Distinct Metabolic Flow in Response to Temperature in Thermotolerant Kluyveromyces marxianus

Abstract

The intrinsic mechanism of the thermotolerance of Kluyveromyces marxianus was investigated by comparison of its physiological and metabolic properties at high and low temperatures. After glucose consumption, the conversion of ethanol to acetic acid became gradually prominent only at a high temperature (45°C) and eventually caused a decline in viability, which was prevented by exogenous glutathione. Distinct levels of reactive oxygen species (ROS), glutathione, and NADPH suggest a greater accumulation of ROS and enhanced ROS-scavenging activity at a high temperature. Fusion and fission forms of mitochondria were dominantly observed at 30°C and 45°C, respectively. Consistent results were obtained by temperature upshift experiments, including transcriptomic and enzymatic analyses, suggesting a change of metabolic flow from glycolysis to the pentose phosphate pathway. The results of this study suggest that K. marxianus survives at a high temperature by scavenging ROS via metabolic change for a period until a critical concentration of acetate is reached. IMPORTANCE Kluyveromyces marxianus, a thermotolerant yeast, can grow well at temperatures over 45°C, unlike Kluyveromyces lactis, which belongs to the same genus, or Saccharomyces cerevisiae, which is a closely related yeast. K. marxianus may thus bear an intrinsic mechanism to survive at high temperatures. This study revealed the thermotolerant mechanism of the yeast, including ROS scavenging with NADPH, which is generated by changes in metabolic flow.

Keywords: Kluyveromyces marxianus; NADPH; acetic acid; reactive oxygen species; thermotolerance; thermotolerant yeast; transcriptome analysis.

FIG 1

Ethanol fermentation of K. marxianus in long-term cultivation at 30°C and 45°C. Cells were cultivated in 2% YPD medium at 30°C (open circles) and 45°C (open triangles) under shaking conditions. (A) The cell density was estimated by measuring the turbidity at the OD₆₆₀. (B to D) Concentrations of glucose (B), ethanol (C), and acetate (D) were determined by HPLC. (E and F) pH (E) and CFU (F) were also determined as described in Materials and Methods. Error bars represent standard deviations (SD) for triplicate experiments.

FIG 2

Effects of acetic acid on cell viability and cellular levels of ROS of K. marxianus at 45°C. Cells were cultivated in 2% YPD medium supplemented with acids at 45°C under shaking conditions at 160 rpm. Six hours after inoculation, acids, including 0.118% (wt/vol) HCl (open circles), 0.2% (wt/vol) acetic acid (closed squares), and 0.4% (wt/vol) acetic acid (open squares), were added or not added (closed circles). (A and B) CFU (A) and pH (B) were determined as described in the legend of Fig. 1. (C) Relative cellular levels of ROS were measured at log phase (6 h) as described in Materials and Methods. Error bars represent SD for triplicate experiments. *, P value of <0.05.

FIG 3

Cellular and mitochondrial levels of ROS and levels of glutathione and NADPH of K. marxianus at 30°C and 45°C. Cells were cultivated in 2% YPD medium at 30°C and 45°C under shaking conditions for 6 h or 36 h. (A and B) Cellular (A) and mitochondrial (B) levels of ROS at 6 h or 36 h were estimated as described in the legend of Fig. 2. White and gray columns represent levels of ROS at 30°C and 45°C, respectively. (C to H) GSH (C), GSSG (D), the GSH/GSSG ratio (E), NADP⁺ (F), NADPH (G), and the NADPH/NADP⁺ ratio (H) were determined as described in Materials and Methods. Error bars represent SD for triplicate experiments. *, P value of <0.05.

FIG 4

Distinct characteristics of K. marxianus at 30°C and 45°C. Cells were cultivated in 2% YPD medium at 30°C and 45°C under shaking conditions. (A) The DOC was measured by a DO sensor (open circles, 30°C; open triangles, 45°C). (B) NADH oxidase activity was determined with samples taken at 6 h and 24 h. Error bars represent SD for triplicate experiments. *, P value of <0.05.

FIG 5

Ethanol fermentation of K. marxianus during short-term cultivation at 30°C and 45°C and effects of temperature upshift on K. marxianus. Cells were cultivated in 2% YPD medium at 30°C (open circles), 45°C (open triangles), or 30°C to 45°C (temperature upshift) (open squares) under shaking conditions. The temperature upshift was performed by transferring flasks from a 30°C incubator to a 45°C incubator 4 h after incubation at 30°C. (A to D) Cell density (A) and concentrations of glucose (B), ethanol (C), and acetate (D) (percent, wt/vol) were determined as described in the legend of Fig. 1. (E) The DOC was measured by a DO sensor. Error bars represent SD for triplicate experiments.

FIG 6

Effects of temperature upshift on cellular levels of ROS and mitochondrial morphology. Cells were cultivated in 2% YPD medium at 30°C, 45°C, or 30°C to 45°C under shaking conditions. The temperature upshift was performed as described in the legend of Fig. 5. (A) Relative levels of ROS were determined 30 min (30-45°C, 30 min) or 2 h (30-45°C, 2 h) after the temperature upshift. (B) Mitochondrial morphology was observed 2 h (30-45°C, 2 h) after the temperature upshift, and the ratios were determined by observation of 250 cells. White and gray columns represent fusion and fission forms of mitochondria, respectively. Determination of the cellular levels of ROS and observation of mitochondrial morphology were performed as described in Materials and Methods. Error bars represent SD for triplicate experiments. *, P value of <0.05.

FIG 7

Transcriptome analysis of the effect of temperature upshift on K. marxianus. Cells were cultivated in YPD medium at 30°C for 4 h under shaking conditions, and flasks were transferred from a 30°C incubator to a 45°C incubator for further culture for 30 min (4H30-30M45) (orange) or 2 h (4H30-2H45) (red). The control was the culture at 30°C for 6 h under shaking conditions (6H30) (yellow). Total RNA was then isolated, purified, and subjected to RNA-Seq analysis. The y axis of each graph represents the reads per kilobase of exon per million (RPKM) of each gene under the indicated conditions. Data for central metabolism (A) and the TCA cycle (B) are shown. The RPKM value of each gene represents the average value from two independent experiments. G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FDP, fructose 1,6 bisphosphate; DHAP, dihydroxyacetone phosphate; DHA, dihydroxyacetone; GAP, glyceraldehyde-3-phosphate; 1,3-DPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; 6P1,5R, 6-phospho-D-glucono-1,5-lactone; 6PG, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; Xu5P, xylulose-5-phosphate; E4P, erythrose-4-phosphate; S7P, sedoheptulose-7-phosphate; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; Q, quinone; ATP, adenosine triphosphate; ADP, adenosine diphosphate.

FIG 8

Enzyme activity analysis of the effect of temperature upshift on K. marxianus. Cells were cultivated in YPD medium at 30°C for 4 h under shaking conditions, and flasks were transferred from a 30°C incubator to a 45°C incubator for further culture for 30 min (4H30-30M45) or 2 h (4H30-2H45). Cells were also cultivated in YPD medium at 30°C for 6 h (6H30) or at 45°C for 6 h (6H45) under shaking conditions. Cells were then harvested, and ultracentrifugation supernatants were prepared for enzyme assays as described in Materials and Methods. The activities of glucose-6-phosphate dehydrogenase (A) and 6-phosphogluconate dehydrogenase (B) in the PPP, of phosphoglucose isomerase (C) and glyceraldehyde-3-phosphate dehydrogenase (D) in glycolysis, and of fructose-1,6-diphosphatase in gluconeogenesis (E) were determined spectrophotometrically. Error bars represent SD for triplicate experiments. *, P value of <0.05.

FIG 9

Models of metabolic flow at a high temperature (HT) in K. marxianus. (A) Log phase, in which glucose is present in the culture. (B) Acetate accumulation phase, in which there is no glucose in the culture.