Less research has been carried out to date in catabolite repression of non-conventional yeasts compared to that of S.cerevisiae. It is believed that catabolite repression in the Kluyveromyces species is conserved, but not identical, to that of S.cerevisiae. Kluyveromyces lactis (K.lactis) is the sister species of K.marxianus, and therefore, the two species are closely related. K.lactis is useful due to its possibilities as a utilizer of residual whey proteins.
The RAG5 gene in K. lactis encodes a protein which is homologous to that of hexokinase in S.cerevisiae. Until the work of Kettner et al., it was believed the only glucose phosphorylating enzyme in K.lactis appeared to be that of RAG 5, whereas S.cerevisiae contains 3 phosphorylating enzymes. It has since been proven that K.lactis also contains a glucokinase, referred to as KlGlk1, encoded by ORF KLLA0C01. As can be seen with the rag5 gene, KlGlk1 also phosphorylates on glucose. Also as with rag5, KlGlk1 does not act on fructose. The single hexokinase gene in K.lactis controls transcription of the glucose carrier gene RAG1. In rag5 mutants, the inducible transcription of RAG1 transporter gene which is inducible by glucose is blocked. Glucose transport in K.lactis appears to take place by facilitated fusion. Also, 2 different genes encoding transporters with different affinities for glucose have been identified, that is HGT1 and RAG1. HGT1 is associated with high affinity glucose repression, and RAG1 is associated with a transporter with low affinity for glucose transport.