Date Published: July 19, 2011
Author(s): Noppon Lertwattanasakul, Nadchanok Rodrussamee, Savitree Limtong, Pornthap Thanonkeo, Tomoyuki Kosaka, Mamoru Yamada.
Kluyveromyces marxianus possesses a useful potential to assimilate a wide variety of substrates at a high temperature, but the negative effect by coexisting glucose is critical for utilization of biomass containing various sugars. Such a negative effect on the activity of inulinase, which is the sole enzyme to hydrolyze sucrose, raffinose and inulin, has been demonstrated in K. marxianus without analysis at the gene level. To clarify the utilization capability of sucrose, raffinose and inulin and the glucose effect on inulinase in K. marxianus DMKU 3-1042, its growth and metabolite profiles on these sugars were examined with or without glucose under a static condition, in which glucose repression evidently occurs. Consumption of sucrose was not influenced by glucose or 2-deoxyglucose. On the other hand, raffinose and inulin consumption was hampered by glucose at 30°C but hardly hampered at 45°C. Unlike Saccharomyces cerevisiae, increase in glucose concentration had no effect on sucrose utilization. These sugar-specific glucose effects were consistent with the level of inulinase activity but not with that of the KmINU1 transcript, which was repressed in the presence of glucose via KmMig1p. This inconsistency may be due to sufficient activity of inulinase even when glucose is present. Our results encourage us to apply K. marxianus DMKU 3-1042 to high-temperature ethanol fermentation with biomass containing these sugars with glucose.
Glucose-mediated negative control in the budding yeast Saccharomyces cerevisiae is a model system for transcriptional repression (Ronne 1995; Entian and Schuller 1997; Gancedo 1998). This control, called glucose repression, physiologically occurs when glucose coexists as one of carbon sources, by which cells shut down the transcription of a specific set of genes for respiration, gluconeogenesis and the metabolism of alternative carbon sources, which may allow cells to perform rational energy consumption.
Results presented in this paper showed the utilization capability of Suc, Raf and Inu at a high temperature in K. marxianus DMKU 3-1042, which is the most thermotolerant among strains available (Nonklang et al. 2008) and efficiently utilizes hexose and pentose sugars (Rodrussamee et al. 2011), as well as the glucose effects on consumption of these sugars and on the expression of KmINU1 for inulinase responsible for their hydrolysis. This work thus also provides an insight into the fundamental mechanism of glucose repression in K. marxianus. The strain can assimilate the three sugars at a high temperature even under a static condition, though the respiratory yeast exhibits a sugar assimilation activity much higher under a shaking condition than that under a static condition (Rodrussamee et al. 2011). The hydrolysis and consumption of Suc, Raf or Inu in the presence of Glc were found to be preferable at a high temperature, and no detectable effect of glucose repression on Suc consumption was observed. Therefore, this strain is applicable for high-temperature ethanol fermentation with a biomass such as sugar cane juice containing mainly Suc, Glc and Frt.
YP: yeast extract and peptone; Glc: glucose; Suc: sucrose; Raf: raffinose; Inu: inulin; Frt: fructose; Mel: melibiose; Gal: galactose; 2-DOG: 2-deoxyglucose; GH32: glycoside hydrolase family 32; SD: standard deviation; RT-PCR: reverse transcriptase-polymerase chain reaction; NITE: National Institute of Technology and Evaluation; NBRC: NITE Biological Resource Center
The authors declare that they have no competing interests.