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首页> 外文期刊>Applied Microbiology >Single-Cell Measurements of Enzyme Levels as a Predictive Tool for Cellular Fates during Organic Acid Production
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Single-Cell Measurements of Enzyme Levels as a Predictive Tool for Cellular Fates during Organic Acid Production

机译：酶水平的单细胞测量作为生产有机酸过程中细胞命运的预测工具

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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.
机译：在许多应用中，源自工程微生物的有机酸可替代源自化石的化学物质。真菌宿主是有机酸生产的首选，因为它们可以耐受木质纤维素水解产物且pH值低，从而可以经济地生产和回收游离酸。但是，由胞质酸化引起的细胞死亡限制了生产率。胞质酸化作用异步影响细胞，表明酸生产率和/或抗毒性方面存在潜在的细胞间异质性。我们使用荧光显微镜研究了酿酒酵母中合成木糖醛酸的酶浓度，细胞质pH值和单细胞水平活力之间的关系。我们发现产生木糖醛酸的培养物会积聚细胞溶质的pH值低于5的细胞（此处称为“酸化”）。使用活细胞时间过程，我们发现酸化的可能性与木糖脱氢酶的初始水平有关，并且从0.2急剧增加到0.8，而酶的丰度仅增加了60％（希尔系数，> 6）。这种“开关状”关系可能是由于酶水平阈值所引起的，在该阈值以上，所产生的酸会使细胞的pH缓冲能力不堪重负。与此假设相符，我们表明，相对于具有可变拷贝数质粒的酶表达，从染色体基因座表达木糖脱氢酶产生的酸化细胞减少了约20倍，而木糖酸的产生则增加了约2倍。这些结果表明，进一步降低酶水平细胞间异质性的策略可能会导致木糖酸生产率的进一步提高。我们的结果证明了一种通用方法，该方法利用克隆群体的细胞间差异来揭示工程途径毒性中的因果关系。

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