Understanding the impact of the cofactor swapping of isocitrate dehydrogenase over the growth phenotype of Escherichia coli on acetate by using constraint-based modeling
Understanding the impact of the cofactor swapping of isocitrate dehydrogenase over the growth phenotype of Escherichia coli on acetate by using constraint-based modeling
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DOI：
10.1371/journal.pone.0196182
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发表时间：
2018-04-20
期刊：
影响因子：
3.7
通讯作者：
Cabrera, Ricardo
中科院分区：
文献类型：
--
作者：
Armingol, Erick;Tobar, Eduardo;Cabrera, Ricardo
已经提出，异柠檬酸脱氢酶（ICDH）的NADP（+）-特异性进化为微生物在乙酸盐作为唯一碳源和能量源上生长的适应。在大肠杆菌中，将ICDH的辅因子特异性从NADP（+）改变为NAD（+）（辅因子交换）降低了乙酸盐上的生长速率。然而，这种表型的代谢基础尚未分析。在这项工作中，我们使用基于约束的建模来研究ICDH的辅因子交换在能量生产，NADPH替代来源的响应以及ICDH和异柠檬酸裂解酶（ICL）之间的通量分配方面的影响-当细菌在乙酸盐上生长时，这是一个至关重要的分叉-。我们生成了E。表达NAD（+）-特异性ICDH而不是天然酶，并带有产生NADPH的转氢酶PntAB的缺失的大肠杆菌菌株。我们测量了它们的生长速率和乙酸吸收速率，通过通量平衡分析（FBA）模拟了代谢通量的分布，并量化了中央途径中产生NADPH的脱氢酶的比活性。ICDH的辅因子交换导致生物量产量降低三分之一，而与PntAB的存在无关。根据我们的模拟，在辅因子交换后观察到的生长速率的减少可以解释为NADPH的总产量减少一半和生物合成的碳的可用性降低，因为在异柠檬酸分叉处的分区的变化。再加上ATP总产量的增加，这种情况导致不用于生长目的的ATP流量增加了10倍。PntAB被确定为主要的NADPH平衡反应，其中戊糖磷酸途径氧化分支的脱氢酶和苹果酸酶在其不存在的情况下发挥作用。我们建议在E.在乙酸盐上生长的大肠杆菌中，ICDH的NADP（+）特异性是一种不仅影响NADPH产生，而且影响碳和能量有效分配的性状。
It has been proposed that NADP(+)-specificity of isocitrate dehydrogenase (ICDH) evolved as an adaptation of microorganisms to grow on acetate as the sole source of carbon and energy. In Escherichia coli, changing the cofactor specificity of ICDH from NADP(+) to NAD(+) (cofactor swapping) decreases the growth rate on acetate. However, the metabolic basis of this phenotype has not been analyzed. In this work, we used constraint-based modeling to investigate the effect of the cofactor swapping of ICDH in terms of energy production, response of alternative sources of NADPH, and partitioning of fluxes between ICDH and isocitrate lyase (ICL) -a crucial bifurcation when the bacterium grows on acetate-. We generated E. coli strains expressing NAD(+)-specific ICDH instead of the native enzyme, and bearing the deletion of the NADPH-producing transhydrogenase PntAB. We measured their growth rate and acetate uptake rate, modeled the distribution of metabolic fluxes by Flux Balance Analysis (FBA), and quantified the specific activities of NADPH-producing dehydrogenases in central pathways. The cofactor swapping of ICDH led to one-third decrease in biomass yield, irrespective of the presence of PntAB. According to our simulations, the diminution in growth rate observed upon cofactor swapping could be explained by one-half decrease in the total production of NADPH and a lower availability of carbon for biosynthesis because of a change in the partition at the isocitrate bifurcation. Together with an increased total ATP production, this scenario resulted in a 10-fold increment in the flux of ATP not used for growing purposes. PntAB was identified as the primary NADPH balancing response, with the dehydrogenases of the oxidative branch of the Pentose Phosphate Pathway and the malic enzyme playing a role in its absence. We propose that in the context of E. coli growing on acetate, the NADP(+)-specificity of ICDH is a trait that impacts not only NADPH production, but also the efficient allocation of carbon and energy.