Effect of different levels of NADH availability on metabolite distribution in Escherichia coli fermentation in minimal and complex media
Effect of different levels of NADH availability on metabolite distribution in Escherichia coli fermentation in minimal and complex media
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DOI：
10.1007/s00253-004-1609-3
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发表时间：
2004-09-01
影响因子：
5
通讯作者：
San, KY
中科院分区：
文献类型：
--
作者：
Berríos-Rivera, SJ;Sánchez, AM;San, KY
通过结合外部和遗传策略，实现了一系列细胞内NADH的利用。在缺氧条件下，在微量和复杂的介质中评估了这些操作对大肠杆菌中代谢物分布的影响。我们的提高细胞内NADH利用率的体内系统在大肠杆菌中表达了来自假丝酵母的异源NAD(+)依赖的甲酸脱氢酶(FDH)。异源FDH途径将I摩尔甲酸盐转化为I摩尔NADH和二氧化碳，而本地FDH途径不存在辅因子参与。以前，我们发现这种NADH再生系统使NADH的最大产量增加了一倍，从2molNADH/mol葡萄糖消耗增加到4molNADH/mol葡萄糖消耗。在目前的研究中，我们发现当碳源比葡萄糖更少的碳源与我们体内的NADH再生系统相结合时，NADH的产量可以超过4molNADH。本文通过实验证明，用不同氧化状态的碳源喂养细胞和通过异源脱氢酶途径再生NADH的策略相结合，可以达到不同水平的NADH利用率。对于简单和复杂的媒体，数据的总体趋势基本上是相似的。获得的NADH利用率与最终培养中减少的副产物比例呈正相关。在过量表达异源FDH途径的菌株中，乙醇的最大理论产量来自葡萄糖和山梨醇。
A range, of intracellular NADH availability was achieved by combining external and genetic strategies. The effect of these manipulations on the distribution of metabolites in Escherichia coli was assessed in minimal and complex medium under anoxic conditions. Our in vivo system to increase intracellular NADH availability expressed a heterologous NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii in E. coli. The heterologous FDH pathway converted I mol formate into I mol NADH and carbon dioxide, in contrast to the native FDH where cofactor involvement was not present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 mol to 4 mol NADH/mol glucose consumed. In the current study, we found that yields of greater than 4 mol NADH were achieved when carbon sources more reduced than glucose were combined with our in vivo NADH regeneration system. This paper demonstrates experimentally that different levels of NADH availability can be achieved by combining the strategies of feeding the cells with carbon sources which have different oxidation states and regenerating NADH through the heterologous FDH pathway. The general trend of the data is substantially similar for minimal and complex media. The NADH availability obtained positively correlates with the proportion of reduced by-products in the final culture. The maximum theoretical yield for ethanol is obtained from glucose and sorbitol in strains overexpressing the heterologous FDH pathway.