Single-cell measurements of enzyme levels as a predictive tool for cellular fates during organic acid production

Stefan Zdraljevic, Drew Wagner, Kevin Cheng, Laura Ruohonen, Jussi Jäntti, Merja Penttilä, Orna Resnekov, C. Gustavo Pesce

Research output: Contribution to journalArticleScientificpeer-review

5 Citations (Scopus)

Abstract

Organic 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 ~20 times fewer acidified cells and ~2-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.

Original languageEnglish
Pages (from-to)7569-7582
JournalApplied and Environmental Microbiology
Volume79
Issue number24
DOIs
Publication statusPublished - 1 Dec 2013
MoE publication typeA1 Journal article-refereed

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organic acid
organic acids and salts
enzyme
Acids
acid
Enzymes
enzymes
acidification
cells
acids
productivity
toxicity
Xylose
xylose
buffering
plasmid
Oxidoreductases
microscopy
viability
fluorescence

Cite this

Zdraljevic, Stefan ; Wagner, Drew ; Cheng, Kevin ; Ruohonen, Laura ; Jäntti, Jussi ; Penttilä, Merja ; Resnekov, Orna ; Pesce, C. Gustavo. / Single-cell measurements of enzyme levels as a predictive tool for cellular fates during organic acid production. In: Applied and Environmental Microbiology. 2013 ; Vol. 79, No. 24. pp. 7569-7582.
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Single-cell measurements of enzyme levels as a predictive tool for cellular fates during organic acid production. / Zdraljevic, Stefan; Wagner, Drew; Cheng, Kevin; Ruohonen, Laura; Jäntti, Jussi; Penttilä, Merja; Resnekov, Orna; Pesce, C. Gustavo.

In: Applied and Environmental Microbiology, Vol. 79, No. 24, 01.12.2013, p. 7569-7582.

Research output: Contribution to journalArticleScientificpeer-review

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AU - Zdraljevic, Stefan

AU - Wagner, Drew

AU - Cheng, Kevin

AU - Ruohonen, Laura

AU - Jäntti, Jussi

AU - Penttilä, Merja

AU - Resnekov, Orna

AU - Pesce, C. Gustavo

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AB - Organic 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 ~20 times fewer acidified cells and ~2-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.

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