Repression of mitochondrial metabolism for cytosolic pyruvate-derived chemical production in Saccharomyces cerevisiae

Abstract

Background Saccharomyces cerevisiae is a suitable host for the industrial production of pyruvate-derived chemicals such as ethanol and 2,3-butanediol (23BD). For the improvement of the productivity of these chemicals, it is essential to suppress the unnecessary pyruvate consumption in S. cerevisiae to redirect the metabolic flux toward the target chemical production. In this study, mitochondrial pyruvate transporter gene (MPC1) or the essential gene for mitophagy (ATG32) was knocked-out to repress the mitochondrial metabolism and improve the production of pyruvate-derived chemical in S. cerevisiae. Results The growth rates of both aforementioned strains were 1.6-fold higher than that of the control strain. 13C-metabolic flux analysis revealed that both strains presented similar flux distributions and successfully decreased the tricarboxylic acid cycle fluxes by 50% compared to the control strain. Nevertheless, the intracellular metabolite pool sizes were completely different, suggesting distinct metabolic effects of gene knockouts in both strains. This difference was also observed in the test-tube culture for 23BD production. Knockout of ATG32 revealed a 23.6-fold increase in 23BD titer (557.0 ± 20.6 mg/L) compared to the control strain (23.5 ± 12.8 mg/L), whereas the knockout of MPC1 revealed only 14.3-fold increase (336.4 ± 113.5 mg/L). Further investigation using the anaerobic high-density fermentation test revealed that the MPC1 knockout was more effective for ethanol production than the 23BD production. Conclusion These results suggest that the engineering of the mitochondrial transporters and membrane dynamics were effective in controlling the mitochondrial metabolism to improve the productivities of chemicals in yeast cytosol.

Effect of glucose levels on carbon flow rate, antioxidant status, and enzyme activity of yeast during fermentation

“…Pyruvate produced by glycolysis is a key precursor for the biosynthesis of various chemicals. In S. cerevisiae cells, large amounts of pyruvate are consumed by the synthesis of cellular components, the production of ethanol, and mitochondrial respiration 60 . The metabolic intensity and bias of yeast can be analyzed by comparing the pyruvate content in yeast cells with the enzyme activity of the upstream and downstream processes and the product determination.…”

Control of topoisomerase II activity and chemotherapeutic inhibition by TCA cycle metabolites

“…We first assessed the effects of lowered TCA metabolite abundance by deleting mpc1 , a subunit of the mitochondrial pyruvate carrier (MPC), in the ED background. Transport of pyruvate into the mitochondrial matrix is necessary to replenish TCA cycle intermediates; loss of mpc1 leads to a significant decrease in the concentration of TCA intermediates (Herzig et al, 2012; Morita et al, 2019). Given our biochemical data, we hypothesized that topo II activity levels should be lower in the mpc1Δ strain as compared to the wildtype MPC1 strain on account of the diminished metabolite levels ( Figure 6A ).…”

Blocking mitophagy does not improve fuel ethanol production in Saccharomyces cerevisiae

“…The mechanisms by which the improvements are brought about have not been explored, but this increased ethanol production in the absence of mitophagy could be related to a lack of recycled nutrients normally provided by mitophagy, which cells use for biosynthesis. A combination of the absence of these internal building blocks and the concomitant excess of the external carbon and energy source – the sugar in the cultivation medium – would channel more of this carbon towards the fermentative pathway (36). In a different work, Jing and co-workers (35) report that deletion of ATG32 not only decreased tolerance to ethanol stress in haploid BY 4742, but also led to a lower ethanol titre (7.25 %v/v) compared to the WT (7.5 %v/v) in standard YPD medium, which is nutrient rich.…”

“…However, these authors did not observe higher ethanol yields or specific production rates in the atg32 mutant, with respect to the values displayed by the parental strain, although deletion of ATG32 significantly increased the maximum specific growth rate. It should be noted that these experiments were performed using a commercial synthetic yeast medium with 20 g/L initial glucose, meaning that nutrient limitation is much less prone to occur, when compared to the use of the same medium base, but with 150 g/L initial glucose, as performed by Shiroma et al (16, 35,36).…”

“…In addition, the different analytical techniques employed prevents direct comparison between studies of any claims regarding the absolute ethanol titres. Morita and co-workers (36) report that deletion of ATG32 increased the titres of 2,3-butanediol in engineered haploid BY4742 yeast, when compared to the parental strain. However, these authors did not observe higher ethanol yields or specific production rates in the atg32 mutant, with respect to the values displayed by the parental strain, although deletion of ATG32 significantly increased the maximum specific growth rate.…”