The growing concerns about the depletion of fossil fuels and their environmental impact are the driving forces to find alternative energy sources. Bioethanol produced from renewable materials offers a promising alternative fuel, as its combustion releases fewer nitrogen oxides and solid particles into the environment. In particular, second-generation bioethanol, made from lignocellulosic biomass, derived from agricultural and forestry waste, makes it a more sustainable option since it utilizes non-food raw materials [1].
Xylose, the second most abundant monosaccharide after glucose and the most prevalent pentose sugar, is a key component of hemicelluloses. More than half of the world’s agricultural plant biomass consists of lignocellulosic crop residues [2]. By utilizing xylose, the bioethanol yield from raw materials can be increased, and maximizing xylose uptake by yeast cells is crucial for improving its use in lignocellulosic bioethanol production.
This Ogataea polymorpha, a thermotolerant methylotrophic yeast, is capable of fermenting xylose at elevated temperatures. However, transporting xylose into yeast cells in the presence of both glucose and xylose remains a challenge for efficient lignocellulosic ethanol production. In xylose-utilizing wild-type yeast cultures, xylose consumption begins only after glucose is depleted, leading to longer fermentation periods and incomplete sugar conversion from lignocellulose hydrolysates [3]. This issue arises because transporters responsible for xylose uptake have a stronger affinity for glucose than for xylose. Moreover, some potential xylose transporters are removed from the yeast plasma membrane when glucose levels are low, making it essential to modify these transporters to enhance their stability [4]. For this reason, transporters found in Saccharomyces cerevisiae Gal2 and Hxt7 or O. polymorpha Hxt1 are seen as potential xylose carriers, and mutagenesis could improve xylose uptake and its utilization in alcoholic fermentation. This research project aims to investigate the importance of the O. polymorpha Hxt1 transporter and heterologous modifications of the S. cerevisiae Gal2 or Hxt7 transporters in O. polymorpha yeast, particularly for high-temperature alcoholic fermentation. Registration of the activity of energy metabolism (respiration, glycolysis, ATP synthesis) allows to determine the rate of glucose and xylose uptake into the cells.
In our experiments, respiration and glycolysis of O. polymorpha cells were evaluated in different environmental conditions, such as reduced or increased sugar concentrations, different temperatures, in salt buffers and/or the growth media. Results of the experiments showed that a high concentration of one sugar (glucose or xylose) inhibited the entry of the other sugar into O. polymorpha cells.