Single-Cell Measurements of Enzyme Levels as a Predictive Tool for Cellular Fates during Organic Acid Production

Abstract

bOrganic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as "acidified"). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This "switch-like" relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell's pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ϳ20 times fewer acidified cells and ϳ2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways.

Effects of glucose, ethanol and acetic acid on regulation of ADH2 gene fromLachancea fermentati

“…Acetic acid co-production can influence yeast ADH2 activity during cell growth via the metabolic oxidation of carbon sources that generates large amounts of organic acids. To manage the cytosolic pH homeostasis, the production of acids must be kept in equilibrium with their utilizations ( Zdraljevic et al, 2013 ). As ADH2 involved in the oxidation of ethanol and acetic acid production, it was relevant to determine the correlation between the production of organic acid, particularly of acetic acid, and the changes of ADH2 activity.…”

Transcriptome of Saccharomyces cerevisiae during production of D-xylonate

“…The PKA pathway is one of the major regulators of the cell cycle and of general stress responses [ 53 ], but D-xylonate producing cells appear to have an activated PKA pathway during stationary phase, when they should not. Acidification of the cytosol, which has been observed in D-xylonate producing cells [ 9 , 54 ], may lead to accumulation of cAMP and activation of the PKA pathway [ 51 , 53 , 55 ], although measurable increase in cAMP levels was not observed in D-xylonate producing cells. None-the-less, some of the negative regulators of the PKA pathway ( IRA1 , IRA2 and RGS2 ) were less expressed in the production than in the control strain, and the lack of down-regulation of Rap1 targets [ 41 ] (Additional file 7 ) may also reflect PKA activation.…”