Evaluation of Pyrophosphate-Driven Proton Pumps in Saccharomyces cerevisiae under Stress Conditions

Abstract

In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H⁺-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H⁺-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PP_i) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H⁺-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pH_i) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L^-1 acetic acid stress was observed in the vacuolar membrane H⁺-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H⁺-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry.

Keywords: ATP; Saccharomyces cerevisiae; acetic acid; glucose; mQueen-2m; pH homeostasis; pHluorin; proton translocating pyrophosphatase (H+-PPase); proton-translocating ATPase (H+-ATPase); xylose.

Figure 1

Growth characteristics of the control (TMB 3504), vacuolar membrane H⁺-PPase (TMB_KS_S02), and cell membrane H⁺-PPase (TMB_KS_S03) strains grown in a Verduyn mineral media with 20 g·L⁻¹ glucose and 0, 3, and 6 g·L⁻¹ of acetic acid at an initial pH of 5 and 3.7 in microtiter plates. (A) Growth rates of the three strains over three biological replicates at different concentrations of acetic acid in minimal media at two different pHs (OD₆₂₀ = optical density at 620 nm). The error bars represent the standard deviation between the biological replicates. [NS represents a p-value greater than 0.1, (.) represents a p-value between 0.05 and 0.1, (*) represents a p-value between 0.01 and 0.05, (**) represents a p-value between 0.001 and 0.01, (***) represents a p-value between 0 and 0.001]. (B) The growth profiles of the parent strain (TMB 3504) (Grey) and the vacuolar membrane H⁺-PPase strain (TMB_KS_S02) (Green) under the most stressful condition tested (pH 3.7, 6 g·L⁻¹ acetic acid). The square, diamond and the triangle shapes are biological replicates with three technical replicates (three individual wells inoculated from separate colonies obtained from a single clone) represented as the standard deviations for each biological replicate. The solid lines are the logistic models fitted through the technical replicates for each biological replicate. ND = not determined.

Figure 2

Observed factors for anaerobic glucose fermentations. (A) Growth rates of the various strains [error bars represent the standard deviations between biological duplicates], (B) pH_i determined using pHluorin biosensor [the box represents the quartiles of all the pH readings obtained across the biological duplicates and the outliers are represented as dots]. (C) Intracellular ATP concentration detected with QUEEN-2m. pHluorin and QUEEN-2m measurements are made across two biological duplicates (represented as standard deviations) at distinct time points during glucose fermentation. The dashed grey line in (C) represents the ATP depletion condition established by resuspending the cells in mineral media with 0.5 g·L⁻¹ of 2-deoxy-D-glucose for 1 h. TMB_3504 is the parent strain, and TMB_KS_S02 and TMB_KS_S03 are its derivatives with the proton pump targeted to the vacuolar and cytosolic membrane, respectively. TMB_KS_S04, TMB_KS_S06 and TMB_KS_S08 are the derivatives of TMB_3504, TMB_KS_S02 and TMB_KS_S03, respectively, with the pHluorin biosensor. TMB_KS_S05, TMB_KS_S07 and TMB_KS_S09 are the derivatives of TMB_3504, TMB_KS_S02 and TMB_KS_S03, respectively, with the QUEEN-2m biosensor.

Figure 3

Growth rates of the various strains during anaerobic xylose fermentations [error bars represent the standard deviations between biological duplicates]. ND = not determined.

Figure 4

Time series of cell dry weights (CDW) for anaerobic fermentations on xylose 50 g·L⁻¹ in bioreactors. (A) The parent strain, the vacuolar, and the cytosolic strain. (B) Derivative strains of (A) with pHluorin. (C) Derivative strains of (A) with QUEEN-2m. The metabolic profiles for these fermentations are shown in Supplementary Figures S7–S9. The error bars are the standard deviations obtained from biological duplicates.

Figure 5

Specific productivity (q), volumetric productivity (Q), and yield (Y) of the various strains grown on 50 g·L⁻¹ xylose in bioreactors. [Standard deviations between replicates are represented as error bars]. (A) The q_xylose, q_xylitol, and q_ethanol calculated during logarithmic growth. (B) The Q_xylose, Q_xylitol, and Q_ethanol calculated during logarithmic growth. (C) The Y_xylose, Y_xylitol, and Y_ethanol over the entire fermentation period. TMB 3504 is the parent strain, and TMB_KS_S02 and TMB_KS_S03 are its derivatives with the proton pump targeted to the vacuolar and cytosolic membrane, respectively. TMB_KS_S04, TMB_KS_S06 and TMB_KS_S08 are the derivatives of TMB_3504, TMB_KS_S02 and TMB_KS_S03, respectively, with the pHluorin biosensor. TMB_KS_S05, TMB_KS_S07, and TMB_KS_S09 are the derivatives of TMB_3504, TMB_KS_S02, and TMB_KS_S03, respectively, with the QUEEN-2m biosensor. NA = not applicable.

Figure 6

Time course of the carbon dioxide (CO₂) production profiles for each of the biological replicates for various fermentations carried out in bioreactors (measured at either 5 or 10 s intervals (Section 2.6)). The first ~18 h of the fermentation was carried out without sparging and hence removed from the image. (A) CO₂ production profile for the parent strain (TMB 3504) and the parent strain with pHluorin (TMB_KS_S04) normalized to the parent strain (TMB 3504). (B) CO₂ production profile for the vacuolar membrane H⁺-PPase strain (TMB_KS_S02) and the vacuolar membrane H⁺-PPase strain with pHluorin (TMB_KS_S06) normalized to the vacuolar membrane H⁺-PPase strain (TMB_KS_S02). The CO₂ production profiles for the strains with pHluorin are normalized to its ancestor strain.

Figure 7

(A) Intracellular pH determined using pHluorin [the box represents the quartiles of all the pH readings obtained across the biological duplicates and the outliers are represented as dots]. (B) Intracellular pH determined using pHrodo^® green [the box represents the quartiles of all the pH readings obtained across the biological duplicates]. (C) Intracellular ATP concentration detected with QUEEN-2m. pHluorin and QUEEN-2m measurements are made across two biological duplicates (represented as standard deviations) at distinct time points during glucose fermentation. The dashed grey line defines the ATP depletion condition. (TMB_3504 is the parent strain, and TMB_KS_S02 and TMB_KS_S03 are its derivatives with the proton pump targeted to the vacuolar and cytosolic membrane, respectively. TMB_KS_S04, TMB_KS_S06 and TMB_KS_S08 are the derivatives of TMB_3504, TMB_KS_S02, and TMB_KS_S03, respectively, with the pHluorin biosensor. TMB_KS_S05 and TMB_KS_S07 are the derivatives of TMB_3504 and TMB_KS_S02, respectively, with the QUEEN-2m biosensor).