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.
journal_name
Appl Environ Microbioljournal_title
Applied and environmental microbiologyauthors
Zdraljevic S,Wagner D,Cheng K,Ruohonen L,Jäntti J,Penttilä M,Resnekov O,Pesce CGdoi
10.1128/AEM.01749-13subject
Has Abstractpub_date
2013-12-01 00:00:00pages
7569-82issue
24eissn
0099-2240issn
1098-5336pii
AEM.01749-13journal_volume
79pub_type
杂志文章abstract::Nontoxigenic strains of Clostridium botulinum types C and D are converted to toxigenic strains by infection with specific Tox+ bacteriophages. The nucleic acids were extracted from five converting phages, c-st, c-468, c-203, c-d6f, and d-1873, and one nonconverting phage, c-n71, and treated with nucleases. The nucleic...
journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.54.1.69-73.1988
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doi:10.1128/AEM.01647-06
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pub_type: 杂志文章
doi:10.1128/AEM.45.4.1277-1282.1983
更新日期:1983-04-01 00:00:00
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pub_type: 杂志文章
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pub_type: 杂志文章
doi:10.1128/AEM.71.2.774-781.2005
更新日期:2005-02-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.59.5.1289-1293.1993
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.59.8.2411-2417.1993
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pub_type: 杂志文章
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更新日期:2004-03-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/aem.69.8.4697-4705.2003
更新日期:2003-08-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
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更新日期:1995-11-01 00:00:00
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doi:10.1128/AEM.57.2.568-572.1991
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journal_title:Applied and environmental microbiology
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doi:10.1128/AEM.67.2.1008-1010.2001
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更新日期:2009-09-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.01866-09
更新日期:2010-03-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
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更新日期:2012-11-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.54.11.2871-2873.1988
更新日期:1988-11-01 00:00:00