title: Single-cell measurements of enzyme levels as a predictive tool for cellular fates during organic acid production.
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.
authors.affiliation: VTT/MSI Molecular Sciences Institute, Berkeley, California, USA.
authors.forename: Stefan
authors.initials: S
authors.lastname: Zdraljevic
authors.forename: Drew
authors.initials: D
authors.lastname: Wagner
authors.forename: Kevin
authors.initials: K
authors.lastname: Cheng
authors.forename: Laura
authors.initials: L
authors.lastname: Ruohonen
authors.forename: Jussi
authors.initials: J
authors.lastname: Jäntti
authors.forename: Merja
authors.initials: M
authors.lastname: Penttilä
authors.forename: Orna
authors.initials: O
authors.lastname: Resnekov
authors.forename: C Gustavo
authors.initials: CG
authors.lastname: Pesce
mesh_terms: D002237:Carbohydrate Dehydrogenases / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism* / Q000633:toxicity*; D003600:Cytosol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D050296:Microbial Viability / Q000187:drug effects; D008856:Microscopy, Fluorescence; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*
chemical_list: D002264:Carboxylic Acids; D002237:Carbohydrate Dehydrogenases; C082601:aldose dehydrogenase (pyrroloquinoline-quinone)
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